Review ArticleThemes and Variations Regulation of RpoN-DependentFlagellar Genes across Diverse Bacterial Species

Jennifer Tsang and Timothy R Hoover

Department of Microbiology University of Georgia Athens GA 30602 USA

Correspondence should be addressed to Timothy R Hoover trhooverugaedu

Received 4 November 2013 Accepted 16 December 2013 Published 2 January 2014

Academic Editors A S Lang P Soultanas and O Tsodikov

Copyright copy 2014 J Tsang and T R Hoover This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Flagellar biogenesis in bacteria is a complex process in which the transcription of dozens of structural and regulatory genes iscoordinated with the assembly of the flagellum Although the overall process of flagellar biogenesis is conserved among bacteriathe mechanisms used to regulate flagellar gene expression vary greatly among different bacterial species Many bacteria use thealternative sigma factor 12059054 (also known as RpoN) to transcribe specific sets of flagellar genes These bacteria include members ofthe Epsilonproteobacteria (egHelicobacter pylori andCampylobacter jejuni) Gammaproteobacteria (egVibrio and Pseudomonasspecies) and Alphaproteobacteria (eg Caulobacter crescentus) This review characterizes the flagellar transcriptional hierarchiesin these bacteria and examines what is known about how flagellar gene regulation is linked with other processes including growthphase quorum sensing and host colonization

1 Introduction

The flagellum is an exquisitely complex nanomachine that isthe primary means for motility in many bacteria Given thatmotility plays a vital role in important microbial processessuch as chemotaxis host colonization and biofilm formationunderstanding how bacteria regulate flagellar biogenesis iscritical for developing new strategies for the control ofharmful microbes and manipulation of useful ones Flagellarbiogenesis is a highly ordered process that involves thecoordinated regulation of dozens of structural and regulatorygenes with the assembly of the flagellum As expected forchoreographing such an intricate process flagellar biogenesisinvolves some of the most sophisticated regulatory mecha-nisms found in microbiology

Although the structure of the flagellum differs slightlybetween Gram-negative type and Gram-positive type bac-teria in all cases the bacterial flagellum is comprised ofthree main parts the basal body hook and filament [1]Components of the basal body are located within or areclosely associated with the cell envelope The basal bodyconsists of several distinct structures including the C ringa type III secretion system known as the flagellar protein

export apparatus the flagellar motor the rod and ringsthat anchor the flagellum to the membrane (Figure 1(a))BothGram-negative andGram-positive type bacteria possessMS and P rings located in the inner membrane and pep-tidoglycan layer respectively Gram-negative type bacteriapossess another ring known as the L ring located in the outermembraneThese rings provide support for the rod as it goesthrough the cell envelopeDepending on the bacterial speciesthe C ring consists of a complex of three or four differenttypes of protein subunits assembled around the export poreof the flagellar protein export apparatus and extending intothe cytoplasm The C ring has a role in switching therotational direction of the flagellum but has an additionalrole in directing protein substrates to the flagellar proteinexport apparatus for transport [2] The flagellar proteinexport apparatus (Figure 1(b)) transports axial componentsof the flagellum (ie proteins that constitute the rod hookand filament) across the cell membrane where they areincorporated at the distal end of the nascent flagellum [1 3 4]

Despite the commonality in flagellar architecture theways in which bacteria regulate expression of their flagellargenes vary remarkably (reviewed by Smith and Hoover [5]and Anderson et al [6]) This review focuses on systems

Hindawi Publishing CorporationScientificaVolume 2014 Article ID 681754 14 pageshttpdxdoiorg1011552014681754

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Rod

IM

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(a)

FlhA FlhB R P MS ring

Inner membrane

Rod

Lumen

CytoplasmFliI FliH FliH

C ring

FliE

Q O

(b)

Figure 1 Structure of the flagellum (a) An overview of the structure of the flagellum in Gram-negative bacteria Abbreviations are as followsL ring (L) P ring (P) outer membrane (OM) inner membrane (IM) and C ring (C) (b) Components of the flagellar basal body Exportapparatus proteins are shown in light blue Abbreviations of the export apparatus proteins are as follows FliR (R) FliP (P) FliO (O) and FliQ(Q) Details of the organization of the export apparatus proteins are not known although results from genetic studies suggest associationsbetween FlhA and the MS ring [10] FlhB and FliR [11] and FliO and FliP [12] FliI is an ATPase which forms a heterotrimer with FliHTheseproteins function together with other chaperones (not shown) to shuttle protein substrates to the export pore Upon docking with a platformformed by the large cytoplasmic domains of FlhA and FlhB a larger FliI

6FliH12complex is formed Inmost bacteria the C ring is composed of

three different types of protein subunits (FliG FliM and FliN)The C ring in Salmonella contains an estimated 26 copies of FliG 34 copies ofFliM and sim136 copies of FliN FliG is closest to the membrane and interacts with the MS ring while FliN is the most distal to the membraneand FliM is situated between FliG and FliN The H pylori C ring contains FliG FliM and FliN plus an additional protein subunit (FliY)that shares homology with FliN [13] Additional information on the bacterial flagellar protein export apparatus can be found in reviews byMinamino et al [1] and Macnab [14]

which utilize the alternative sigma factor 12059054 (also known asRpoN) for regulating transcription of specific sets of flagellargenes Sigma factors bind core RNApolymerase and allow theresulting RNA polymerase holoenzyme to recognize specificpromoter sequences [7] All bacteria possess a primary sigmafactor (RpoD referred to as 12059070 in E coli) and most alsoutilize one or more alternative sigma factors for transcrip-tion of specific sets of genes A unique feature of RpoN-dependent transcription is the requirement of an activatorto stimulate the transition of a closed complex between12059054-RNA polymerase holoenzyme (12059054-holoenzyme) and the

promoter to an open promoter complex that is able to initiatetranscription [8 9] Escherichia coli and Salmonella are thearchetypes for flagellar biogenesis and gene regulation butthese bacteria do not use RpoN for transcription of flagellargenes Nevertheless we refer to the E coliSalmonella systemsthroughout the review to make inferences for other systemsthat are not as well characterized The bacteria that utilizeRpoN in flagellar gene expression constitute a diverse groupof microorganisms some of which are human or animalpathogens We discuss aspects of flagellar gene regulationin response to host and environmental stimuli and potentialramifications of these responses for survival and persistenceof the bacteria

2 Transcriptional Hierarchies GoverningFlagellar Gene Expression

Though microorganisms utilize various mechanisms to con-trol flagellar gene expression in most (if not all) bacteria

the flagellar genes are transcribed in an organized fashionwhere genes that encode components needed early in flagellarbiogenesis are transcribed before genes encoding proteinsrequired later in the assembly process (Figure 2) To a certainextent these transcriptional hierarchies are governed by theorganization of the flagellar genes into different regulonsbased on the sigma factors required for their transcriptionThese transcriptional hierarchies are further coordinated byregulatory proteins that control expression of various setsor classes of genes However the mechanism by which sucha hierarchy is regulated in a given bacterial species is notalways understood and varies among organisms A masterregulator(s) initiates the flagellar gene transcription cascadeand is generally thought to couple flagellar biogenesis tothe cell cycle For some bacteria however master regulatorshave yet to be identified The master regulator stimulatestranscription of the early genes which generally encodecomponents of the basal body as well as an array of regulatoryproteins which depending on the bacterium includes RpoNFliA (12059028) FlgM (an anti-12059028 factor) or other proteinsFollowing completion of the basal body and hook late genesencoding components of the filament are expressed

21 H pylori and C jejuni The best studied membersof the Epsilonproteobacteria are Campylobacter jejuni andHelicobacter pylori C jejuni is a food-borne pathogen thatcolonizes the intestinal tract where it causes severe diarrheawhile H pylori colonizes the stomach where it can causepeptic ulcer disease which can progress into gastric cancerif left untreated Both organisms synthesize polar flagella that

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Rod IM

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H pyloriC jejuni

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Export apparatus rings FlgS FlgR

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Export apparatus motor filament cap rings FleN

FleS FleR

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hook cap

Hook

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FlaAflagellar

cap

Alternative flagellins FlgM

motor

FliC FleL FlgM FlgN chemotaxis

proteins

Flagellins

Masterregulator

Early Middle Late

Figure 2 Diversity in flagellar gene transcription hierarchies Transcriptional hierarchies are compared between the EpsilonproteobacteriaVibrio Pseudomonas and C crescentus Regulatory proteins and sigma factors involved in controlling the transcriptional hierarchies areindicated above the arrows Early middle and late genes are indicated between the arrows

are required for colonization of the host [15ndash18] Moreoverthe degree of motility appears to be important for H pylorivirulence as more motile strains are able to maintain higherbacterial density and inflammation in the stomach cardiathan those that are less motile [19]

In H pylori and C jejuni flagellar gene regulationinvolves the primary sigma factor RpoD as well as thealternative sigma factors RpoN and FliA (Figure 2) A masterregulator which activates transcription of the early flagellargenes has not been identified for H pylori or C jejuni It ispossible that these bacteria lack a true master regulator offlagellar biogenesis a possibility that was suggested byNiehusand coworkers since motility is required for the obligateparasitic lifestyle of H pylori [20] Alternatively H pylorior C jejuni could possess a master regulator for flagellarbiogenesis that has other essential roles which might explainwhy it has not been identified from mutagenesis screens

The organization of flagellar genes into regulons basedon the sigma factor required for transcription is very similarthough not identical in H pylori and C jejuni RpoD isrequired for transcription of genes encoding components ofthe basal body as well as regulatory proteins that controltranscription of genes required at later stages of flagellarbiogenesis [20] Other genes regulated by RpoD include flhF

and flhG whose products influence the localization of flagellato the cell pole and number of flagella per cell respectively[21ndash23] It is not known if transcription of the early flagellargenes is temporally regulated and intimately associated withthe cell cycle as occurs in E coli and Salmonella [24 25] orif these genes are transcribed constitutively Distinguishingbetween these two options would require one to followtemporal changes of early flagellar genes transcription insynchronous cultures which has not been reported

Transcription of genes needed midway through flagellarbiogenesis is dependent on RpoN These genes encode theproximal rod proteins the hook protein hook-associatedproteins the hook-length control protein (FliK) a minorflagellin (FlaB) and enzymes required for glycosylation ofthe flagellins [20 26 27] The transcription of the RpoN-dependent genes is activated by a two-component regulatorysystem consisting of the sensor kinase FlgS and the responseregulator FlgR [26 28ndash30] FlgS differs from most sensorkinases in that it is not membrane bound The signal orcellular cue to which FlgS responds has yet to be identifiedbut several studies (see below) have implicated the flagellarprotein export apparatus in this process FlgS uses ATP toautophosphorylate a specific histidine residue in responseto the cellular cue and this phosphate is subsequently

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transferred to a specific aspartate residue in FlgR to activateit Based on studies with other RpoN-dependent activators(reviewed by Bush and Dixon [31]) phosphorylation ofFlgR likely stimulates oligomerization of the protein froma dimer to a hexamer capable of activating transcriptionRpoN-dependent activators typically possess an N-terminalregulatory domain (often a response regulator domain as isthe case in FlgR) an AAA+ ATPase domain that engages12059054 and couples the hydrolysis of ATP with open complex

formation and aC-terminalDNA-binding domain that bindsan enhancer sequence (reviewed by Schumacher et al [32])H pylori FlgR is unusual in that it lacks a C-terminal DNA-binding domain and activates transcription by contacting12059054-holoenzyme in the closed promoter complex without

binding DNA [29] Although C jejuni FlgR possesses a C-terminal domain it is not needed for activation of the RpoNregulon suggesting that like its counterpart in H pyloriC jejuni FlgR engages the closed promoter complex fromsolution [33] While the C-terminal domain of C jejuni FlgRdoes not appear to play a role inDNAbinding it does preventphosphorylation of the protein by acetyl phosphatewhich caninterfere with normal flagellation [34]

The flagellar protein export apparatus has a major rolein regulating expression of the RpoN regulon of H pyloriand C jejuni as evidenced by the observation that mutationswhich interfere with formation of the export apparatusinhibit expression of RpoN-dependent flagellar genes Por-wollik and coworkers showed that inactivation of fliI orfliQ (encode a cytosolic ATPase component and integralmembrane component of the export apparatus resp) inH pylori results in reduced levels of the minor flagellinFlaB and the hook protein FlgE both of which dependon RpoN for their expression [35] Allan and colleaguesshowed that mutations in flhB (encodes an integral mem-brane component of the export apparatus) result in reducedlevels of FlaB and FlgE inH pylori [36] Smith and coworkerssubsequently demonstrated that disrupting flhB inhibitedexpression of RpoN-dependent reporter genes [37] UsingDNAmicroarrays Niehus and coworkers [20] demonstratedthat FlhA (an integral membrane component of the exportapparatus) is required for transcription of the entire RpoNregulon Hendrixson et al [38] reported that deleting anyone of several integral membrane components of the exportapparatus (FlhA FlhB FliP or FliR) inhibited expressionof an RpoN-dependent flagellar reporter gene in C jejuniStudies by Tsang and coworkers revealed thatH pylori strainswhich stably express truncated forms of FlhA are able totranscribe RpoN-dependent genes at levels that are close towild type but are unable to export flagellar protein substrates[39]Thus anymodel for how the export apparatus influencestranscription of theRpoN regulonmust take into account thatthe export apparatus need not transport protein substrates tostimulate the RpoN regulon Boll and Hendrixson recentlyshowed that loss of the MS ring protein FliF or the Cring protein FliG inhibits transcription of RpoN-dependentflagellar genes in C jejuni [40] The MS ring is the firstflagellar structure assembled and in its absence the exportapparatus presumably is not formedwhich would account for

the inhibition of the RpoN regulon in the fliF mutant FliGmay facilitate autophosphorylation of FlgS either by directlystimulating FlgS or by promoting assembly of the exportapparatus into a conformation that stimulates FlgS activity[40]

The export apparatus is an ideal indicator for the statusof flagellar assembly as it undergoes a major conformationalchange upon completion of the hook-basal body structureIn the early stages of flagellar biogenesis the export apparatusis in a conformation that transports rod- and hook-typesubstrates Upon completion of the mature hook-basal bodystructure the export apparatus undergoes a conformationalchange which is accompanied by a switch in substrate speci-ficity to filament-type substrates [41] This conformationalchange involves an autocleavage event in the C-terminalcytoplasmic domain of the export apparatus protein FlhB(referred to as FlhBC) and interactions between the C-terminal domain of the hook-length control protein FliKand FlhBC [42] The change in conformation and substratespecificity of the export apparatus may serve as a cellular cuefor the temporal regulation of flagellar genes Indeed in Ecoli and Salmonella the anti-12059028 factor FlgM is a filament-type substrate and is secreted by the export apparatus uponcompletion of the mature hook-basal body structure whichallows transcription of the 12059028-dependent flagellar genes tooccur [43]

Transcription of the RpoN-dependent flagellar genes inH pylori also requires the putative RpoN chaperone FlgZ(HP0958) [44 45] FlgZ was first implicated in having a rolein flagellar biogenesis in H pylori when it was identifiedas interacting with RpoN in a high-throughput screen of ayeast two-hybrid system (httpspimhybrigenicscom) [46]In the absence of FlgZ RpoN is turned over rapidly in Hpyloriwith a half-life of about 30 minutes compared to a half-life greater than 4 hours in wild type [45] The molecularbasis for how FlgZ protects RpoN from rapid turnover is notknown but it is possible that FlgZ binds RpoN to protectit from proteolysis or that FlgZ facilitates the association ofRpoN with core RNA polymerase which serves to protectRpoN from degradation Overexpression of RpoN restoresmotility in a H pylori flgZ mutant indicating that FlgZ isnot essential for flagellar biogenesis [45] In addition toits potential role as an RpoN chaperone FlgZ may haveadditional roles in flagellar biogenesis as it was also shown tointeract with the flagellar export apparatus protein FliH in theyeast two-hybrid system (httpspimhybrigenicscom) [46]In addition Douillard and coworkers demonstrated that FlgZbinds the flaA transcript and is needed for optimal expressionof FlaA [47] These researchers proposed that FlgZ functionswith FliH to direct flaA transcripts to the flagellar proteinexport apparatus where translation of the transcripts couldbe coupled with secretion of the nascent FlaA [47]

Transcription of genes needed late in flagellar biogenesisin H pylori and C jejuni which includes genes encodingthe major flagellin filament cap and associated chaper-ones is dependent on 12059028 (FliA) [20] Similar to E coliand Salmonella the H pylori FliA regulon is negativelyregulated by FlgM [48] Unlike E coli and Salmonella

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the inhibitory effect of FlgM on FliA in H pylori is thoughtto be alleviated via interactions between FlgM and the C-terminal cytoplasmic domain of FlhA (FlhAC) rather thanby secretion of FlgM via the export apparatus [49] C jejunialso possesses a FlgM homolog [50] but unlike H pyloriC jejuni FlgM appears to be secreted from the cell via theflagellar export apparatus [51] Interestingly interaction of Cjejuni FlgM with FliA is temperature dependent occurringat 42∘C (the optimal growth temperature for C jejuni) butnot 37∘C [51] The primary function of C jejuni FlgM is notto inhibit FliA activity during formation of the hook-basalbody structure but rather to limit the length of the flagellarfilament by suppressing expression of the FliA-dependentflagellin (FlaA) and the RpoN-dependent flagellin (FlaB) [51]The mechanism by which FlgM represses transcription ofRpoN-dependent genes in C jejuni is unknown but Wostenand coworkers have speculated that accumulation of theFliAFlgM complex may inhibit phosphorylation of FlgSandor FlgR [51] FlgM also appears to inhibit transcriptionof RpoN-dependent genes inH pylori but so far this has onlybeen demonstrated in an flhAmutant background [20]

22 Vibrio Species The vibrios are typically saltwater micro-organisms and several species cause food-borne illnessesVibrio species vary greatly in flagella morphology Vibriocholerae has a single polar sheathed flagellum while Vibrioparahaemolyticus and Vibrio alginolyticus produce a singlepolar sheathed flagellum and lateral peritrichous flagella andVibrio fischeri has a tuft of polar sheathed flagella Lateralflagella promote swarming and colonization of surfaces andthereby enhance biofilm formation upon encountering highlyviscous media or solid surfaces

The flagellar gene transcriptional hierarchy in Vibriocholerae is governed by RpoN and FliA [52] flrA encodesan RpoN dependent activator and is the master regulatorfor the flagellar gene transcriptional hierarchy [52] FlrA isregulated by cyclic di-GMP a secondarymessenger with rolesin biofilm formation and virulence which binds to FlrA andprevents it from binding the flrBC promoter [53] Early andmiddle genes are RpoN-dependent but they rely on differenttranscriptional activators for their transcription FlrA alongwith RpoN is required for transcription of the early geneswhich encode the components of the MS ring C ring andexport apparatus Other early genes encode the regulatoryproteins FlrB FlrC and FliA FlrB and FlrC constitutethe sensor kinase and response regulator respectively of atwo-component system that regulates transcription of themiddle genes in conjunction with RpoN [54] The middlegenes encode the basal body-hook structures and the coreflagellin FlaA The late genes are dependent on FliA fortheir transcription and encode alternative flagellins FlgMand components of the flagellar motor As in SalmonellaFlgM is secreted via the flagellar protein export apparatusto relieve the inhibition on FliA activity in V cholerae [55]flgA is located upstream of flgM and its promoter (RpoNindependent and FliA independent) may drive transcriptionof flgM such that FliA is repressed until formation of themature hook-basal body structure [52]

The signal(s) that stimulates the FlrBFlrC two-component system is not known FlrD is necessary fortranscription of middle and late flagellar genes making ita candidate for regulating the FlrBFlrC two-componentsystem [56] Additionally FlrD contains HAMP domains(domain present in histidine kinases adenyl cyclasesmethyl-accepting proteins and phosphatases) that areusually found in integral membrane proteins that are part ofsignal transduction pathways [56 57] Moisi and coworkersshowed that FlrD is inserted into the inner membrane andproposed that it senses the completion of the MS ring-switch-export apparatus structure and communicates thisinformation to FlrB [56]

The regulation of flagellar genes inV parahaemolyticus ismore complex since the bacterium is capable of producinglateral flagella in addition to polar flagella (reviewed byMerino et al [58] and McCarter [59]) The polar flagellarsystem (Fla) is constitutively expressed while the lateral flag-ellar system (Laf) is expressed upon impedance of the polarflagellaThe lateral flagella are used for swarmingmotility andenhance biofilm formation and host colonization by the bac-terium [60 61] When grown planktonically the bacteriumproduces a polar flagellum but when grown on highly viscousor solid medium [58] or during iron-limiting conditions[62] the bacterium produces lateral flagella Thus the polarflagellum acts as a mechanosensor to regulate expression ofthe lateral flagella genes through a mechanism that has yet tobe defined The dual flagellar system suited for locomotionunder different conditions allows V parahaemolyticus to behighly adaptive to changing habitats including planktonicenvironments surfaces and biofilms

The polar and lateral flagellar systems do not share anystructural or regulatory components (aside from RpoN) butthe regulatory networks that control polar and lateral flagellarsystems in V parahaemolyticus are similar to that of Vcholerae The lateral flagellar genes are activated by LafKand like the polar flagellar system lateral flagellar genes areboth RpoN dependent and FliA dependent [63] AlthoughLafK is homologous to master regulators of other flagellarsystems LafK does not appear to be amaster regulator for thelateral flagellar system as the 119891119897119894119872L operon (contains genesencoding components of the C ring and export apparatus) isnot LafK dependent [64] This suggests that there is anotherlevel of regulation preceding expression of these genes

23 Pseudomonas Species Pseudomonas aeruginosa is anaerobic Gram-negative Gammaproteobacterium that causesopportunistic infections particularly in patients with cysticfibrosis where it results in inflammation and sepsis (reviewedby Veesenmeyer et al [65] and Balasubramanian et al[66]) P aeruginosa which thrives on surfaces and formsbiofilms is a prevalent agent of nosocomial infections Wild-type P aeruginosa cells form biofilms which can causechronic infections P aeruginosa possesses a single polarflagellum that is required for virulence [67] Motile strainsof P aeruginosa induce activation of the inflammasome (amultiprotein oligomer responsible for the activation of theinflammatory response) whereas nonmotile strains have amarkedly reduced ability to induce inflammasome activation

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[68] Nonmotile strains of P aeruginosamay have an advan-tage during chronic infection by evading stimulation of theinnate immune responses However a nonmotile flgKmutantis defective in surface attachment suggesting a role for flagellaandor motility in the initial cell-to-surface interaction [69]Mutants in fliM and cheY are also nonmotile and are unableto form biofilm cap structures on initial biofilm colonies [70]Other Pseudomonas species include Pseudomonas fluorescenswhich possesses multiple flagella and is found in the soil andwater Some P fluorescens strains have been shown to protectplant roots against fungal infections [71] P fluorescens doesnot typically cause disease in humans but it is an opportunis-tic pathogen in immunocompromised individuals [72 73]

Like the Epsilonproteobacteria and Vibrio P aeruginosapossesses a four-tiered transcriptional hierarchy for regulat-ing flagellar gene expression FleQ is the master regulatorfor flagellar synthesis and fleQ is regulated by global factorsoutside the flagellar regulons such as cyclic di-GMP asignaling molecule important in modulating the transitionbetween planktonic and biofilm lifestyles [74] The earlyflagellar genes are dependent on FleQ and RpoN for theirtranscription and encode components of the basal body aswell as the filament cap which is somewhat surprising sinceit is not needed until late in flagellar biogenesis [75] Theearly genes also encode regulatory proteins which includeflhF fleN fleS and fleR [75 76] FleN is an antiactivator ofFleQ that maintains the normal flagellum copy number ofone per cell by downregulating genes encoding early flagellarcomponents via a negative feedbackmechanism [77 78] FleSand FleR form a two-component system that is required forthe transcription of the middle RpoN-dependent genes [75]The signal(s) required for FleS activation is unknown Themiddle genes encode components of the basal body includingrod L ring hook hook cap and hook-filament junctionproteins [75] The late genes are FliA dependent and encodeFliC (flagellin) FleL (filament length control protein) FlgMFlgN (a protein required for initiation of filament assembly)and some chemotaxis proteins [75] Transcription of fliA isconstitutive [75] and FlgM (whose transcription is also FleQdependent but not RpoN dependent) represses the activityof FliA until completion of the hook-basal body structure atwhich point it is secreted from the cytoplasm via the exportapparatus which alleviates its inhibition of FliA

24 Caulobacter crescentus Caulobacter crescentus is anAlphaproteobacterium found in freshwater that is a modelorganism for cell cycle studies C crescentus divides asym-metrically to produce two cells with distinct morphologiesa swarmer cell and a stalked cell The nonmotile stalked cellcontains a polar stalk which secretes an exopolysaccharidethat facilitates adhesion to surfaces In addition the stalkedcell initiatesDNA replication upon the start of cell division Incontrast the swarmer cell is motile via a polar flagellum andDNA replication is repressed for a defined length of time untilthe cell differentiates into a stalked cell Flagellar biogenesis iscoordinated with the cell division cycle such that all progenyswarmer cells possess a functional flagellum

Flagellar genes inC crescentus are regulated by two sigmafactors The initiation of DNA replication in the swarmer cellresults in expression and activation of the global transcriptionfactor CtrA CtrA also acts as themaster regulator for flagellargene expression by activating transcription of the early genes(encode MS ring C ring and export apparatus FlbD FliX)Additionally CtrA controls expression of rpoD [79] and isneeded for transcription of the early flagellar genes [80]FlbD and FliX are transcriptional regulators that regulateexpression of the early and middle genes The middle andlate genes which encode the hook protein and flagellins aretranscribed only after the components encoded by the earlygenes have been assembled into the nascent flagellum FlbDis an RpoN-dependent transcriptional activator which bindsftr (flagella transcriptional regulation) sequence motifs toregulate gene expression Once the early genes are expressedFlbD binds the ftr sequence motifs in the promoter regionsof early flagellar genes to inhibit transcription of these genesFor example FlbD binds the ftr4 site located upstream ofthe early gene fliF to repress its transcription once the earlygenes have been expressed [81] In contrast FlbD stimulatestranscription of the middle genes by binding to the ftrsites of the middle and late genes [81 82] FlbD activity isregulated by FliX which acts to either inhibit or stimulateFlbD In the absence of early flagellar structures FliX inhibitsFlbD activity by binding FlbD preventing it from bindingftr sites [83] Once the early flagellar structures are formedFliX stimulates FlbD The mechanism for this stimulationis unknown but it may involve another factor such asan assembled component of the flagellum which convertsFliX into a positive regulator or it may involve covalentmodifications of FliX or FlbD [83] Although FlbD possessesan N-terminal response regulator domain FlbD activity doesnot appear to be regulated by phosphorylation via a sensorkinase The exact cellular cues that regulate FlbD are notknown but FlbD activity is linked to cell division [84]

Unlike the bacteria discussed thus far flagellar biogenesisin C crescentus does not involve FliA Rather the six flagellingenes found in C crescentus are RpoN dependent Theexpression of the flagellin genes is regulated by FlbD and FlbTwhich binds the 51015840 UTR of flagellin transcripts and repressestranslation until the hook-basal body structure is assembled[85] Though the mechanism for this temporal regulation isunknown one possibility is that FlbT represses translationof flagellin genes until a positive factor binds the 51015840 UTRregion of flagellins to promote translation upon assembly ofthe mature hook-basal body structure [85]

3 Coupling Flagellar Biosynthesis withOther Cellular Activities

Though flagellar biogenesis is a complex process in itselfit is further complicated by its coordination with othercellular processes Flagellar biosynthesis is timed with othercellular activities such as cell replication and quorum sensingEnvironmental stimuli also play a role in regulating flagellarbiogenesis as bacteria respond to changes in the environmentby altering behavior needed for survival Many of these

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responses involve altering the state of flagellar biogenesiswithin these different contexts

31 Modulating Flagellar Gene Expression during the CellCycle Flagellar biosynthesis is linked to the cell cyclethrough various mechanisms and involves coordinating flag-ellar gene expression with growth phase and cell division InV cholerae chemotaxis and motility genes are upregulatedas the population enters stationary phase Of the 114 genesidentified with roles in chemotaxis and flagellar biogen-esis 72 genes were upregulated more than 2-fold duringstationary phase compared to mid-exponential phase [86]The upregulation of 60 of the 72 chemotaxis and flagellargenes is dependent on RpoS indicating a correlation betweenflagellar biogenesis and entry into stationary phase [86]The coordination between entry into stationary phase andflagellar biogenesis may aid the bacterium in exiting the hostwhen nutrients become depleted and prepare the bacteriumfor colonization of other niches or survival in environmentalreservoirs

C crescentus is a particularly good organism for studyinghow flagellar biogenesis is integrated with the cell cyclebecause of its two distinct cell morphologies The flagellarfilament is assembled just prior to cell division and allprogeny swarmer cells possess a fully functional flagellumThe synthesis of the flagellum is intimately linked to the cellcycle through regulatory proteins such as CtrA FlbD andFliX that are involved in both the cell cycle and flagellarbiogenesis This dual functionality may exist to ensure thateach swarmer cell is flagellated The master regulator forflagellar biogenesis CtrA regulates at least 95 genes [87]including the cell-division genes ftsZ [88] and ftsQA [89]Cells of a fliX mutant become filamentous as they enterlate log phase [90] demonstrating a role for this flagellarregulatory protein in cell division Flagellar biogenesis is alsolinked to the cell cycle via FlbT and FlaF FlaF is required forflagellar biogenesis and motility [91] FlbT protein levels areconstant throughout the cell cycle while FlaF levels correlateto flagellin levels both of which peak just prior to cell division[91] Since FlbT is a negative regulator for flagellin translationand peaks in flagellin levels correlate with FlaF levels thisobservation suggests that FlaF may temporally modulateFlbT activity [91]

Expression of Vibrio vulnificus FlhF which is needed forpolar localization of the flagellum is regulated by the quorumsensing master regulator SmcR flhF transcript levels in asmcRmutant are higher than wild-type levels indicating thatSmcR represses flhF expression [92] In wild-type cells flhFtranscript levels are highest during exponential phase anddecrease upon entry into stationary phase [92] In contrasttranscript levels of smcR increase as cultures enter stationaryphase suggesting that SmcR plays a major role for growthphase-dependent variation of flhF expression [92] Thisgrowth phase-dependent gene expression may be importantin host colonization as during the initial infection bacterialcell densities are low but at later stages of colonizationwhen bacterial cell densities are higher motility may not benecessary

Expression of flagellar genes is regulated in P fluorescensthroughout the growth phases by the Gac (GacAGasS) two-component system which limits flagellar biosynthesis duringexponential growth by downregulating transcription of fleQ(encodes the master regulator) [93] The Gac system posi-tively regulates production of virulence factors and quorumsensing molecules and is required for full virulence in animaland plant hosts [94] Levels of the Gac system componentsare regulated in response to growth phase as gacA and gacStranscript levels are highest at mid-exponential growth phase[95] The concomitant upregulation of virulence factors andthe downregulation of flagellar genes by the Gac system maybe a way to coordinate virulence and motility with growthphase so that virulence factors are expressed at high celldensities during host colonization and flagellar genes areturned off to facilitate attachment to the host

32 Connections between Quorum Sensing Host Colonizationand Flagellar Gene Expression In many pathogenic bacte-ria flagella aid in surface attachment and colonization Insuch cases successful colonization may require cooperationbetween quorum sensing and flagellar synthesis to integratesurface attachment and virulence gene expression at highcell densities in pathogens Surface colonization can leadto biofilm formation which is generally more resistant tohost defenses and antimicrobials Pathogens use differentmechanisms to couple expression of virulence genes withflagellar gene expression in the context of the host Thisincludes modulating quorum sensing and host colonizationbased on the status of the flagellum or modulating flagellarbiogenesis based on quorum sensing signals

Quorum sensing is used to regulate gene expression inresponse to cell densityThis is achieved through the secretionof a signaling molecule called an autoinducer (AI) AIs areproduced by bacteria and travel across the cell membrane andaccumulate in the environment Upon reaching a critical con-centration the AIs stimulate transcription of genes requiredfor group behavior luxS is responsible for the productionof AI-2 [96] and an H pylori mutant in luxS exhibitsdecreasedmotility compared towild type [97]flhA transcriptlevels are decreased in the luxS mutant but addition of 45-dihydroxy-23-pentanedione (functions as AI-2) into thegrowth medium restores flhA transcript levels to wild-typelevels [97] Like the H pylori luxS mutant a C jejuni luxSmutant is less motile than wild type [98] and the mutantdisplays reduced transcript levels of flaA [99] Using DNAmicroarrays He and coworkers found that a majority of theflagellar genes are downregulated in a C jejuni luxS mutant[98] Expression of chemotaxis genes however is unchangedin the luxS mutant indicating that the motility defect of theluxS mutant is due to defects in flagellar biogenesis and notchemotaxis [98] A rationale for the link between cell densityand flagellar biogenesis is that it may aid in colonization ofhost tissue by ensuring that a sufficient number of flagellatedbacteria are present to establish a successful infection

Regulatory proteins involved in flagellar biogenesis canregulate quorum sensing and colonization or they themselvescan be regulated by quorum sensing and colonization factorsFor example the RpoN-dependent activator FlrC not only

8 Scientifica

is required for flagellar biogenesis but also appears to play arole in host colonization by V cholerae Mutants expressinga FlrC variant incapable of being phosphorylated fail toproduce flagella and are therefore defective in colonization[54] Interestingly a mutant expressing a constitutively activeform of FlrC is also deficient in colonization even though thecells are motile and produce flagella [54] This observationdemonstrates the need for coordinating flagellar biogenesisand pathogenesis for successful colonization ConverselyVfr a major quorum sensing and virulence regulator inP aeruginosa downregulates expression of flagellar genesby repressing transcription from the fleQ promoter [100]Inhibition of flagellar biogenesis by Vfr may aid P aeruginosain persisting at the site of infection to achieve high celldensities and virulence gene expression

HapR is a transcriptional regulator involved in quorumsensing in V cholerae which also regulates expression ofvirulence genes HapR represses expression of several vir-ulence genes [101] and hapR expression is regulated by anunknown mechanism involving FliA [102] hapR expres-sion is depressed in flgM and flgD mutants (both of thesemutations alleviate FlgM repression on FliA activity) whiledeletion of fliA results in elevated hapR expression [102] Theauthors of this study speculated that expression of HapR isregulated by a regulator or small noncoding RNA (sRNA)that is part of the FliA regulon FliA-mediated regulation ofhapR appears to be linked to the shearing of flagella frombacteria that penetrate the mucin layer to infect the hostThis shearing of the flagella leads to secretion of FlgM viathe flagellar protein export apparatus and results in increasedexpression of FliA-dependent genes and repression of hapRexpression [102] The outcome of such a mechanism is thatvirulence genes which are negatively regulated by HapRare expressed specifically when V cholerae penetrates theintestinal mucous layer hapR production is also regulatedby the quorum sensing protein LuxO further linking viru-lence to cell density LuxO is an RpoN-dependent activator[103] which along with HapR regulates motility proteaseproduction and biofilm formation [101] High cell densitiesare common during late stages of infection and the HapR-mediated repression of virulence genes may aidV cholerae indetaching and finding new colonization sites [86 101]

V parahaemolyticus uses its polar flagellum to detectsurfaces by sensing impedance to the polar flagellum rotationrate [104] Once a surface is encountered expression of thelateral flagellar genes is induced Lateral flagella are importantfor host colonization as mutants that do not produce lateralflagella are deficient in adherence to HeLa cells and biofilmformation [61] Gode-Potratz and coworkers identified sim70genes that are surface responsive most of which are positivelyregulated [64] Some of the surface-regulated genes that areupregulated are virulence genes such as a putative GbpAhomolog and components of a type III secretion system [64]GbpA from V cholerae promotes adherence to chitinoussurfaces of zooplankton and human epithelia cells [105]which is consistent with the observed upregulation of gbpAin V parahaemolyticus when it encounters a surface

The P aeruginosa flagellum is an important determinantin the susceptibility of the bacterium to host defenses

P aeruginosa fliCmutants are unable to upregulate transcrip-tion of lasI and rhlI which encode enzymes that synthesizequorum sensing homoserine lactones [106] These quo-rum sensing molecules regulate production of exoproteaseswhich degrade surfactant protein-A (SP-A) an antimicro-bial that opsonizes and permeabilizes membranes of lungpathogens [106] In the absence of these exoproteases theP aeruginosa fliC mutants are cleared from the lung [106]Though it is not known why fliC mutants are unable toupregulate transcription of lasI and rhlI it is not due tothe inability to sense the environment [106] P aeruginosafliE flgE fliC and fliD mutants are also attenuated in LPSbiosynthesis which compromises the integrity of their outermembranes making them more susceptible to SP-A [107]Additionally flgE and fliC mutants make less pyocyanin aredox-active toxic secondary metabolite that is crucial forlung infection [108 109]

33 Reacting to pH Changes by Gastrointestinal PathogensGastrointestinal pathogens encounter varying environmentalconditions as they travel from the mouth to the stomach andthen to the intestines The gastric pathogen H pylori has theadded burden of long-term survival in the acidic environ-ment of the stomach Pathogens that colonize the intestinaltract must also survive passage through the stomach in highenough numbers to be able to colonize their target organsThus it is not surprising that gastrointestinal pathogens oftenmodulate gene expression in response to acidic conditions

Merrell and coworkers found that expression of approxi-mately 7 of theH pylori genes is altered by a shift to low pH[110] Many of these genes are involved in flagellar synthesiswith the majority of those being RpoN dependent [110] FliA-dependent genes such as flgM in H pylori [110] and flaAin C jejuni [111] are also upregulated at low pH conditionsThe upregulation of flagellar genes is critical for survival ofH pylori as the bacterium is not able to survive at low pHbut must swim across the mucous layer to the underlyinggastric epithelium to establish a successful infection Inaddition to enhancing transcription of flagellar genes acidicenvironments also stimulate motility and swimming speedspotentially in response to increased concentration of protonsin the stomach [110]

The basis for upregulation of the RpoN-dependent flag-ellar genes in H pylori likely involves a stimulation of FlgSactivity at low pHWen and coworkers reported thatH pylorirequires FlgS to survive acid stress (30-minute exposure topH25) [112]These researchers found that besides the knownRpoN-dependent flagellar genes FlgS was required for theupregulation of 86 genes in response to acid stress many ofwhich were previously known to be required for acid stresssurvival [112] It is not known if FlgS responds directly toacid stress or if the stress signal is mediated through anotherfactor

34 Altering Flagellar Biogenesis by Phase Variation In somebacteria flagellar biogenesis is subject to phase variationevents that affect expression of specific flagellar genes Phasevariation causes changes in phenotypes at frequencies that

Scientifica 9

are much higher than random mutations and contribute toheterogeneity within a population The ability of H pylori topersist in the host may involve the phase-variable dependentexpression of flagella that can serve as an advantage foradaptation to the host and for evading the host immuneresponse C jejuni utilizes phase variation to turn on andoff production of flagella a strategy that increases com-mensal colonization in poultry [113] Many phase variationevents occur by random reversible changes in the length ofshort DNA sequence repeats resulting from slipped-strandmispairing These events can take place within the codingsequence or in the promoter of a gene C jejuni phasevariants in which expression of flgR is in the OFF stateresult from the addition or removal of a nucleotide withinone of two homopolymeric nucleotide tracts consisting ofadenine and thymine These nucleotide tracts are locatedwithin the coding sequence of flgR causing the sequence toshift out of frame [113] Revertants to the ON state ariseupon the addition or removal of a nucleotide within theoriginal mutated homopolymeric nucleotide tract to restorethe wild-type sequence Alternatively pseudorevertants canarise in which nucleotides are removed or added close to theoriginal mutated homopolymeric nucleotide tract to restorethe correct reading frame but which result in changes in theamino acid sequence of the protein [113] flgS is also regulatedby phase variation in C jejuni providing multiple levels ofphase-variable control for theRpoN regulon [114 115] Similarmechanisms of phase variation also exist for flhA in C coli[116] and fliP in H pylori [117] which leads to the formationof truncated proteins that prevent flagellar biogenesis

A second type of phase variation in flagellar biogen-esis is mediated through alterations in DNA methylationwhich impact the expression of specific flagellar genesHost-adapted bacterial pathogens such as H pylori oftenpossess DNA methyltransferases and type III restriction-modification (R-M) systems that contain simple tandemDNA repeats which undergo slipped-strand mispairing dur-ing DNA replication that causes frameshift mutations andphase variation [118] R-M systems widespread among manybacteria confer protection from invasion by foreign DNAType III R-M systems are composed of a methyltransferase(mod) and an endonuclease (res) gene whose gene prod-ucts typically function together The switching between ONand OFF states of a DNA methyltransferase can regulateexpression of a ldquophasevarionrdquo via differential methylationof the genome in ON and OFF states [119] Many of thesephase-variable methyltransferase genes are associated withan inactive res gene resulting from frameshift mutationor are orphans that are not associated with a res genesuggesting that DNA restriction is not the primary roleof these methyltransferases (reviewed by Fox et al [120])For example H pylori modH encodes a phase-variableDNA methyltransferase and the cognate res gene containsa nonsense mutation frameshift mutation or is absent invarious H pylori strains [119] modH OFF strains havedecreased expression of flaA and fliK compared to themodHON strain [119] FlaA has a low ability to activate innateimmunity via the Toll-like receptor 5 [121] and modulatingexpression of the flagellin may be advantageous in evading

the host response Another H pylori methyltransferase withan inactive res gene is hpyAVIBM [122 123] hpyAVIBMencodes a C5 cytosine methyltransferase and contains AGrepeats in its open reading frame making it susceptible tophase variation via frame shift mutations [124] For instancehpyAVIBM from strain 26695 and hpyAVIBM from strainHPAG1 have five AG repeats whereas the four AG repeats instrain San 74 result in the translation of a truncated protein[124] Deletion of hpyAVIBM results in both the upregulationand downregulation of rpoN fliR fliD fliS motA fliK andflgK depending on the strain used [124] indicating that phasevariation of hpyAVIBM can alter flagellar biogenesis

P aeruginosa populations can possess two phenotypicvariants one which forms small rough colonies (S) andanother which forms large flat (L) colonies S variants formbiofilms in nonagitated liquid cultures whereas the L variantsdo not [125] S variants possess defects in flagellum-mediatedswimming flagellum-mediated swarming and type IV-pilus-mediated twitching but are able to revert back to the Lphenotype at relatively high frequencies suggesting that theshift between phenotypes is regulated by phase variation[125] Phase variation also occurs in P fluorescens whichcolonizes the alfalfa rhizosphere Phenotypic variants ariseduring rhizosphere colonization and are dependent on theactivity of a site-specific recombinase [126] C variants havewild-type colony morphology while F and S variants havea translucent and diffuse colony morphology [126] F andS variants which colonize distal parts of the roots andswim faster than the C variants overproduce fliC (flagellin)transcripts and synthesize flagellin filaments that aresim3 timeslonger than those of the C variant [126]

4 Conclusions and Future Directions

Flagellar biogenesis is a carefully choreographed processin which many different components of the flagellum aremade as they are needed for assembly into the nascentflagellum Bacteria have evolved elaborate and sundry reg-ulatory networks to couple flagellar gene expression withassembly A driving force for the evolution of such regulatorynetworks is undoubtedly the environmental and ecologicalconstraints imposed by a particular bacteriumrsquos niche Inaddition the degree to which a given bacterial species needsto couple flagellar biogenesis to other cellular processes suchas cell division has also likely shaped the evolution of theseregulatory networks We focused here on bacteria whichutilize RpoN for flagellar biogenesis yet even among thesebacteria the mechanisms used to regulate expression of theRpoN-dependent flagellar genes vary greatly For most of thesystems described in this review we have only scratched thesurface in our understanding of how they operate For each ofthese systems fundamental questions remain to be answeredsuch as the following are all of the flagellar genes of a givensystem expressed as part of a transcription hierarchy or aresome genes expressed constitutively Are there regulatorymechanisms within RpoN flagellar regulons that fine-tunegene expression so that components of the flagellum aremadeprecisely when needed What are the cellular cues sensed by

10 Scientifica

the regulatory proteins that control expression of the RpoN-dependent flagellar genes How does the flagellar exportapparatus control expression of RpoN-dependent flagellargenes in various bacteria Does FlgZ have a regulatoryrole in flagellar biogenesis in H pylori and other bacteriaAs our understanding of the molecular genetics of diversebacterial species expands novel regulatory mechanisms thatcontrol expression of flagellar genes within these bacteria willundoubtedly come to light For example it was only recentlythat sRNAs were identified in H pylori some of whichare potentially FliA dependent [127] and may have roles inflagellar biogenesis Finally with increasingly sophisticatedmethods in bioinformatics genomics and proteomics fur-ther characterization of flagellar regulatory networks willprovide important insights into the mechanisms governinggene expression and identify new regulatory proteins andmechanisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by award MCB-1244242 from theNational Science Foundation

References

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[2] J L McMurry J W Murphy and B Gonzalez-Pedrajo ldquoTheFliN-FliH interaction mediates localization of flagellar exportATPase FliI to the C ring complexrdquo Biochemistry vol 45 no39 pp 11790ndash11798 2006

[3] R M Macnab Ed Flagella and Motility ASM Press 1996[4] T Minamino K Imada and K Namba ldquoMolecular motors of

the bacterial flagellardquoCurrent Opinion in Structural Biology vol18 no 6 pp 693ndash701 2008

[5] T G Smith and T R Hoover ldquoDeciphering bacterial flagellargene regulatory networks in the genomic erardquo Advances inApplied Microbiology vol 67 pp 257ndash295 2009

[6] J K Anderson T G Smith and T R Hoover ldquoSense andsensibility flagellum-mediated gene regulationrdquo Trends inMicrobiology vol 18 no 1 pp 30ndash37 2010

[7] J D Helmann ldquoAlternative sigma factors and the regulation offlagellar gene expressionrdquoMolecular Microbiology vol 5 no 12pp 2875ndash2882 1991

[8] D L Popham D Szeto J Keener and S Kustu ldquoFunctionof a bacterial activator protein that binds to transcriptionalenhancersrdquo Science vol 243 no 4891 pp 629ndash635 1989

[9] S Sasse-Dwight and J D Gralla ldquoProbing the EscherichiacoliglnALG upstream activation mechanism in vivordquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8934ndash8938 1988

[10] M Kihara T Minamino S Yamaguchi and R M MacnabldquoIntergenic suppression between the flagellar MS ring protein

FliF of Salmonella and FlhA a membrane component of itsexport apparatusrdquo Journal of Bacteriology vol 183 no 5 pp1655ndash1662 2001

[11] J S van Arnam J L McMurry M Kihara and R M MacnabldquoAnalysis of an engineered Salmonella flagellar fusion proteinFliR-FlhBrdquo Journal of Bacteriology vol 186 no 8 pp 2495ndash2498 2004

[12] C S Barker I V Meshcheryakova A S Kostyukova and FA Samatey ldquoFliO regulation of FliP in the formation of theSalmonella enterica flagellumrdquo PLoS Genetics vol 6 no 9Article ID e1001143 2010

[13] A C Lowenthal M Hill L K Sycuro K Mehmood N RSalama and K M Ottemann ldquoFunctional analysis of the Heli-cobacter pylori flagellar switch proteinsrdquo Journal of Bacteriologyvol 191 no 23 pp 7147ndash7156 2009

[14] R M Macnab ldquoType III flagellar protein export and flagellarassemblyrdquo Biochimica et Biophysica Acta vol 1694 no 1ndash3 pp207ndash217 2004

[15] K A Eaton D R Morgan and S Krakowka ldquoCampylobacterpylori virulence factors in gnotobiotic pigletsrdquo Infection andImmunity vol 57 no 4 pp 1119ndash1125 1989

[16] K A Eaton D R Morgan and S Krakowka ldquoMotility as afactor in the colonisation of gnotobiotic piglets by Helicobacterpylorirdquo Journal of Medical Microbiology vol 37 no 2 pp 123ndash127 1992

[17] D G Newell H McBride and J M Dolby ldquoInvestigationson the role of flagella in the colonization of infant mice withCampylobacter jejuni and attachment of Campylobacter jejunito human epithelial cell linesrdquo Journal of Hygiene vol 95 no 2pp 217ndash227 1985

[18] TMorooka A Umeda andK Amako ldquoMotility as an intestinalcolonization factor forCampylobacter jejunirdquo Journal of GeneralMicrobiology vol 131 no 8 pp 1973ndash1980 1985

[19] C Y Kao B S Sheu SM Sheu et al ldquoHighermotility enhancesbacterial density and inflammatory response in dyspepticpatients infected with Helicobacter pylorirdquo Helicobacter vol 17no 6 pp 411ndash416 2012

[20] E Niehus H Gressmann F Ye et al ldquoGenome-wide analysisof transcriptional hierarchy and feedback regulation in theflagellar system of Helicobacter pylorirdquoMolecular Microbiologyvol 52 no 4 pp 947ndash961 2004

[21] M Balaban S N Joslin and D R Hendrixson ldquoFlhF and itsGTPase activity are required for distinct processes in flagellargene regulation and biosynthesis in Campylobacter jejunirdquoJournal of Bacteriology vol 191 no 21 pp 6602ndash6611 2009

[22] A Kusumoto A Shinohara H Terashima S Kojima TYakushi and M Homma ldquoCollaboration of FlhF and FlhGto regulate polarflagella number and localization in VibrioalginolyticusrdquoMicrobiology vol 154 no 5 pp 1390ndash1399 2008

[23] A Kusumoto K Kamisaka T Yakushi H Terashima AShinohara and M Homma ldquoRegulation of polar flagellarnumber by the flhF and flhG genes in Vibrio alginolyticusrdquoJournal of Biochemistry vol 139 no 1 pp 113ndash121 2006

[24] B M Pruszlig and P Matsumura ldquoA regulator of the flagellarregulon of Escherichia coli flhD also affects cell divisionrdquoJournal of Bacteriology vol 178 no 3 pp 668ndash674 1996

[25] B M Pruszlig and P Matsumura ldquoCell cycle regulation of flagellargenesrdquo Journal of Bacteriology vol 179 no 17 pp 5602ndash56041997

[26] G Spohn and V Scarlato ldquoMotility of Helicobacter pylori iscoordinately regulated by the transcriptional activator FlgR an

Scientifica 11

NtrC homologrdquo Journal of Bacteriology vol 181 no 2 pp 593ndash599 1999

[27] M Schirm E C Soo A J Aubry J Austin P Thibault and SM Logan ldquoStructural genetic and functional characterizationof the flagellin glycosylation process in Helicobacter pylorirdquoMolecular Microbiology vol 48 no 6 pp 1579ndash1592 2003

[28] D Beier and R Frank ldquoMolecular characterization of two-component systems of Helicobacter pylorirdquo Journal of Bacteri-ology vol 182 no 8 pp 2068ndash2076 2000

[29] P Brahmachary M G Dashti J W Olson and T R HooverldquoHelicobacter pylori FlgR is an enhancer-independent activatorof 12059054-RNA polymerase holoenzymerdquo Journal of Bacteriologyvol 186 no 14 pp 4535ndash4542 2004

[30] M M S M Wosten J A Wagenaar and J P M van PuttenldquoThe FlgSFlgR two-component signal transduction systemregulates the fla regulon in Campylobacter jejunirdquo The Journalof Biological Chemistry vol 279 no 16 pp 16214ndash16222 2004

[31] M Bush and R Dixon ldquoThe role of bacterial enhancer bindingproteins as specialized activators of 12059054-dependent transcrip-tionrdquo MicroBiology and Molecular Biology Reviews vol 76 no3 pp 497ndash529 2012

[32] J Schumacher N Joly M Rappas X Zhang and M BuckldquoStructures and organisation of AAA+ enhancer binding pro-teins in transcriptional activationrdquo Journal of Structural Biologyvol 156 no 1 pp 190ndash199 2006

[33] S N Joslin and D R Hendrixson ldquoAnalysis of the Campy-lobacter jejuni FlgR response regulator suggests integration ofdiverse mechanisms to activate an NtrC-like proteinrdquo Journalof Bacteriology vol 190 no 7 pp 2422ndash2433 2008

[34] J M Boll and D R Hendrixson ldquoA specificity determinant forphosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphaterdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 50 pp 20160ndash20165 2011

[35] S Porwollik B Noonan and P W OrsquoToole ldquoMolecular char-acterization of a flagellar export locus of Helicobacter pylorirdquoInfection and Immunity vol 67 no 5 pp 2060ndash2070 1999

[36] E Allan N Dorrell S Foynes M Anyim and B W WrenldquoMutational analysis of genes encoding the early flagellarcomponents of Helicobacter pylori evidence for transcriptionalregulation of flagellin A biosynthesisrdquo Journal of Bacteriologyvol 182 no 18 pp 5274ndash5277 2000

[37] T G Smith L Pereira and T R Hoover ldquoHelicobacter pyloriFlhB processing-deficient variants affect flagellar assembly butnot flagellar gene expressionrdquo Microbiology vol 155 no 4 pp1170ndash1180 2009

[38] D R Hendrixson B J Akerley and V J DiRita ldquoTranspo-son mutagenesis of Campylobacter jejuni identifies a bipartiteenergy taxis system required for motilityrdquoMolecular Microbiol-ogy vol 40 no 1 pp 214ndash224 2001

[39] J Tsang T G Smith L E Pereira and T R Hoover ldquoInsertionmutations inHelicobacter pylori flhA reveal strain differences inRpoN-dependent gene expressionrdquo Microbiology vol 159 part1 pp 58ndash67 2013

[40] J M Boll and D R Hendrixson ldquoA regulatory checkpointduring flagellar biogenesis in Campylobacter jejuni initiatessignal transduction to activate transcription of flagellar genesrdquoMBio vol 4 no 5 Article ID e00432-13 2013

[41] T Minamino H Doi and K Kutsukake ldquoSubstrate specificityswitching of the flagellum-specific export apparatus during flag-ellar morphogenesis in Salmonella typhimuriumrdquo Bioscience

Biotechnology and Biochemistry vol 63 no 7 pp 1301ndash13031999

[42] M Erhardt H M Singer D H Wee J P Keener and K THughes ldquoAn infrequent molecular ruler controls flagellar hooklength in Salmonella entericardquo EMBO Journal vol 30 no 14 pp2948ndash2961 2011

[43] K T Hughes K L Gillen M J Semon and J E KarlinseyldquoSensing structural intermediates in bacterial flagellar assemblyby export of a negative regulatorrdquo Science vol 262 no 5137 pp1277ndash1280 1993

[44] K A Ryan N Karim M Worku S A Moore C W Pennand P W OrsquoToole ldquoHP0958 is an essential motility gene inHelicobacter pylorirdquo FEMS Microbiology Letters vol 248 no 1pp 47ndash55 2005

[45] L Pereira and T R Hoover ldquoStable accumulation of 12059054 inHelicobacter pylori requires the novel protein HP0958rdquo Journalof Bacteriology vol 187 no 13 pp 4463ndash4469 2005

[46] J C Rain L Selig H de Reuse et al ldquoThe protein-proteininteraction map of Helicobacter pylorirdquo Nature vol 409 no6817 pp 211ndash215 2001

[47] F P Douillard K A Ryan D L Caly et al ldquoPosttranscriptionalregulation of flagellin synthesis in Helicobacter pylori by theRpoN chaperone HP0958rdquo Journal of Bacteriology vol 190 no24 pp 7975ndash7984 2008

[48] C Josenhans E Niehus S Amersbach et al ldquoFunctionalcharacterization of the antagonistic flagellar late regulators FliAand FlgM ofHelicobacter pylori and their effects on theH pyloritranscriptomerdquoMolecular Microbiology vol 43 no 2 pp 307ndash322 2002

[49] M Rust S Borchert E Niehus et al ldquoThe Helicobacter pylorianti-sigma factor FlgM is predominantly cytoplasmic andcooperates with the flagellar basal body protein FlhArdquo Journalof Bacteriology vol 191 no 15 pp 4824ndash4834 2009

[50] D R Hendrixson and V J DiRita ldquoTranscription of 12059054-dependent but not 12059028-dependent flagellar genes in Campy-lobacter jejuni is associated with formation of the flagellarsecretory apparatusrdquoMolecular Microbiology vol 50 no 2 pp687ndash702 2003

[51] M M S M Wosten L van Dijk A K J Veenendaal MR de Zoete N M C Bleumink-Pluijm and J P M vanPutten ldquoTemperature-dependent FlgMFliA complex forma-tion regulates Campylobacter jejuni flagella lengthrdquo MolecularMicrobiology vol 75 no 6 pp 1577ndash1591 2010

[52] M G Prouty N E Correa and K E Klose ldquoThe novel 12059054- and12059028-dependent flagellar gene transcription hierarchy of Vibrio

choleraerdquo Molecular Microbiology vol 39 no 6 pp 1595ndash16092001

[53] D Srivastava M L Hsieh A Khataokar M B Neiditch and CM Waters ldquoCyclic di-GMP inhibits Vibrio choleraemotility byrepressing induction of transcription and inducing extracellularpolysaccharide productionrdquo Molecular Microbiology vol 90no 6 pp 1262ndash1276 2013

[54] N E Correa C M Lauriano R McGee and K E KloseldquoPhosphorylation of the flagellar regulatory protein FlrC is nec-essary for Vibrio cholerae motility and enhanced colonizationrdquoMolecular Microbiology vol 35 no 4 pp 743ndash755 2000

[55] N E Correa J R Barker and K E Klose ldquoThe Vibrio choleraeFlgM homologue is an anti-12059028 factor that is secreted throughthe sheathed polar flagellumrdquo Journal of Bacteriology vol 186no 14 pp 4613ndash4619 2004

12 Scientifica

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[58] SMerino J G Shaw and JM Tomas ldquoBacterial lateral flagellaan inducible flagella systemrdquo FEMS Microbiology Letters vol263 no 2 pp 127ndash135 2006

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[60] S Ulitzur ldquoInduction of swarming in Vibrio parahaemolyticusrdquoArchives of Microbiology vol 101 no 4 pp 357ndash363 1974

[61] K S Park M Arita T Iida and T Honda ldquovpaH a geneencoding a novel histone-like nucleoid structure-like proteinthat was possibly horizontally acquired regulates the biogen-esis of lateral flagella in trh-positive Vibrio parahaemolyticusTH3996rdquo Infection and Immunity vol 73 no 9 pp 5754ndash57612005

[62] LMcCarter andM Silverman ldquoIron regulation of swarmer celldifferentiation ofVibrio parahaemolyticusrdquo Journal of Bacteriol-ogy vol 171 no 2 pp 731ndash736 1989

[63] B J Stewart and L L McCarter ldquoLateral flagellar gene systemofVibrio parahaemolyticusrdquo Journal of Bacteriology vol 185 no15 pp 4508ndash4518 2003

[64] C J Gode-Potratz R J Kustusch P J Breheny D S Weissand L LMcCarter ldquoSurface sensing inVibrio parahaemolyticustriggers a programme of gene expression that promotes colo-nization and virulencerdquo Molecular Microbiology vol 79 no 1pp 240ndash263 2011

[65] J L Veesenmeyer A R Hauser T Lisboa and J Rello ldquoPseu-domonas aeruginosa virulence and therapy evolving transla-tional strategiesrdquo Critical Care Medicine vol 37 no 5 pp 1777ndash1786 2009

[66] D Balasubramanian L Schneper H Kumari and K MatheeldquoA dynamic and intricate regulatory network determines Pseu-domonas aeruginosa virulencerdquo Nucleic Acids Research vol 41no 1 pp 1ndash20 2013

[67] T C Montie D D Huntzinger R C Craven and I A HolderldquoLoss of virulence associated with absence of flagellum in anisogenic mutant of Pseudomonas aeruginosa in the burned-mouse modelrdquo Infection and Immunity vol 38 no 3 pp 1296ndash1298 1982

[68] Y R Patankar R R Lovewell M E Poynter J Jyot BI Kazmierczak and B Berwin ldquoFlagellar motility is a keydeterminant of themagnitude of the inflammasome response toPseudomonas aeruginosardquo Infection and Immunity vol 81 no 6pp 2043ndash2052 2013

[69] G A OrsquoToole and R Kolter ldquoFlagellar and twitching motilityare necessary for Pseudomonas aeruginosa biofilm develop-mentrdquoMolecular Microbiology vol 30 no 2 pp 295ndash304 1998

[70] K B Barken S J Pamp L Yang et al ldquoRoles of type IVpili flagellum-mediated motility and extracellular DNA in theformation of mature multicellular structures in Pseudomonasaeruginosa biofilmsrdquo Environmental Microbiology vol 10 no 9pp 2331ndash2343 2008

[71] M Leeman J A van Pelt F M den Ouden M Heinsbroek PA Bakker and B Schippers ldquoInduction of systemic resistance

against fusarium wilt of radish by lipopolysaccharides of Pseu-domonas fluorescensrdquo Phytopathology vol 85 no 9 pp 1021ndash1027 1995

[72] P Hsueh L Teng H Pan et al ldquoOutbreak of Pseudomonasfluorescens bacteremia among oncology patientsrdquo Journal ofClinical Microbiology vol 36 no 10 pp 2914ndash2917 1998

[73] VWongK Levi B Baddal J Turton andTC Boswell ldquoSpreadof Pseudomonas fluorescens due to contaminated drinkingwater in a bone marrow transplant unitrdquo Journal of ClinicalMicrobiology vol 49 no 6 pp 2093ndash2096 2011

[74] J W Hickman and C S Harwood ldquoIdentification of FleQ fromPseudomonas aeruginosa as a c-di-GMP-responsive transcrip-tion factorrdquoMolecular Microbiology vol 69 no 2 pp 376ndash3892008

[75] N Dasgupta M C Wolfgang A L Goodman et al ldquoA four-tiered transcriptional regulatory circuit controls flagellar bio-genesis in Pseudomonas aeruginosardquo Molecular Microbiologyvol 50 no 3 pp 809ndash824 2003

[76] S K Arora B W Ritchings E C Almira S Lory and RRamphal ldquoA transcriptional activator FleQ regulates mucinadhesion and flagellar gene expression in Pseudomonas aerugi-nosa in a cascade mannerrdquo Journal of Bacteriology vol 179 no17 pp 5574ndash5581 1997

[77] N Dasgupta and R Ramphal ldquoInteraction of the antiactivatorFleN with the transcriptional activator FleQ regulates flagellarnumber in Pseudomonas aeruginosardquo Journal of Bacteriologyvol 183 no 22 pp 6636ndash6644 2001

[78] N Dasgupta S K Arora and R Ramphal ldquofleN a gene thatregulates flagellar number in Pseudomonas aeruginosardquo Journalof Bacteriology vol 182 no 2 pp 357ndash364 2000

[79] J Malakooti and B Ely ldquoPrincipal sigma subunit of theCaulobacter crescentus RNA polymeraserdquo Journal of Bacteriol-ogy vol 177 no 23 pp 6854ndash6860 1995

[80] M T Laub H H McAdams T Feldblyum C M Fraser andL Shapiro ldquoGlobal analysis of the genetic network controllinga bacterial cell cyclerdquo Science vol 290 no 5499 pp 2144ndash21482000

[81] A K Benson G Ramakrishnan N Ohta J Feng A J Ninfaand A Newton ldquoThe Caulobacter crescentus FlbD proteinacts at ftr sequence elements both to activate and to represstranscription of cell cycle-regulated flagellar genesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 91 no 11 pp 4989ndash4993 1994

[82] D A Mullin S M van Way C A Blankenship and A HMullin ldquoFlbD has a DNA-binding activity near its carboxyterminus that recognizes ftr sequences involved in positive andnegative regulation of flagellar gene transcription in Caulobac-ter crescentusrdquo Journal of Bacteriology vol 176 no 19 pp 5971ndash5981 1994

[83] R J Dutton Z Xu and J W Gober ldquoLinking structural assem-bly to gene expression a novel mechanism for regulating theactivity of a 12059054 transcription factorrdquo Molecular Microbiologyvol 58 no 3 pp 743ndash757 2005

[84] R E Muir J Easter and J W Gober ldquoThe trans-acting flagellarregulatory proteins FliX and FlbD play a central role in linkingflagellar biogenesis and cytokinesis in Caulobacter crescentusrdquoMicrobiology vol 151 no 11 pp 3699ndash3711 2005

[85] P E Anderson and J W Gober ldquoFlbT the post-transcriptionalregulator of flagellin synthesis in Caulobacter crescentus inter-acts with the 51015840 untranslated region of flagellin mRNArdquoMolec-ular Microbiology vol 38 no 1 pp 41ndash52 2000

Scientifica 13

[86] A T Nielsen N A Dolganov G Otto M C Miller C Y WuandGK Schoolnik ldquoRpoS controls theVibrio choleraemucosalescape responserdquo PLoS Pathogens vol 2 no 10 article e1092006

[87] M T Laub S L Chen L Shapiro and H H McAdamsldquoGenes directly controlled by CtrA a master regulator of theCaulobacter cell cyclerdquo Proceedings of the National Academy ofSciences of the United States of America vol 99 no 7 pp 4632ndash4637 2002

[88] A J Kelly M J Sackett N Din E Quardokus and Y VBrun ldquoCell cycle-dependent transcriptional and proteolyticregulation of FtsZ in Caulobacterrdquo Genes and Development vol12 no 6 pp 880ndash893 1998

[89] M Wortinger M J Sackett and Y V Brun ldquoCtrA mediatesa DNA replication checkpoint that prevents cell division inCaulobacter crescentusrdquo EMBO Journal vol 19 no 17 pp 4503ndash4512 2000

[90] R E Muir T M OrsquoBrien and J W Gober ldquoThe Caulobactercrescentus flagellar gene fliX encodes a novel trans-acting fac-tor that couples flagellar assembly to transcriptionrdquo MolecularMicrobiology vol 39 no 6 pp 1623ndash1637 2001

[91] M Llewellyn R J Dutton J Easter D OrsquoDonnol and J WGober ldquoThe conserved flaF gene has a critical role in couplingflagellin translation and assembly in Caulobacter crescentusrdquoMolecular Microbiology vol 57 no 4 pp 1127ndash1142 2005

[92] S M Kim D H Lee and S H Choi ldquoEvidence that the Vibriovulnificus flagellar regulator FlhF is regulated by a quorumsensing master regulator SmcRrdquo Microbiology vol 158 part 8pp 2017ndash2025 2012

[93] F Martınez-Granero A Navazo E Barahona M Redondo-Nieto R Rivilla andMMartın ldquoTheGac-Rsm and SadB signaltransduction pathways converge on Algu to downregulatemotility in Pseudomonas fluorescensrdquo PLoS ONE vol 7 no 2Article ID e31765 2012

[94] C Reimmann M Beyeler A Latifi et al ldquoThe global activatorGacA of Pseudomonas aeruginosa PAO positively controls theproduction of the autoinducer N-butyryl-homoserine lactoneand the formation of the virulence factors pyocyanin cyanideand lipaserdquo Molecular Microbiology vol 24 no 2 pp 309ndash3191997

[95] J Dubern and G V Bloemberg ldquoInfluence of environmentalconditions on putisolvins I and II production in Pseudomonasputida strain PCL1445rdquo FEMSMicrobiology Letters vol 263 no2 pp 169ndash175 2006

[96] M G Surette M B Miller and B L Bassler ldquoQuorum sensingin Escherichia coli Salmonella typhimurium andVibrio harveyia new family of genes responsible for autoinducer productionrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 96 no 4 pp 1639ndash1644 1999

[97] B A Rader S R Campagna M F Semmelhack B L Basslerand K Guillemin ldquoThe quorum-sensing molecule autoinducer2 regulates motility and flagellar morphogenesis inHelicobacterpylorirdquo Journal of Bacteriology vol 189 no 17 pp 6109ndash61172007

[98] YHe J G Frye T P Strobaugh Jr andCChen ldquoAnalysis of AI-2LuxS-dependent transcription in Campylobacter jejuni strain81-176rdquo Foodborne Pathogens and Disease vol 5 no 4 pp 399ndash415 2008

[99] B Jeon K Itoh N Misawa and S Ryu ldquoEffects of quorumsensing on flaA transcription and autoagglutination in Campy-lobacter jejunirdquoMicrobiology and Immunology vol 47 no 11 pp833ndash839 2003

[100] N Dasgupta E P Ferrell K J Kanack S E H West andR Ramphal ldquofleQ the gene encoding the major flagellarregulator of Pseudomonas aeruginosa is 12059070 dependent and isdownregulated by Vfr a homolog of Escherichia coli cyclic AMPreceptor proteinrdquo Journal of Bacteriology vol 184 no 19 pp5240ndash5250 2002

[101] J Zhu M B Miller R E Vance M Dziejman B L Bassler andJ J Mekalanos ldquoQuorum-sensing regulators control virulencegene expression in Vibrio choleraerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no5 pp 3129ndash3134 2002

[102] Z Liu T Miyashiro A Tsou A Hsiao M Goulian and JZhu ldquoMucosal penetration primes Vibrio cholerae for hostcolonization by repressing quorum sensingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 28 pp 9769ndash9774 2008

[103] K E Klose V Novik and J J Mekalanos ldquoIdentificationof multiple 12059054-dependent transcriptional activators in Vibriocholeraerdquo Journal of Bacteriology vol 180 no 19 pp 5256ndash52591998

[104] I Kawagishi M Imagawa Y Imae L McCarter and MHomma ldquoThe sodium-driven polar flagellar motor of marineVibrio as the mechanosensor that regulates lateral flagellarexpressionrdquoMolecular Microbiology vol 20 no 4 pp 693ndash6991996

[105] T J Kirn B A Jude and R K Taylor ldquoA colonizationfactor links Vibrio cholerae environmental survival and humaninfectionrdquo Nature vol 438 no 7069 pp 863ndash866 2005

[106] Z Kuang YHao S Hwang et al ldquoThe Pseudomonas aeruginosaflagellum confers resistance to pulmonary surfactant protein-Aby impacting the production of exoproteases through quorum-sensingrdquo Molecular Microbiology vol 79 no 5 pp 1220ndash12352011

[107] S Zhang F X McCormack R C Levesque G A OrsquoTooleand G W Lau ldquoThe flagellum of Pseudomonas aeruginosa isrequired for resistance to clearance by surfactant protein ArdquoPLoS ONE vol 2 no 6 article e564 2007

[108] G W Lau D J Hassett H Ran and F Kong ldquoThe role ofpyocyanin in Pseudomonas aeruginosa infectionrdquo Trends inMolecular Medicine vol 10 no 12 pp 599ndash606 2004

[109] GW LauH Ran F Kong D J Hassett andDMavrodi ldquoPseu-domonas aeruginosa pyocyanin is critical for lung infection inmicerdquo Infection and Immunity vol 72 no 7 pp 4275ndash42782004

[110] D S Merrell M L Goodrich G Otto L S Tompkins and SFalkow ldquopH-regulated gene expression of the gastric pathogenHelicobacter pylorirdquo Infection and Immunity vol 71 no 6 pp3529ndash3539 2003

[111] K J Allen and M W Griffiths ldquoEffect of environmental andchemotactic stimuli on the activity of the Campylobacter jejuniflaA 12059028 promoterrdquo FEMS Microbiology Letters vol 205 no 1pp 43ndash48 2001

[112] Y Wen J Feng D R Scott E A Marcus and G SachsldquoThe pH-responsive regulon of HP0244 (FlgS) the cytoplasmichistidine kinase of Helicobacter pylorirdquo Journal of Bacteriologyvol 191 no 2 pp 449ndash460 2009

[113] D R Hendrixson ldquoA phase-variable mechanism controllingthe Campylobacter jejuni FlgR response regulator influencescommensalismrdquo Molecular Microbiology vol 61 no 6 pp1646ndash1659 2006

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 Scientifica

Rod

IM

OM

C

MS ring

Hook Filament

Export apparatus

P

L

(a)

FlhA FlhB R P MS ring

Inner membrane

Rod

Lumen

CytoplasmFliI FliH FliH

C ring

FliE

Q O

(b)

Figure 1 Structure of the flagellum (a) An overview of the structure of the flagellum in Gram-negative bacteria Abbreviations are as followsL ring (L) P ring (P) outer membrane (OM) inner membrane (IM) and C ring (C) (b) Components of the flagellar basal body Exportapparatus proteins are shown in light blue Abbreviations of the export apparatus proteins are as follows FliR (R) FliP (P) FliO (O) and FliQ(Q) Details of the organization of the export apparatus proteins are not known although results from genetic studies suggest associationsbetween FlhA and the MS ring [10] FlhB and FliR [11] and FliO and FliP [12] FliI is an ATPase which forms a heterotrimer with FliHTheseproteins function together with other chaperones (not shown) to shuttle protein substrates to the export pore Upon docking with a platformformed by the large cytoplasmic domains of FlhA and FlhB a larger FliI

6FliH12complex is formed Inmost bacteria the C ring is composed of

three different types of protein subunits (FliG FliM and FliN)The C ring in Salmonella contains an estimated 26 copies of FliG 34 copies ofFliM and sim136 copies of FliN FliG is closest to the membrane and interacts with the MS ring while FliN is the most distal to the membraneand FliM is situated between FliG and FliN The H pylori C ring contains FliG FliM and FliN plus an additional protein subunit (FliY)that shares homology with FliN [13] Additional information on the bacterial flagellar protein export apparatus can be found in reviews byMinamino et al [1] and Macnab [14]

which utilize the alternative sigma factor 12059054 (also known asRpoN) for regulating transcription of specific sets of flagellargenes Sigma factors bind core RNApolymerase and allow theresulting RNA polymerase holoenzyme to recognize specificpromoter sequences [7] All bacteria possess a primary sigmafactor (RpoD referred to as 12059070 in E coli) and most alsoutilize one or more alternative sigma factors for transcrip-tion of specific sets of genes A unique feature of RpoN-dependent transcription is the requirement of an activatorto stimulate the transition of a closed complex between12059054-RNA polymerase holoenzyme (12059054-holoenzyme) and the

promoter to an open promoter complex that is able to initiatetranscription [8 9] Escherichia coli and Salmonella are thearchetypes for flagellar biogenesis and gene regulation butthese bacteria do not use RpoN for transcription of flagellargenes Nevertheless we refer to the E coliSalmonella systemsthroughout the review to make inferences for other systemsthat are not as well characterized The bacteria that utilizeRpoN in flagellar gene expression constitute a diverse groupof microorganisms some of which are human or animalpathogens We discuss aspects of flagellar gene regulationin response to host and environmental stimuli and potentialramifications of these responses for survival and persistenceof the bacteria

2 Transcriptional Hierarchies GoverningFlagellar Gene Expression

Though microorganisms utilize various mechanisms to con-trol flagellar gene expression in most (if not all) bacteria

the flagellar genes are transcribed in an organized fashionwhere genes that encode components needed early in flagellarbiogenesis are transcribed before genes encoding proteinsrequired later in the assembly process (Figure 2) To a certainextent these transcriptional hierarchies are governed by theorganization of the flagellar genes into different regulonsbased on the sigma factors required for their transcriptionThese transcriptional hierarchies are further coordinated byregulatory proteins that control expression of various setsor classes of genes However the mechanism by which sucha hierarchy is regulated in a given bacterial species is notalways understood and varies among organisms A masterregulator(s) initiates the flagellar gene transcription cascadeand is generally thought to couple flagellar biogenesis tothe cell cycle For some bacteria however master regulatorshave yet to be identified The master regulator stimulatestranscription of the early genes which generally encodecomponents of the basal body as well as an array of regulatoryproteins which depending on the bacterium includes RpoNFliA (12059028) FlgM (an anti-12059028 factor) or other proteinsFollowing completion of the basal body and hook late genesencoding components of the filament are expressed

21 H pylori and C jejuni The best studied membersof the Epsilonproteobacteria are Campylobacter jejuni andHelicobacter pylori C jejuni is a food-borne pathogen thatcolonizes the intestinal tract where it causes severe diarrheawhile H pylori colonizes the stomach where it can causepeptic ulcer disease which can progress into gastric cancerif left untreated Both organisms synthesize polar flagella that

Scientifica 3

Rod IM

OM

C

MS ring

Hook Filament

Export apparatus

Rod IM

OM

C

MS ring

Hook

Export apparatus

Rod IM

OM

C

MS ring Export

apparatus

H pyloriC jejuni

Vibriospecies

Pseudomonasspecies

C crescentus

FlrA

RpoD

FlrARpoN

FleQRpoN

FleQ

CtrARpoD

Export apparatus rings FlgS FlgR

FlgM rod

Export apparatus rings FlrB FlrC

FliA

Export apparatus motor filament cap rings FleN

FleS FleR

Export apparatus rings FlbD FliX

FlgSFlgRRpoN

FlrBFlrCRpoN

FleSFleRRpoN

FliXFlbDRpoN

Rodhook

FliK FlaB

Hook FlaA

Rod L ring hook

hook cap

Hook

FliA

FliA

FliA

FlbDFlbTRpoN

FlaAflagellar

cap

Alternative flagellins FlgM

motor

FliC FleL FlgM FlgN chemotaxis

proteins

Flagellins

Masterregulator

Early Middle Late

Figure 2 Diversity in flagellar gene transcription hierarchies Transcriptional hierarchies are compared between the EpsilonproteobacteriaVibrio Pseudomonas and C crescentus Regulatory proteins and sigma factors involved in controlling the transcriptional hierarchies areindicated above the arrows Early middle and late genes are indicated between the arrows

are required for colonization of the host [15ndash18] Moreoverthe degree of motility appears to be important for H pylorivirulence as more motile strains are able to maintain higherbacterial density and inflammation in the stomach cardiathan those that are less motile [19]

In H pylori and C jejuni flagellar gene regulationinvolves the primary sigma factor RpoD as well as thealternative sigma factors RpoN and FliA (Figure 2) A masterregulator which activates transcription of the early flagellargenes has not been identified for H pylori or C jejuni It ispossible that these bacteria lack a true master regulator offlagellar biogenesis a possibility that was suggested byNiehusand coworkers since motility is required for the obligateparasitic lifestyle of H pylori [20] Alternatively H pylorior C jejuni could possess a master regulator for flagellarbiogenesis that has other essential roles which might explainwhy it has not been identified from mutagenesis screens

The organization of flagellar genes into regulons basedon the sigma factor required for transcription is very similarthough not identical in H pylori and C jejuni RpoD isrequired for transcription of genes encoding components ofthe basal body as well as regulatory proteins that controltranscription of genes required at later stages of flagellarbiogenesis [20] Other genes regulated by RpoD include flhF

and flhG whose products influence the localization of flagellato the cell pole and number of flagella per cell respectively[21ndash23] It is not known if transcription of the early flagellargenes is temporally regulated and intimately associated withthe cell cycle as occurs in E coli and Salmonella [24 25] orif these genes are transcribed constitutively Distinguishingbetween these two options would require one to followtemporal changes of early flagellar genes transcription insynchronous cultures which has not been reported

Transcription of genes needed midway through flagellarbiogenesis is dependent on RpoN These genes encode theproximal rod proteins the hook protein hook-associatedproteins the hook-length control protein (FliK) a minorflagellin (FlaB) and enzymes required for glycosylation ofthe flagellins [20 26 27] The transcription of the RpoN-dependent genes is activated by a two-component regulatorysystem consisting of the sensor kinase FlgS and the responseregulator FlgR [26 28ndash30] FlgS differs from most sensorkinases in that it is not membrane bound The signal orcellular cue to which FlgS responds has yet to be identifiedbut several studies (see below) have implicated the flagellarprotein export apparatus in this process FlgS uses ATP toautophosphorylate a specific histidine residue in responseto the cellular cue and this phosphate is subsequently

4 Scientifica

transferred to a specific aspartate residue in FlgR to activateit Based on studies with other RpoN-dependent activators(reviewed by Bush and Dixon [31]) phosphorylation ofFlgR likely stimulates oligomerization of the protein froma dimer to a hexamer capable of activating transcriptionRpoN-dependent activators typically possess an N-terminalregulatory domain (often a response regulator domain as isthe case in FlgR) an AAA+ ATPase domain that engages12059054 and couples the hydrolysis of ATP with open complex

formation and aC-terminalDNA-binding domain that bindsan enhancer sequence (reviewed by Schumacher et al [32])H pylori FlgR is unusual in that it lacks a C-terminal DNA-binding domain and activates transcription by contacting12059054-holoenzyme in the closed promoter complex without

binding DNA [29] Although C jejuni FlgR possesses a C-terminal domain it is not needed for activation of the RpoNregulon suggesting that like its counterpart in H pyloriC jejuni FlgR engages the closed promoter complex fromsolution [33] While the C-terminal domain of C jejuni FlgRdoes not appear to play a role inDNAbinding it does preventphosphorylation of the protein by acetyl phosphatewhich caninterfere with normal flagellation [34]

The flagellar protein export apparatus has a major rolein regulating expression of the RpoN regulon of H pyloriand C jejuni as evidenced by the observation that mutationswhich interfere with formation of the export apparatusinhibit expression of RpoN-dependent flagellar genes Por-wollik and coworkers showed that inactivation of fliI orfliQ (encode a cytosolic ATPase component and integralmembrane component of the export apparatus resp) inH pylori results in reduced levels of the minor flagellinFlaB and the hook protein FlgE both of which dependon RpoN for their expression [35] Allan and colleaguesshowed that mutations in flhB (encodes an integral mem-brane component of the export apparatus) result in reducedlevels of FlaB and FlgE inH pylori [36] Smith and coworkerssubsequently demonstrated that disrupting flhB inhibitedexpression of RpoN-dependent reporter genes [37] UsingDNAmicroarrays Niehus and coworkers [20] demonstratedthat FlhA (an integral membrane component of the exportapparatus) is required for transcription of the entire RpoNregulon Hendrixson et al [38] reported that deleting anyone of several integral membrane components of the exportapparatus (FlhA FlhB FliP or FliR) inhibited expressionof an RpoN-dependent flagellar reporter gene in C jejuniStudies by Tsang and coworkers revealed thatH pylori strainswhich stably express truncated forms of FlhA are able totranscribe RpoN-dependent genes at levels that are close towild type but are unable to export flagellar protein substrates[39]Thus anymodel for how the export apparatus influencestranscription of theRpoN regulonmust take into account thatthe export apparatus need not transport protein substrates tostimulate the RpoN regulon Boll and Hendrixson recentlyshowed that loss of the MS ring protein FliF or the Cring protein FliG inhibits transcription of RpoN-dependentflagellar genes in C jejuni [40] The MS ring is the firstflagellar structure assembled and in its absence the exportapparatus presumably is not formedwhich would account for

the inhibition of the RpoN regulon in the fliF mutant FliGmay facilitate autophosphorylation of FlgS either by directlystimulating FlgS or by promoting assembly of the exportapparatus into a conformation that stimulates FlgS activity[40]

The export apparatus is an ideal indicator for the statusof flagellar assembly as it undergoes a major conformationalchange upon completion of the hook-basal body structureIn the early stages of flagellar biogenesis the export apparatusis in a conformation that transports rod- and hook-typesubstrates Upon completion of the mature hook-basal bodystructure the export apparatus undergoes a conformationalchange which is accompanied by a switch in substrate speci-ficity to filament-type substrates [41] This conformationalchange involves an autocleavage event in the C-terminalcytoplasmic domain of the export apparatus protein FlhB(referred to as FlhBC) and interactions between the C-terminal domain of the hook-length control protein FliKand FlhBC [42] The change in conformation and substratespecificity of the export apparatus may serve as a cellular cuefor the temporal regulation of flagellar genes Indeed in Ecoli and Salmonella the anti-12059028 factor FlgM is a filament-type substrate and is secreted by the export apparatus uponcompletion of the mature hook-basal body structure whichallows transcription of the 12059028-dependent flagellar genes tooccur [43]

Transcription of the RpoN-dependent flagellar genes inH pylori also requires the putative RpoN chaperone FlgZ(HP0958) [44 45] FlgZ was first implicated in having a rolein flagellar biogenesis in H pylori when it was identifiedas interacting with RpoN in a high-throughput screen of ayeast two-hybrid system (httpspimhybrigenicscom) [46]In the absence of FlgZ RpoN is turned over rapidly in Hpyloriwith a half-life of about 30 minutes compared to a half-life greater than 4 hours in wild type [45] The molecularbasis for how FlgZ protects RpoN from rapid turnover is notknown but it is possible that FlgZ binds RpoN to protectit from proteolysis or that FlgZ facilitates the association ofRpoN with core RNA polymerase which serves to protectRpoN from degradation Overexpression of RpoN restoresmotility in a H pylori flgZ mutant indicating that FlgZ isnot essential for flagellar biogenesis [45] In addition toits potential role as an RpoN chaperone FlgZ may haveadditional roles in flagellar biogenesis as it was also shown tointeract with the flagellar export apparatus protein FliH in theyeast two-hybrid system (httpspimhybrigenicscom) [46]In addition Douillard and coworkers demonstrated that FlgZbinds the flaA transcript and is needed for optimal expressionof FlaA [47] These researchers proposed that FlgZ functionswith FliH to direct flaA transcripts to the flagellar proteinexport apparatus where translation of the transcripts couldbe coupled with secretion of the nascent FlaA [47]

Transcription of genes needed late in flagellar biogenesisin H pylori and C jejuni which includes genes encodingthe major flagellin filament cap and associated chaper-ones is dependent on 12059028 (FliA) [20] Similar to E coliand Salmonella the H pylori FliA regulon is negativelyregulated by FlgM [48] Unlike E coli and Salmonella

Scientifica 5

the inhibitory effect of FlgM on FliA in H pylori is thoughtto be alleviated via interactions between FlgM and the C-terminal cytoplasmic domain of FlhA (FlhAC) rather thanby secretion of FlgM via the export apparatus [49] C jejunialso possesses a FlgM homolog [50] but unlike H pyloriC jejuni FlgM appears to be secreted from the cell via theflagellar export apparatus [51] Interestingly interaction of Cjejuni FlgM with FliA is temperature dependent occurringat 42∘C (the optimal growth temperature for C jejuni) butnot 37∘C [51] The primary function of C jejuni FlgM is notto inhibit FliA activity during formation of the hook-basalbody structure but rather to limit the length of the flagellarfilament by suppressing expression of the FliA-dependentflagellin (FlaA) and the RpoN-dependent flagellin (FlaB) [51]The mechanism by which FlgM represses transcription ofRpoN-dependent genes in C jejuni is unknown but Wostenand coworkers have speculated that accumulation of theFliAFlgM complex may inhibit phosphorylation of FlgSandor FlgR [51] FlgM also appears to inhibit transcriptionof RpoN-dependent genes inH pylori but so far this has onlybeen demonstrated in an flhAmutant background [20]

22 Vibrio Species The vibrios are typically saltwater micro-organisms and several species cause food-borne illnessesVibrio species vary greatly in flagella morphology Vibriocholerae has a single polar sheathed flagellum while Vibrioparahaemolyticus and Vibrio alginolyticus produce a singlepolar sheathed flagellum and lateral peritrichous flagella andVibrio fischeri has a tuft of polar sheathed flagella Lateralflagella promote swarming and colonization of surfaces andthereby enhance biofilm formation upon encountering highlyviscous media or solid surfaces

The flagellar gene transcriptional hierarchy in Vibriocholerae is governed by RpoN and FliA [52] flrA encodesan RpoN dependent activator and is the master regulatorfor the flagellar gene transcriptional hierarchy [52] FlrA isregulated by cyclic di-GMP a secondarymessenger with rolesin biofilm formation and virulence which binds to FlrA andprevents it from binding the flrBC promoter [53] Early andmiddle genes are RpoN-dependent but they rely on differenttranscriptional activators for their transcription FlrA alongwith RpoN is required for transcription of the early geneswhich encode the components of the MS ring C ring andexport apparatus Other early genes encode the regulatoryproteins FlrB FlrC and FliA FlrB and FlrC constitutethe sensor kinase and response regulator respectively of atwo-component system that regulates transcription of themiddle genes in conjunction with RpoN [54] The middlegenes encode the basal body-hook structures and the coreflagellin FlaA The late genes are dependent on FliA fortheir transcription and encode alternative flagellins FlgMand components of the flagellar motor As in SalmonellaFlgM is secreted via the flagellar protein export apparatusto relieve the inhibition on FliA activity in V cholerae [55]flgA is located upstream of flgM and its promoter (RpoNindependent and FliA independent) may drive transcriptionof flgM such that FliA is repressed until formation of themature hook-basal body structure [52]

The signal(s) that stimulates the FlrBFlrC two-component system is not known FlrD is necessary fortranscription of middle and late flagellar genes making ita candidate for regulating the FlrBFlrC two-componentsystem [56] Additionally FlrD contains HAMP domains(domain present in histidine kinases adenyl cyclasesmethyl-accepting proteins and phosphatases) that areusually found in integral membrane proteins that are part ofsignal transduction pathways [56 57] Moisi and coworkersshowed that FlrD is inserted into the inner membrane andproposed that it senses the completion of the MS ring-switch-export apparatus structure and communicates thisinformation to FlrB [56]

The regulation of flagellar genes inV parahaemolyticus ismore complex since the bacterium is capable of producinglateral flagella in addition to polar flagella (reviewed byMerino et al [58] and McCarter [59]) The polar flagellarsystem (Fla) is constitutively expressed while the lateral flag-ellar system (Laf) is expressed upon impedance of the polarflagellaThe lateral flagella are used for swarmingmotility andenhance biofilm formation and host colonization by the bac-terium [60 61] When grown planktonically the bacteriumproduces a polar flagellum but when grown on highly viscousor solid medium [58] or during iron-limiting conditions[62] the bacterium produces lateral flagella Thus the polarflagellum acts as a mechanosensor to regulate expression ofthe lateral flagella genes through a mechanism that has yet tobe defined The dual flagellar system suited for locomotionunder different conditions allows V parahaemolyticus to behighly adaptive to changing habitats including planktonicenvironments surfaces and biofilms

The polar and lateral flagellar systems do not share anystructural or regulatory components (aside from RpoN) butthe regulatory networks that control polar and lateral flagellarsystems in V parahaemolyticus are similar to that of Vcholerae The lateral flagellar genes are activated by LafKand like the polar flagellar system lateral flagellar genes areboth RpoN dependent and FliA dependent [63] AlthoughLafK is homologous to master regulators of other flagellarsystems LafK does not appear to be amaster regulator for thelateral flagellar system as the 119891119897119894119872L operon (contains genesencoding components of the C ring and export apparatus) isnot LafK dependent [64] This suggests that there is anotherlevel of regulation preceding expression of these genes

23 Pseudomonas Species Pseudomonas aeruginosa is anaerobic Gram-negative Gammaproteobacterium that causesopportunistic infections particularly in patients with cysticfibrosis where it results in inflammation and sepsis (reviewedby Veesenmeyer et al [65] and Balasubramanian et al[66]) P aeruginosa which thrives on surfaces and formsbiofilms is a prevalent agent of nosocomial infections Wild-type P aeruginosa cells form biofilms which can causechronic infections P aeruginosa possesses a single polarflagellum that is required for virulence [67] Motile strainsof P aeruginosa induce activation of the inflammasome (amultiprotein oligomer responsible for the activation of theinflammatory response) whereas nonmotile strains have amarkedly reduced ability to induce inflammasome activation

6 Scientifica

[68] Nonmotile strains of P aeruginosamay have an advan-tage during chronic infection by evading stimulation of theinnate immune responses However a nonmotile flgKmutantis defective in surface attachment suggesting a role for flagellaandor motility in the initial cell-to-surface interaction [69]Mutants in fliM and cheY are also nonmotile and are unableto form biofilm cap structures on initial biofilm colonies [70]Other Pseudomonas species include Pseudomonas fluorescenswhich possesses multiple flagella and is found in the soil andwater Some P fluorescens strains have been shown to protectplant roots against fungal infections [71] P fluorescens doesnot typically cause disease in humans but it is an opportunis-tic pathogen in immunocompromised individuals [72 73]

Like the Epsilonproteobacteria and Vibrio P aeruginosapossesses a four-tiered transcriptional hierarchy for regulat-ing flagellar gene expression FleQ is the master regulatorfor flagellar synthesis and fleQ is regulated by global factorsoutside the flagellar regulons such as cyclic di-GMP asignaling molecule important in modulating the transitionbetween planktonic and biofilm lifestyles [74] The earlyflagellar genes are dependent on FleQ and RpoN for theirtranscription and encode components of the basal body aswell as the filament cap which is somewhat surprising sinceit is not needed until late in flagellar biogenesis [75] Theearly genes also encode regulatory proteins which includeflhF fleN fleS and fleR [75 76] FleN is an antiactivator ofFleQ that maintains the normal flagellum copy number ofone per cell by downregulating genes encoding early flagellarcomponents via a negative feedbackmechanism [77 78] FleSand FleR form a two-component system that is required forthe transcription of the middle RpoN-dependent genes [75]The signal(s) required for FleS activation is unknown Themiddle genes encode components of the basal body includingrod L ring hook hook cap and hook-filament junctionproteins [75] The late genes are FliA dependent and encodeFliC (flagellin) FleL (filament length control protein) FlgMFlgN (a protein required for initiation of filament assembly)and some chemotaxis proteins [75] Transcription of fliA isconstitutive [75] and FlgM (whose transcription is also FleQdependent but not RpoN dependent) represses the activityof FliA until completion of the hook-basal body structure atwhich point it is secreted from the cytoplasm via the exportapparatus which alleviates its inhibition of FliA

24 Caulobacter crescentus Caulobacter crescentus is anAlphaproteobacterium found in freshwater that is a modelorganism for cell cycle studies C crescentus divides asym-metrically to produce two cells with distinct morphologiesa swarmer cell and a stalked cell The nonmotile stalked cellcontains a polar stalk which secretes an exopolysaccharidethat facilitates adhesion to surfaces In addition the stalkedcell initiatesDNA replication upon the start of cell division Incontrast the swarmer cell is motile via a polar flagellum andDNA replication is repressed for a defined length of time untilthe cell differentiates into a stalked cell Flagellar biogenesis iscoordinated with the cell division cycle such that all progenyswarmer cells possess a functional flagellum

Flagellar genes inC crescentus are regulated by two sigmafactors The initiation of DNA replication in the swarmer cellresults in expression and activation of the global transcriptionfactor CtrA CtrA also acts as themaster regulator for flagellargene expression by activating transcription of the early genes(encode MS ring C ring and export apparatus FlbD FliX)Additionally CtrA controls expression of rpoD [79] and isneeded for transcription of the early flagellar genes [80]FlbD and FliX are transcriptional regulators that regulateexpression of the early and middle genes The middle andlate genes which encode the hook protein and flagellins aretranscribed only after the components encoded by the earlygenes have been assembled into the nascent flagellum FlbDis an RpoN-dependent transcriptional activator which bindsftr (flagella transcriptional regulation) sequence motifs toregulate gene expression Once the early genes are expressedFlbD binds the ftr sequence motifs in the promoter regionsof early flagellar genes to inhibit transcription of these genesFor example FlbD binds the ftr4 site located upstream ofthe early gene fliF to repress its transcription once the earlygenes have been expressed [81] In contrast FlbD stimulatestranscription of the middle genes by binding to the ftrsites of the middle and late genes [81 82] FlbD activity isregulated by FliX which acts to either inhibit or stimulateFlbD In the absence of early flagellar structures FliX inhibitsFlbD activity by binding FlbD preventing it from bindingftr sites [83] Once the early flagellar structures are formedFliX stimulates FlbD The mechanism for this stimulationis unknown but it may involve another factor such asan assembled component of the flagellum which convertsFliX into a positive regulator or it may involve covalentmodifications of FliX or FlbD [83] Although FlbD possessesan N-terminal response regulator domain FlbD activity doesnot appear to be regulated by phosphorylation via a sensorkinase The exact cellular cues that regulate FlbD are notknown but FlbD activity is linked to cell division [84]

Unlike the bacteria discussed thus far flagellar biogenesisin C crescentus does not involve FliA Rather the six flagellingenes found in C crescentus are RpoN dependent Theexpression of the flagellin genes is regulated by FlbD and FlbTwhich binds the 51015840 UTR of flagellin transcripts and repressestranslation until the hook-basal body structure is assembled[85] Though the mechanism for this temporal regulation isunknown one possibility is that FlbT represses translationof flagellin genes until a positive factor binds the 51015840 UTRregion of flagellins to promote translation upon assembly ofthe mature hook-basal body structure [85]

3 Coupling Flagellar Biosynthesis withOther Cellular Activities

Though flagellar biogenesis is a complex process in itselfit is further complicated by its coordination with othercellular processes Flagellar biosynthesis is timed with othercellular activities such as cell replication and quorum sensingEnvironmental stimuli also play a role in regulating flagellarbiogenesis as bacteria respond to changes in the environmentby altering behavior needed for survival Many of these

Scientifica 7

responses involve altering the state of flagellar biogenesiswithin these different contexts

31 Modulating Flagellar Gene Expression during the CellCycle Flagellar biosynthesis is linked to the cell cyclethrough various mechanisms and involves coordinating flag-ellar gene expression with growth phase and cell division InV cholerae chemotaxis and motility genes are upregulatedas the population enters stationary phase Of the 114 genesidentified with roles in chemotaxis and flagellar biogen-esis 72 genes were upregulated more than 2-fold duringstationary phase compared to mid-exponential phase [86]The upregulation of 60 of the 72 chemotaxis and flagellargenes is dependent on RpoS indicating a correlation betweenflagellar biogenesis and entry into stationary phase [86]The coordination between entry into stationary phase andflagellar biogenesis may aid the bacterium in exiting the hostwhen nutrients become depleted and prepare the bacteriumfor colonization of other niches or survival in environmentalreservoirs

C crescentus is a particularly good organism for studyinghow flagellar biogenesis is integrated with the cell cyclebecause of its two distinct cell morphologies The flagellarfilament is assembled just prior to cell division and allprogeny swarmer cells possess a fully functional flagellumThe synthesis of the flagellum is intimately linked to the cellcycle through regulatory proteins such as CtrA FlbD andFliX that are involved in both the cell cycle and flagellarbiogenesis This dual functionality may exist to ensure thateach swarmer cell is flagellated The master regulator forflagellar biogenesis CtrA regulates at least 95 genes [87]including the cell-division genes ftsZ [88] and ftsQA [89]Cells of a fliX mutant become filamentous as they enterlate log phase [90] demonstrating a role for this flagellarregulatory protein in cell division Flagellar biogenesis is alsolinked to the cell cycle via FlbT and FlaF FlaF is required forflagellar biogenesis and motility [91] FlbT protein levels areconstant throughout the cell cycle while FlaF levels correlateto flagellin levels both of which peak just prior to cell division[91] Since FlbT is a negative regulator for flagellin translationand peaks in flagellin levels correlate with FlaF levels thisobservation suggests that FlaF may temporally modulateFlbT activity [91]

Expression of Vibrio vulnificus FlhF which is needed forpolar localization of the flagellum is regulated by the quorumsensing master regulator SmcR flhF transcript levels in asmcRmutant are higher than wild-type levels indicating thatSmcR represses flhF expression [92] In wild-type cells flhFtranscript levels are highest during exponential phase anddecrease upon entry into stationary phase [92] In contrasttranscript levels of smcR increase as cultures enter stationaryphase suggesting that SmcR plays a major role for growthphase-dependent variation of flhF expression [92] Thisgrowth phase-dependent gene expression may be importantin host colonization as during the initial infection bacterialcell densities are low but at later stages of colonizationwhen bacterial cell densities are higher motility may not benecessary

Expression of flagellar genes is regulated in P fluorescensthroughout the growth phases by the Gac (GacAGasS) two-component system which limits flagellar biosynthesis duringexponential growth by downregulating transcription of fleQ(encodes the master regulator) [93] The Gac system posi-tively regulates production of virulence factors and quorumsensing molecules and is required for full virulence in animaland plant hosts [94] Levels of the Gac system componentsare regulated in response to growth phase as gacA and gacStranscript levels are highest at mid-exponential growth phase[95] The concomitant upregulation of virulence factors andthe downregulation of flagellar genes by the Gac system maybe a way to coordinate virulence and motility with growthphase so that virulence factors are expressed at high celldensities during host colonization and flagellar genes areturned off to facilitate attachment to the host

32 Connections between Quorum Sensing Host Colonizationand Flagellar Gene Expression In many pathogenic bacte-ria flagella aid in surface attachment and colonization Insuch cases successful colonization may require cooperationbetween quorum sensing and flagellar synthesis to integratesurface attachment and virulence gene expression at highcell densities in pathogens Surface colonization can leadto biofilm formation which is generally more resistant tohost defenses and antimicrobials Pathogens use differentmechanisms to couple expression of virulence genes withflagellar gene expression in the context of the host Thisincludes modulating quorum sensing and host colonizationbased on the status of the flagellum or modulating flagellarbiogenesis based on quorum sensing signals

Quorum sensing is used to regulate gene expression inresponse to cell densityThis is achieved through the secretionof a signaling molecule called an autoinducer (AI) AIs areproduced by bacteria and travel across the cell membrane andaccumulate in the environment Upon reaching a critical con-centration the AIs stimulate transcription of genes requiredfor group behavior luxS is responsible for the productionof AI-2 [96] and an H pylori mutant in luxS exhibitsdecreasedmotility compared towild type [97]flhA transcriptlevels are decreased in the luxS mutant but addition of 45-dihydroxy-23-pentanedione (functions as AI-2) into thegrowth medium restores flhA transcript levels to wild-typelevels [97] Like the H pylori luxS mutant a C jejuni luxSmutant is less motile than wild type [98] and the mutantdisplays reduced transcript levels of flaA [99] Using DNAmicroarrays He and coworkers found that a majority of theflagellar genes are downregulated in a C jejuni luxS mutant[98] Expression of chemotaxis genes however is unchangedin the luxS mutant indicating that the motility defect of theluxS mutant is due to defects in flagellar biogenesis and notchemotaxis [98] A rationale for the link between cell densityand flagellar biogenesis is that it may aid in colonization ofhost tissue by ensuring that a sufficient number of flagellatedbacteria are present to establish a successful infection

Regulatory proteins involved in flagellar biogenesis canregulate quorum sensing and colonization or they themselvescan be regulated by quorum sensing and colonization factorsFor example the RpoN-dependent activator FlrC not only

8 Scientifica

is required for flagellar biogenesis but also appears to play arole in host colonization by V cholerae Mutants expressinga FlrC variant incapable of being phosphorylated fail toproduce flagella and are therefore defective in colonization[54] Interestingly a mutant expressing a constitutively activeform of FlrC is also deficient in colonization even though thecells are motile and produce flagella [54] This observationdemonstrates the need for coordinating flagellar biogenesisand pathogenesis for successful colonization ConverselyVfr a major quorum sensing and virulence regulator inP aeruginosa downregulates expression of flagellar genesby repressing transcription from the fleQ promoter [100]Inhibition of flagellar biogenesis by Vfr may aid P aeruginosain persisting at the site of infection to achieve high celldensities and virulence gene expression

HapR is a transcriptional regulator involved in quorumsensing in V cholerae which also regulates expression ofvirulence genes HapR represses expression of several vir-ulence genes [101] and hapR expression is regulated by anunknown mechanism involving FliA [102] hapR expres-sion is depressed in flgM and flgD mutants (both of thesemutations alleviate FlgM repression on FliA activity) whiledeletion of fliA results in elevated hapR expression [102] Theauthors of this study speculated that expression of HapR isregulated by a regulator or small noncoding RNA (sRNA)that is part of the FliA regulon FliA-mediated regulation ofhapR appears to be linked to the shearing of flagella frombacteria that penetrate the mucin layer to infect the hostThis shearing of the flagella leads to secretion of FlgM viathe flagellar protein export apparatus and results in increasedexpression of FliA-dependent genes and repression of hapRexpression [102] The outcome of such a mechanism is thatvirulence genes which are negatively regulated by HapRare expressed specifically when V cholerae penetrates theintestinal mucous layer hapR production is also regulatedby the quorum sensing protein LuxO further linking viru-lence to cell density LuxO is an RpoN-dependent activator[103] which along with HapR regulates motility proteaseproduction and biofilm formation [101] High cell densitiesare common during late stages of infection and the HapR-mediated repression of virulence genes may aidV cholerae indetaching and finding new colonization sites [86 101]

V parahaemolyticus uses its polar flagellum to detectsurfaces by sensing impedance to the polar flagellum rotationrate [104] Once a surface is encountered expression of thelateral flagellar genes is induced Lateral flagella are importantfor host colonization as mutants that do not produce lateralflagella are deficient in adherence to HeLa cells and biofilmformation [61] Gode-Potratz and coworkers identified sim70genes that are surface responsive most of which are positivelyregulated [64] Some of the surface-regulated genes that areupregulated are virulence genes such as a putative GbpAhomolog and components of a type III secretion system [64]GbpA from V cholerae promotes adherence to chitinoussurfaces of zooplankton and human epithelia cells [105]which is consistent with the observed upregulation of gbpAin V parahaemolyticus when it encounters a surface

The P aeruginosa flagellum is an important determinantin the susceptibility of the bacterium to host defenses

P aeruginosa fliCmutants are unable to upregulate transcrip-tion of lasI and rhlI which encode enzymes that synthesizequorum sensing homoserine lactones [106] These quo-rum sensing molecules regulate production of exoproteaseswhich degrade surfactant protein-A (SP-A) an antimicro-bial that opsonizes and permeabilizes membranes of lungpathogens [106] In the absence of these exoproteases theP aeruginosa fliC mutants are cleared from the lung [106]Though it is not known why fliC mutants are unable toupregulate transcription of lasI and rhlI it is not due tothe inability to sense the environment [106] P aeruginosafliE flgE fliC and fliD mutants are also attenuated in LPSbiosynthesis which compromises the integrity of their outermembranes making them more susceptible to SP-A [107]Additionally flgE and fliC mutants make less pyocyanin aredox-active toxic secondary metabolite that is crucial forlung infection [108 109]

33 Reacting to pH Changes by Gastrointestinal PathogensGastrointestinal pathogens encounter varying environmentalconditions as they travel from the mouth to the stomach andthen to the intestines The gastric pathogen H pylori has theadded burden of long-term survival in the acidic environ-ment of the stomach Pathogens that colonize the intestinaltract must also survive passage through the stomach in highenough numbers to be able to colonize their target organsThus it is not surprising that gastrointestinal pathogens oftenmodulate gene expression in response to acidic conditions

Merrell and coworkers found that expression of approxi-mately 7 of theH pylori genes is altered by a shift to low pH[110] Many of these genes are involved in flagellar synthesiswith the majority of those being RpoN dependent [110] FliA-dependent genes such as flgM in H pylori [110] and flaAin C jejuni [111] are also upregulated at low pH conditionsThe upregulation of flagellar genes is critical for survival ofH pylori as the bacterium is not able to survive at low pHbut must swim across the mucous layer to the underlyinggastric epithelium to establish a successful infection Inaddition to enhancing transcription of flagellar genes acidicenvironments also stimulate motility and swimming speedspotentially in response to increased concentration of protonsin the stomach [110]

The basis for upregulation of the RpoN-dependent flag-ellar genes in H pylori likely involves a stimulation of FlgSactivity at low pHWen and coworkers reported thatH pylorirequires FlgS to survive acid stress (30-minute exposure topH25) [112]These researchers found that besides the knownRpoN-dependent flagellar genes FlgS was required for theupregulation of 86 genes in response to acid stress many ofwhich were previously known to be required for acid stresssurvival [112] It is not known if FlgS responds directly toacid stress or if the stress signal is mediated through anotherfactor

34 Altering Flagellar Biogenesis by Phase Variation In somebacteria flagellar biogenesis is subject to phase variationevents that affect expression of specific flagellar genes Phasevariation causes changes in phenotypes at frequencies that

Scientifica 9

are much higher than random mutations and contribute toheterogeneity within a population The ability of H pylori topersist in the host may involve the phase-variable dependentexpression of flagella that can serve as an advantage foradaptation to the host and for evading the host immuneresponse C jejuni utilizes phase variation to turn on andoff production of flagella a strategy that increases com-mensal colonization in poultry [113] Many phase variationevents occur by random reversible changes in the length ofshort DNA sequence repeats resulting from slipped-strandmispairing These events can take place within the codingsequence or in the promoter of a gene C jejuni phasevariants in which expression of flgR is in the OFF stateresult from the addition or removal of a nucleotide withinone of two homopolymeric nucleotide tracts consisting ofadenine and thymine These nucleotide tracts are locatedwithin the coding sequence of flgR causing the sequence toshift out of frame [113] Revertants to the ON state ariseupon the addition or removal of a nucleotide within theoriginal mutated homopolymeric nucleotide tract to restorethe wild-type sequence Alternatively pseudorevertants canarise in which nucleotides are removed or added close to theoriginal mutated homopolymeric nucleotide tract to restorethe correct reading frame but which result in changes in theamino acid sequence of the protein [113] flgS is also regulatedby phase variation in C jejuni providing multiple levels ofphase-variable control for theRpoN regulon [114 115] Similarmechanisms of phase variation also exist for flhA in C coli[116] and fliP in H pylori [117] which leads to the formationof truncated proteins that prevent flagellar biogenesis

A second type of phase variation in flagellar biogen-esis is mediated through alterations in DNA methylationwhich impact the expression of specific flagellar genesHost-adapted bacterial pathogens such as H pylori oftenpossess DNA methyltransferases and type III restriction-modification (R-M) systems that contain simple tandemDNA repeats which undergo slipped-strand mispairing dur-ing DNA replication that causes frameshift mutations andphase variation [118] R-M systems widespread among manybacteria confer protection from invasion by foreign DNAType III R-M systems are composed of a methyltransferase(mod) and an endonuclease (res) gene whose gene prod-ucts typically function together The switching between ONand OFF states of a DNA methyltransferase can regulateexpression of a ldquophasevarionrdquo via differential methylationof the genome in ON and OFF states [119] Many of thesephase-variable methyltransferase genes are associated withan inactive res gene resulting from frameshift mutationor are orphans that are not associated with a res genesuggesting that DNA restriction is not the primary roleof these methyltransferases (reviewed by Fox et al [120])For example H pylori modH encodes a phase-variableDNA methyltransferase and the cognate res gene containsa nonsense mutation frameshift mutation or is absent invarious H pylori strains [119] modH OFF strains havedecreased expression of flaA and fliK compared to themodHON strain [119] FlaA has a low ability to activate innateimmunity via the Toll-like receptor 5 [121] and modulatingexpression of the flagellin may be advantageous in evading

the host response Another H pylori methyltransferase withan inactive res gene is hpyAVIBM [122 123] hpyAVIBMencodes a C5 cytosine methyltransferase and contains AGrepeats in its open reading frame making it susceptible tophase variation via frame shift mutations [124] For instancehpyAVIBM from strain 26695 and hpyAVIBM from strainHPAG1 have five AG repeats whereas the four AG repeats instrain San 74 result in the translation of a truncated protein[124] Deletion of hpyAVIBM results in both the upregulationand downregulation of rpoN fliR fliD fliS motA fliK andflgK depending on the strain used [124] indicating that phasevariation of hpyAVIBM can alter flagellar biogenesis

P aeruginosa populations can possess two phenotypicvariants one which forms small rough colonies (S) andanother which forms large flat (L) colonies S variants formbiofilms in nonagitated liquid cultures whereas the L variantsdo not [125] S variants possess defects in flagellum-mediatedswimming flagellum-mediated swarming and type IV-pilus-mediated twitching but are able to revert back to the Lphenotype at relatively high frequencies suggesting that theshift between phenotypes is regulated by phase variation[125] Phase variation also occurs in P fluorescens whichcolonizes the alfalfa rhizosphere Phenotypic variants ariseduring rhizosphere colonization and are dependent on theactivity of a site-specific recombinase [126] C variants havewild-type colony morphology while F and S variants havea translucent and diffuse colony morphology [126] F andS variants which colonize distal parts of the roots andswim faster than the C variants overproduce fliC (flagellin)transcripts and synthesize flagellin filaments that aresim3 timeslonger than those of the C variant [126]

4 Conclusions and Future Directions

Flagellar biogenesis is a carefully choreographed processin which many different components of the flagellum aremade as they are needed for assembly into the nascentflagellum Bacteria have evolved elaborate and sundry reg-ulatory networks to couple flagellar gene expression withassembly A driving force for the evolution of such regulatorynetworks is undoubtedly the environmental and ecologicalconstraints imposed by a particular bacteriumrsquos niche Inaddition the degree to which a given bacterial species needsto couple flagellar biogenesis to other cellular processes suchas cell division has also likely shaped the evolution of theseregulatory networks We focused here on bacteria whichutilize RpoN for flagellar biogenesis yet even among thesebacteria the mechanisms used to regulate expression of theRpoN-dependent flagellar genes vary greatly For most of thesystems described in this review we have only scratched thesurface in our understanding of how they operate For each ofthese systems fundamental questions remain to be answeredsuch as the following are all of the flagellar genes of a givensystem expressed as part of a transcription hierarchy or aresome genes expressed constitutively Are there regulatorymechanisms within RpoN flagellar regulons that fine-tunegene expression so that components of the flagellum aremadeprecisely when needed What are the cellular cues sensed by

10 Scientifica

the regulatory proteins that control expression of the RpoN-dependent flagellar genes How does the flagellar exportapparatus control expression of RpoN-dependent flagellargenes in various bacteria Does FlgZ have a regulatoryrole in flagellar biogenesis in H pylori and other bacteriaAs our understanding of the molecular genetics of diversebacterial species expands novel regulatory mechanisms thatcontrol expression of flagellar genes within these bacteria willundoubtedly come to light For example it was only recentlythat sRNAs were identified in H pylori some of whichare potentially FliA dependent [127] and may have roles inflagellar biogenesis Finally with increasingly sophisticatedmethods in bioinformatics genomics and proteomics fur-ther characterization of flagellar regulatory networks willprovide important insights into the mechanisms governinggene expression and identify new regulatory proteins andmechanisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by award MCB-1244242 from theNational Science Foundation

References

[1] T Minamino K Imada and K Namba ldquoMechanisms of typeIII protein export for bacterial flagellar assemblyrdquo MolecularBioSystems vol 4 no 11 pp 1105ndash1115 2008

[2] J L McMurry J W Murphy and B Gonzalez-Pedrajo ldquoTheFliN-FliH interaction mediates localization of flagellar exportATPase FliI to the C ring complexrdquo Biochemistry vol 45 no39 pp 11790ndash11798 2006

[3] R M Macnab Ed Flagella and Motility ASM Press 1996[4] T Minamino K Imada and K Namba ldquoMolecular motors of

the bacterial flagellardquoCurrent Opinion in Structural Biology vol18 no 6 pp 693ndash701 2008

[5] T G Smith and T R Hoover ldquoDeciphering bacterial flagellargene regulatory networks in the genomic erardquo Advances inApplied Microbiology vol 67 pp 257ndash295 2009

[6] J K Anderson T G Smith and T R Hoover ldquoSense andsensibility flagellum-mediated gene regulationrdquo Trends inMicrobiology vol 18 no 1 pp 30ndash37 2010

[7] J D Helmann ldquoAlternative sigma factors and the regulation offlagellar gene expressionrdquoMolecular Microbiology vol 5 no 12pp 2875ndash2882 1991

[8] D L Popham D Szeto J Keener and S Kustu ldquoFunctionof a bacterial activator protein that binds to transcriptionalenhancersrdquo Science vol 243 no 4891 pp 629ndash635 1989

[9] S Sasse-Dwight and J D Gralla ldquoProbing the EscherichiacoliglnALG upstream activation mechanism in vivordquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8934ndash8938 1988

[10] M Kihara T Minamino S Yamaguchi and R M MacnabldquoIntergenic suppression between the flagellar MS ring protein

FliF of Salmonella and FlhA a membrane component of itsexport apparatusrdquo Journal of Bacteriology vol 183 no 5 pp1655ndash1662 2001

[11] J S van Arnam J L McMurry M Kihara and R M MacnabldquoAnalysis of an engineered Salmonella flagellar fusion proteinFliR-FlhBrdquo Journal of Bacteriology vol 186 no 8 pp 2495ndash2498 2004

[12] C S Barker I V Meshcheryakova A S Kostyukova and FA Samatey ldquoFliO regulation of FliP in the formation of theSalmonella enterica flagellumrdquo PLoS Genetics vol 6 no 9Article ID e1001143 2010

[13] A C Lowenthal M Hill L K Sycuro K Mehmood N RSalama and K M Ottemann ldquoFunctional analysis of the Heli-cobacter pylori flagellar switch proteinsrdquo Journal of Bacteriologyvol 191 no 23 pp 7147ndash7156 2009

[14] R M Macnab ldquoType III flagellar protein export and flagellarassemblyrdquo Biochimica et Biophysica Acta vol 1694 no 1ndash3 pp207ndash217 2004

[15] K A Eaton D R Morgan and S Krakowka ldquoCampylobacterpylori virulence factors in gnotobiotic pigletsrdquo Infection andImmunity vol 57 no 4 pp 1119ndash1125 1989

[16] K A Eaton D R Morgan and S Krakowka ldquoMotility as afactor in the colonisation of gnotobiotic piglets by Helicobacterpylorirdquo Journal of Medical Microbiology vol 37 no 2 pp 123ndash127 1992

[17] D G Newell H McBride and J M Dolby ldquoInvestigationson the role of flagella in the colonization of infant mice withCampylobacter jejuni and attachment of Campylobacter jejunito human epithelial cell linesrdquo Journal of Hygiene vol 95 no 2pp 217ndash227 1985

[18] TMorooka A Umeda andK Amako ldquoMotility as an intestinalcolonization factor forCampylobacter jejunirdquo Journal of GeneralMicrobiology vol 131 no 8 pp 1973ndash1980 1985

[19] C Y Kao B S Sheu SM Sheu et al ldquoHighermotility enhancesbacterial density and inflammatory response in dyspepticpatients infected with Helicobacter pylorirdquo Helicobacter vol 17no 6 pp 411ndash416 2012

[20] E Niehus H Gressmann F Ye et al ldquoGenome-wide analysisof transcriptional hierarchy and feedback regulation in theflagellar system of Helicobacter pylorirdquoMolecular Microbiologyvol 52 no 4 pp 947ndash961 2004

[21] M Balaban S N Joslin and D R Hendrixson ldquoFlhF and itsGTPase activity are required for distinct processes in flagellargene regulation and biosynthesis in Campylobacter jejunirdquoJournal of Bacteriology vol 191 no 21 pp 6602ndash6611 2009

[22] A Kusumoto A Shinohara H Terashima S Kojima TYakushi and M Homma ldquoCollaboration of FlhF and FlhGto regulate polarflagella number and localization in VibrioalginolyticusrdquoMicrobiology vol 154 no 5 pp 1390ndash1399 2008

[23] A Kusumoto K Kamisaka T Yakushi H Terashima AShinohara and M Homma ldquoRegulation of polar flagellarnumber by the flhF and flhG genes in Vibrio alginolyticusrdquoJournal of Biochemistry vol 139 no 1 pp 113ndash121 2006

[24] B M Pruszlig and P Matsumura ldquoA regulator of the flagellarregulon of Escherichia coli flhD also affects cell divisionrdquoJournal of Bacteriology vol 178 no 3 pp 668ndash674 1996

[25] B M Pruszlig and P Matsumura ldquoCell cycle regulation of flagellargenesrdquo Journal of Bacteriology vol 179 no 17 pp 5602ndash56041997

[26] G Spohn and V Scarlato ldquoMotility of Helicobacter pylori iscoordinately regulated by the transcriptional activator FlgR an

Scientifica 11

NtrC homologrdquo Journal of Bacteriology vol 181 no 2 pp 593ndash599 1999

[27] M Schirm E C Soo A J Aubry J Austin P Thibault and SM Logan ldquoStructural genetic and functional characterizationof the flagellin glycosylation process in Helicobacter pylorirdquoMolecular Microbiology vol 48 no 6 pp 1579ndash1592 2003

[28] D Beier and R Frank ldquoMolecular characterization of two-component systems of Helicobacter pylorirdquo Journal of Bacteri-ology vol 182 no 8 pp 2068ndash2076 2000

[29] P Brahmachary M G Dashti J W Olson and T R HooverldquoHelicobacter pylori FlgR is an enhancer-independent activatorof 12059054-RNA polymerase holoenzymerdquo Journal of Bacteriologyvol 186 no 14 pp 4535ndash4542 2004

[30] M M S M Wosten J A Wagenaar and J P M van PuttenldquoThe FlgSFlgR two-component signal transduction systemregulates the fla regulon in Campylobacter jejunirdquo The Journalof Biological Chemistry vol 279 no 16 pp 16214ndash16222 2004

[31] M Bush and R Dixon ldquoThe role of bacterial enhancer bindingproteins as specialized activators of 12059054-dependent transcrip-tionrdquo MicroBiology and Molecular Biology Reviews vol 76 no3 pp 497ndash529 2012

[32] J Schumacher N Joly M Rappas X Zhang and M BuckldquoStructures and organisation of AAA+ enhancer binding pro-teins in transcriptional activationrdquo Journal of Structural Biologyvol 156 no 1 pp 190ndash199 2006

[33] S N Joslin and D R Hendrixson ldquoAnalysis of the Campy-lobacter jejuni FlgR response regulator suggests integration ofdiverse mechanisms to activate an NtrC-like proteinrdquo Journalof Bacteriology vol 190 no 7 pp 2422ndash2433 2008

[34] J M Boll and D R Hendrixson ldquoA specificity determinant forphosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphaterdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 50 pp 20160ndash20165 2011

[35] S Porwollik B Noonan and P W OrsquoToole ldquoMolecular char-acterization of a flagellar export locus of Helicobacter pylorirdquoInfection and Immunity vol 67 no 5 pp 2060ndash2070 1999

[36] E Allan N Dorrell S Foynes M Anyim and B W WrenldquoMutational analysis of genes encoding the early flagellarcomponents of Helicobacter pylori evidence for transcriptionalregulation of flagellin A biosynthesisrdquo Journal of Bacteriologyvol 182 no 18 pp 5274ndash5277 2000

[37] T G Smith L Pereira and T R Hoover ldquoHelicobacter pyloriFlhB processing-deficient variants affect flagellar assembly butnot flagellar gene expressionrdquo Microbiology vol 155 no 4 pp1170ndash1180 2009

[38] D R Hendrixson B J Akerley and V J DiRita ldquoTranspo-son mutagenesis of Campylobacter jejuni identifies a bipartiteenergy taxis system required for motilityrdquoMolecular Microbiol-ogy vol 40 no 1 pp 214ndash224 2001

[39] J Tsang T G Smith L E Pereira and T R Hoover ldquoInsertionmutations inHelicobacter pylori flhA reveal strain differences inRpoN-dependent gene expressionrdquo Microbiology vol 159 part1 pp 58ndash67 2013

[40] J M Boll and D R Hendrixson ldquoA regulatory checkpointduring flagellar biogenesis in Campylobacter jejuni initiatessignal transduction to activate transcription of flagellar genesrdquoMBio vol 4 no 5 Article ID e00432-13 2013

[41] T Minamino H Doi and K Kutsukake ldquoSubstrate specificityswitching of the flagellum-specific export apparatus during flag-ellar morphogenesis in Salmonella typhimuriumrdquo Bioscience

Biotechnology and Biochemistry vol 63 no 7 pp 1301ndash13031999

[42] M Erhardt H M Singer D H Wee J P Keener and K THughes ldquoAn infrequent molecular ruler controls flagellar hooklength in Salmonella entericardquo EMBO Journal vol 30 no 14 pp2948ndash2961 2011

[43] K T Hughes K L Gillen M J Semon and J E KarlinseyldquoSensing structural intermediates in bacterial flagellar assemblyby export of a negative regulatorrdquo Science vol 262 no 5137 pp1277ndash1280 1993

[44] K A Ryan N Karim M Worku S A Moore C W Pennand P W OrsquoToole ldquoHP0958 is an essential motility gene inHelicobacter pylorirdquo FEMS Microbiology Letters vol 248 no 1pp 47ndash55 2005

[45] L Pereira and T R Hoover ldquoStable accumulation of 12059054 inHelicobacter pylori requires the novel protein HP0958rdquo Journalof Bacteriology vol 187 no 13 pp 4463ndash4469 2005

[46] J C Rain L Selig H de Reuse et al ldquoThe protein-proteininteraction map of Helicobacter pylorirdquo Nature vol 409 no6817 pp 211ndash215 2001

[47] F P Douillard K A Ryan D L Caly et al ldquoPosttranscriptionalregulation of flagellin synthesis in Helicobacter pylori by theRpoN chaperone HP0958rdquo Journal of Bacteriology vol 190 no24 pp 7975ndash7984 2008

[48] C Josenhans E Niehus S Amersbach et al ldquoFunctionalcharacterization of the antagonistic flagellar late regulators FliAand FlgM ofHelicobacter pylori and their effects on theH pyloritranscriptomerdquoMolecular Microbiology vol 43 no 2 pp 307ndash322 2002

[49] M Rust S Borchert E Niehus et al ldquoThe Helicobacter pylorianti-sigma factor FlgM is predominantly cytoplasmic andcooperates with the flagellar basal body protein FlhArdquo Journalof Bacteriology vol 191 no 15 pp 4824ndash4834 2009

[50] D R Hendrixson and V J DiRita ldquoTranscription of 12059054-dependent but not 12059028-dependent flagellar genes in Campy-lobacter jejuni is associated with formation of the flagellarsecretory apparatusrdquoMolecular Microbiology vol 50 no 2 pp687ndash702 2003

[51] M M S M Wosten L van Dijk A K J Veenendaal MR de Zoete N M C Bleumink-Pluijm and J P M vanPutten ldquoTemperature-dependent FlgMFliA complex forma-tion regulates Campylobacter jejuni flagella lengthrdquo MolecularMicrobiology vol 75 no 6 pp 1577ndash1591 2010

[52] M G Prouty N E Correa and K E Klose ldquoThe novel 12059054- and12059028-dependent flagellar gene transcription hierarchy of Vibrio

choleraerdquo Molecular Microbiology vol 39 no 6 pp 1595ndash16092001

[53] D Srivastava M L Hsieh A Khataokar M B Neiditch and CM Waters ldquoCyclic di-GMP inhibits Vibrio choleraemotility byrepressing induction of transcription and inducing extracellularpolysaccharide productionrdquo Molecular Microbiology vol 90no 6 pp 1262ndash1276 2013

[54] N E Correa C M Lauriano R McGee and K E KloseldquoPhosphorylation of the flagellar regulatory protein FlrC is nec-essary for Vibrio cholerae motility and enhanced colonizationrdquoMolecular Microbiology vol 35 no 4 pp 743ndash755 2000

[55] N E Correa J R Barker and K E Klose ldquoThe Vibrio choleraeFlgM homologue is an anti-12059028 factor that is secreted throughthe sheathed polar flagellumrdquo Journal of Bacteriology vol 186no 14 pp 4613ndash4619 2004

12 Scientifica

[56] MMoisi C Jenul SM Butler et al ldquoA novel regulatory proteininvolved in motility of Vibrio choleraerdquo Journal of Bacteriologyvol 191 no 22 pp 7027ndash7038 2009

[57] L Aravind and C P Ponting ldquoThe cytoplasmic helical linkerdomain of receptor histidine kinase and methyl-acceptingproteins is common to many prokaryotic signalling proteinsrdquoFEMS Microbiology Letters vol 176 no 1 pp 111ndash116 1999

[58] SMerino J G Shaw and JM Tomas ldquoBacterial lateral flagellaan inducible flagella systemrdquo FEMS Microbiology Letters vol263 no 2 pp 127ndash135 2006

[59] L L McCarter ldquoDual flagellar systems enable motility underdifferent circumstancesrdquo Journal of Molecular Microbiology andBiotechnology vol 7 no 1-2 pp 18ndash29 2004

[60] S Ulitzur ldquoInduction of swarming in Vibrio parahaemolyticusrdquoArchives of Microbiology vol 101 no 4 pp 357ndash363 1974

[61] K S Park M Arita T Iida and T Honda ldquovpaH a geneencoding a novel histone-like nucleoid structure-like proteinthat was possibly horizontally acquired regulates the biogen-esis of lateral flagella in trh-positive Vibrio parahaemolyticusTH3996rdquo Infection and Immunity vol 73 no 9 pp 5754ndash57612005

[62] LMcCarter andM Silverman ldquoIron regulation of swarmer celldifferentiation ofVibrio parahaemolyticusrdquo Journal of Bacteriol-ogy vol 171 no 2 pp 731ndash736 1989

[63] B J Stewart and L L McCarter ldquoLateral flagellar gene systemofVibrio parahaemolyticusrdquo Journal of Bacteriology vol 185 no15 pp 4508ndash4518 2003

[64] C J Gode-Potratz R J Kustusch P J Breheny D S Weissand L LMcCarter ldquoSurface sensing inVibrio parahaemolyticustriggers a programme of gene expression that promotes colo-nization and virulencerdquo Molecular Microbiology vol 79 no 1pp 240ndash263 2011

[65] J L Veesenmeyer A R Hauser T Lisboa and J Rello ldquoPseu-domonas aeruginosa virulence and therapy evolving transla-tional strategiesrdquo Critical Care Medicine vol 37 no 5 pp 1777ndash1786 2009

[66] D Balasubramanian L Schneper H Kumari and K MatheeldquoA dynamic and intricate regulatory network determines Pseu-domonas aeruginosa virulencerdquo Nucleic Acids Research vol 41no 1 pp 1ndash20 2013

[67] T C Montie D D Huntzinger R C Craven and I A HolderldquoLoss of virulence associated with absence of flagellum in anisogenic mutant of Pseudomonas aeruginosa in the burned-mouse modelrdquo Infection and Immunity vol 38 no 3 pp 1296ndash1298 1982

[68] Y R Patankar R R Lovewell M E Poynter J Jyot BI Kazmierczak and B Berwin ldquoFlagellar motility is a keydeterminant of themagnitude of the inflammasome response toPseudomonas aeruginosardquo Infection and Immunity vol 81 no 6pp 2043ndash2052 2013

[69] G A OrsquoToole and R Kolter ldquoFlagellar and twitching motilityare necessary for Pseudomonas aeruginosa biofilm develop-mentrdquoMolecular Microbiology vol 30 no 2 pp 295ndash304 1998

[70] K B Barken S J Pamp L Yang et al ldquoRoles of type IVpili flagellum-mediated motility and extracellular DNA in theformation of mature multicellular structures in Pseudomonasaeruginosa biofilmsrdquo Environmental Microbiology vol 10 no 9pp 2331ndash2343 2008

[71] M Leeman J A van Pelt F M den Ouden M Heinsbroek PA Bakker and B Schippers ldquoInduction of systemic resistance

against fusarium wilt of radish by lipopolysaccharides of Pseu-domonas fluorescensrdquo Phytopathology vol 85 no 9 pp 1021ndash1027 1995

[72] P Hsueh L Teng H Pan et al ldquoOutbreak of Pseudomonasfluorescens bacteremia among oncology patientsrdquo Journal ofClinical Microbiology vol 36 no 10 pp 2914ndash2917 1998

[73] VWongK Levi B Baddal J Turton andTC Boswell ldquoSpreadof Pseudomonas fluorescens due to contaminated drinkingwater in a bone marrow transplant unitrdquo Journal of ClinicalMicrobiology vol 49 no 6 pp 2093ndash2096 2011

[74] J W Hickman and C S Harwood ldquoIdentification of FleQ fromPseudomonas aeruginosa as a c-di-GMP-responsive transcrip-tion factorrdquoMolecular Microbiology vol 69 no 2 pp 376ndash3892008

[75] N Dasgupta M C Wolfgang A L Goodman et al ldquoA four-tiered transcriptional regulatory circuit controls flagellar bio-genesis in Pseudomonas aeruginosardquo Molecular Microbiologyvol 50 no 3 pp 809ndash824 2003

[76] S K Arora B W Ritchings E C Almira S Lory and RRamphal ldquoA transcriptional activator FleQ regulates mucinadhesion and flagellar gene expression in Pseudomonas aerugi-nosa in a cascade mannerrdquo Journal of Bacteriology vol 179 no17 pp 5574ndash5581 1997

[77] N Dasgupta and R Ramphal ldquoInteraction of the antiactivatorFleN with the transcriptional activator FleQ regulates flagellarnumber in Pseudomonas aeruginosardquo Journal of Bacteriologyvol 183 no 22 pp 6636ndash6644 2001

[78] N Dasgupta S K Arora and R Ramphal ldquofleN a gene thatregulates flagellar number in Pseudomonas aeruginosardquo Journalof Bacteriology vol 182 no 2 pp 357ndash364 2000

[79] J Malakooti and B Ely ldquoPrincipal sigma subunit of theCaulobacter crescentus RNA polymeraserdquo Journal of Bacteriol-ogy vol 177 no 23 pp 6854ndash6860 1995

[80] M T Laub H H McAdams T Feldblyum C M Fraser andL Shapiro ldquoGlobal analysis of the genetic network controllinga bacterial cell cyclerdquo Science vol 290 no 5499 pp 2144ndash21482000

[81] A K Benson G Ramakrishnan N Ohta J Feng A J Ninfaand A Newton ldquoThe Caulobacter crescentus FlbD proteinacts at ftr sequence elements both to activate and to represstranscription of cell cycle-regulated flagellar genesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 91 no 11 pp 4989ndash4993 1994

[82] D A Mullin S M van Way C A Blankenship and A HMullin ldquoFlbD has a DNA-binding activity near its carboxyterminus that recognizes ftr sequences involved in positive andnegative regulation of flagellar gene transcription in Caulobac-ter crescentusrdquo Journal of Bacteriology vol 176 no 19 pp 5971ndash5981 1994

[83] R J Dutton Z Xu and J W Gober ldquoLinking structural assem-bly to gene expression a novel mechanism for regulating theactivity of a 12059054 transcription factorrdquo Molecular Microbiologyvol 58 no 3 pp 743ndash757 2005

[84] R E Muir J Easter and J W Gober ldquoThe trans-acting flagellarregulatory proteins FliX and FlbD play a central role in linkingflagellar biogenesis and cytokinesis in Caulobacter crescentusrdquoMicrobiology vol 151 no 11 pp 3699ndash3711 2005

[85] P E Anderson and J W Gober ldquoFlbT the post-transcriptionalregulator of flagellin synthesis in Caulobacter crescentus inter-acts with the 51015840 untranslated region of flagellin mRNArdquoMolec-ular Microbiology vol 38 no 1 pp 41ndash52 2000

Scientifica 13

[86] A T Nielsen N A Dolganov G Otto M C Miller C Y WuandGK Schoolnik ldquoRpoS controls theVibrio choleraemucosalescape responserdquo PLoS Pathogens vol 2 no 10 article e1092006

[87] M T Laub S L Chen L Shapiro and H H McAdamsldquoGenes directly controlled by CtrA a master regulator of theCaulobacter cell cyclerdquo Proceedings of the National Academy ofSciences of the United States of America vol 99 no 7 pp 4632ndash4637 2002

[88] A J Kelly M J Sackett N Din E Quardokus and Y VBrun ldquoCell cycle-dependent transcriptional and proteolyticregulation of FtsZ in Caulobacterrdquo Genes and Development vol12 no 6 pp 880ndash893 1998

[89] M Wortinger M J Sackett and Y V Brun ldquoCtrA mediatesa DNA replication checkpoint that prevents cell division inCaulobacter crescentusrdquo EMBO Journal vol 19 no 17 pp 4503ndash4512 2000

[90] R E Muir T M OrsquoBrien and J W Gober ldquoThe Caulobactercrescentus flagellar gene fliX encodes a novel trans-acting fac-tor that couples flagellar assembly to transcriptionrdquo MolecularMicrobiology vol 39 no 6 pp 1623ndash1637 2001

[91] M Llewellyn R J Dutton J Easter D OrsquoDonnol and J WGober ldquoThe conserved flaF gene has a critical role in couplingflagellin translation and assembly in Caulobacter crescentusrdquoMolecular Microbiology vol 57 no 4 pp 1127ndash1142 2005

[92] S M Kim D H Lee and S H Choi ldquoEvidence that the Vibriovulnificus flagellar regulator FlhF is regulated by a quorumsensing master regulator SmcRrdquo Microbiology vol 158 part 8pp 2017ndash2025 2012

[93] F Martınez-Granero A Navazo E Barahona M Redondo-Nieto R Rivilla andMMartın ldquoTheGac-Rsm and SadB signaltransduction pathways converge on Algu to downregulatemotility in Pseudomonas fluorescensrdquo PLoS ONE vol 7 no 2Article ID e31765 2012

[94] C Reimmann M Beyeler A Latifi et al ldquoThe global activatorGacA of Pseudomonas aeruginosa PAO positively controls theproduction of the autoinducer N-butyryl-homoserine lactoneand the formation of the virulence factors pyocyanin cyanideand lipaserdquo Molecular Microbiology vol 24 no 2 pp 309ndash3191997

[95] J Dubern and G V Bloemberg ldquoInfluence of environmentalconditions on putisolvins I and II production in Pseudomonasputida strain PCL1445rdquo FEMSMicrobiology Letters vol 263 no2 pp 169ndash175 2006

[96] M G Surette M B Miller and B L Bassler ldquoQuorum sensingin Escherichia coli Salmonella typhimurium andVibrio harveyia new family of genes responsible for autoinducer productionrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 96 no 4 pp 1639ndash1644 1999

[97] B A Rader S R Campagna M F Semmelhack B L Basslerand K Guillemin ldquoThe quorum-sensing molecule autoinducer2 regulates motility and flagellar morphogenesis inHelicobacterpylorirdquo Journal of Bacteriology vol 189 no 17 pp 6109ndash61172007

[98] YHe J G Frye T P Strobaugh Jr andCChen ldquoAnalysis of AI-2LuxS-dependent transcription in Campylobacter jejuni strain81-176rdquo Foodborne Pathogens and Disease vol 5 no 4 pp 399ndash415 2008

[99] B Jeon K Itoh N Misawa and S Ryu ldquoEffects of quorumsensing on flaA transcription and autoagglutination in Campy-lobacter jejunirdquoMicrobiology and Immunology vol 47 no 11 pp833ndash839 2003

[100] N Dasgupta E P Ferrell K J Kanack S E H West andR Ramphal ldquofleQ the gene encoding the major flagellarregulator of Pseudomonas aeruginosa is 12059070 dependent and isdownregulated by Vfr a homolog of Escherichia coli cyclic AMPreceptor proteinrdquo Journal of Bacteriology vol 184 no 19 pp5240ndash5250 2002

[101] J Zhu M B Miller R E Vance M Dziejman B L Bassler andJ J Mekalanos ldquoQuorum-sensing regulators control virulencegene expression in Vibrio choleraerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no5 pp 3129ndash3134 2002

[102] Z Liu T Miyashiro A Tsou A Hsiao M Goulian and JZhu ldquoMucosal penetration primes Vibrio cholerae for hostcolonization by repressing quorum sensingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 28 pp 9769ndash9774 2008

[103] K E Klose V Novik and J J Mekalanos ldquoIdentificationof multiple 12059054-dependent transcriptional activators in Vibriocholeraerdquo Journal of Bacteriology vol 180 no 19 pp 5256ndash52591998

[104] I Kawagishi M Imagawa Y Imae L McCarter and MHomma ldquoThe sodium-driven polar flagellar motor of marineVibrio as the mechanosensor that regulates lateral flagellarexpressionrdquoMolecular Microbiology vol 20 no 4 pp 693ndash6991996

[105] T J Kirn B A Jude and R K Taylor ldquoA colonizationfactor links Vibrio cholerae environmental survival and humaninfectionrdquo Nature vol 438 no 7069 pp 863ndash866 2005

[106] Z Kuang YHao S Hwang et al ldquoThe Pseudomonas aeruginosaflagellum confers resistance to pulmonary surfactant protein-Aby impacting the production of exoproteases through quorum-sensingrdquo Molecular Microbiology vol 79 no 5 pp 1220ndash12352011

[107] S Zhang F X McCormack R C Levesque G A OrsquoTooleand G W Lau ldquoThe flagellum of Pseudomonas aeruginosa isrequired for resistance to clearance by surfactant protein ArdquoPLoS ONE vol 2 no 6 article e564 2007

[108] G W Lau D J Hassett H Ran and F Kong ldquoThe role ofpyocyanin in Pseudomonas aeruginosa infectionrdquo Trends inMolecular Medicine vol 10 no 12 pp 599ndash606 2004

[109] GW LauH Ran F Kong D J Hassett andDMavrodi ldquoPseu-domonas aeruginosa pyocyanin is critical for lung infection inmicerdquo Infection and Immunity vol 72 no 7 pp 4275ndash42782004

[110] D S Merrell M L Goodrich G Otto L S Tompkins and SFalkow ldquopH-regulated gene expression of the gastric pathogenHelicobacter pylorirdquo Infection and Immunity vol 71 no 6 pp3529ndash3539 2003

[111] K J Allen and M W Griffiths ldquoEffect of environmental andchemotactic stimuli on the activity of the Campylobacter jejuniflaA 12059028 promoterrdquo FEMS Microbiology Letters vol 205 no 1pp 43ndash48 2001

[112] Y Wen J Feng D R Scott E A Marcus and G SachsldquoThe pH-responsive regulon of HP0244 (FlgS) the cytoplasmichistidine kinase of Helicobacter pylorirdquo Journal of Bacteriologyvol 191 no 2 pp 449ndash460 2009

[113] D R Hendrixson ldquoA phase-variable mechanism controllingthe Campylobacter jejuni FlgR response regulator influencescommensalismrdquo Molecular Microbiology vol 61 no 6 pp1646ndash1659 2006

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

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Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Scientifica 3

Rod IM

OM

C

MS ring

Hook Filament

Export apparatus

Rod IM

OM

C

MS ring

Hook

Export apparatus

Rod IM

OM

C

MS ring Export

apparatus

H pyloriC jejuni

Vibriospecies

Pseudomonasspecies

C crescentus

FlrA

RpoD

FlrARpoN

FleQRpoN

FleQ

CtrARpoD

Export apparatus rings FlgS FlgR

FlgM rod

Export apparatus rings FlrB FlrC

FliA

Export apparatus motor filament cap rings FleN

FleS FleR

Export apparatus rings FlbD FliX

FlgSFlgRRpoN

FlrBFlrCRpoN

FleSFleRRpoN

FliXFlbDRpoN

Rodhook

FliK FlaB

Hook FlaA

Rod L ring hook

hook cap

Hook

FliA

FliA

FliA

FlbDFlbTRpoN

FlaAflagellar

cap

Alternative flagellins FlgM

motor

FliC FleL FlgM FlgN chemotaxis

proteins

Flagellins

Masterregulator

Early Middle Late

Figure 2 Diversity in flagellar gene transcription hierarchies Transcriptional hierarchies are compared between the EpsilonproteobacteriaVibrio Pseudomonas and C crescentus Regulatory proteins and sigma factors involved in controlling the transcriptional hierarchies areindicated above the arrows Early middle and late genes are indicated between the arrows

are required for colonization of the host [15ndash18] Moreoverthe degree of motility appears to be important for H pylorivirulence as more motile strains are able to maintain higherbacterial density and inflammation in the stomach cardiathan those that are less motile [19]

In H pylori and C jejuni flagellar gene regulationinvolves the primary sigma factor RpoD as well as thealternative sigma factors RpoN and FliA (Figure 2) A masterregulator which activates transcription of the early flagellargenes has not been identified for H pylori or C jejuni It ispossible that these bacteria lack a true master regulator offlagellar biogenesis a possibility that was suggested byNiehusand coworkers since motility is required for the obligateparasitic lifestyle of H pylori [20] Alternatively H pylorior C jejuni could possess a master regulator for flagellarbiogenesis that has other essential roles which might explainwhy it has not been identified from mutagenesis screens

The organization of flagellar genes into regulons basedon the sigma factor required for transcription is very similarthough not identical in H pylori and C jejuni RpoD isrequired for transcription of genes encoding components ofthe basal body as well as regulatory proteins that controltranscription of genes required at later stages of flagellarbiogenesis [20] Other genes regulated by RpoD include flhF

and flhG whose products influence the localization of flagellato the cell pole and number of flagella per cell respectively[21ndash23] It is not known if transcription of the early flagellargenes is temporally regulated and intimately associated withthe cell cycle as occurs in E coli and Salmonella [24 25] orif these genes are transcribed constitutively Distinguishingbetween these two options would require one to followtemporal changes of early flagellar genes transcription insynchronous cultures which has not been reported

Transcription of genes needed midway through flagellarbiogenesis is dependent on RpoN These genes encode theproximal rod proteins the hook protein hook-associatedproteins the hook-length control protein (FliK) a minorflagellin (FlaB) and enzymes required for glycosylation ofthe flagellins [20 26 27] The transcription of the RpoN-dependent genes is activated by a two-component regulatorysystem consisting of the sensor kinase FlgS and the responseregulator FlgR [26 28ndash30] FlgS differs from most sensorkinases in that it is not membrane bound The signal orcellular cue to which FlgS responds has yet to be identifiedbut several studies (see below) have implicated the flagellarprotein export apparatus in this process FlgS uses ATP toautophosphorylate a specific histidine residue in responseto the cellular cue and this phosphate is subsequently

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transferred to a specific aspartate residue in FlgR to activateit Based on studies with other RpoN-dependent activators(reviewed by Bush and Dixon [31]) phosphorylation ofFlgR likely stimulates oligomerization of the protein froma dimer to a hexamer capable of activating transcriptionRpoN-dependent activators typically possess an N-terminalregulatory domain (often a response regulator domain as isthe case in FlgR) an AAA+ ATPase domain that engages12059054 and couples the hydrolysis of ATP with open complex

formation and aC-terminalDNA-binding domain that bindsan enhancer sequence (reviewed by Schumacher et al [32])H pylori FlgR is unusual in that it lacks a C-terminal DNA-binding domain and activates transcription by contacting12059054-holoenzyme in the closed promoter complex without

binding DNA [29] Although C jejuni FlgR possesses a C-terminal domain it is not needed for activation of the RpoNregulon suggesting that like its counterpart in H pyloriC jejuni FlgR engages the closed promoter complex fromsolution [33] While the C-terminal domain of C jejuni FlgRdoes not appear to play a role inDNAbinding it does preventphosphorylation of the protein by acetyl phosphatewhich caninterfere with normal flagellation [34]

The flagellar protein export apparatus has a major rolein regulating expression of the RpoN regulon of H pyloriand C jejuni as evidenced by the observation that mutationswhich interfere with formation of the export apparatusinhibit expression of RpoN-dependent flagellar genes Por-wollik and coworkers showed that inactivation of fliI orfliQ (encode a cytosolic ATPase component and integralmembrane component of the export apparatus resp) inH pylori results in reduced levels of the minor flagellinFlaB and the hook protein FlgE both of which dependon RpoN for their expression [35] Allan and colleaguesshowed that mutations in flhB (encodes an integral mem-brane component of the export apparatus) result in reducedlevels of FlaB and FlgE inH pylori [36] Smith and coworkerssubsequently demonstrated that disrupting flhB inhibitedexpression of RpoN-dependent reporter genes [37] UsingDNAmicroarrays Niehus and coworkers [20] demonstratedthat FlhA (an integral membrane component of the exportapparatus) is required for transcription of the entire RpoNregulon Hendrixson et al [38] reported that deleting anyone of several integral membrane components of the exportapparatus (FlhA FlhB FliP or FliR) inhibited expressionof an RpoN-dependent flagellar reporter gene in C jejuniStudies by Tsang and coworkers revealed thatH pylori strainswhich stably express truncated forms of FlhA are able totranscribe RpoN-dependent genes at levels that are close towild type but are unable to export flagellar protein substrates[39]Thus anymodel for how the export apparatus influencestranscription of theRpoN regulonmust take into account thatthe export apparatus need not transport protein substrates tostimulate the RpoN regulon Boll and Hendrixson recentlyshowed that loss of the MS ring protein FliF or the Cring protein FliG inhibits transcription of RpoN-dependentflagellar genes in C jejuni [40] The MS ring is the firstflagellar structure assembled and in its absence the exportapparatus presumably is not formedwhich would account for

the inhibition of the RpoN regulon in the fliF mutant FliGmay facilitate autophosphorylation of FlgS either by directlystimulating FlgS or by promoting assembly of the exportapparatus into a conformation that stimulates FlgS activity[40]

The export apparatus is an ideal indicator for the statusof flagellar assembly as it undergoes a major conformationalchange upon completion of the hook-basal body structureIn the early stages of flagellar biogenesis the export apparatusis in a conformation that transports rod- and hook-typesubstrates Upon completion of the mature hook-basal bodystructure the export apparatus undergoes a conformationalchange which is accompanied by a switch in substrate speci-ficity to filament-type substrates [41] This conformationalchange involves an autocleavage event in the C-terminalcytoplasmic domain of the export apparatus protein FlhB(referred to as FlhBC) and interactions between the C-terminal domain of the hook-length control protein FliKand FlhBC [42] The change in conformation and substratespecificity of the export apparatus may serve as a cellular cuefor the temporal regulation of flagellar genes Indeed in Ecoli and Salmonella the anti-12059028 factor FlgM is a filament-type substrate and is secreted by the export apparatus uponcompletion of the mature hook-basal body structure whichallows transcription of the 12059028-dependent flagellar genes tooccur [43]

Transcription of the RpoN-dependent flagellar genes inH pylori also requires the putative RpoN chaperone FlgZ(HP0958) [44 45] FlgZ was first implicated in having a rolein flagellar biogenesis in H pylori when it was identifiedas interacting with RpoN in a high-throughput screen of ayeast two-hybrid system (httpspimhybrigenicscom) [46]In the absence of FlgZ RpoN is turned over rapidly in Hpyloriwith a half-life of about 30 minutes compared to a half-life greater than 4 hours in wild type [45] The molecularbasis for how FlgZ protects RpoN from rapid turnover is notknown but it is possible that FlgZ binds RpoN to protectit from proteolysis or that FlgZ facilitates the association ofRpoN with core RNA polymerase which serves to protectRpoN from degradation Overexpression of RpoN restoresmotility in a H pylori flgZ mutant indicating that FlgZ isnot essential for flagellar biogenesis [45] In addition toits potential role as an RpoN chaperone FlgZ may haveadditional roles in flagellar biogenesis as it was also shown tointeract with the flagellar export apparatus protein FliH in theyeast two-hybrid system (httpspimhybrigenicscom) [46]In addition Douillard and coworkers demonstrated that FlgZbinds the flaA transcript and is needed for optimal expressionof FlaA [47] These researchers proposed that FlgZ functionswith FliH to direct flaA transcripts to the flagellar proteinexport apparatus where translation of the transcripts couldbe coupled with secretion of the nascent FlaA [47]

Transcription of genes needed late in flagellar biogenesisin H pylori and C jejuni which includes genes encodingthe major flagellin filament cap and associated chaper-ones is dependent on 12059028 (FliA) [20] Similar to E coliand Salmonella the H pylori FliA regulon is negativelyregulated by FlgM [48] Unlike E coli and Salmonella

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the inhibitory effect of FlgM on FliA in H pylori is thoughtto be alleviated via interactions between FlgM and the C-terminal cytoplasmic domain of FlhA (FlhAC) rather thanby secretion of FlgM via the export apparatus [49] C jejunialso possesses a FlgM homolog [50] but unlike H pyloriC jejuni FlgM appears to be secreted from the cell via theflagellar export apparatus [51] Interestingly interaction of Cjejuni FlgM with FliA is temperature dependent occurringat 42∘C (the optimal growth temperature for C jejuni) butnot 37∘C [51] The primary function of C jejuni FlgM is notto inhibit FliA activity during formation of the hook-basalbody structure but rather to limit the length of the flagellarfilament by suppressing expression of the FliA-dependentflagellin (FlaA) and the RpoN-dependent flagellin (FlaB) [51]The mechanism by which FlgM represses transcription ofRpoN-dependent genes in C jejuni is unknown but Wostenand coworkers have speculated that accumulation of theFliAFlgM complex may inhibit phosphorylation of FlgSandor FlgR [51] FlgM also appears to inhibit transcriptionof RpoN-dependent genes inH pylori but so far this has onlybeen demonstrated in an flhAmutant background [20]

22 Vibrio Species The vibrios are typically saltwater micro-organisms and several species cause food-borne illnessesVibrio species vary greatly in flagella morphology Vibriocholerae has a single polar sheathed flagellum while Vibrioparahaemolyticus and Vibrio alginolyticus produce a singlepolar sheathed flagellum and lateral peritrichous flagella andVibrio fischeri has a tuft of polar sheathed flagella Lateralflagella promote swarming and colonization of surfaces andthereby enhance biofilm formation upon encountering highlyviscous media or solid surfaces

The flagellar gene transcriptional hierarchy in Vibriocholerae is governed by RpoN and FliA [52] flrA encodesan RpoN dependent activator and is the master regulatorfor the flagellar gene transcriptional hierarchy [52] FlrA isregulated by cyclic di-GMP a secondarymessenger with rolesin biofilm formation and virulence which binds to FlrA andprevents it from binding the flrBC promoter [53] Early andmiddle genes are RpoN-dependent but they rely on differenttranscriptional activators for their transcription FlrA alongwith RpoN is required for transcription of the early geneswhich encode the components of the MS ring C ring andexport apparatus Other early genes encode the regulatoryproteins FlrB FlrC and FliA FlrB and FlrC constitutethe sensor kinase and response regulator respectively of atwo-component system that regulates transcription of themiddle genes in conjunction with RpoN [54] The middlegenes encode the basal body-hook structures and the coreflagellin FlaA The late genes are dependent on FliA fortheir transcription and encode alternative flagellins FlgMand components of the flagellar motor As in SalmonellaFlgM is secreted via the flagellar protein export apparatusto relieve the inhibition on FliA activity in V cholerae [55]flgA is located upstream of flgM and its promoter (RpoNindependent and FliA independent) may drive transcriptionof flgM such that FliA is repressed until formation of themature hook-basal body structure [52]

The signal(s) that stimulates the FlrBFlrC two-component system is not known FlrD is necessary fortranscription of middle and late flagellar genes making ita candidate for regulating the FlrBFlrC two-componentsystem [56] Additionally FlrD contains HAMP domains(domain present in histidine kinases adenyl cyclasesmethyl-accepting proteins and phosphatases) that areusually found in integral membrane proteins that are part ofsignal transduction pathways [56 57] Moisi and coworkersshowed that FlrD is inserted into the inner membrane andproposed that it senses the completion of the MS ring-switch-export apparatus structure and communicates thisinformation to FlrB [56]

The regulation of flagellar genes inV parahaemolyticus ismore complex since the bacterium is capable of producinglateral flagella in addition to polar flagella (reviewed byMerino et al [58] and McCarter [59]) The polar flagellarsystem (Fla) is constitutively expressed while the lateral flag-ellar system (Laf) is expressed upon impedance of the polarflagellaThe lateral flagella are used for swarmingmotility andenhance biofilm formation and host colonization by the bac-terium [60 61] When grown planktonically the bacteriumproduces a polar flagellum but when grown on highly viscousor solid medium [58] or during iron-limiting conditions[62] the bacterium produces lateral flagella Thus the polarflagellum acts as a mechanosensor to regulate expression ofthe lateral flagella genes through a mechanism that has yet tobe defined The dual flagellar system suited for locomotionunder different conditions allows V parahaemolyticus to behighly adaptive to changing habitats including planktonicenvironments surfaces and biofilms

The polar and lateral flagellar systems do not share anystructural or regulatory components (aside from RpoN) butthe regulatory networks that control polar and lateral flagellarsystems in V parahaemolyticus are similar to that of Vcholerae The lateral flagellar genes are activated by LafKand like the polar flagellar system lateral flagellar genes areboth RpoN dependent and FliA dependent [63] AlthoughLafK is homologous to master regulators of other flagellarsystems LafK does not appear to be amaster regulator for thelateral flagellar system as the 119891119897119894119872L operon (contains genesencoding components of the C ring and export apparatus) isnot LafK dependent [64] This suggests that there is anotherlevel of regulation preceding expression of these genes

23 Pseudomonas Species Pseudomonas aeruginosa is anaerobic Gram-negative Gammaproteobacterium that causesopportunistic infections particularly in patients with cysticfibrosis where it results in inflammation and sepsis (reviewedby Veesenmeyer et al [65] and Balasubramanian et al[66]) P aeruginosa which thrives on surfaces and formsbiofilms is a prevalent agent of nosocomial infections Wild-type P aeruginosa cells form biofilms which can causechronic infections P aeruginosa possesses a single polarflagellum that is required for virulence [67] Motile strainsof P aeruginosa induce activation of the inflammasome (amultiprotein oligomer responsible for the activation of theinflammatory response) whereas nonmotile strains have amarkedly reduced ability to induce inflammasome activation

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[68] Nonmotile strains of P aeruginosamay have an advan-tage during chronic infection by evading stimulation of theinnate immune responses However a nonmotile flgKmutantis defective in surface attachment suggesting a role for flagellaandor motility in the initial cell-to-surface interaction [69]Mutants in fliM and cheY are also nonmotile and are unableto form biofilm cap structures on initial biofilm colonies [70]Other Pseudomonas species include Pseudomonas fluorescenswhich possesses multiple flagella and is found in the soil andwater Some P fluorescens strains have been shown to protectplant roots against fungal infections [71] P fluorescens doesnot typically cause disease in humans but it is an opportunis-tic pathogen in immunocompromised individuals [72 73]

Like the Epsilonproteobacteria and Vibrio P aeruginosapossesses a four-tiered transcriptional hierarchy for regulat-ing flagellar gene expression FleQ is the master regulatorfor flagellar synthesis and fleQ is regulated by global factorsoutside the flagellar regulons such as cyclic di-GMP asignaling molecule important in modulating the transitionbetween planktonic and biofilm lifestyles [74] The earlyflagellar genes are dependent on FleQ and RpoN for theirtranscription and encode components of the basal body aswell as the filament cap which is somewhat surprising sinceit is not needed until late in flagellar biogenesis [75] Theearly genes also encode regulatory proteins which includeflhF fleN fleS and fleR [75 76] FleN is an antiactivator ofFleQ that maintains the normal flagellum copy number ofone per cell by downregulating genes encoding early flagellarcomponents via a negative feedbackmechanism [77 78] FleSand FleR form a two-component system that is required forthe transcription of the middle RpoN-dependent genes [75]The signal(s) required for FleS activation is unknown Themiddle genes encode components of the basal body includingrod L ring hook hook cap and hook-filament junctionproteins [75] The late genes are FliA dependent and encodeFliC (flagellin) FleL (filament length control protein) FlgMFlgN (a protein required for initiation of filament assembly)and some chemotaxis proteins [75] Transcription of fliA isconstitutive [75] and FlgM (whose transcription is also FleQdependent but not RpoN dependent) represses the activityof FliA until completion of the hook-basal body structure atwhich point it is secreted from the cytoplasm via the exportapparatus which alleviates its inhibition of FliA

24 Caulobacter crescentus Caulobacter crescentus is anAlphaproteobacterium found in freshwater that is a modelorganism for cell cycle studies C crescentus divides asym-metrically to produce two cells with distinct morphologiesa swarmer cell and a stalked cell The nonmotile stalked cellcontains a polar stalk which secretes an exopolysaccharidethat facilitates adhesion to surfaces In addition the stalkedcell initiatesDNA replication upon the start of cell division Incontrast the swarmer cell is motile via a polar flagellum andDNA replication is repressed for a defined length of time untilthe cell differentiates into a stalked cell Flagellar biogenesis iscoordinated with the cell division cycle such that all progenyswarmer cells possess a functional flagellum

Flagellar genes inC crescentus are regulated by two sigmafactors The initiation of DNA replication in the swarmer cellresults in expression and activation of the global transcriptionfactor CtrA CtrA also acts as themaster regulator for flagellargene expression by activating transcription of the early genes(encode MS ring C ring and export apparatus FlbD FliX)Additionally CtrA controls expression of rpoD [79] and isneeded for transcription of the early flagellar genes [80]FlbD and FliX are transcriptional regulators that regulateexpression of the early and middle genes The middle andlate genes which encode the hook protein and flagellins aretranscribed only after the components encoded by the earlygenes have been assembled into the nascent flagellum FlbDis an RpoN-dependent transcriptional activator which bindsftr (flagella transcriptional regulation) sequence motifs toregulate gene expression Once the early genes are expressedFlbD binds the ftr sequence motifs in the promoter regionsof early flagellar genes to inhibit transcription of these genesFor example FlbD binds the ftr4 site located upstream ofthe early gene fliF to repress its transcription once the earlygenes have been expressed [81] In contrast FlbD stimulatestranscription of the middle genes by binding to the ftrsites of the middle and late genes [81 82] FlbD activity isregulated by FliX which acts to either inhibit or stimulateFlbD In the absence of early flagellar structures FliX inhibitsFlbD activity by binding FlbD preventing it from bindingftr sites [83] Once the early flagellar structures are formedFliX stimulates FlbD The mechanism for this stimulationis unknown but it may involve another factor such asan assembled component of the flagellum which convertsFliX into a positive regulator or it may involve covalentmodifications of FliX or FlbD [83] Although FlbD possessesan N-terminal response regulator domain FlbD activity doesnot appear to be regulated by phosphorylation via a sensorkinase The exact cellular cues that regulate FlbD are notknown but FlbD activity is linked to cell division [84]

Unlike the bacteria discussed thus far flagellar biogenesisin C crescentus does not involve FliA Rather the six flagellingenes found in C crescentus are RpoN dependent Theexpression of the flagellin genes is regulated by FlbD and FlbTwhich binds the 51015840 UTR of flagellin transcripts and repressestranslation until the hook-basal body structure is assembled[85] Though the mechanism for this temporal regulation isunknown one possibility is that FlbT represses translationof flagellin genes until a positive factor binds the 51015840 UTRregion of flagellins to promote translation upon assembly ofthe mature hook-basal body structure [85]

3 Coupling Flagellar Biosynthesis withOther Cellular Activities

Though flagellar biogenesis is a complex process in itselfit is further complicated by its coordination with othercellular processes Flagellar biosynthesis is timed with othercellular activities such as cell replication and quorum sensingEnvironmental stimuli also play a role in regulating flagellarbiogenesis as bacteria respond to changes in the environmentby altering behavior needed for survival Many of these

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responses involve altering the state of flagellar biogenesiswithin these different contexts

31 Modulating Flagellar Gene Expression during the CellCycle Flagellar biosynthesis is linked to the cell cyclethrough various mechanisms and involves coordinating flag-ellar gene expression with growth phase and cell division InV cholerae chemotaxis and motility genes are upregulatedas the population enters stationary phase Of the 114 genesidentified with roles in chemotaxis and flagellar biogen-esis 72 genes were upregulated more than 2-fold duringstationary phase compared to mid-exponential phase [86]The upregulation of 60 of the 72 chemotaxis and flagellargenes is dependent on RpoS indicating a correlation betweenflagellar biogenesis and entry into stationary phase [86]The coordination between entry into stationary phase andflagellar biogenesis may aid the bacterium in exiting the hostwhen nutrients become depleted and prepare the bacteriumfor colonization of other niches or survival in environmentalreservoirs

C crescentus is a particularly good organism for studyinghow flagellar biogenesis is integrated with the cell cyclebecause of its two distinct cell morphologies The flagellarfilament is assembled just prior to cell division and allprogeny swarmer cells possess a fully functional flagellumThe synthesis of the flagellum is intimately linked to the cellcycle through regulatory proteins such as CtrA FlbD andFliX that are involved in both the cell cycle and flagellarbiogenesis This dual functionality may exist to ensure thateach swarmer cell is flagellated The master regulator forflagellar biogenesis CtrA regulates at least 95 genes [87]including the cell-division genes ftsZ [88] and ftsQA [89]Cells of a fliX mutant become filamentous as they enterlate log phase [90] demonstrating a role for this flagellarregulatory protein in cell division Flagellar biogenesis is alsolinked to the cell cycle via FlbT and FlaF FlaF is required forflagellar biogenesis and motility [91] FlbT protein levels areconstant throughout the cell cycle while FlaF levels correlateto flagellin levels both of which peak just prior to cell division[91] Since FlbT is a negative regulator for flagellin translationand peaks in flagellin levels correlate with FlaF levels thisobservation suggests that FlaF may temporally modulateFlbT activity [91]

Expression of Vibrio vulnificus FlhF which is needed forpolar localization of the flagellum is regulated by the quorumsensing master regulator SmcR flhF transcript levels in asmcRmutant are higher than wild-type levels indicating thatSmcR represses flhF expression [92] In wild-type cells flhFtranscript levels are highest during exponential phase anddecrease upon entry into stationary phase [92] In contrasttranscript levels of smcR increase as cultures enter stationaryphase suggesting that SmcR plays a major role for growthphase-dependent variation of flhF expression [92] Thisgrowth phase-dependent gene expression may be importantin host colonization as during the initial infection bacterialcell densities are low but at later stages of colonizationwhen bacterial cell densities are higher motility may not benecessary

Expression of flagellar genes is regulated in P fluorescensthroughout the growth phases by the Gac (GacAGasS) two-component system which limits flagellar biosynthesis duringexponential growth by downregulating transcription of fleQ(encodes the master regulator) [93] The Gac system posi-tively regulates production of virulence factors and quorumsensing molecules and is required for full virulence in animaland plant hosts [94] Levels of the Gac system componentsare regulated in response to growth phase as gacA and gacStranscript levels are highest at mid-exponential growth phase[95] The concomitant upregulation of virulence factors andthe downregulation of flagellar genes by the Gac system maybe a way to coordinate virulence and motility with growthphase so that virulence factors are expressed at high celldensities during host colonization and flagellar genes areturned off to facilitate attachment to the host

32 Connections between Quorum Sensing Host Colonizationand Flagellar Gene Expression In many pathogenic bacte-ria flagella aid in surface attachment and colonization Insuch cases successful colonization may require cooperationbetween quorum sensing and flagellar synthesis to integratesurface attachment and virulence gene expression at highcell densities in pathogens Surface colonization can leadto biofilm formation which is generally more resistant tohost defenses and antimicrobials Pathogens use differentmechanisms to couple expression of virulence genes withflagellar gene expression in the context of the host Thisincludes modulating quorum sensing and host colonizationbased on the status of the flagellum or modulating flagellarbiogenesis based on quorum sensing signals

Quorum sensing is used to regulate gene expression inresponse to cell densityThis is achieved through the secretionof a signaling molecule called an autoinducer (AI) AIs areproduced by bacteria and travel across the cell membrane andaccumulate in the environment Upon reaching a critical con-centration the AIs stimulate transcription of genes requiredfor group behavior luxS is responsible for the productionof AI-2 [96] and an H pylori mutant in luxS exhibitsdecreasedmotility compared towild type [97]flhA transcriptlevels are decreased in the luxS mutant but addition of 45-dihydroxy-23-pentanedione (functions as AI-2) into thegrowth medium restores flhA transcript levels to wild-typelevels [97] Like the H pylori luxS mutant a C jejuni luxSmutant is less motile than wild type [98] and the mutantdisplays reduced transcript levels of flaA [99] Using DNAmicroarrays He and coworkers found that a majority of theflagellar genes are downregulated in a C jejuni luxS mutant[98] Expression of chemotaxis genes however is unchangedin the luxS mutant indicating that the motility defect of theluxS mutant is due to defects in flagellar biogenesis and notchemotaxis [98] A rationale for the link between cell densityand flagellar biogenesis is that it may aid in colonization ofhost tissue by ensuring that a sufficient number of flagellatedbacteria are present to establish a successful infection

Regulatory proteins involved in flagellar biogenesis canregulate quorum sensing and colonization or they themselvescan be regulated by quorum sensing and colonization factorsFor example the RpoN-dependent activator FlrC not only

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is required for flagellar biogenesis but also appears to play arole in host colonization by V cholerae Mutants expressinga FlrC variant incapable of being phosphorylated fail toproduce flagella and are therefore defective in colonization[54] Interestingly a mutant expressing a constitutively activeform of FlrC is also deficient in colonization even though thecells are motile and produce flagella [54] This observationdemonstrates the need for coordinating flagellar biogenesisand pathogenesis for successful colonization ConverselyVfr a major quorum sensing and virulence regulator inP aeruginosa downregulates expression of flagellar genesby repressing transcription from the fleQ promoter [100]Inhibition of flagellar biogenesis by Vfr may aid P aeruginosain persisting at the site of infection to achieve high celldensities and virulence gene expression

HapR is a transcriptional regulator involved in quorumsensing in V cholerae which also regulates expression ofvirulence genes HapR represses expression of several vir-ulence genes [101] and hapR expression is regulated by anunknown mechanism involving FliA [102] hapR expres-sion is depressed in flgM and flgD mutants (both of thesemutations alleviate FlgM repression on FliA activity) whiledeletion of fliA results in elevated hapR expression [102] Theauthors of this study speculated that expression of HapR isregulated by a regulator or small noncoding RNA (sRNA)that is part of the FliA regulon FliA-mediated regulation ofhapR appears to be linked to the shearing of flagella frombacteria that penetrate the mucin layer to infect the hostThis shearing of the flagella leads to secretion of FlgM viathe flagellar protein export apparatus and results in increasedexpression of FliA-dependent genes and repression of hapRexpression [102] The outcome of such a mechanism is thatvirulence genes which are negatively regulated by HapRare expressed specifically when V cholerae penetrates theintestinal mucous layer hapR production is also regulatedby the quorum sensing protein LuxO further linking viru-lence to cell density LuxO is an RpoN-dependent activator[103] which along with HapR regulates motility proteaseproduction and biofilm formation [101] High cell densitiesare common during late stages of infection and the HapR-mediated repression of virulence genes may aidV cholerae indetaching and finding new colonization sites [86 101]

V parahaemolyticus uses its polar flagellum to detectsurfaces by sensing impedance to the polar flagellum rotationrate [104] Once a surface is encountered expression of thelateral flagellar genes is induced Lateral flagella are importantfor host colonization as mutants that do not produce lateralflagella are deficient in adherence to HeLa cells and biofilmformation [61] Gode-Potratz and coworkers identified sim70genes that are surface responsive most of which are positivelyregulated [64] Some of the surface-regulated genes that areupregulated are virulence genes such as a putative GbpAhomolog and components of a type III secretion system [64]GbpA from V cholerae promotes adherence to chitinoussurfaces of zooplankton and human epithelia cells [105]which is consistent with the observed upregulation of gbpAin V parahaemolyticus when it encounters a surface

The P aeruginosa flagellum is an important determinantin the susceptibility of the bacterium to host defenses

P aeruginosa fliCmutants are unable to upregulate transcrip-tion of lasI and rhlI which encode enzymes that synthesizequorum sensing homoserine lactones [106] These quo-rum sensing molecules regulate production of exoproteaseswhich degrade surfactant protein-A (SP-A) an antimicro-bial that opsonizes and permeabilizes membranes of lungpathogens [106] In the absence of these exoproteases theP aeruginosa fliC mutants are cleared from the lung [106]Though it is not known why fliC mutants are unable toupregulate transcription of lasI and rhlI it is not due tothe inability to sense the environment [106] P aeruginosafliE flgE fliC and fliD mutants are also attenuated in LPSbiosynthesis which compromises the integrity of their outermembranes making them more susceptible to SP-A [107]Additionally flgE and fliC mutants make less pyocyanin aredox-active toxic secondary metabolite that is crucial forlung infection [108 109]

33 Reacting to pH Changes by Gastrointestinal PathogensGastrointestinal pathogens encounter varying environmentalconditions as they travel from the mouth to the stomach andthen to the intestines The gastric pathogen H pylori has theadded burden of long-term survival in the acidic environ-ment of the stomach Pathogens that colonize the intestinaltract must also survive passage through the stomach in highenough numbers to be able to colonize their target organsThus it is not surprising that gastrointestinal pathogens oftenmodulate gene expression in response to acidic conditions

Merrell and coworkers found that expression of approxi-mately 7 of theH pylori genes is altered by a shift to low pH[110] Many of these genes are involved in flagellar synthesiswith the majority of those being RpoN dependent [110] FliA-dependent genes such as flgM in H pylori [110] and flaAin C jejuni [111] are also upregulated at low pH conditionsThe upregulation of flagellar genes is critical for survival ofH pylori as the bacterium is not able to survive at low pHbut must swim across the mucous layer to the underlyinggastric epithelium to establish a successful infection Inaddition to enhancing transcription of flagellar genes acidicenvironments also stimulate motility and swimming speedspotentially in response to increased concentration of protonsin the stomach [110]

The basis for upregulation of the RpoN-dependent flag-ellar genes in H pylori likely involves a stimulation of FlgSactivity at low pHWen and coworkers reported thatH pylorirequires FlgS to survive acid stress (30-minute exposure topH25) [112]These researchers found that besides the knownRpoN-dependent flagellar genes FlgS was required for theupregulation of 86 genes in response to acid stress many ofwhich were previously known to be required for acid stresssurvival [112] It is not known if FlgS responds directly toacid stress or if the stress signal is mediated through anotherfactor

34 Altering Flagellar Biogenesis by Phase Variation In somebacteria flagellar biogenesis is subject to phase variationevents that affect expression of specific flagellar genes Phasevariation causes changes in phenotypes at frequencies that

Scientifica 9

are much higher than random mutations and contribute toheterogeneity within a population The ability of H pylori topersist in the host may involve the phase-variable dependentexpression of flagella that can serve as an advantage foradaptation to the host and for evading the host immuneresponse C jejuni utilizes phase variation to turn on andoff production of flagella a strategy that increases com-mensal colonization in poultry [113] Many phase variationevents occur by random reversible changes in the length ofshort DNA sequence repeats resulting from slipped-strandmispairing These events can take place within the codingsequence or in the promoter of a gene C jejuni phasevariants in which expression of flgR is in the OFF stateresult from the addition or removal of a nucleotide withinone of two homopolymeric nucleotide tracts consisting ofadenine and thymine These nucleotide tracts are locatedwithin the coding sequence of flgR causing the sequence toshift out of frame [113] Revertants to the ON state ariseupon the addition or removal of a nucleotide within theoriginal mutated homopolymeric nucleotide tract to restorethe wild-type sequence Alternatively pseudorevertants canarise in which nucleotides are removed or added close to theoriginal mutated homopolymeric nucleotide tract to restorethe correct reading frame but which result in changes in theamino acid sequence of the protein [113] flgS is also regulatedby phase variation in C jejuni providing multiple levels ofphase-variable control for theRpoN regulon [114 115] Similarmechanisms of phase variation also exist for flhA in C coli[116] and fliP in H pylori [117] which leads to the formationof truncated proteins that prevent flagellar biogenesis

A second type of phase variation in flagellar biogen-esis is mediated through alterations in DNA methylationwhich impact the expression of specific flagellar genesHost-adapted bacterial pathogens such as H pylori oftenpossess DNA methyltransferases and type III restriction-modification (R-M) systems that contain simple tandemDNA repeats which undergo slipped-strand mispairing dur-ing DNA replication that causes frameshift mutations andphase variation [118] R-M systems widespread among manybacteria confer protection from invasion by foreign DNAType III R-M systems are composed of a methyltransferase(mod) and an endonuclease (res) gene whose gene prod-ucts typically function together The switching between ONand OFF states of a DNA methyltransferase can regulateexpression of a ldquophasevarionrdquo via differential methylationof the genome in ON and OFF states [119] Many of thesephase-variable methyltransferase genes are associated withan inactive res gene resulting from frameshift mutationor are orphans that are not associated with a res genesuggesting that DNA restriction is not the primary roleof these methyltransferases (reviewed by Fox et al [120])For example H pylori modH encodes a phase-variableDNA methyltransferase and the cognate res gene containsa nonsense mutation frameshift mutation or is absent invarious H pylori strains [119] modH OFF strains havedecreased expression of flaA and fliK compared to themodHON strain [119] FlaA has a low ability to activate innateimmunity via the Toll-like receptor 5 [121] and modulatingexpression of the flagellin may be advantageous in evading

the host response Another H pylori methyltransferase withan inactive res gene is hpyAVIBM [122 123] hpyAVIBMencodes a C5 cytosine methyltransferase and contains AGrepeats in its open reading frame making it susceptible tophase variation via frame shift mutations [124] For instancehpyAVIBM from strain 26695 and hpyAVIBM from strainHPAG1 have five AG repeats whereas the four AG repeats instrain San 74 result in the translation of a truncated protein[124] Deletion of hpyAVIBM results in both the upregulationand downregulation of rpoN fliR fliD fliS motA fliK andflgK depending on the strain used [124] indicating that phasevariation of hpyAVIBM can alter flagellar biogenesis

P aeruginosa populations can possess two phenotypicvariants one which forms small rough colonies (S) andanother which forms large flat (L) colonies S variants formbiofilms in nonagitated liquid cultures whereas the L variantsdo not [125] S variants possess defects in flagellum-mediatedswimming flagellum-mediated swarming and type IV-pilus-mediated twitching but are able to revert back to the Lphenotype at relatively high frequencies suggesting that theshift between phenotypes is regulated by phase variation[125] Phase variation also occurs in P fluorescens whichcolonizes the alfalfa rhizosphere Phenotypic variants ariseduring rhizosphere colonization and are dependent on theactivity of a site-specific recombinase [126] C variants havewild-type colony morphology while F and S variants havea translucent and diffuse colony morphology [126] F andS variants which colonize distal parts of the roots andswim faster than the C variants overproduce fliC (flagellin)transcripts and synthesize flagellin filaments that aresim3 timeslonger than those of the C variant [126]

4 Conclusions and Future Directions

Flagellar biogenesis is a carefully choreographed processin which many different components of the flagellum aremade as they are needed for assembly into the nascentflagellum Bacteria have evolved elaborate and sundry reg-ulatory networks to couple flagellar gene expression withassembly A driving force for the evolution of such regulatorynetworks is undoubtedly the environmental and ecologicalconstraints imposed by a particular bacteriumrsquos niche Inaddition the degree to which a given bacterial species needsto couple flagellar biogenesis to other cellular processes suchas cell division has also likely shaped the evolution of theseregulatory networks We focused here on bacteria whichutilize RpoN for flagellar biogenesis yet even among thesebacteria the mechanisms used to regulate expression of theRpoN-dependent flagellar genes vary greatly For most of thesystems described in this review we have only scratched thesurface in our understanding of how they operate For each ofthese systems fundamental questions remain to be answeredsuch as the following are all of the flagellar genes of a givensystem expressed as part of a transcription hierarchy or aresome genes expressed constitutively Are there regulatorymechanisms within RpoN flagellar regulons that fine-tunegene expression so that components of the flagellum aremadeprecisely when needed What are the cellular cues sensed by

10 Scientifica

the regulatory proteins that control expression of the RpoN-dependent flagellar genes How does the flagellar exportapparatus control expression of RpoN-dependent flagellargenes in various bacteria Does FlgZ have a regulatoryrole in flagellar biogenesis in H pylori and other bacteriaAs our understanding of the molecular genetics of diversebacterial species expands novel regulatory mechanisms thatcontrol expression of flagellar genes within these bacteria willundoubtedly come to light For example it was only recentlythat sRNAs were identified in H pylori some of whichare potentially FliA dependent [127] and may have roles inflagellar biogenesis Finally with increasingly sophisticatedmethods in bioinformatics genomics and proteomics fur-ther characterization of flagellar regulatory networks willprovide important insights into the mechanisms governinggene expression and identify new regulatory proteins andmechanisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by award MCB-1244242 from theNational Science Foundation

References

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[2] J L McMurry J W Murphy and B Gonzalez-Pedrajo ldquoTheFliN-FliH interaction mediates localization of flagellar exportATPase FliI to the C ring complexrdquo Biochemistry vol 45 no39 pp 11790ndash11798 2006

[3] R M Macnab Ed Flagella and Motility ASM Press 1996[4] T Minamino K Imada and K Namba ldquoMolecular motors of

the bacterial flagellardquoCurrent Opinion in Structural Biology vol18 no 6 pp 693ndash701 2008

[5] T G Smith and T R Hoover ldquoDeciphering bacterial flagellargene regulatory networks in the genomic erardquo Advances inApplied Microbiology vol 67 pp 257ndash295 2009

[6] J K Anderson T G Smith and T R Hoover ldquoSense andsensibility flagellum-mediated gene regulationrdquo Trends inMicrobiology vol 18 no 1 pp 30ndash37 2010

[7] J D Helmann ldquoAlternative sigma factors and the regulation offlagellar gene expressionrdquoMolecular Microbiology vol 5 no 12pp 2875ndash2882 1991

[8] D L Popham D Szeto J Keener and S Kustu ldquoFunctionof a bacterial activator protein that binds to transcriptionalenhancersrdquo Science vol 243 no 4891 pp 629ndash635 1989

[9] S Sasse-Dwight and J D Gralla ldquoProbing the EscherichiacoliglnALG upstream activation mechanism in vivordquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8934ndash8938 1988

[10] M Kihara T Minamino S Yamaguchi and R M MacnabldquoIntergenic suppression between the flagellar MS ring protein

FliF of Salmonella and FlhA a membrane component of itsexport apparatusrdquo Journal of Bacteriology vol 183 no 5 pp1655ndash1662 2001

[11] J S van Arnam J L McMurry M Kihara and R M MacnabldquoAnalysis of an engineered Salmonella flagellar fusion proteinFliR-FlhBrdquo Journal of Bacteriology vol 186 no 8 pp 2495ndash2498 2004

[12] C S Barker I V Meshcheryakova A S Kostyukova and FA Samatey ldquoFliO regulation of FliP in the formation of theSalmonella enterica flagellumrdquo PLoS Genetics vol 6 no 9Article ID e1001143 2010

[13] A C Lowenthal M Hill L K Sycuro K Mehmood N RSalama and K M Ottemann ldquoFunctional analysis of the Heli-cobacter pylori flagellar switch proteinsrdquo Journal of Bacteriologyvol 191 no 23 pp 7147ndash7156 2009

[14] R M Macnab ldquoType III flagellar protein export and flagellarassemblyrdquo Biochimica et Biophysica Acta vol 1694 no 1ndash3 pp207ndash217 2004

[15] K A Eaton D R Morgan and S Krakowka ldquoCampylobacterpylori virulence factors in gnotobiotic pigletsrdquo Infection andImmunity vol 57 no 4 pp 1119ndash1125 1989

[16] K A Eaton D R Morgan and S Krakowka ldquoMotility as afactor in the colonisation of gnotobiotic piglets by Helicobacterpylorirdquo Journal of Medical Microbiology vol 37 no 2 pp 123ndash127 1992

[17] D G Newell H McBride and J M Dolby ldquoInvestigationson the role of flagella in the colonization of infant mice withCampylobacter jejuni and attachment of Campylobacter jejunito human epithelial cell linesrdquo Journal of Hygiene vol 95 no 2pp 217ndash227 1985

[18] TMorooka A Umeda andK Amako ldquoMotility as an intestinalcolonization factor forCampylobacter jejunirdquo Journal of GeneralMicrobiology vol 131 no 8 pp 1973ndash1980 1985

[19] C Y Kao B S Sheu SM Sheu et al ldquoHighermotility enhancesbacterial density and inflammatory response in dyspepticpatients infected with Helicobacter pylorirdquo Helicobacter vol 17no 6 pp 411ndash416 2012

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[21] M Balaban S N Joslin and D R Hendrixson ldquoFlhF and itsGTPase activity are required for distinct processes in flagellargene regulation and biosynthesis in Campylobacter jejunirdquoJournal of Bacteriology vol 191 no 21 pp 6602ndash6611 2009

[22] A Kusumoto A Shinohara H Terashima S Kojima TYakushi and M Homma ldquoCollaboration of FlhF and FlhGto regulate polarflagella number and localization in VibrioalginolyticusrdquoMicrobiology vol 154 no 5 pp 1390ndash1399 2008

[23] A Kusumoto K Kamisaka T Yakushi H Terashima AShinohara and M Homma ldquoRegulation of polar flagellarnumber by the flhF and flhG genes in Vibrio alginolyticusrdquoJournal of Biochemistry vol 139 no 1 pp 113ndash121 2006

[24] B M Pruszlig and P Matsumura ldquoA regulator of the flagellarregulon of Escherichia coli flhD also affects cell divisionrdquoJournal of Bacteriology vol 178 no 3 pp 668ndash674 1996

[25] B M Pruszlig and P Matsumura ldquoCell cycle regulation of flagellargenesrdquo Journal of Bacteriology vol 179 no 17 pp 5602ndash56041997

[26] G Spohn and V Scarlato ldquoMotility of Helicobacter pylori iscoordinately regulated by the transcriptional activator FlgR an

Scientifica 11

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[28] D Beier and R Frank ldquoMolecular characterization of two-component systems of Helicobacter pylorirdquo Journal of Bacteri-ology vol 182 no 8 pp 2068ndash2076 2000

[29] P Brahmachary M G Dashti J W Olson and T R HooverldquoHelicobacter pylori FlgR is an enhancer-independent activatorof 12059054-RNA polymerase holoenzymerdquo Journal of Bacteriologyvol 186 no 14 pp 4535ndash4542 2004

[30] M M S M Wosten J A Wagenaar and J P M van PuttenldquoThe FlgSFlgR two-component signal transduction systemregulates the fla regulon in Campylobacter jejunirdquo The Journalof Biological Chemistry vol 279 no 16 pp 16214ndash16222 2004

[31] M Bush and R Dixon ldquoThe role of bacterial enhancer bindingproteins as specialized activators of 12059054-dependent transcrip-tionrdquo MicroBiology and Molecular Biology Reviews vol 76 no3 pp 497ndash529 2012

[32] J Schumacher N Joly M Rappas X Zhang and M BuckldquoStructures and organisation of AAA+ enhancer binding pro-teins in transcriptional activationrdquo Journal of Structural Biologyvol 156 no 1 pp 190ndash199 2006

[33] S N Joslin and D R Hendrixson ldquoAnalysis of the Campy-lobacter jejuni FlgR response regulator suggests integration ofdiverse mechanisms to activate an NtrC-like proteinrdquo Journalof Bacteriology vol 190 no 7 pp 2422ndash2433 2008

[34] J M Boll and D R Hendrixson ldquoA specificity determinant forphosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphaterdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 50 pp 20160ndash20165 2011

[35] S Porwollik B Noonan and P W OrsquoToole ldquoMolecular char-acterization of a flagellar export locus of Helicobacter pylorirdquoInfection and Immunity vol 67 no 5 pp 2060ndash2070 1999

[36] E Allan N Dorrell S Foynes M Anyim and B W WrenldquoMutational analysis of genes encoding the early flagellarcomponents of Helicobacter pylori evidence for transcriptionalregulation of flagellin A biosynthesisrdquo Journal of Bacteriologyvol 182 no 18 pp 5274ndash5277 2000

[37] T G Smith L Pereira and T R Hoover ldquoHelicobacter pyloriFlhB processing-deficient variants affect flagellar assembly butnot flagellar gene expressionrdquo Microbiology vol 155 no 4 pp1170ndash1180 2009

[38] D R Hendrixson B J Akerley and V J DiRita ldquoTranspo-son mutagenesis of Campylobacter jejuni identifies a bipartiteenergy taxis system required for motilityrdquoMolecular Microbiol-ogy vol 40 no 1 pp 214ndash224 2001

[39] J Tsang T G Smith L E Pereira and T R Hoover ldquoInsertionmutations inHelicobacter pylori flhA reveal strain differences inRpoN-dependent gene expressionrdquo Microbiology vol 159 part1 pp 58ndash67 2013

[40] J M Boll and D R Hendrixson ldquoA regulatory checkpointduring flagellar biogenesis in Campylobacter jejuni initiatessignal transduction to activate transcription of flagellar genesrdquoMBio vol 4 no 5 Article ID e00432-13 2013

[41] T Minamino H Doi and K Kutsukake ldquoSubstrate specificityswitching of the flagellum-specific export apparatus during flag-ellar morphogenesis in Salmonella typhimuriumrdquo Bioscience

Biotechnology and Biochemistry vol 63 no 7 pp 1301ndash13031999

[42] M Erhardt H M Singer D H Wee J P Keener and K THughes ldquoAn infrequent molecular ruler controls flagellar hooklength in Salmonella entericardquo EMBO Journal vol 30 no 14 pp2948ndash2961 2011

[43] K T Hughes K L Gillen M J Semon and J E KarlinseyldquoSensing structural intermediates in bacterial flagellar assemblyby export of a negative regulatorrdquo Science vol 262 no 5137 pp1277ndash1280 1993

[44] K A Ryan N Karim M Worku S A Moore C W Pennand P W OrsquoToole ldquoHP0958 is an essential motility gene inHelicobacter pylorirdquo FEMS Microbiology Letters vol 248 no 1pp 47ndash55 2005

[45] L Pereira and T R Hoover ldquoStable accumulation of 12059054 inHelicobacter pylori requires the novel protein HP0958rdquo Journalof Bacteriology vol 187 no 13 pp 4463ndash4469 2005

[46] J C Rain L Selig H de Reuse et al ldquoThe protein-proteininteraction map of Helicobacter pylorirdquo Nature vol 409 no6817 pp 211ndash215 2001

[47] F P Douillard K A Ryan D L Caly et al ldquoPosttranscriptionalregulation of flagellin synthesis in Helicobacter pylori by theRpoN chaperone HP0958rdquo Journal of Bacteriology vol 190 no24 pp 7975ndash7984 2008

[48] C Josenhans E Niehus S Amersbach et al ldquoFunctionalcharacterization of the antagonistic flagellar late regulators FliAand FlgM ofHelicobacter pylori and their effects on theH pyloritranscriptomerdquoMolecular Microbiology vol 43 no 2 pp 307ndash322 2002

[49] M Rust S Borchert E Niehus et al ldquoThe Helicobacter pylorianti-sigma factor FlgM is predominantly cytoplasmic andcooperates with the flagellar basal body protein FlhArdquo Journalof Bacteriology vol 191 no 15 pp 4824ndash4834 2009

[50] D R Hendrixson and V J DiRita ldquoTranscription of 12059054-dependent but not 12059028-dependent flagellar genes in Campy-lobacter jejuni is associated with formation of the flagellarsecretory apparatusrdquoMolecular Microbiology vol 50 no 2 pp687ndash702 2003

[51] M M S M Wosten L van Dijk A K J Veenendaal MR de Zoete N M C Bleumink-Pluijm and J P M vanPutten ldquoTemperature-dependent FlgMFliA complex forma-tion regulates Campylobacter jejuni flagella lengthrdquo MolecularMicrobiology vol 75 no 6 pp 1577ndash1591 2010

[52] M G Prouty N E Correa and K E Klose ldquoThe novel 12059054- and12059028-dependent flagellar gene transcription hierarchy of Vibrio

choleraerdquo Molecular Microbiology vol 39 no 6 pp 1595ndash16092001

[53] D Srivastava M L Hsieh A Khataokar M B Neiditch and CM Waters ldquoCyclic di-GMP inhibits Vibrio choleraemotility byrepressing induction of transcription and inducing extracellularpolysaccharide productionrdquo Molecular Microbiology vol 90no 6 pp 1262ndash1276 2013

[54] N E Correa C M Lauriano R McGee and K E KloseldquoPhosphorylation of the flagellar regulatory protein FlrC is nec-essary for Vibrio cholerae motility and enhanced colonizationrdquoMolecular Microbiology vol 35 no 4 pp 743ndash755 2000

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12 Scientifica

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against fusarium wilt of radish by lipopolysaccharides of Pseu-domonas fluorescensrdquo Phytopathology vol 85 no 9 pp 1021ndash1027 1995

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[79] J Malakooti and B Ely ldquoPrincipal sigma subunit of theCaulobacter crescentus RNA polymeraserdquo Journal of Bacteriol-ogy vol 177 no 23 pp 6854ndash6860 1995

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[81] A K Benson G Ramakrishnan N Ohta J Feng A J Ninfaand A Newton ldquoThe Caulobacter crescentus FlbD proteinacts at ftr sequence elements both to activate and to represstranscription of cell cycle-regulated flagellar genesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 91 no 11 pp 4989ndash4993 1994

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[85] P E Anderson and J W Gober ldquoFlbT the post-transcriptionalregulator of flagellin synthesis in Caulobacter crescentus inter-acts with the 51015840 untranslated region of flagellin mRNArdquoMolec-ular Microbiology vol 38 no 1 pp 41ndash52 2000

Scientifica 13

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[91] M Llewellyn R J Dutton J Easter D OrsquoDonnol and J WGober ldquoThe conserved flaF gene has a critical role in couplingflagellin translation and assembly in Caulobacter crescentusrdquoMolecular Microbiology vol 57 no 4 pp 1127ndash1142 2005

[92] S M Kim D H Lee and S H Choi ldquoEvidence that the Vibriovulnificus flagellar regulator FlhF is regulated by a quorumsensing master regulator SmcRrdquo Microbiology vol 158 part 8pp 2017ndash2025 2012

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[97] B A Rader S R Campagna M F Semmelhack B L Basslerand K Guillemin ldquoThe quorum-sensing molecule autoinducer2 regulates motility and flagellar morphogenesis inHelicobacterpylorirdquo Journal of Bacteriology vol 189 no 17 pp 6109ndash61172007

[98] YHe J G Frye T P Strobaugh Jr andCChen ldquoAnalysis of AI-2LuxS-dependent transcription in Campylobacter jejuni strain81-176rdquo Foodborne Pathogens and Disease vol 5 no 4 pp 399ndash415 2008

[99] B Jeon K Itoh N Misawa and S Ryu ldquoEffects of quorumsensing on flaA transcription and autoagglutination in Campy-lobacter jejunirdquoMicrobiology and Immunology vol 47 no 11 pp833ndash839 2003

[100] N Dasgupta E P Ferrell K J Kanack S E H West andR Ramphal ldquofleQ the gene encoding the major flagellarregulator of Pseudomonas aeruginosa is 12059070 dependent and isdownregulated by Vfr a homolog of Escherichia coli cyclic AMPreceptor proteinrdquo Journal of Bacteriology vol 184 no 19 pp5240ndash5250 2002

[101] J Zhu M B Miller R E Vance M Dziejman B L Bassler andJ J Mekalanos ldquoQuorum-sensing regulators control virulencegene expression in Vibrio choleraerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no5 pp 3129ndash3134 2002

[102] Z Liu T Miyashiro A Tsou A Hsiao M Goulian and JZhu ldquoMucosal penetration primes Vibrio cholerae for hostcolonization by repressing quorum sensingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 28 pp 9769ndash9774 2008

[103] K E Klose V Novik and J J Mekalanos ldquoIdentificationof multiple 12059054-dependent transcriptional activators in Vibriocholeraerdquo Journal of Bacteriology vol 180 no 19 pp 5256ndash52591998

[104] I Kawagishi M Imagawa Y Imae L McCarter and MHomma ldquoThe sodium-driven polar flagellar motor of marineVibrio as the mechanosensor that regulates lateral flagellarexpressionrdquoMolecular Microbiology vol 20 no 4 pp 693ndash6991996

[105] T J Kirn B A Jude and R K Taylor ldquoA colonizationfactor links Vibrio cholerae environmental survival and humaninfectionrdquo Nature vol 438 no 7069 pp 863ndash866 2005

[106] Z Kuang YHao S Hwang et al ldquoThe Pseudomonas aeruginosaflagellum confers resistance to pulmonary surfactant protein-Aby impacting the production of exoproteases through quorum-sensingrdquo Molecular Microbiology vol 79 no 5 pp 1220ndash12352011

[107] S Zhang F X McCormack R C Levesque G A OrsquoTooleand G W Lau ldquoThe flagellum of Pseudomonas aeruginosa isrequired for resistance to clearance by surfactant protein ArdquoPLoS ONE vol 2 no 6 article e564 2007

[108] G W Lau D J Hassett H Ran and F Kong ldquoThe role ofpyocyanin in Pseudomonas aeruginosa infectionrdquo Trends inMolecular Medicine vol 10 no 12 pp 599ndash606 2004

[109] GW LauH Ran F Kong D J Hassett andDMavrodi ldquoPseu-domonas aeruginosa pyocyanin is critical for lung infection inmicerdquo Infection and Immunity vol 72 no 7 pp 4275ndash42782004

[110] D S Merrell M L Goodrich G Otto L S Tompkins and SFalkow ldquopH-regulated gene expression of the gastric pathogenHelicobacter pylorirdquo Infection and Immunity vol 71 no 6 pp3529ndash3539 2003

[111] K J Allen and M W Griffiths ldquoEffect of environmental andchemotactic stimuli on the activity of the Campylobacter jejuniflaA 12059028 promoterrdquo FEMS Microbiology Letters vol 205 no 1pp 43ndash48 2001

[112] Y Wen J Feng D R Scott E A Marcus and G SachsldquoThe pH-responsive regulon of HP0244 (FlgS) the cytoplasmichistidine kinase of Helicobacter pylorirdquo Journal of Bacteriologyvol 191 no 2 pp 449ndash460 2009

[113] D R Hendrixson ldquoA phase-variable mechanism controllingthe Campylobacter jejuni FlgR response regulator influencescommensalismrdquo Molecular Microbiology vol 61 no 6 pp1646ndash1659 2006

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

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4 Scientifica

transferred to a specific aspartate residue in FlgR to activateit Based on studies with other RpoN-dependent activators(reviewed by Bush and Dixon [31]) phosphorylation ofFlgR likely stimulates oligomerization of the protein froma dimer to a hexamer capable of activating transcriptionRpoN-dependent activators typically possess an N-terminalregulatory domain (often a response regulator domain as isthe case in FlgR) an AAA+ ATPase domain that engages12059054 and couples the hydrolysis of ATP with open complex

formation and aC-terminalDNA-binding domain that bindsan enhancer sequence (reviewed by Schumacher et al [32])H pylori FlgR is unusual in that it lacks a C-terminal DNA-binding domain and activates transcription by contacting12059054-holoenzyme in the closed promoter complex without

binding DNA [29] Although C jejuni FlgR possesses a C-terminal domain it is not needed for activation of the RpoNregulon suggesting that like its counterpart in H pyloriC jejuni FlgR engages the closed promoter complex fromsolution [33] While the C-terminal domain of C jejuni FlgRdoes not appear to play a role inDNAbinding it does preventphosphorylation of the protein by acetyl phosphatewhich caninterfere with normal flagellation [34]

The flagellar protein export apparatus has a major rolein regulating expression of the RpoN regulon of H pyloriand C jejuni as evidenced by the observation that mutationswhich interfere with formation of the export apparatusinhibit expression of RpoN-dependent flagellar genes Por-wollik and coworkers showed that inactivation of fliI orfliQ (encode a cytosolic ATPase component and integralmembrane component of the export apparatus resp) inH pylori results in reduced levels of the minor flagellinFlaB and the hook protein FlgE both of which dependon RpoN for their expression [35] Allan and colleaguesshowed that mutations in flhB (encodes an integral mem-brane component of the export apparatus) result in reducedlevels of FlaB and FlgE inH pylori [36] Smith and coworkerssubsequently demonstrated that disrupting flhB inhibitedexpression of RpoN-dependent reporter genes [37] UsingDNAmicroarrays Niehus and coworkers [20] demonstratedthat FlhA (an integral membrane component of the exportapparatus) is required for transcription of the entire RpoNregulon Hendrixson et al [38] reported that deleting anyone of several integral membrane components of the exportapparatus (FlhA FlhB FliP or FliR) inhibited expressionof an RpoN-dependent flagellar reporter gene in C jejuniStudies by Tsang and coworkers revealed thatH pylori strainswhich stably express truncated forms of FlhA are able totranscribe RpoN-dependent genes at levels that are close towild type but are unable to export flagellar protein substrates[39]Thus anymodel for how the export apparatus influencestranscription of theRpoN regulonmust take into account thatthe export apparatus need not transport protein substrates tostimulate the RpoN regulon Boll and Hendrixson recentlyshowed that loss of the MS ring protein FliF or the Cring protein FliG inhibits transcription of RpoN-dependentflagellar genes in C jejuni [40] The MS ring is the firstflagellar structure assembled and in its absence the exportapparatus presumably is not formedwhich would account for

the inhibition of the RpoN regulon in the fliF mutant FliGmay facilitate autophosphorylation of FlgS either by directlystimulating FlgS or by promoting assembly of the exportapparatus into a conformation that stimulates FlgS activity[40]

The export apparatus is an ideal indicator for the statusof flagellar assembly as it undergoes a major conformationalchange upon completion of the hook-basal body structureIn the early stages of flagellar biogenesis the export apparatusis in a conformation that transports rod- and hook-typesubstrates Upon completion of the mature hook-basal bodystructure the export apparatus undergoes a conformationalchange which is accompanied by a switch in substrate speci-ficity to filament-type substrates [41] This conformationalchange involves an autocleavage event in the C-terminalcytoplasmic domain of the export apparatus protein FlhB(referred to as FlhBC) and interactions between the C-terminal domain of the hook-length control protein FliKand FlhBC [42] The change in conformation and substratespecificity of the export apparatus may serve as a cellular cuefor the temporal regulation of flagellar genes Indeed in Ecoli and Salmonella the anti-12059028 factor FlgM is a filament-type substrate and is secreted by the export apparatus uponcompletion of the mature hook-basal body structure whichallows transcription of the 12059028-dependent flagellar genes tooccur [43]

Transcription of the RpoN-dependent flagellar genes inH pylori also requires the putative RpoN chaperone FlgZ(HP0958) [44 45] FlgZ was first implicated in having a rolein flagellar biogenesis in H pylori when it was identifiedas interacting with RpoN in a high-throughput screen of ayeast two-hybrid system (httpspimhybrigenicscom) [46]In the absence of FlgZ RpoN is turned over rapidly in Hpyloriwith a half-life of about 30 minutes compared to a half-life greater than 4 hours in wild type [45] The molecularbasis for how FlgZ protects RpoN from rapid turnover is notknown but it is possible that FlgZ binds RpoN to protectit from proteolysis or that FlgZ facilitates the association ofRpoN with core RNA polymerase which serves to protectRpoN from degradation Overexpression of RpoN restoresmotility in a H pylori flgZ mutant indicating that FlgZ isnot essential for flagellar biogenesis [45] In addition toits potential role as an RpoN chaperone FlgZ may haveadditional roles in flagellar biogenesis as it was also shown tointeract with the flagellar export apparatus protein FliH in theyeast two-hybrid system (httpspimhybrigenicscom) [46]In addition Douillard and coworkers demonstrated that FlgZbinds the flaA transcript and is needed for optimal expressionof FlaA [47] These researchers proposed that FlgZ functionswith FliH to direct flaA transcripts to the flagellar proteinexport apparatus where translation of the transcripts couldbe coupled with secretion of the nascent FlaA [47]

Transcription of genes needed late in flagellar biogenesisin H pylori and C jejuni which includes genes encodingthe major flagellin filament cap and associated chaper-ones is dependent on 12059028 (FliA) [20] Similar to E coliand Salmonella the H pylori FliA regulon is negativelyregulated by FlgM [48] Unlike E coli and Salmonella

Scientifica 5

the inhibitory effect of FlgM on FliA in H pylori is thoughtto be alleviated via interactions between FlgM and the C-terminal cytoplasmic domain of FlhA (FlhAC) rather thanby secretion of FlgM via the export apparatus [49] C jejunialso possesses a FlgM homolog [50] but unlike H pyloriC jejuni FlgM appears to be secreted from the cell via theflagellar export apparatus [51] Interestingly interaction of Cjejuni FlgM with FliA is temperature dependent occurringat 42∘C (the optimal growth temperature for C jejuni) butnot 37∘C [51] The primary function of C jejuni FlgM is notto inhibit FliA activity during formation of the hook-basalbody structure but rather to limit the length of the flagellarfilament by suppressing expression of the FliA-dependentflagellin (FlaA) and the RpoN-dependent flagellin (FlaB) [51]The mechanism by which FlgM represses transcription ofRpoN-dependent genes in C jejuni is unknown but Wostenand coworkers have speculated that accumulation of theFliAFlgM complex may inhibit phosphorylation of FlgSandor FlgR [51] FlgM also appears to inhibit transcriptionof RpoN-dependent genes inH pylori but so far this has onlybeen demonstrated in an flhAmutant background [20]

22 Vibrio Species The vibrios are typically saltwater micro-organisms and several species cause food-borne illnessesVibrio species vary greatly in flagella morphology Vibriocholerae has a single polar sheathed flagellum while Vibrioparahaemolyticus and Vibrio alginolyticus produce a singlepolar sheathed flagellum and lateral peritrichous flagella andVibrio fischeri has a tuft of polar sheathed flagella Lateralflagella promote swarming and colonization of surfaces andthereby enhance biofilm formation upon encountering highlyviscous media or solid surfaces

The flagellar gene transcriptional hierarchy in Vibriocholerae is governed by RpoN and FliA [52] flrA encodesan RpoN dependent activator and is the master regulatorfor the flagellar gene transcriptional hierarchy [52] FlrA isregulated by cyclic di-GMP a secondarymessenger with rolesin biofilm formation and virulence which binds to FlrA andprevents it from binding the flrBC promoter [53] Early andmiddle genes are RpoN-dependent but they rely on differenttranscriptional activators for their transcription FlrA alongwith RpoN is required for transcription of the early geneswhich encode the components of the MS ring C ring andexport apparatus Other early genes encode the regulatoryproteins FlrB FlrC and FliA FlrB and FlrC constitutethe sensor kinase and response regulator respectively of atwo-component system that regulates transcription of themiddle genes in conjunction with RpoN [54] The middlegenes encode the basal body-hook structures and the coreflagellin FlaA The late genes are dependent on FliA fortheir transcription and encode alternative flagellins FlgMand components of the flagellar motor As in SalmonellaFlgM is secreted via the flagellar protein export apparatusto relieve the inhibition on FliA activity in V cholerae [55]flgA is located upstream of flgM and its promoter (RpoNindependent and FliA independent) may drive transcriptionof flgM such that FliA is repressed until formation of themature hook-basal body structure [52]

The signal(s) that stimulates the FlrBFlrC two-component system is not known FlrD is necessary fortranscription of middle and late flagellar genes making ita candidate for regulating the FlrBFlrC two-componentsystem [56] Additionally FlrD contains HAMP domains(domain present in histidine kinases adenyl cyclasesmethyl-accepting proteins and phosphatases) that areusually found in integral membrane proteins that are part ofsignal transduction pathways [56 57] Moisi and coworkersshowed that FlrD is inserted into the inner membrane andproposed that it senses the completion of the MS ring-switch-export apparatus structure and communicates thisinformation to FlrB [56]

The regulation of flagellar genes inV parahaemolyticus ismore complex since the bacterium is capable of producinglateral flagella in addition to polar flagella (reviewed byMerino et al [58] and McCarter [59]) The polar flagellarsystem (Fla) is constitutively expressed while the lateral flag-ellar system (Laf) is expressed upon impedance of the polarflagellaThe lateral flagella are used for swarmingmotility andenhance biofilm formation and host colonization by the bac-terium [60 61] When grown planktonically the bacteriumproduces a polar flagellum but when grown on highly viscousor solid medium [58] or during iron-limiting conditions[62] the bacterium produces lateral flagella Thus the polarflagellum acts as a mechanosensor to regulate expression ofthe lateral flagella genes through a mechanism that has yet tobe defined The dual flagellar system suited for locomotionunder different conditions allows V parahaemolyticus to behighly adaptive to changing habitats including planktonicenvironments surfaces and biofilms

The polar and lateral flagellar systems do not share anystructural or regulatory components (aside from RpoN) butthe regulatory networks that control polar and lateral flagellarsystems in V parahaemolyticus are similar to that of Vcholerae The lateral flagellar genes are activated by LafKand like the polar flagellar system lateral flagellar genes areboth RpoN dependent and FliA dependent [63] AlthoughLafK is homologous to master regulators of other flagellarsystems LafK does not appear to be amaster regulator for thelateral flagellar system as the 119891119897119894119872L operon (contains genesencoding components of the C ring and export apparatus) isnot LafK dependent [64] This suggests that there is anotherlevel of regulation preceding expression of these genes

23 Pseudomonas Species Pseudomonas aeruginosa is anaerobic Gram-negative Gammaproteobacterium that causesopportunistic infections particularly in patients with cysticfibrosis where it results in inflammation and sepsis (reviewedby Veesenmeyer et al [65] and Balasubramanian et al[66]) P aeruginosa which thrives on surfaces and formsbiofilms is a prevalent agent of nosocomial infections Wild-type P aeruginosa cells form biofilms which can causechronic infections P aeruginosa possesses a single polarflagellum that is required for virulence [67] Motile strainsof P aeruginosa induce activation of the inflammasome (amultiprotein oligomer responsible for the activation of theinflammatory response) whereas nonmotile strains have amarkedly reduced ability to induce inflammasome activation

6 Scientifica

[68] Nonmotile strains of P aeruginosamay have an advan-tage during chronic infection by evading stimulation of theinnate immune responses However a nonmotile flgKmutantis defective in surface attachment suggesting a role for flagellaandor motility in the initial cell-to-surface interaction [69]Mutants in fliM and cheY are also nonmotile and are unableto form biofilm cap structures on initial biofilm colonies [70]Other Pseudomonas species include Pseudomonas fluorescenswhich possesses multiple flagella and is found in the soil andwater Some P fluorescens strains have been shown to protectplant roots against fungal infections [71] P fluorescens doesnot typically cause disease in humans but it is an opportunis-tic pathogen in immunocompromised individuals [72 73]

Like the Epsilonproteobacteria and Vibrio P aeruginosapossesses a four-tiered transcriptional hierarchy for regulat-ing flagellar gene expression FleQ is the master regulatorfor flagellar synthesis and fleQ is regulated by global factorsoutside the flagellar regulons such as cyclic di-GMP asignaling molecule important in modulating the transitionbetween planktonic and biofilm lifestyles [74] The earlyflagellar genes are dependent on FleQ and RpoN for theirtranscription and encode components of the basal body aswell as the filament cap which is somewhat surprising sinceit is not needed until late in flagellar biogenesis [75] Theearly genes also encode regulatory proteins which includeflhF fleN fleS and fleR [75 76] FleN is an antiactivator ofFleQ that maintains the normal flagellum copy number ofone per cell by downregulating genes encoding early flagellarcomponents via a negative feedbackmechanism [77 78] FleSand FleR form a two-component system that is required forthe transcription of the middle RpoN-dependent genes [75]The signal(s) required for FleS activation is unknown Themiddle genes encode components of the basal body includingrod L ring hook hook cap and hook-filament junctionproteins [75] The late genes are FliA dependent and encodeFliC (flagellin) FleL (filament length control protein) FlgMFlgN (a protein required for initiation of filament assembly)and some chemotaxis proteins [75] Transcription of fliA isconstitutive [75] and FlgM (whose transcription is also FleQdependent but not RpoN dependent) represses the activityof FliA until completion of the hook-basal body structure atwhich point it is secreted from the cytoplasm via the exportapparatus which alleviates its inhibition of FliA

24 Caulobacter crescentus Caulobacter crescentus is anAlphaproteobacterium found in freshwater that is a modelorganism for cell cycle studies C crescentus divides asym-metrically to produce two cells with distinct morphologiesa swarmer cell and a stalked cell The nonmotile stalked cellcontains a polar stalk which secretes an exopolysaccharidethat facilitates adhesion to surfaces In addition the stalkedcell initiatesDNA replication upon the start of cell division Incontrast the swarmer cell is motile via a polar flagellum andDNA replication is repressed for a defined length of time untilthe cell differentiates into a stalked cell Flagellar biogenesis iscoordinated with the cell division cycle such that all progenyswarmer cells possess a functional flagellum

Flagellar genes inC crescentus are regulated by two sigmafactors The initiation of DNA replication in the swarmer cellresults in expression and activation of the global transcriptionfactor CtrA CtrA also acts as themaster regulator for flagellargene expression by activating transcription of the early genes(encode MS ring C ring and export apparatus FlbD FliX)Additionally CtrA controls expression of rpoD [79] and isneeded for transcription of the early flagellar genes [80]FlbD and FliX are transcriptional regulators that regulateexpression of the early and middle genes The middle andlate genes which encode the hook protein and flagellins aretranscribed only after the components encoded by the earlygenes have been assembled into the nascent flagellum FlbDis an RpoN-dependent transcriptional activator which bindsftr (flagella transcriptional regulation) sequence motifs toregulate gene expression Once the early genes are expressedFlbD binds the ftr sequence motifs in the promoter regionsof early flagellar genes to inhibit transcription of these genesFor example FlbD binds the ftr4 site located upstream ofthe early gene fliF to repress its transcription once the earlygenes have been expressed [81] In contrast FlbD stimulatestranscription of the middle genes by binding to the ftrsites of the middle and late genes [81 82] FlbD activity isregulated by FliX which acts to either inhibit or stimulateFlbD In the absence of early flagellar structures FliX inhibitsFlbD activity by binding FlbD preventing it from bindingftr sites [83] Once the early flagellar structures are formedFliX stimulates FlbD The mechanism for this stimulationis unknown but it may involve another factor such asan assembled component of the flagellum which convertsFliX into a positive regulator or it may involve covalentmodifications of FliX or FlbD [83] Although FlbD possessesan N-terminal response regulator domain FlbD activity doesnot appear to be regulated by phosphorylation via a sensorkinase The exact cellular cues that regulate FlbD are notknown but FlbD activity is linked to cell division [84]

Unlike the bacteria discussed thus far flagellar biogenesisin C crescentus does not involve FliA Rather the six flagellingenes found in C crescentus are RpoN dependent Theexpression of the flagellin genes is regulated by FlbD and FlbTwhich binds the 51015840 UTR of flagellin transcripts and repressestranslation until the hook-basal body structure is assembled[85] Though the mechanism for this temporal regulation isunknown one possibility is that FlbT represses translationof flagellin genes until a positive factor binds the 51015840 UTRregion of flagellins to promote translation upon assembly ofthe mature hook-basal body structure [85]

3 Coupling Flagellar Biosynthesis withOther Cellular Activities

Though flagellar biogenesis is a complex process in itselfit is further complicated by its coordination with othercellular processes Flagellar biosynthesis is timed with othercellular activities such as cell replication and quorum sensingEnvironmental stimuli also play a role in regulating flagellarbiogenesis as bacteria respond to changes in the environmentby altering behavior needed for survival Many of these

Scientifica 7

responses involve altering the state of flagellar biogenesiswithin these different contexts

31 Modulating Flagellar Gene Expression during the CellCycle Flagellar biosynthesis is linked to the cell cyclethrough various mechanisms and involves coordinating flag-ellar gene expression with growth phase and cell division InV cholerae chemotaxis and motility genes are upregulatedas the population enters stationary phase Of the 114 genesidentified with roles in chemotaxis and flagellar biogen-esis 72 genes were upregulated more than 2-fold duringstationary phase compared to mid-exponential phase [86]The upregulation of 60 of the 72 chemotaxis and flagellargenes is dependent on RpoS indicating a correlation betweenflagellar biogenesis and entry into stationary phase [86]The coordination between entry into stationary phase andflagellar biogenesis may aid the bacterium in exiting the hostwhen nutrients become depleted and prepare the bacteriumfor colonization of other niches or survival in environmentalreservoirs

C crescentus is a particularly good organism for studyinghow flagellar biogenesis is integrated with the cell cyclebecause of its two distinct cell morphologies The flagellarfilament is assembled just prior to cell division and allprogeny swarmer cells possess a fully functional flagellumThe synthesis of the flagellum is intimately linked to the cellcycle through regulatory proteins such as CtrA FlbD andFliX that are involved in both the cell cycle and flagellarbiogenesis This dual functionality may exist to ensure thateach swarmer cell is flagellated The master regulator forflagellar biogenesis CtrA regulates at least 95 genes [87]including the cell-division genes ftsZ [88] and ftsQA [89]Cells of a fliX mutant become filamentous as they enterlate log phase [90] demonstrating a role for this flagellarregulatory protein in cell division Flagellar biogenesis is alsolinked to the cell cycle via FlbT and FlaF FlaF is required forflagellar biogenesis and motility [91] FlbT protein levels areconstant throughout the cell cycle while FlaF levels correlateto flagellin levels both of which peak just prior to cell division[91] Since FlbT is a negative regulator for flagellin translationand peaks in flagellin levels correlate with FlaF levels thisobservation suggests that FlaF may temporally modulateFlbT activity [91]

Expression of Vibrio vulnificus FlhF which is needed forpolar localization of the flagellum is regulated by the quorumsensing master regulator SmcR flhF transcript levels in asmcRmutant are higher than wild-type levels indicating thatSmcR represses flhF expression [92] In wild-type cells flhFtranscript levels are highest during exponential phase anddecrease upon entry into stationary phase [92] In contrasttranscript levels of smcR increase as cultures enter stationaryphase suggesting that SmcR plays a major role for growthphase-dependent variation of flhF expression [92] Thisgrowth phase-dependent gene expression may be importantin host colonization as during the initial infection bacterialcell densities are low but at later stages of colonizationwhen bacterial cell densities are higher motility may not benecessary

Expression of flagellar genes is regulated in P fluorescensthroughout the growth phases by the Gac (GacAGasS) two-component system which limits flagellar biosynthesis duringexponential growth by downregulating transcription of fleQ(encodes the master regulator) [93] The Gac system posi-tively regulates production of virulence factors and quorumsensing molecules and is required for full virulence in animaland plant hosts [94] Levels of the Gac system componentsare regulated in response to growth phase as gacA and gacStranscript levels are highest at mid-exponential growth phase[95] The concomitant upregulation of virulence factors andthe downregulation of flagellar genes by the Gac system maybe a way to coordinate virulence and motility with growthphase so that virulence factors are expressed at high celldensities during host colonization and flagellar genes areturned off to facilitate attachment to the host

32 Connections between Quorum Sensing Host Colonizationand Flagellar Gene Expression In many pathogenic bacte-ria flagella aid in surface attachment and colonization Insuch cases successful colonization may require cooperationbetween quorum sensing and flagellar synthesis to integratesurface attachment and virulence gene expression at highcell densities in pathogens Surface colonization can leadto biofilm formation which is generally more resistant tohost defenses and antimicrobials Pathogens use differentmechanisms to couple expression of virulence genes withflagellar gene expression in the context of the host Thisincludes modulating quorum sensing and host colonizationbased on the status of the flagellum or modulating flagellarbiogenesis based on quorum sensing signals

Quorum sensing is used to regulate gene expression inresponse to cell densityThis is achieved through the secretionof a signaling molecule called an autoinducer (AI) AIs areproduced by bacteria and travel across the cell membrane andaccumulate in the environment Upon reaching a critical con-centration the AIs stimulate transcription of genes requiredfor group behavior luxS is responsible for the productionof AI-2 [96] and an H pylori mutant in luxS exhibitsdecreasedmotility compared towild type [97]flhA transcriptlevels are decreased in the luxS mutant but addition of 45-dihydroxy-23-pentanedione (functions as AI-2) into thegrowth medium restores flhA transcript levels to wild-typelevels [97] Like the H pylori luxS mutant a C jejuni luxSmutant is less motile than wild type [98] and the mutantdisplays reduced transcript levels of flaA [99] Using DNAmicroarrays He and coworkers found that a majority of theflagellar genes are downregulated in a C jejuni luxS mutant[98] Expression of chemotaxis genes however is unchangedin the luxS mutant indicating that the motility defect of theluxS mutant is due to defects in flagellar biogenesis and notchemotaxis [98] A rationale for the link between cell densityand flagellar biogenesis is that it may aid in colonization ofhost tissue by ensuring that a sufficient number of flagellatedbacteria are present to establish a successful infection

Regulatory proteins involved in flagellar biogenesis canregulate quorum sensing and colonization or they themselvescan be regulated by quorum sensing and colonization factorsFor example the RpoN-dependent activator FlrC not only

8 Scientifica

is required for flagellar biogenesis but also appears to play arole in host colonization by V cholerae Mutants expressinga FlrC variant incapable of being phosphorylated fail toproduce flagella and are therefore defective in colonization[54] Interestingly a mutant expressing a constitutively activeform of FlrC is also deficient in colonization even though thecells are motile and produce flagella [54] This observationdemonstrates the need for coordinating flagellar biogenesisand pathogenesis for successful colonization ConverselyVfr a major quorum sensing and virulence regulator inP aeruginosa downregulates expression of flagellar genesby repressing transcription from the fleQ promoter [100]Inhibition of flagellar biogenesis by Vfr may aid P aeruginosain persisting at the site of infection to achieve high celldensities and virulence gene expression

HapR is a transcriptional regulator involved in quorumsensing in V cholerae which also regulates expression ofvirulence genes HapR represses expression of several vir-ulence genes [101] and hapR expression is regulated by anunknown mechanism involving FliA [102] hapR expres-sion is depressed in flgM and flgD mutants (both of thesemutations alleviate FlgM repression on FliA activity) whiledeletion of fliA results in elevated hapR expression [102] Theauthors of this study speculated that expression of HapR isregulated by a regulator or small noncoding RNA (sRNA)that is part of the FliA regulon FliA-mediated regulation ofhapR appears to be linked to the shearing of flagella frombacteria that penetrate the mucin layer to infect the hostThis shearing of the flagella leads to secretion of FlgM viathe flagellar protein export apparatus and results in increasedexpression of FliA-dependent genes and repression of hapRexpression [102] The outcome of such a mechanism is thatvirulence genes which are negatively regulated by HapRare expressed specifically when V cholerae penetrates theintestinal mucous layer hapR production is also regulatedby the quorum sensing protein LuxO further linking viru-lence to cell density LuxO is an RpoN-dependent activator[103] which along with HapR regulates motility proteaseproduction and biofilm formation [101] High cell densitiesare common during late stages of infection and the HapR-mediated repression of virulence genes may aidV cholerae indetaching and finding new colonization sites [86 101]

V parahaemolyticus uses its polar flagellum to detectsurfaces by sensing impedance to the polar flagellum rotationrate [104] Once a surface is encountered expression of thelateral flagellar genes is induced Lateral flagella are importantfor host colonization as mutants that do not produce lateralflagella are deficient in adherence to HeLa cells and biofilmformation [61] Gode-Potratz and coworkers identified sim70genes that are surface responsive most of which are positivelyregulated [64] Some of the surface-regulated genes that areupregulated are virulence genes such as a putative GbpAhomolog and components of a type III secretion system [64]GbpA from V cholerae promotes adherence to chitinoussurfaces of zooplankton and human epithelia cells [105]which is consistent with the observed upregulation of gbpAin V parahaemolyticus when it encounters a surface

The P aeruginosa flagellum is an important determinantin the susceptibility of the bacterium to host defenses

P aeruginosa fliCmutants are unable to upregulate transcrip-tion of lasI and rhlI which encode enzymes that synthesizequorum sensing homoserine lactones [106] These quo-rum sensing molecules regulate production of exoproteaseswhich degrade surfactant protein-A (SP-A) an antimicro-bial that opsonizes and permeabilizes membranes of lungpathogens [106] In the absence of these exoproteases theP aeruginosa fliC mutants are cleared from the lung [106]Though it is not known why fliC mutants are unable toupregulate transcription of lasI and rhlI it is not due tothe inability to sense the environment [106] P aeruginosafliE flgE fliC and fliD mutants are also attenuated in LPSbiosynthesis which compromises the integrity of their outermembranes making them more susceptible to SP-A [107]Additionally flgE and fliC mutants make less pyocyanin aredox-active toxic secondary metabolite that is crucial forlung infection [108 109]

33 Reacting to pH Changes by Gastrointestinal PathogensGastrointestinal pathogens encounter varying environmentalconditions as they travel from the mouth to the stomach andthen to the intestines The gastric pathogen H pylori has theadded burden of long-term survival in the acidic environ-ment of the stomach Pathogens that colonize the intestinaltract must also survive passage through the stomach in highenough numbers to be able to colonize their target organsThus it is not surprising that gastrointestinal pathogens oftenmodulate gene expression in response to acidic conditions

Merrell and coworkers found that expression of approxi-mately 7 of theH pylori genes is altered by a shift to low pH[110] Many of these genes are involved in flagellar synthesiswith the majority of those being RpoN dependent [110] FliA-dependent genes such as flgM in H pylori [110] and flaAin C jejuni [111] are also upregulated at low pH conditionsThe upregulation of flagellar genes is critical for survival ofH pylori as the bacterium is not able to survive at low pHbut must swim across the mucous layer to the underlyinggastric epithelium to establish a successful infection Inaddition to enhancing transcription of flagellar genes acidicenvironments also stimulate motility and swimming speedspotentially in response to increased concentration of protonsin the stomach [110]

The basis for upregulation of the RpoN-dependent flag-ellar genes in H pylori likely involves a stimulation of FlgSactivity at low pHWen and coworkers reported thatH pylorirequires FlgS to survive acid stress (30-minute exposure topH25) [112]These researchers found that besides the knownRpoN-dependent flagellar genes FlgS was required for theupregulation of 86 genes in response to acid stress many ofwhich were previously known to be required for acid stresssurvival [112] It is not known if FlgS responds directly toacid stress or if the stress signal is mediated through anotherfactor

34 Altering Flagellar Biogenesis by Phase Variation In somebacteria flagellar biogenesis is subject to phase variationevents that affect expression of specific flagellar genes Phasevariation causes changes in phenotypes at frequencies that

Scientifica 9

are much higher than random mutations and contribute toheterogeneity within a population The ability of H pylori topersist in the host may involve the phase-variable dependentexpression of flagella that can serve as an advantage foradaptation to the host and for evading the host immuneresponse C jejuni utilizes phase variation to turn on andoff production of flagella a strategy that increases com-mensal colonization in poultry [113] Many phase variationevents occur by random reversible changes in the length ofshort DNA sequence repeats resulting from slipped-strandmispairing These events can take place within the codingsequence or in the promoter of a gene C jejuni phasevariants in which expression of flgR is in the OFF stateresult from the addition or removal of a nucleotide withinone of two homopolymeric nucleotide tracts consisting ofadenine and thymine These nucleotide tracts are locatedwithin the coding sequence of flgR causing the sequence toshift out of frame [113] Revertants to the ON state ariseupon the addition or removal of a nucleotide within theoriginal mutated homopolymeric nucleotide tract to restorethe wild-type sequence Alternatively pseudorevertants canarise in which nucleotides are removed or added close to theoriginal mutated homopolymeric nucleotide tract to restorethe correct reading frame but which result in changes in theamino acid sequence of the protein [113] flgS is also regulatedby phase variation in C jejuni providing multiple levels ofphase-variable control for theRpoN regulon [114 115] Similarmechanisms of phase variation also exist for flhA in C coli[116] and fliP in H pylori [117] which leads to the formationof truncated proteins that prevent flagellar biogenesis

A second type of phase variation in flagellar biogen-esis is mediated through alterations in DNA methylationwhich impact the expression of specific flagellar genesHost-adapted bacterial pathogens such as H pylori oftenpossess DNA methyltransferases and type III restriction-modification (R-M) systems that contain simple tandemDNA repeats which undergo slipped-strand mispairing dur-ing DNA replication that causes frameshift mutations andphase variation [118] R-M systems widespread among manybacteria confer protection from invasion by foreign DNAType III R-M systems are composed of a methyltransferase(mod) and an endonuclease (res) gene whose gene prod-ucts typically function together The switching between ONand OFF states of a DNA methyltransferase can regulateexpression of a ldquophasevarionrdquo via differential methylationof the genome in ON and OFF states [119] Many of thesephase-variable methyltransferase genes are associated withan inactive res gene resulting from frameshift mutationor are orphans that are not associated with a res genesuggesting that DNA restriction is not the primary roleof these methyltransferases (reviewed by Fox et al [120])For example H pylori modH encodes a phase-variableDNA methyltransferase and the cognate res gene containsa nonsense mutation frameshift mutation or is absent invarious H pylori strains [119] modH OFF strains havedecreased expression of flaA and fliK compared to themodHON strain [119] FlaA has a low ability to activate innateimmunity via the Toll-like receptor 5 [121] and modulatingexpression of the flagellin may be advantageous in evading

the host response Another H pylori methyltransferase withan inactive res gene is hpyAVIBM [122 123] hpyAVIBMencodes a C5 cytosine methyltransferase and contains AGrepeats in its open reading frame making it susceptible tophase variation via frame shift mutations [124] For instancehpyAVIBM from strain 26695 and hpyAVIBM from strainHPAG1 have five AG repeats whereas the four AG repeats instrain San 74 result in the translation of a truncated protein[124] Deletion of hpyAVIBM results in both the upregulationand downregulation of rpoN fliR fliD fliS motA fliK andflgK depending on the strain used [124] indicating that phasevariation of hpyAVIBM can alter flagellar biogenesis

P aeruginosa populations can possess two phenotypicvariants one which forms small rough colonies (S) andanother which forms large flat (L) colonies S variants formbiofilms in nonagitated liquid cultures whereas the L variantsdo not [125] S variants possess defects in flagellum-mediatedswimming flagellum-mediated swarming and type IV-pilus-mediated twitching but are able to revert back to the Lphenotype at relatively high frequencies suggesting that theshift between phenotypes is regulated by phase variation[125] Phase variation also occurs in P fluorescens whichcolonizes the alfalfa rhizosphere Phenotypic variants ariseduring rhizosphere colonization and are dependent on theactivity of a site-specific recombinase [126] C variants havewild-type colony morphology while F and S variants havea translucent and diffuse colony morphology [126] F andS variants which colonize distal parts of the roots andswim faster than the C variants overproduce fliC (flagellin)transcripts and synthesize flagellin filaments that aresim3 timeslonger than those of the C variant [126]

4 Conclusions and Future Directions

Flagellar biogenesis is a carefully choreographed processin which many different components of the flagellum aremade as they are needed for assembly into the nascentflagellum Bacteria have evolved elaborate and sundry reg-ulatory networks to couple flagellar gene expression withassembly A driving force for the evolution of such regulatorynetworks is undoubtedly the environmental and ecologicalconstraints imposed by a particular bacteriumrsquos niche Inaddition the degree to which a given bacterial species needsto couple flagellar biogenesis to other cellular processes suchas cell division has also likely shaped the evolution of theseregulatory networks We focused here on bacteria whichutilize RpoN for flagellar biogenesis yet even among thesebacteria the mechanisms used to regulate expression of theRpoN-dependent flagellar genes vary greatly For most of thesystems described in this review we have only scratched thesurface in our understanding of how they operate For each ofthese systems fundamental questions remain to be answeredsuch as the following are all of the flagellar genes of a givensystem expressed as part of a transcription hierarchy or aresome genes expressed constitutively Are there regulatorymechanisms within RpoN flagellar regulons that fine-tunegene expression so that components of the flagellum aremadeprecisely when needed What are the cellular cues sensed by

10 Scientifica

the regulatory proteins that control expression of the RpoN-dependent flagellar genes How does the flagellar exportapparatus control expression of RpoN-dependent flagellargenes in various bacteria Does FlgZ have a regulatoryrole in flagellar biogenesis in H pylori and other bacteriaAs our understanding of the molecular genetics of diversebacterial species expands novel regulatory mechanisms thatcontrol expression of flagellar genes within these bacteria willundoubtedly come to light For example it was only recentlythat sRNAs were identified in H pylori some of whichare potentially FliA dependent [127] and may have roles inflagellar biogenesis Finally with increasingly sophisticatedmethods in bioinformatics genomics and proteomics fur-ther characterization of flagellar regulatory networks willprovide important insights into the mechanisms governinggene expression and identify new regulatory proteins andmechanisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by award MCB-1244242 from theNational Science Foundation

References

[1] T Minamino K Imada and K Namba ldquoMechanisms of typeIII protein export for bacterial flagellar assemblyrdquo MolecularBioSystems vol 4 no 11 pp 1105ndash1115 2008

[2] J L McMurry J W Murphy and B Gonzalez-Pedrajo ldquoTheFliN-FliH interaction mediates localization of flagellar exportATPase FliI to the C ring complexrdquo Biochemistry vol 45 no39 pp 11790ndash11798 2006

[3] R M Macnab Ed Flagella and Motility ASM Press 1996[4] T Minamino K Imada and K Namba ldquoMolecular motors of

the bacterial flagellardquoCurrent Opinion in Structural Biology vol18 no 6 pp 693ndash701 2008

[5] T G Smith and T R Hoover ldquoDeciphering bacterial flagellargene regulatory networks in the genomic erardquo Advances inApplied Microbiology vol 67 pp 257ndash295 2009

[6] J K Anderson T G Smith and T R Hoover ldquoSense andsensibility flagellum-mediated gene regulationrdquo Trends inMicrobiology vol 18 no 1 pp 30ndash37 2010

[7] J D Helmann ldquoAlternative sigma factors and the regulation offlagellar gene expressionrdquoMolecular Microbiology vol 5 no 12pp 2875ndash2882 1991

[8] D L Popham D Szeto J Keener and S Kustu ldquoFunctionof a bacterial activator protein that binds to transcriptionalenhancersrdquo Science vol 243 no 4891 pp 629ndash635 1989

[9] S Sasse-Dwight and J D Gralla ldquoProbing the EscherichiacoliglnALG upstream activation mechanism in vivordquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8934ndash8938 1988

[10] M Kihara T Minamino S Yamaguchi and R M MacnabldquoIntergenic suppression between the flagellar MS ring protein

FliF of Salmonella and FlhA a membrane component of itsexport apparatusrdquo Journal of Bacteriology vol 183 no 5 pp1655ndash1662 2001

[11] J S van Arnam J L McMurry M Kihara and R M MacnabldquoAnalysis of an engineered Salmonella flagellar fusion proteinFliR-FlhBrdquo Journal of Bacteriology vol 186 no 8 pp 2495ndash2498 2004

[12] C S Barker I V Meshcheryakova A S Kostyukova and FA Samatey ldquoFliO regulation of FliP in the formation of theSalmonella enterica flagellumrdquo PLoS Genetics vol 6 no 9Article ID e1001143 2010

[13] A C Lowenthal M Hill L K Sycuro K Mehmood N RSalama and K M Ottemann ldquoFunctional analysis of the Heli-cobacter pylori flagellar switch proteinsrdquo Journal of Bacteriologyvol 191 no 23 pp 7147ndash7156 2009

[14] R M Macnab ldquoType III flagellar protein export and flagellarassemblyrdquo Biochimica et Biophysica Acta vol 1694 no 1ndash3 pp207ndash217 2004

[15] K A Eaton D R Morgan and S Krakowka ldquoCampylobacterpylori virulence factors in gnotobiotic pigletsrdquo Infection andImmunity vol 57 no 4 pp 1119ndash1125 1989

[16] K A Eaton D R Morgan and S Krakowka ldquoMotility as afactor in the colonisation of gnotobiotic piglets by Helicobacterpylorirdquo Journal of Medical Microbiology vol 37 no 2 pp 123ndash127 1992

[17] D G Newell H McBride and J M Dolby ldquoInvestigationson the role of flagella in the colonization of infant mice withCampylobacter jejuni and attachment of Campylobacter jejunito human epithelial cell linesrdquo Journal of Hygiene vol 95 no 2pp 217ndash227 1985

[18] TMorooka A Umeda andK Amako ldquoMotility as an intestinalcolonization factor forCampylobacter jejunirdquo Journal of GeneralMicrobiology vol 131 no 8 pp 1973ndash1980 1985

[19] C Y Kao B S Sheu SM Sheu et al ldquoHighermotility enhancesbacterial density and inflammatory response in dyspepticpatients infected with Helicobacter pylorirdquo Helicobacter vol 17no 6 pp 411ndash416 2012

[20] E Niehus H Gressmann F Ye et al ldquoGenome-wide analysisof transcriptional hierarchy and feedback regulation in theflagellar system of Helicobacter pylorirdquoMolecular Microbiologyvol 52 no 4 pp 947ndash961 2004

[21] M Balaban S N Joslin and D R Hendrixson ldquoFlhF and itsGTPase activity are required for distinct processes in flagellargene regulation and biosynthesis in Campylobacter jejunirdquoJournal of Bacteriology vol 191 no 21 pp 6602ndash6611 2009

[22] A Kusumoto A Shinohara H Terashima S Kojima TYakushi and M Homma ldquoCollaboration of FlhF and FlhGto regulate polarflagella number and localization in VibrioalginolyticusrdquoMicrobiology vol 154 no 5 pp 1390ndash1399 2008

[23] A Kusumoto K Kamisaka T Yakushi H Terashima AShinohara and M Homma ldquoRegulation of polar flagellarnumber by the flhF and flhG genes in Vibrio alginolyticusrdquoJournal of Biochemistry vol 139 no 1 pp 113ndash121 2006

[24] B M Pruszlig and P Matsumura ldquoA regulator of the flagellarregulon of Escherichia coli flhD also affects cell divisionrdquoJournal of Bacteriology vol 178 no 3 pp 668ndash674 1996

[25] B M Pruszlig and P Matsumura ldquoCell cycle regulation of flagellargenesrdquo Journal of Bacteriology vol 179 no 17 pp 5602ndash56041997

[26] G Spohn and V Scarlato ldquoMotility of Helicobacter pylori iscoordinately regulated by the transcriptional activator FlgR an

Scientifica 11

NtrC homologrdquo Journal of Bacteriology vol 181 no 2 pp 593ndash599 1999

[27] M Schirm E C Soo A J Aubry J Austin P Thibault and SM Logan ldquoStructural genetic and functional characterizationof the flagellin glycosylation process in Helicobacter pylorirdquoMolecular Microbiology vol 48 no 6 pp 1579ndash1592 2003

[28] D Beier and R Frank ldquoMolecular characterization of two-component systems of Helicobacter pylorirdquo Journal of Bacteri-ology vol 182 no 8 pp 2068ndash2076 2000

[29] P Brahmachary M G Dashti J W Olson and T R HooverldquoHelicobacter pylori FlgR is an enhancer-independent activatorof 12059054-RNA polymerase holoenzymerdquo Journal of Bacteriologyvol 186 no 14 pp 4535ndash4542 2004

[30] M M S M Wosten J A Wagenaar and J P M van PuttenldquoThe FlgSFlgR two-component signal transduction systemregulates the fla regulon in Campylobacter jejunirdquo The Journalof Biological Chemistry vol 279 no 16 pp 16214ndash16222 2004

[31] M Bush and R Dixon ldquoThe role of bacterial enhancer bindingproteins as specialized activators of 12059054-dependent transcrip-tionrdquo MicroBiology and Molecular Biology Reviews vol 76 no3 pp 497ndash529 2012

[32] J Schumacher N Joly M Rappas X Zhang and M BuckldquoStructures and organisation of AAA+ enhancer binding pro-teins in transcriptional activationrdquo Journal of Structural Biologyvol 156 no 1 pp 190ndash199 2006

[33] S N Joslin and D R Hendrixson ldquoAnalysis of the Campy-lobacter jejuni FlgR response regulator suggests integration ofdiverse mechanisms to activate an NtrC-like proteinrdquo Journalof Bacteriology vol 190 no 7 pp 2422ndash2433 2008

[34] J M Boll and D R Hendrixson ldquoA specificity determinant forphosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphaterdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 50 pp 20160ndash20165 2011

[35] S Porwollik B Noonan and P W OrsquoToole ldquoMolecular char-acterization of a flagellar export locus of Helicobacter pylorirdquoInfection and Immunity vol 67 no 5 pp 2060ndash2070 1999

[36] E Allan N Dorrell S Foynes M Anyim and B W WrenldquoMutational analysis of genes encoding the early flagellarcomponents of Helicobacter pylori evidence for transcriptionalregulation of flagellin A biosynthesisrdquo Journal of Bacteriologyvol 182 no 18 pp 5274ndash5277 2000

[37] T G Smith L Pereira and T R Hoover ldquoHelicobacter pyloriFlhB processing-deficient variants affect flagellar assembly butnot flagellar gene expressionrdquo Microbiology vol 155 no 4 pp1170ndash1180 2009

[38] D R Hendrixson B J Akerley and V J DiRita ldquoTranspo-son mutagenesis of Campylobacter jejuni identifies a bipartiteenergy taxis system required for motilityrdquoMolecular Microbiol-ogy vol 40 no 1 pp 214ndash224 2001

[39] J Tsang T G Smith L E Pereira and T R Hoover ldquoInsertionmutations inHelicobacter pylori flhA reveal strain differences inRpoN-dependent gene expressionrdquo Microbiology vol 159 part1 pp 58ndash67 2013

[40] J M Boll and D R Hendrixson ldquoA regulatory checkpointduring flagellar biogenesis in Campylobacter jejuni initiatessignal transduction to activate transcription of flagellar genesrdquoMBio vol 4 no 5 Article ID e00432-13 2013

[41] T Minamino H Doi and K Kutsukake ldquoSubstrate specificityswitching of the flagellum-specific export apparatus during flag-ellar morphogenesis in Salmonella typhimuriumrdquo Bioscience

Biotechnology and Biochemistry vol 63 no 7 pp 1301ndash13031999

[42] M Erhardt H M Singer D H Wee J P Keener and K THughes ldquoAn infrequent molecular ruler controls flagellar hooklength in Salmonella entericardquo EMBO Journal vol 30 no 14 pp2948ndash2961 2011

[43] K T Hughes K L Gillen M J Semon and J E KarlinseyldquoSensing structural intermediates in bacterial flagellar assemblyby export of a negative regulatorrdquo Science vol 262 no 5137 pp1277ndash1280 1993

[44] K A Ryan N Karim M Worku S A Moore C W Pennand P W OrsquoToole ldquoHP0958 is an essential motility gene inHelicobacter pylorirdquo FEMS Microbiology Letters vol 248 no 1pp 47ndash55 2005

[45] L Pereira and T R Hoover ldquoStable accumulation of 12059054 inHelicobacter pylori requires the novel protein HP0958rdquo Journalof Bacteriology vol 187 no 13 pp 4463ndash4469 2005

[46] J C Rain L Selig H de Reuse et al ldquoThe protein-proteininteraction map of Helicobacter pylorirdquo Nature vol 409 no6817 pp 211ndash215 2001

[47] F P Douillard K A Ryan D L Caly et al ldquoPosttranscriptionalregulation of flagellin synthesis in Helicobacter pylori by theRpoN chaperone HP0958rdquo Journal of Bacteriology vol 190 no24 pp 7975ndash7984 2008

[48] C Josenhans E Niehus S Amersbach et al ldquoFunctionalcharacterization of the antagonistic flagellar late regulators FliAand FlgM ofHelicobacter pylori and their effects on theH pyloritranscriptomerdquoMolecular Microbiology vol 43 no 2 pp 307ndash322 2002

[49] M Rust S Borchert E Niehus et al ldquoThe Helicobacter pylorianti-sigma factor FlgM is predominantly cytoplasmic andcooperates with the flagellar basal body protein FlhArdquo Journalof Bacteriology vol 191 no 15 pp 4824ndash4834 2009

[50] D R Hendrixson and V J DiRita ldquoTranscription of 12059054-dependent but not 12059028-dependent flagellar genes in Campy-lobacter jejuni is associated with formation of the flagellarsecretory apparatusrdquoMolecular Microbiology vol 50 no 2 pp687ndash702 2003

[51] M M S M Wosten L van Dijk A K J Veenendaal MR de Zoete N M C Bleumink-Pluijm and J P M vanPutten ldquoTemperature-dependent FlgMFliA complex forma-tion regulates Campylobacter jejuni flagella lengthrdquo MolecularMicrobiology vol 75 no 6 pp 1577ndash1591 2010

[52] M G Prouty N E Correa and K E Klose ldquoThe novel 12059054- and12059028-dependent flagellar gene transcription hierarchy of Vibrio

choleraerdquo Molecular Microbiology vol 39 no 6 pp 1595ndash16092001

[53] D Srivastava M L Hsieh A Khataokar M B Neiditch and CM Waters ldquoCyclic di-GMP inhibits Vibrio choleraemotility byrepressing induction of transcription and inducing extracellularpolysaccharide productionrdquo Molecular Microbiology vol 90no 6 pp 1262ndash1276 2013

[54] N E Correa C M Lauriano R McGee and K E KloseldquoPhosphorylation of the flagellar regulatory protein FlrC is nec-essary for Vibrio cholerae motility and enhanced colonizationrdquoMolecular Microbiology vol 35 no 4 pp 743ndash755 2000

[55] N E Correa J R Barker and K E Klose ldquoThe Vibrio choleraeFlgM homologue is an anti-12059028 factor that is secreted throughthe sheathed polar flagellumrdquo Journal of Bacteriology vol 186no 14 pp 4613ndash4619 2004

12 Scientifica

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[60] S Ulitzur ldquoInduction of swarming in Vibrio parahaemolyticusrdquoArchives of Microbiology vol 101 no 4 pp 357ndash363 1974

[61] K S Park M Arita T Iida and T Honda ldquovpaH a geneencoding a novel histone-like nucleoid structure-like proteinthat was possibly horizontally acquired regulates the biogen-esis of lateral flagella in trh-positive Vibrio parahaemolyticusTH3996rdquo Infection and Immunity vol 73 no 9 pp 5754ndash57612005

[62] LMcCarter andM Silverman ldquoIron regulation of swarmer celldifferentiation ofVibrio parahaemolyticusrdquo Journal of Bacteriol-ogy vol 171 no 2 pp 731ndash736 1989

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[64] C J Gode-Potratz R J Kustusch P J Breheny D S Weissand L LMcCarter ldquoSurface sensing inVibrio parahaemolyticustriggers a programme of gene expression that promotes colo-nization and virulencerdquo Molecular Microbiology vol 79 no 1pp 240ndash263 2011

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[71] M Leeman J A van Pelt F M den Ouden M Heinsbroek PA Bakker and B Schippers ldquoInduction of systemic resistance

against fusarium wilt of radish by lipopolysaccharides of Pseu-domonas fluorescensrdquo Phytopathology vol 85 no 9 pp 1021ndash1027 1995

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[77] N Dasgupta and R Ramphal ldquoInteraction of the antiactivatorFleN with the transcriptional activator FleQ regulates flagellarnumber in Pseudomonas aeruginosardquo Journal of Bacteriologyvol 183 no 22 pp 6636ndash6644 2001

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[79] J Malakooti and B Ely ldquoPrincipal sigma subunit of theCaulobacter crescentus RNA polymeraserdquo Journal of Bacteriol-ogy vol 177 no 23 pp 6854ndash6860 1995

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[81] A K Benson G Ramakrishnan N Ohta J Feng A J Ninfaand A Newton ldquoThe Caulobacter crescentus FlbD proteinacts at ftr sequence elements both to activate and to represstranscription of cell cycle-regulated flagellar genesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 91 no 11 pp 4989ndash4993 1994

[82] D A Mullin S M van Way C A Blankenship and A HMullin ldquoFlbD has a DNA-binding activity near its carboxyterminus that recognizes ftr sequences involved in positive andnegative regulation of flagellar gene transcription in Caulobac-ter crescentusrdquo Journal of Bacteriology vol 176 no 19 pp 5971ndash5981 1994

[83] R J Dutton Z Xu and J W Gober ldquoLinking structural assem-bly to gene expression a novel mechanism for regulating theactivity of a 12059054 transcription factorrdquo Molecular Microbiologyvol 58 no 3 pp 743ndash757 2005

[84] R E Muir J Easter and J W Gober ldquoThe trans-acting flagellarregulatory proteins FliX and FlbD play a central role in linkingflagellar biogenesis and cytokinesis in Caulobacter crescentusrdquoMicrobiology vol 151 no 11 pp 3699ndash3711 2005

[85] P E Anderson and J W Gober ldquoFlbT the post-transcriptionalregulator of flagellin synthesis in Caulobacter crescentus inter-acts with the 51015840 untranslated region of flagellin mRNArdquoMolec-ular Microbiology vol 38 no 1 pp 41ndash52 2000

Scientifica 13

[86] A T Nielsen N A Dolganov G Otto M C Miller C Y WuandGK Schoolnik ldquoRpoS controls theVibrio choleraemucosalescape responserdquo PLoS Pathogens vol 2 no 10 article e1092006

[87] M T Laub S L Chen L Shapiro and H H McAdamsldquoGenes directly controlled by CtrA a master regulator of theCaulobacter cell cyclerdquo Proceedings of the National Academy ofSciences of the United States of America vol 99 no 7 pp 4632ndash4637 2002

[88] A J Kelly M J Sackett N Din E Quardokus and Y VBrun ldquoCell cycle-dependent transcriptional and proteolyticregulation of FtsZ in Caulobacterrdquo Genes and Development vol12 no 6 pp 880ndash893 1998

[89] M Wortinger M J Sackett and Y V Brun ldquoCtrA mediatesa DNA replication checkpoint that prevents cell division inCaulobacter crescentusrdquo EMBO Journal vol 19 no 17 pp 4503ndash4512 2000

[90] R E Muir T M OrsquoBrien and J W Gober ldquoThe Caulobactercrescentus flagellar gene fliX encodes a novel trans-acting fac-tor that couples flagellar assembly to transcriptionrdquo MolecularMicrobiology vol 39 no 6 pp 1623ndash1637 2001

[91] M Llewellyn R J Dutton J Easter D OrsquoDonnol and J WGober ldquoThe conserved flaF gene has a critical role in couplingflagellin translation and assembly in Caulobacter crescentusrdquoMolecular Microbiology vol 57 no 4 pp 1127ndash1142 2005

[92] S M Kim D H Lee and S H Choi ldquoEvidence that the Vibriovulnificus flagellar regulator FlhF is regulated by a quorumsensing master regulator SmcRrdquo Microbiology vol 158 part 8pp 2017ndash2025 2012

[93] F Martınez-Granero A Navazo E Barahona M Redondo-Nieto R Rivilla andMMartın ldquoTheGac-Rsm and SadB signaltransduction pathways converge on Algu to downregulatemotility in Pseudomonas fluorescensrdquo PLoS ONE vol 7 no 2Article ID e31765 2012

[94] C Reimmann M Beyeler A Latifi et al ldquoThe global activatorGacA of Pseudomonas aeruginosa PAO positively controls theproduction of the autoinducer N-butyryl-homoserine lactoneand the formation of the virulence factors pyocyanin cyanideand lipaserdquo Molecular Microbiology vol 24 no 2 pp 309ndash3191997

[95] J Dubern and G V Bloemberg ldquoInfluence of environmentalconditions on putisolvins I and II production in Pseudomonasputida strain PCL1445rdquo FEMSMicrobiology Letters vol 263 no2 pp 169ndash175 2006

[96] M G Surette M B Miller and B L Bassler ldquoQuorum sensingin Escherichia coli Salmonella typhimurium andVibrio harveyia new family of genes responsible for autoinducer productionrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 96 no 4 pp 1639ndash1644 1999

[97] B A Rader S R Campagna M F Semmelhack B L Basslerand K Guillemin ldquoThe quorum-sensing molecule autoinducer2 regulates motility and flagellar morphogenesis inHelicobacterpylorirdquo Journal of Bacteriology vol 189 no 17 pp 6109ndash61172007

[98] YHe J G Frye T P Strobaugh Jr andCChen ldquoAnalysis of AI-2LuxS-dependent transcription in Campylobacter jejuni strain81-176rdquo Foodborne Pathogens and Disease vol 5 no 4 pp 399ndash415 2008

[99] B Jeon K Itoh N Misawa and S Ryu ldquoEffects of quorumsensing on flaA transcription and autoagglutination in Campy-lobacter jejunirdquoMicrobiology and Immunology vol 47 no 11 pp833ndash839 2003

[100] N Dasgupta E P Ferrell K J Kanack S E H West andR Ramphal ldquofleQ the gene encoding the major flagellarregulator of Pseudomonas aeruginosa is 12059070 dependent and isdownregulated by Vfr a homolog of Escherichia coli cyclic AMPreceptor proteinrdquo Journal of Bacteriology vol 184 no 19 pp5240ndash5250 2002

[101] J Zhu M B Miller R E Vance M Dziejman B L Bassler andJ J Mekalanos ldquoQuorum-sensing regulators control virulencegene expression in Vibrio choleraerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no5 pp 3129ndash3134 2002

[102] Z Liu T Miyashiro A Tsou A Hsiao M Goulian and JZhu ldquoMucosal penetration primes Vibrio cholerae for hostcolonization by repressing quorum sensingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 28 pp 9769ndash9774 2008

[103] K E Klose V Novik and J J Mekalanos ldquoIdentificationof multiple 12059054-dependent transcriptional activators in Vibriocholeraerdquo Journal of Bacteriology vol 180 no 19 pp 5256ndash52591998

[104] I Kawagishi M Imagawa Y Imae L McCarter and MHomma ldquoThe sodium-driven polar flagellar motor of marineVibrio as the mechanosensor that regulates lateral flagellarexpressionrdquoMolecular Microbiology vol 20 no 4 pp 693ndash6991996

[105] T J Kirn B A Jude and R K Taylor ldquoA colonizationfactor links Vibrio cholerae environmental survival and humaninfectionrdquo Nature vol 438 no 7069 pp 863ndash866 2005

[106] Z Kuang YHao S Hwang et al ldquoThe Pseudomonas aeruginosaflagellum confers resistance to pulmonary surfactant protein-Aby impacting the production of exoproteases through quorum-sensingrdquo Molecular Microbiology vol 79 no 5 pp 1220ndash12352011

[107] S Zhang F X McCormack R C Levesque G A OrsquoTooleand G W Lau ldquoThe flagellum of Pseudomonas aeruginosa isrequired for resistance to clearance by surfactant protein ArdquoPLoS ONE vol 2 no 6 article e564 2007

[108] G W Lau D J Hassett H Ran and F Kong ldquoThe role ofpyocyanin in Pseudomonas aeruginosa infectionrdquo Trends inMolecular Medicine vol 10 no 12 pp 599ndash606 2004

[109] GW LauH Ran F Kong D J Hassett andDMavrodi ldquoPseu-domonas aeruginosa pyocyanin is critical for lung infection inmicerdquo Infection and Immunity vol 72 no 7 pp 4275ndash42782004

[110] D S Merrell M L Goodrich G Otto L S Tompkins and SFalkow ldquopH-regulated gene expression of the gastric pathogenHelicobacter pylorirdquo Infection and Immunity vol 71 no 6 pp3529ndash3539 2003

[111] K J Allen and M W Griffiths ldquoEffect of environmental andchemotactic stimuli on the activity of the Campylobacter jejuniflaA 12059028 promoterrdquo FEMS Microbiology Letters vol 205 no 1pp 43ndash48 2001

[112] Y Wen J Feng D R Scott E A Marcus and G SachsldquoThe pH-responsive regulon of HP0244 (FlgS) the cytoplasmichistidine kinase of Helicobacter pylorirdquo Journal of Bacteriologyvol 191 no 2 pp 449ndash460 2009

[113] D R Hendrixson ldquoA phase-variable mechanism controllingthe Campylobacter jejuni FlgR response regulator influencescommensalismrdquo Molecular Microbiology vol 61 no 6 pp1646ndash1659 2006

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Advances in

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Enzyme Research

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International Journal of

Microbiology

Scientifica 5

the inhibitory effect of FlgM on FliA in H pylori is thoughtto be alleviated via interactions between FlgM and the C-terminal cytoplasmic domain of FlhA (FlhAC) rather thanby secretion of FlgM via the export apparatus [49] C jejunialso possesses a FlgM homolog [50] but unlike H pyloriC jejuni FlgM appears to be secreted from the cell via theflagellar export apparatus [51] Interestingly interaction of Cjejuni FlgM with FliA is temperature dependent occurringat 42∘C (the optimal growth temperature for C jejuni) butnot 37∘C [51] The primary function of C jejuni FlgM is notto inhibit FliA activity during formation of the hook-basalbody structure but rather to limit the length of the flagellarfilament by suppressing expression of the FliA-dependentflagellin (FlaA) and the RpoN-dependent flagellin (FlaB) [51]The mechanism by which FlgM represses transcription ofRpoN-dependent genes in C jejuni is unknown but Wostenand coworkers have speculated that accumulation of theFliAFlgM complex may inhibit phosphorylation of FlgSandor FlgR [51] FlgM also appears to inhibit transcriptionof RpoN-dependent genes inH pylori but so far this has onlybeen demonstrated in an flhAmutant background [20]

22 Vibrio Species The vibrios are typically saltwater micro-organisms and several species cause food-borne illnessesVibrio species vary greatly in flagella morphology Vibriocholerae has a single polar sheathed flagellum while Vibrioparahaemolyticus and Vibrio alginolyticus produce a singlepolar sheathed flagellum and lateral peritrichous flagella andVibrio fischeri has a tuft of polar sheathed flagella Lateralflagella promote swarming and colonization of surfaces andthereby enhance biofilm formation upon encountering highlyviscous media or solid surfaces

The flagellar gene transcriptional hierarchy in Vibriocholerae is governed by RpoN and FliA [52] flrA encodesan RpoN dependent activator and is the master regulatorfor the flagellar gene transcriptional hierarchy [52] FlrA isregulated by cyclic di-GMP a secondarymessenger with rolesin biofilm formation and virulence which binds to FlrA andprevents it from binding the flrBC promoter [53] Early andmiddle genes are RpoN-dependent but they rely on differenttranscriptional activators for their transcription FlrA alongwith RpoN is required for transcription of the early geneswhich encode the components of the MS ring C ring andexport apparatus Other early genes encode the regulatoryproteins FlrB FlrC and FliA FlrB and FlrC constitutethe sensor kinase and response regulator respectively of atwo-component system that regulates transcription of themiddle genes in conjunction with RpoN [54] The middlegenes encode the basal body-hook structures and the coreflagellin FlaA The late genes are dependent on FliA fortheir transcription and encode alternative flagellins FlgMand components of the flagellar motor As in SalmonellaFlgM is secreted via the flagellar protein export apparatusto relieve the inhibition on FliA activity in V cholerae [55]flgA is located upstream of flgM and its promoter (RpoNindependent and FliA independent) may drive transcriptionof flgM such that FliA is repressed until formation of themature hook-basal body structure [52]

The signal(s) that stimulates the FlrBFlrC two-component system is not known FlrD is necessary fortranscription of middle and late flagellar genes making ita candidate for regulating the FlrBFlrC two-componentsystem [56] Additionally FlrD contains HAMP domains(domain present in histidine kinases adenyl cyclasesmethyl-accepting proteins and phosphatases) that areusually found in integral membrane proteins that are part ofsignal transduction pathways [56 57] Moisi and coworkersshowed that FlrD is inserted into the inner membrane andproposed that it senses the completion of the MS ring-switch-export apparatus structure and communicates thisinformation to FlrB [56]

The regulation of flagellar genes inV parahaemolyticus ismore complex since the bacterium is capable of producinglateral flagella in addition to polar flagella (reviewed byMerino et al [58] and McCarter [59]) The polar flagellarsystem (Fla) is constitutively expressed while the lateral flag-ellar system (Laf) is expressed upon impedance of the polarflagellaThe lateral flagella are used for swarmingmotility andenhance biofilm formation and host colonization by the bac-terium [60 61] When grown planktonically the bacteriumproduces a polar flagellum but when grown on highly viscousor solid medium [58] or during iron-limiting conditions[62] the bacterium produces lateral flagella Thus the polarflagellum acts as a mechanosensor to regulate expression ofthe lateral flagella genes through a mechanism that has yet tobe defined The dual flagellar system suited for locomotionunder different conditions allows V parahaemolyticus to behighly adaptive to changing habitats including planktonicenvironments surfaces and biofilms

The polar and lateral flagellar systems do not share anystructural or regulatory components (aside from RpoN) butthe regulatory networks that control polar and lateral flagellarsystems in V parahaemolyticus are similar to that of Vcholerae The lateral flagellar genes are activated by LafKand like the polar flagellar system lateral flagellar genes areboth RpoN dependent and FliA dependent [63] AlthoughLafK is homologous to master regulators of other flagellarsystems LafK does not appear to be amaster regulator for thelateral flagellar system as the 119891119897119894119872L operon (contains genesencoding components of the C ring and export apparatus) isnot LafK dependent [64] This suggests that there is anotherlevel of regulation preceding expression of these genes

23 Pseudomonas Species Pseudomonas aeruginosa is anaerobic Gram-negative Gammaproteobacterium that causesopportunistic infections particularly in patients with cysticfibrosis where it results in inflammation and sepsis (reviewedby Veesenmeyer et al [65] and Balasubramanian et al[66]) P aeruginosa which thrives on surfaces and formsbiofilms is a prevalent agent of nosocomial infections Wild-type P aeruginosa cells form biofilms which can causechronic infections P aeruginosa possesses a single polarflagellum that is required for virulence [67] Motile strainsof P aeruginosa induce activation of the inflammasome (amultiprotein oligomer responsible for the activation of theinflammatory response) whereas nonmotile strains have amarkedly reduced ability to induce inflammasome activation

6 Scientifica

[68] Nonmotile strains of P aeruginosamay have an advan-tage during chronic infection by evading stimulation of theinnate immune responses However a nonmotile flgKmutantis defective in surface attachment suggesting a role for flagellaandor motility in the initial cell-to-surface interaction [69]Mutants in fliM and cheY are also nonmotile and are unableto form biofilm cap structures on initial biofilm colonies [70]Other Pseudomonas species include Pseudomonas fluorescenswhich possesses multiple flagella and is found in the soil andwater Some P fluorescens strains have been shown to protectplant roots against fungal infections [71] P fluorescens doesnot typically cause disease in humans but it is an opportunis-tic pathogen in immunocompromised individuals [72 73]

Like the Epsilonproteobacteria and Vibrio P aeruginosapossesses a four-tiered transcriptional hierarchy for regulat-ing flagellar gene expression FleQ is the master regulatorfor flagellar synthesis and fleQ is regulated by global factorsoutside the flagellar regulons such as cyclic di-GMP asignaling molecule important in modulating the transitionbetween planktonic and biofilm lifestyles [74] The earlyflagellar genes are dependent on FleQ and RpoN for theirtranscription and encode components of the basal body aswell as the filament cap which is somewhat surprising sinceit is not needed until late in flagellar biogenesis [75] Theearly genes also encode regulatory proteins which includeflhF fleN fleS and fleR [75 76] FleN is an antiactivator ofFleQ that maintains the normal flagellum copy number ofone per cell by downregulating genes encoding early flagellarcomponents via a negative feedbackmechanism [77 78] FleSand FleR form a two-component system that is required forthe transcription of the middle RpoN-dependent genes [75]The signal(s) required for FleS activation is unknown Themiddle genes encode components of the basal body includingrod L ring hook hook cap and hook-filament junctionproteins [75] The late genes are FliA dependent and encodeFliC (flagellin) FleL (filament length control protein) FlgMFlgN (a protein required for initiation of filament assembly)and some chemotaxis proteins [75] Transcription of fliA isconstitutive [75] and FlgM (whose transcription is also FleQdependent but not RpoN dependent) represses the activityof FliA until completion of the hook-basal body structure atwhich point it is secreted from the cytoplasm via the exportapparatus which alleviates its inhibition of FliA

24 Caulobacter crescentus Caulobacter crescentus is anAlphaproteobacterium found in freshwater that is a modelorganism for cell cycle studies C crescentus divides asym-metrically to produce two cells with distinct morphologiesa swarmer cell and a stalked cell The nonmotile stalked cellcontains a polar stalk which secretes an exopolysaccharidethat facilitates adhesion to surfaces In addition the stalkedcell initiatesDNA replication upon the start of cell division Incontrast the swarmer cell is motile via a polar flagellum andDNA replication is repressed for a defined length of time untilthe cell differentiates into a stalked cell Flagellar biogenesis iscoordinated with the cell division cycle such that all progenyswarmer cells possess a functional flagellum

Flagellar genes inC crescentus are regulated by two sigmafactors The initiation of DNA replication in the swarmer cellresults in expression and activation of the global transcriptionfactor CtrA CtrA also acts as themaster regulator for flagellargene expression by activating transcription of the early genes(encode MS ring C ring and export apparatus FlbD FliX)Additionally CtrA controls expression of rpoD [79] and isneeded for transcription of the early flagellar genes [80]FlbD and FliX are transcriptional regulators that regulateexpression of the early and middle genes The middle andlate genes which encode the hook protein and flagellins aretranscribed only after the components encoded by the earlygenes have been assembled into the nascent flagellum FlbDis an RpoN-dependent transcriptional activator which bindsftr (flagella transcriptional regulation) sequence motifs toregulate gene expression Once the early genes are expressedFlbD binds the ftr sequence motifs in the promoter regionsof early flagellar genes to inhibit transcription of these genesFor example FlbD binds the ftr4 site located upstream ofthe early gene fliF to repress its transcription once the earlygenes have been expressed [81] In contrast FlbD stimulatestranscription of the middle genes by binding to the ftrsites of the middle and late genes [81 82] FlbD activity isregulated by FliX which acts to either inhibit or stimulateFlbD In the absence of early flagellar structures FliX inhibitsFlbD activity by binding FlbD preventing it from bindingftr sites [83] Once the early flagellar structures are formedFliX stimulates FlbD The mechanism for this stimulationis unknown but it may involve another factor such asan assembled component of the flagellum which convertsFliX into a positive regulator or it may involve covalentmodifications of FliX or FlbD [83] Although FlbD possessesan N-terminal response regulator domain FlbD activity doesnot appear to be regulated by phosphorylation via a sensorkinase The exact cellular cues that regulate FlbD are notknown but FlbD activity is linked to cell division [84]

Unlike the bacteria discussed thus far flagellar biogenesisin C crescentus does not involve FliA Rather the six flagellingenes found in C crescentus are RpoN dependent Theexpression of the flagellin genes is regulated by FlbD and FlbTwhich binds the 51015840 UTR of flagellin transcripts and repressestranslation until the hook-basal body structure is assembled[85] Though the mechanism for this temporal regulation isunknown one possibility is that FlbT represses translationof flagellin genes until a positive factor binds the 51015840 UTRregion of flagellins to promote translation upon assembly ofthe mature hook-basal body structure [85]

3 Coupling Flagellar Biosynthesis withOther Cellular Activities

Though flagellar biogenesis is a complex process in itselfit is further complicated by its coordination with othercellular processes Flagellar biosynthesis is timed with othercellular activities such as cell replication and quorum sensingEnvironmental stimuli also play a role in regulating flagellarbiogenesis as bacteria respond to changes in the environmentby altering behavior needed for survival Many of these

Scientifica 7

responses involve altering the state of flagellar biogenesiswithin these different contexts

31 Modulating Flagellar Gene Expression during the CellCycle Flagellar biosynthesis is linked to the cell cyclethrough various mechanisms and involves coordinating flag-ellar gene expression with growth phase and cell division InV cholerae chemotaxis and motility genes are upregulatedas the population enters stationary phase Of the 114 genesidentified with roles in chemotaxis and flagellar biogen-esis 72 genes were upregulated more than 2-fold duringstationary phase compared to mid-exponential phase [86]The upregulation of 60 of the 72 chemotaxis and flagellargenes is dependent on RpoS indicating a correlation betweenflagellar biogenesis and entry into stationary phase [86]The coordination between entry into stationary phase andflagellar biogenesis may aid the bacterium in exiting the hostwhen nutrients become depleted and prepare the bacteriumfor colonization of other niches or survival in environmentalreservoirs

C crescentus is a particularly good organism for studyinghow flagellar biogenesis is integrated with the cell cyclebecause of its two distinct cell morphologies The flagellarfilament is assembled just prior to cell division and allprogeny swarmer cells possess a fully functional flagellumThe synthesis of the flagellum is intimately linked to the cellcycle through regulatory proteins such as CtrA FlbD andFliX that are involved in both the cell cycle and flagellarbiogenesis This dual functionality may exist to ensure thateach swarmer cell is flagellated The master regulator forflagellar biogenesis CtrA regulates at least 95 genes [87]including the cell-division genes ftsZ [88] and ftsQA [89]Cells of a fliX mutant become filamentous as they enterlate log phase [90] demonstrating a role for this flagellarregulatory protein in cell division Flagellar biogenesis is alsolinked to the cell cycle via FlbT and FlaF FlaF is required forflagellar biogenesis and motility [91] FlbT protein levels areconstant throughout the cell cycle while FlaF levels correlateto flagellin levels both of which peak just prior to cell division[91] Since FlbT is a negative regulator for flagellin translationand peaks in flagellin levels correlate with FlaF levels thisobservation suggests that FlaF may temporally modulateFlbT activity [91]

Expression of Vibrio vulnificus FlhF which is needed forpolar localization of the flagellum is regulated by the quorumsensing master regulator SmcR flhF transcript levels in asmcRmutant are higher than wild-type levels indicating thatSmcR represses flhF expression [92] In wild-type cells flhFtranscript levels are highest during exponential phase anddecrease upon entry into stationary phase [92] In contrasttranscript levels of smcR increase as cultures enter stationaryphase suggesting that SmcR plays a major role for growthphase-dependent variation of flhF expression [92] Thisgrowth phase-dependent gene expression may be importantin host colonization as during the initial infection bacterialcell densities are low but at later stages of colonizationwhen bacterial cell densities are higher motility may not benecessary

Expression of flagellar genes is regulated in P fluorescensthroughout the growth phases by the Gac (GacAGasS) two-component system which limits flagellar biosynthesis duringexponential growth by downregulating transcription of fleQ(encodes the master regulator) [93] The Gac system posi-tively regulates production of virulence factors and quorumsensing molecules and is required for full virulence in animaland plant hosts [94] Levels of the Gac system componentsare regulated in response to growth phase as gacA and gacStranscript levels are highest at mid-exponential growth phase[95] The concomitant upregulation of virulence factors andthe downregulation of flagellar genes by the Gac system maybe a way to coordinate virulence and motility with growthphase so that virulence factors are expressed at high celldensities during host colonization and flagellar genes areturned off to facilitate attachment to the host

32 Connections between Quorum Sensing Host Colonizationand Flagellar Gene Expression In many pathogenic bacte-ria flagella aid in surface attachment and colonization Insuch cases successful colonization may require cooperationbetween quorum sensing and flagellar synthesis to integratesurface attachment and virulence gene expression at highcell densities in pathogens Surface colonization can leadto biofilm formation which is generally more resistant tohost defenses and antimicrobials Pathogens use differentmechanisms to couple expression of virulence genes withflagellar gene expression in the context of the host Thisincludes modulating quorum sensing and host colonizationbased on the status of the flagellum or modulating flagellarbiogenesis based on quorum sensing signals

Quorum sensing is used to regulate gene expression inresponse to cell densityThis is achieved through the secretionof a signaling molecule called an autoinducer (AI) AIs areproduced by bacteria and travel across the cell membrane andaccumulate in the environment Upon reaching a critical con-centration the AIs stimulate transcription of genes requiredfor group behavior luxS is responsible for the productionof AI-2 [96] and an H pylori mutant in luxS exhibitsdecreasedmotility compared towild type [97]flhA transcriptlevels are decreased in the luxS mutant but addition of 45-dihydroxy-23-pentanedione (functions as AI-2) into thegrowth medium restores flhA transcript levels to wild-typelevels [97] Like the H pylori luxS mutant a C jejuni luxSmutant is less motile than wild type [98] and the mutantdisplays reduced transcript levels of flaA [99] Using DNAmicroarrays He and coworkers found that a majority of theflagellar genes are downregulated in a C jejuni luxS mutant[98] Expression of chemotaxis genes however is unchangedin the luxS mutant indicating that the motility defect of theluxS mutant is due to defects in flagellar biogenesis and notchemotaxis [98] A rationale for the link between cell densityand flagellar biogenesis is that it may aid in colonization ofhost tissue by ensuring that a sufficient number of flagellatedbacteria are present to establish a successful infection

Regulatory proteins involved in flagellar biogenesis canregulate quorum sensing and colonization or they themselvescan be regulated by quorum sensing and colonization factorsFor example the RpoN-dependent activator FlrC not only

8 Scientifica

is required for flagellar biogenesis but also appears to play arole in host colonization by V cholerae Mutants expressinga FlrC variant incapable of being phosphorylated fail toproduce flagella and are therefore defective in colonization[54] Interestingly a mutant expressing a constitutively activeform of FlrC is also deficient in colonization even though thecells are motile and produce flagella [54] This observationdemonstrates the need for coordinating flagellar biogenesisand pathogenesis for successful colonization ConverselyVfr a major quorum sensing and virulence regulator inP aeruginosa downregulates expression of flagellar genesby repressing transcription from the fleQ promoter [100]Inhibition of flagellar biogenesis by Vfr may aid P aeruginosain persisting at the site of infection to achieve high celldensities and virulence gene expression

HapR is a transcriptional regulator involved in quorumsensing in V cholerae which also regulates expression ofvirulence genes HapR represses expression of several vir-ulence genes [101] and hapR expression is regulated by anunknown mechanism involving FliA [102] hapR expres-sion is depressed in flgM and flgD mutants (both of thesemutations alleviate FlgM repression on FliA activity) whiledeletion of fliA results in elevated hapR expression [102] Theauthors of this study speculated that expression of HapR isregulated by a regulator or small noncoding RNA (sRNA)that is part of the FliA regulon FliA-mediated regulation ofhapR appears to be linked to the shearing of flagella frombacteria that penetrate the mucin layer to infect the hostThis shearing of the flagella leads to secretion of FlgM viathe flagellar protein export apparatus and results in increasedexpression of FliA-dependent genes and repression of hapRexpression [102] The outcome of such a mechanism is thatvirulence genes which are negatively regulated by HapRare expressed specifically when V cholerae penetrates theintestinal mucous layer hapR production is also regulatedby the quorum sensing protein LuxO further linking viru-lence to cell density LuxO is an RpoN-dependent activator[103] which along with HapR regulates motility proteaseproduction and biofilm formation [101] High cell densitiesare common during late stages of infection and the HapR-mediated repression of virulence genes may aidV cholerae indetaching and finding new colonization sites [86 101]

V parahaemolyticus uses its polar flagellum to detectsurfaces by sensing impedance to the polar flagellum rotationrate [104] Once a surface is encountered expression of thelateral flagellar genes is induced Lateral flagella are importantfor host colonization as mutants that do not produce lateralflagella are deficient in adherence to HeLa cells and biofilmformation [61] Gode-Potratz and coworkers identified sim70genes that are surface responsive most of which are positivelyregulated [64] Some of the surface-regulated genes that areupregulated are virulence genes such as a putative GbpAhomolog and components of a type III secretion system [64]GbpA from V cholerae promotes adherence to chitinoussurfaces of zooplankton and human epithelia cells [105]which is consistent with the observed upregulation of gbpAin V parahaemolyticus when it encounters a surface

The P aeruginosa flagellum is an important determinantin the susceptibility of the bacterium to host defenses

P aeruginosa fliCmutants are unable to upregulate transcrip-tion of lasI and rhlI which encode enzymes that synthesizequorum sensing homoserine lactones [106] These quo-rum sensing molecules regulate production of exoproteaseswhich degrade surfactant protein-A (SP-A) an antimicro-bial that opsonizes and permeabilizes membranes of lungpathogens [106] In the absence of these exoproteases theP aeruginosa fliC mutants are cleared from the lung [106]Though it is not known why fliC mutants are unable toupregulate transcription of lasI and rhlI it is not due tothe inability to sense the environment [106] P aeruginosafliE flgE fliC and fliD mutants are also attenuated in LPSbiosynthesis which compromises the integrity of their outermembranes making them more susceptible to SP-A [107]Additionally flgE and fliC mutants make less pyocyanin aredox-active toxic secondary metabolite that is crucial forlung infection [108 109]

33 Reacting to pH Changes by Gastrointestinal PathogensGastrointestinal pathogens encounter varying environmentalconditions as they travel from the mouth to the stomach andthen to the intestines The gastric pathogen H pylori has theadded burden of long-term survival in the acidic environ-ment of the stomach Pathogens that colonize the intestinaltract must also survive passage through the stomach in highenough numbers to be able to colonize their target organsThus it is not surprising that gastrointestinal pathogens oftenmodulate gene expression in response to acidic conditions

Merrell and coworkers found that expression of approxi-mately 7 of theH pylori genes is altered by a shift to low pH[110] Many of these genes are involved in flagellar synthesiswith the majority of those being RpoN dependent [110] FliA-dependent genes such as flgM in H pylori [110] and flaAin C jejuni [111] are also upregulated at low pH conditionsThe upregulation of flagellar genes is critical for survival ofH pylori as the bacterium is not able to survive at low pHbut must swim across the mucous layer to the underlyinggastric epithelium to establish a successful infection Inaddition to enhancing transcription of flagellar genes acidicenvironments also stimulate motility and swimming speedspotentially in response to increased concentration of protonsin the stomach [110]

The basis for upregulation of the RpoN-dependent flag-ellar genes in H pylori likely involves a stimulation of FlgSactivity at low pHWen and coworkers reported thatH pylorirequires FlgS to survive acid stress (30-minute exposure topH25) [112]These researchers found that besides the knownRpoN-dependent flagellar genes FlgS was required for theupregulation of 86 genes in response to acid stress many ofwhich were previously known to be required for acid stresssurvival [112] It is not known if FlgS responds directly toacid stress or if the stress signal is mediated through anotherfactor

34 Altering Flagellar Biogenesis by Phase Variation In somebacteria flagellar biogenesis is subject to phase variationevents that affect expression of specific flagellar genes Phasevariation causes changes in phenotypes at frequencies that

Scientifica 9

are much higher than random mutations and contribute toheterogeneity within a population The ability of H pylori topersist in the host may involve the phase-variable dependentexpression of flagella that can serve as an advantage foradaptation to the host and for evading the host immuneresponse C jejuni utilizes phase variation to turn on andoff production of flagella a strategy that increases com-mensal colonization in poultry [113] Many phase variationevents occur by random reversible changes in the length ofshort DNA sequence repeats resulting from slipped-strandmispairing These events can take place within the codingsequence or in the promoter of a gene C jejuni phasevariants in which expression of flgR is in the OFF stateresult from the addition or removal of a nucleotide withinone of two homopolymeric nucleotide tracts consisting ofadenine and thymine These nucleotide tracts are locatedwithin the coding sequence of flgR causing the sequence toshift out of frame [113] Revertants to the ON state ariseupon the addition or removal of a nucleotide within theoriginal mutated homopolymeric nucleotide tract to restorethe wild-type sequence Alternatively pseudorevertants canarise in which nucleotides are removed or added close to theoriginal mutated homopolymeric nucleotide tract to restorethe correct reading frame but which result in changes in theamino acid sequence of the protein [113] flgS is also regulatedby phase variation in C jejuni providing multiple levels ofphase-variable control for theRpoN regulon [114 115] Similarmechanisms of phase variation also exist for flhA in C coli[116] and fliP in H pylori [117] which leads to the formationof truncated proteins that prevent flagellar biogenesis

A second type of phase variation in flagellar biogen-esis is mediated through alterations in DNA methylationwhich impact the expression of specific flagellar genesHost-adapted bacterial pathogens such as H pylori oftenpossess DNA methyltransferases and type III restriction-modification (R-M) systems that contain simple tandemDNA repeats which undergo slipped-strand mispairing dur-ing DNA replication that causes frameshift mutations andphase variation [118] R-M systems widespread among manybacteria confer protection from invasion by foreign DNAType III R-M systems are composed of a methyltransferase(mod) and an endonuclease (res) gene whose gene prod-ucts typically function together The switching between ONand OFF states of a DNA methyltransferase can regulateexpression of a ldquophasevarionrdquo via differential methylationof the genome in ON and OFF states [119] Many of thesephase-variable methyltransferase genes are associated withan inactive res gene resulting from frameshift mutationor are orphans that are not associated with a res genesuggesting that DNA restriction is not the primary roleof these methyltransferases (reviewed by Fox et al [120])For example H pylori modH encodes a phase-variableDNA methyltransferase and the cognate res gene containsa nonsense mutation frameshift mutation or is absent invarious H pylori strains [119] modH OFF strains havedecreased expression of flaA and fliK compared to themodHON strain [119] FlaA has a low ability to activate innateimmunity via the Toll-like receptor 5 [121] and modulatingexpression of the flagellin may be advantageous in evading

the host response Another H pylori methyltransferase withan inactive res gene is hpyAVIBM [122 123] hpyAVIBMencodes a C5 cytosine methyltransferase and contains AGrepeats in its open reading frame making it susceptible tophase variation via frame shift mutations [124] For instancehpyAVIBM from strain 26695 and hpyAVIBM from strainHPAG1 have five AG repeats whereas the four AG repeats instrain San 74 result in the translation of a truncated protein[124] Deletion of hpyAVIBM results in both the upregulationand downregulation of rpoN fliR fliD fliS motA fliK andflgK depending on the strain used [124] indicating that phasevariation of hpyAVIBM can alter flagellar biogenesis

P aeruginosa populations can possess two phenotypicvariants one which forms small rough colonies (S) andanother which forms large flat (L) colonies S variants formbiofilms in nonagitated liquid cultures whereas the L variantsdo not [125] S variants possess defects in flagellum-mediatedswimming flagellum-mediated swarming and type IV-pilus-mediated twitching but are able to revert back to the Lphenotype at relatively high frequencies suggesting that theshift between phenotypes is regulated by phase variation[125] Phase variation also occurs in P fluorescens whichcolonizes the alfalfa rhizosphere Phenotypic variants ariseduring rhizosphere colonization and are dependent on theactivity of a site-specific recombinase [126] C variants havewild-type colony morphology while F and S variants havea translucent and diffuse colony morphology [126] F andS variants which colonize distal parts of the roots andswim faster than the C variants overproduce fliC (flagellin)transcripts and synthesize flagellin filaments that aresim3 timeslonger than those of the C variant [126]

4 Conclusions and Future Directions

Flagellar biogenesis is a carefully choreographed processin which many different components of the flagellum aremade as they are needed for assembly into the nascentflagellum Bacteria have evolved elaborate and sundry reg-ulatory networks to couple flagellar gene expression withassembly A driving force for the evolution of such regulatorynetworks is undoubtedly the environmental and ecologicalconstraints imposed by a particular bacteriumrsquos niche Inaddition the degree to which a given bacterial species needsto couple flagellar biogenesis to other cellular processes suchas cell division has also likely shaped the evolution of theseregulatory networks We focused here on bacteria whichutilize RpoN for flagellar biogenesis yet even among thesebacteria the mechanisms used to regulate expression of theRpoN-dependent flagellar genes vary greatly For most of thesystems described in this review we have only scratched thesurface in our understanding of how they operate For each ofthese systems fundamental questions remain to be answeredsuch as the following are all of the flagellar genes of a givensystem expressed as part of a transcription hierarchy or aresome genes expressed constitutively Are there regulatorymechanisms within RpoN flagellar regulons that fine-tunegene expression so that components of the flagellum aremadeprecisely when needed What are the cellular cues sensed by

10 Scientifica

the regulatory proteins that control expression of the RpoN-dependent flagellar genes How does the flagellar exportapparatus control expression of RpoN-dependent flagellargenes in various bacteria Does FlgZ have a regulatoryrole in flagellar biogenesis in H pylori and other bacteriaAs our understanding of the molecular genetics of diversebacterial species expands novel regulatory mechanisms thatcontrol expression of flagellar genes within these bacteria willundoubtedly come to light For example it was only recentlythat sRNAs were identified in H pylori some of whichare potentially FliA dependent [127] and may have roles inflagellar biogenesis Finally with increasingly sophisticatedmethods in bioinformatics genomics and proteomics fur-ther characterization of flagellar regulatory networks willprovide important insights into the mechanisms governinggene expression and identify new regulatory proteins andmechanisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by award MCB-1244242 from theNational Science Foundation

References

[1] T Minamino K Imada and K Namba ldquoMechanisms of typeIII protein export for bacterial flagellar assemblyrdquo MolecularBioSystems vol 4 no 11 pp 1105ndash1115 2008

[2] J L McMurry J W Murphy and B Gonzalez-Pedrajo ldquoTheFliN-FliH interaction mediates localization of flagellar exportATPase FliI to the C ring complexrdquo Biochemistry vol 45 no39 pp 11790ndash11798 2006

[3] R M Macnab Ed Flagella and Motility ASM Press 1996[4] T Minamino K Imada and K Namba ldquoMolecular motors of

the bacterial flagellardquoCurrent Opinion in Structural Biology vol18 no 6 pp 693ndash701 2008

[5] T G Smith and T R Hoover ldquoDeciphering bacterial flagellargene regulatory networks in the genomic erardquo Advances inApplied Microbiology vol 67 pp 257ndash295 2009

[6] J K Anderson T G Smith and T R Hoover ldquoSense andsensibility flagellum-mediated gene regulationrdquo Trends inMicrobiology vol 18 no 1 pp 30ndash37 2010

[7] J D Helmann ldquoAlternative sigma factors and the regulation offlagellar gene expressionrdquoMolecular Microbiology vol 5 no 12pp 2875ndash2882 1991

[8] D L Popham D Szeto J Keener and S Kustu ldquoFunctionof a bacterial activator protein that binds to transcriptionalenhancersrdquo Science vol 243 no 4891 pp 629ndash635 1989

[9] S Sasse-Dwight and J D Gralla ldquoProbing the EscherichiacoliglnALG upstream activation mechanism in vivordquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8934ndash8938 1988

[10] M Kihara T Minamino S Yamaguchi and R M MacnabldquoIntergenic suppression between the flagellar MS ring protein

FliF of Salmonella and FlhA a membrane component of itsexport apparatusrdquo Journal of Bacteriology vol 183 no 5 pp1655ndash1662 2001

[11] J S van Arnam J L McMurry M Kihara and R M MacnabldquoAnalysis of an engineered Salmonella flagellar fusion proteinFliR-FlhBrdquo Journal of Bacteriology vol 186 no 8 pp 2495ndash2498 2004

[12] C S Barker I V Meshcheryakova A S Kostyukova and FA Samatey ldquoFliO regulation of FliP in the formation of theSalmonella enterica flagellumrdquo PLoS Genetics vol 6 no 9Article ID e1001143 2010

[13] A C Lowenthal M Hill L K Sycuro K Mehmood N RSalama and K M Ottemann ldquoFunctional analysis of the Heli-cobacter pylori flagellar switch proteinsrdquo Journal of Bacteriologyvol 191 no 23 pp 7147ndash7156 2009

[14] R M Macnab ldquoType III flagellar protein export and flagellarassemblyrdquo Biochimica et Biophysica Acta vol 1694 no 1ndash3 pp207ndash217 2004

[15] K A Eaton D R Morgan and S Krakowka ldquoCampylobacterpylori virulence factors in gnotobiotic pigletsrdquo Infection andImmunity vol 57 no 4 pp 1119ndash1125 1989

[16] K A Eaton D R Morgan and S Krakowka ldquoMotility as afactor in the colonisation of gnotobiotic piglets by Helicobacterpylorirdquo Journal of Medical Microbiology vol 37 no 2 pp 123ndash127 1992

[17] D G Newell H McBride and J M Dolby ldquoInvestigationson the role of flagella in the colonization of infant mice withCampylobacter jejuni and attachment of Campylobacter jejunito human epithelial cell linesrdquo Journal of Hygiene vol 95 no 2pp 217ndash227 1985

[18] TMorooka A Umeda andK Amako ldquoMotility as an intestinalcolonization factor forCampylobacter jejunirdquo Journal of GeneralMicrobiology vol 131 no 8 pp 1973ndash1980 1985

[19] C Y Kao B S Sheu SM Sheu et al ldquoHighermotility enhancesbacterial density and inflammatory response in dyspepticpatients infected with Helicobacter pylorirdquo Helicobacter vol 17no 6 pp 411ndash416 2012

[20] E Niehus H Gressmann F Ye et al ldquoGenome-wide analysisof transcriptional hierarchy and feedback regulation in theflagellar system of Helicobacter pylorirdquoMolecular Microbiologyvol 52 no 4 pp 947ndash961 2004

[21] M Balaban S N Joslin and D R Hendrixson ldquoFlhF and itsGTPase activity are required for distinct processes in flagellargene regulation and biosynthesis in Campylobacter jejunirdquoJournal of Bacteriology vol 191 no 21 pp 6602ndash6611 2009

[22] A Kusumoto A Shinohara H Terashima S Kojima TYakushi and M Homma ldquoCollaboration of FlhF and FlhGto regulate polarflagella number and localization in VibrioalginolyticusrdquoMicrobiology vol 154 no 5 pp 1390ndash1399 2008

[23] A Kusumoto K Kamisaka T Yakushi H Terashima AShinohara and M Homma ldquoRegulation of polar flagellarnumber by the flhF and flhG genes in Vibrio alginolyticusrdquoJournal of Biochemistry vol 139 no 1 pp 113ndash121 2006

[24] B M Pruszlig and P Matsumura ldquoA regulator of the flagellarregulon of Escherichia coli flhD also affects cell divisionrdquoJournal of Bacteriology vol 178 no 3 pp 668ndash674 1996

[25] B M Pruszlig and P Matsumura ldquoCell cycle regulation of flagellargenesrdquo Journal of Bacteriology vol 179 no 17 pp 5602ndash56041997

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Scientifica 11

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[28] D Beier and R Frank ldquoMolecular characterization of two-component systems of Helicobacter pylorirdquo Journal of Bacteri-ology vol 182 no 8 pp 2068ndash2076 2000

[29] P Brahmachary M G Dashti J W Olson and T R HooverldquoHelicobacter pylori FlgR is an enhancer-independent activatorof 12059054-RNA polymerase holoenzymerdquo Journal of Bacteriologyvol 186 no 14 pp 4535ndash4542 2004

[30] M M S M Wosten J A Wagenaar and J P M van PuttenldquoThe FlgSFlgR two-component signal transduction systemregulates the fla regulon in Campylobacter jejunirdquo The Journalof Biological Chemistry vol 279 no 16 pp 16214ndash16222 2004

[31] M Bush and R Dixon ldquoThe role of bacterial enhancer bindingproteins as specialized activators of 12059054-dependent transcrip-tionrdquo MicroBiology and Molecular Biology Reviews vol 76 no3 pp 497ndash529 2012

[32] J Schumacher N Joly M Rappas X Zhang and M BuckldquoStructures and organisation of AAA+ enhancer binding pro-teins in transcriptional activationrdquo Journal of Structural Biologyvol 156 no 1 pp 190ndash199 2006

[33] S N Joslin and D R Hendrixson ldquoAnalysis of the Campy-lobacter jejuni FlgR response regulator suggests integration ofdiverse mechanisms to activate an NtrC-like proteinrdquo Journalof Bacteriology vol 190 no 7 pp 2422ndash2433 2008

[34] J M Boll and D R Hendrixson ldquoA specificity determinant forphosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphaterdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 50 pp 20160ndash20165 2011

[35] S Porwollik B Noonan and P W OrsquoToole ldquoMolecular char-acterization of a flagellar export locus of Helicobacter pylorirdquoInfection and Immunity vol 67 no 5 pp 2060ndash2070 1999

[36] E Allan N Dorrell S Foynes M Anyim and B W WrenldquoMutational analysis of genes encoding the early flagellarcomponents of Helicobacter pylori evidence for transcriptionalregulation of flagellin A biosynthesisrdquo Journal of Bacteriologyvol 182 no 18 pp 5274ndash5277 2000

[37] T G Smith L Pereira and T R Hoover ldquoHelicobacter pyloriFlhB processing-deficient variants affect flagellar assembly butnot flagellar gene expressionrdquo Microbiology vol 155 no 4 pp1170ndash1180 2009

[38] D R Hendrixson B J Akerley and V J DiRita ldquoTranspo-son mutagenesis of Campylobacter jejuni identifies a bipartiteenergy taxis system required for motilityrdquoMolecular Microbiol-ogy vol 40 no 1 pp 214ndash224 2001

[39] J Tsang T G Smith L E Pereira and T R Hoover ldquoInsertionmutations inHelicobacter pylori flhA reveal strain differences inRpoN-dependent gene expressionrdquo Microbiology vol 159 part1 pp 58ndash67 2013

[40] J M Boll and D R Hendrixson ldquoA regulatory checkpointduring flagellar biogenesis in Campylobacter jejuni initiatessignal transduction to activate transcription of flagellar genesrdquoMBio vol 4 no 5 Article ID e00432-13 2013

[41] T Minamino H Doi and K Kutsukake ldquoSubstrate specificityswitching of the flagellum-specific export apparatus during flag-ellar morphogenesis in Salmonella typhimuriumrdquo Bioscience

Biotechnology and Biochemistry vol 63 no 7 pp 1301ndash13031999

[42] M Erhardt H M Singer D H Wee J P Keener and K THughes ldquoAn infrequent molecular ruler controls flagellar hooklength in Salmonella entericardquo EMBO Journal vol 30 no 14 pp2948ndash2961 2011

[43] K T Hughes K L Gillen M J Semon and J E KarlinseyldquoSensing structural intermediates in bacterial flagellar assemblyby export of a negative regulatorrdquo Science vol 262 no 5137 pp1277ndash1280 1993

[44] K A Ryan N Karim M Worku S A Moore C W Pennand P W OrsquoToole ldquoHP0958 is an essential motility gene inHelicobacter pylorirdquo FEMS Microbiology Letters vol 248 no 1pp 47ndash55 2005

[45] L Pereira and T R Hoover ldquoStable accumulation of 12059054 inHelicobacter pylori requires the novel protein HP0958rdquo Journalof Bacteriology vol 187 no 13 pp 4463ndash4469 2005

[46] J C Rain L Selig H de Reuse et al ldquoThe protein-proteininteraction map of Helicobacter pylorirdquo Nature vol 409 no6817 pp 211ndash215 2001

[47] F P Douillard K A Ryan D L Caly et al ldquoPosttranscriptionalregulation of flagellin synthesis in Helicobacter pylori by theRpoN chaperone HP0958rdquo Journal of Bacteriology vol 190 no24 pp 7975ndash7984 2008

[48] C Josenhans E Niehus S Amersbach et al ldquoFunctionalcharacterization of the antagonistic flagellar late regulators FliAand FlgM ofHelicobacter pylori and their effects on theH pyloritranscriptomerdquoMolecular Microbiology vol 43 no 2 pp 307ndash322 2002

[49] M Rust S Borchert E Niehus et al ldquoThe Helicobacter pylorianti-sigma factor FlgM is predominantly cytoplasmic andcooperates with the flagellar basal body protein FlhArdquo Journalof Bacteriology vol 191 no 15 pp 4824ndash4834 2009

[50] D R Hendrixson and V J DiRita ldquoTranscription of 12059054-dependent but not 12059028-dependent flagellar genes in Campy-lobacter jejuni is associated with formation of the flagellarsecretory apparatusrdquoMolecular Microbiology vol 50 no 2 pp687ndash702 2003

[51] M M S M Wosten L van Dijk A K J Veenendaal MR de Zoete N M C Bleumink-Pluijm and J P M vanPutten ldquoTemperature-dependent FlgMFliA complex forma-tion regulates Campylobacter jejuni flagella lengthrdquo MolecularMicrobiology vol 75 no 6 pp 1577ndash1591 2010

[52] M G Prouty N E Correa and K E Klose ldquoThe novel 12059054- and12059028-dependent flagellar gene transcription hierarchy of Vibrio

choleraerdquo Molecular Microbiology vol 39 no 6 pp 1595ndash16092001

[53] D Srivastava M L Hsieh A Khataokar M B Neiditch and CM Waters ldquoCyclic di-GMP inhibits Vibrio choleraemotility byrepressing induction of transcription and inducing extracellularpolysaccharide productionrdquo Molecular Microbiology vol 90no 6 pp 1262ndash1276 2013

[54] N E Correa C M Lauriano R McGee and K E KloseldquoPhosphorylation of the flagellar regulatory protein FlrC is nec-essary for Vibrio cholerae motility and enhanced colonizationrdquoMolecular Microbiology vol 35 no 4 pp 743ndash755 2000

[55] N E Correa J R Barker and K E Klose ldquoThe Vibrio choleraeFlgM homologue is an anti-12059028 factor that is secreted throughthe sheathed polar flagellumrdquo Journal of Bacteriology vol 186no 14 pp 4613ndash4619 2004

12 Scientifica

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[60] S Ulitzur ldquoInduction of swarming in Vibrio parahaemolyticusrdquoArchives of Microbiology vol 101 no 4 pp 357ndash363 1974

[61] K S Park M Arita T Iida and T Honda ldquovpaH a geneencoding a novel histone-like nucleoid structure-like proteinthat was possibly horizontally acquired regulates the biogen-esis of lateral flagella in trh-positive Vibrio parahaemolyticusTH3996rdquo Infection and Immunity vol 73 no 9 pp 5754ndash57612005

[62] LMcCarter andM Silverman ldquoIron regulation of swarmer celldifferentiation ofVibrio parahaemolyticusrdquo Journal of Bacteriol-ogy vol 171 no 2 pp 731ndash736 1989

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[64] C J Gode-Potratz R J Kustusch P J Breheny D S Weissand L LMcCarter ldquoSurface sensing inVibrio parahaemolyticustriggers a programme of gene expression that promotes colo-nization and virulencerdquo Molecular Microbiology vol 79 no 1pp 240ndash263 2011

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[66] D Balasubramanian L Schneper H Kumari and K MatheeldquoA dynamic and intricate regulatory network determines Pseu-domonas aeruginosa virulencerdquo Nucleic Acids Research vol 41no 1 pp 1ndash20 2013

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[68] Y R Patankar R R Lovewell M E Poynter J Jyot BI Kazmierczak and B Berwin ldquoFlagellar motility is a keydeterminant of themagnitude of the inflammasome response toPseudomonas aeruginosardquo Infection and Immunity vol 81 no 6pp 2043ndash2052 2013

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[71] M Leeman J A van Pelt F M den Ouden M Heinsbroek PA Bakker and B Schippers ldquoInduction of systemic resistance

against fusarium wilt of radish by lipopolysaccharides of Pseu-domonas fluorescensrdquo Phytopathology vol 85 no 9 pp 1021ndash1027 1995

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[75] N Dasgupta M C Wolfgang A L Goodman et al ldquoA four-tiered transcriptional regulatory circuit controls flagellar bio-genesis in Pseudomonas aeruginosardquo Molecular Microbiologyvol 50 no 3 pp 809ndash824 2003

[76] S K Arora B W Ritchings E C Almira S Lory and RRamphal ldquoA transcriptional activator FleQ regulates mucinadhesion and flagellar gene expression in Pseudomonas aerugi-nosa in a cascade mannerrdquo Journal of Bacteriology vol 179 no17 pp 5574ndash5581 1997

[77] N Dasgupta and R Ramphal ldquoInteraction of the antiactivatorFleN with the transcriptional activator FleQ regulates flagellarnumber in Pseudomonas aeruginosardquo Journal of Bacteriologyvol 183 no 22 pp 6636ndash6644 2001

[78] N Dasgupta S K Arora and R Ramphal ldquofleN a gene thatregulates flagellar number in Pseudomonas aeruginosardquo Journalof Bacteriology vol 182 no 2 pp 357ndash364 2000

[79] J Malakooti and B Ely ldquoPrincipal sigma subunit of theCaulobacter crescentus RNA polymeraserdquo Journal of Bacteriol-ogy vol 177 no 23 pp 6854ndash6860 1995

[80] M T Laub H H McAdams T Feldblyum C M Fraser andL Shapiro ldquoGlobal analysis of the genetic network controllinga bacterial cell cyclerdquo Science vol 290 no 5499 pp 2144ndash21482000

[81] A K Benson G Ramakrishnan N Ohta J Feng A J Ninfaand A Newton ldquoThe Caulobacter crescentus FlbD proteinacts at ftr sequence elements both to activate and to represstranscription of cell cycle-regulated flagellar genesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 91 no 11 pp 4989ndash4993 1994

[82] D A Mullin S M van Way C A Blankenship and A HMullin ldquoFlbD has a DNA-binding activity near its carboxyterminus that recognizes ftr sequences involved in positive andnegative regulation of flagellar gene transcription in Caulobac-ter crescentusrdquo Journal of Bacteriology vol 176 no 19 pp 5971ndash5981 1994

[83] R J Dutton Z Xu and J W Gober ldquoLinking structural assem-bly to gene expression a novel mechanism for regulating theactivity of a 12059054 transcription factorrdquo Molecular Microbiologyvol 58 no 3 pp 743ndash757 2005

[84] R E Muir J Easter and J W Gober ldquoThe trans-acting flagellarregulatory proteins FliX and FlbD play a central role in linkingflagellar biogenesis and cytokinesis in Caulobacter crescentusrdquoMicrobiology vol 151 no 11 pp 3699ndash3711 2005

[85] P E Anderson and J W Gober ldquoFlbT the post-transcriptionalregulator of flagellin synthesis in Caulobacter crescentus inter-acts with the 51015840 untranslated region of flagellin mRNArdquoMolec-ular Microbiology vol 38 no 1 pp 41ndash52 2000

Scientifica 13

[86] A T Nielsen N A Dolganov G Otto M C Miller C Y WuandGK Schoolnik ldquoRpoS controls theVibrio choleraemucosalescape responserdquo PLoS Pathogens vol 2 no 10 article e1092006

[87] M T Laub S L Chen L Shapiro and H H McAdamsldquoGenes directly controlled by CtrA a master regulator of theCaulobacter cell cyclerdquo Proceedings of the National Academy ofSciences of the United States of America vol 99 no 7 pp 4632ndash4637 2002

[88] A J Kelly M J Sackett N Din E Quardokus and Y VBrun ldquoCell cycle-dependent transcriptional and proteolyticregulation of FtsZ in Caulobacterrdquo Genes and Development vol12 no 6 pp 880ndash893 1998

[89] M Wortinger M J Sackett and Y V Brun ldquoCtrA mediatesa DNA replication checkpoint that prevents cell division inCaulobacter crescentusrdquo EMBO Journal vol 19 no 17 pp 4503ndash4512 2000

[90] R E Muir T M OrsquoBrien and J W Gober ldquoThe Caulobactercrescentus flagellar gene fliX encodes a novel trans-acting fac-tor that couples flagellar assembly to transcriptionrdquo MolecularMicrobiology vol 39 no 6 pp 1623ndash1637 2001

[91] M Llewellyn R J Dutton J Easter D OrsquoDonnol and J WGober ldquoThe conserved flaF gene has a critical role in couplingflagellin translation and assembly in Caulobacter crescentusrdquoMolecular Microbiology vol 57 no 4 pp 1127ndash1142 2005

[92] S M Kim D H Lee and S H Choi ldquoEvidence that the Vibriovulnificus flagellar regulator FlhF is regulated by a quorumsensing master regulator SmcRrdquo Microbiology vol 158 part 8pp 2017ndash2025 2012

[93] F Martınez-Granero A Navazo E Barahona M Redondo-Nieto R Rivilla andMMartın ldquoTheGac-Rsm and SadB signaltransduction pathways converge on Algu to downregulatemotility in Pseudomonas fluorescensrdquo PLoS ONE vol 7 no 2Article ID e31765 2012

[94] C Reimmann M Beyeler A Latifi et al ldquoThe global activatorGacA of Pseudomonas aeruginosa PAO positively controls theproduction of the autoinducer N-butyryl-homoserine lactoneand the formation of the virulence factors pyocyanin cyanideand lipaserdquo Molecular Microbiology vol 24 no 2 pp 309ndash3191997

[95] J Dubern and G V Bloemberg ldquoInfluence of environmentalconditions on putisolvins I and II production in Pseudomonasputida strain PCL1445rdquo FEMSMicrobiology Letters vol 263 no2 pp 169ndash175 2006

[96] M G Surette M B Miller and B L Bassler ldquoQuorum sensingin Escherichia coli Salmonella typhimurium andVibrio harveyia new family of genes responsible for autoinducer productionrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 96 no 4 pp 1639ndash1644 1999

[97] B A Rader S R Campagna M F Semmelhack B L Basslerand K Guillemin ldquoThe quorum-sensing molecule autoinducer2 regulates motility and flagellar morphogenesis inHelicobacterpylorirdquo Journal of Bacteriology vol 189 no 17 pp 6109ndash61172007

[98] YHe J G Frye T P Strobaugh Jr andCChen ldquoAnalysis of AI-2LuxS-dependent transcription in Campylobacter jejuni strain81-176rdquo Foodborne Pathogens and Disease vol 5 no 4 pp 399ndash415 2008

[99] B Jeon K Itoh N Misawa and S Ryu ldquoEffects of quorumsensing on flaA transcription and autoagglutination in Campy-lobacter jejunirdquoMicrobiology and Immunology vol 47 no 11 pp833ndash839 2003

[100] N Dasgupta E P Ferrell K J Kanack S E H West andR Ramphal ldquofleQ the gene encoding the major flagellarregulator of Pseudomonas aeruginosa is 12059070 dependent and isdownregulated by Vfr a homolog of Escherichia coli cyclic AMPreceptor proteinrdquo Journal of Bacteriology vol 184 no 19 pp5240ndash5250 2002

[101] J Zhu M B Miller R E Vance M Dziejman B L Bassler andJ J Mekalanos ldquoQuorum-sensing regulators control virulencegene expression in Vibrio choleraerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no5 pp 3129ndash3134 2002

[102] Z Liu T Miyashiro A Tsou A Hsiao M Goulian and JZhu ldquoMucosal penetration primes Vibrio cholerae for hostcolonization by repressing quorum sensingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 28 pp 9769ndash9774 2008

[103] K E Klose V Novik and J J Mekalanos ldquoIdentificationof multiple 12059054-dependent transcriptional activators in Vibriocholeraerdquo Journal of Bacteriology vol 180 no 19 pp 5256ndash52591998

[104] I Kawagishi M Imagawa Y Imae L McCarter and MHomma ldquoThe sodium-driven polar flagellar motor of marineVibrio as the mechanosensor that regulates lateral flagellarexpressionrdquoMolecular Microbiology vol 20 no 4 pp 693ndash6991996

[105] T J Kirn B A Jude and R K Taylor ldquoA colonizationfactor links Vibrio cholerae environmental survival and humaninfectionrdquo Nature vol 438 no 7069 pp 863ndash866 2005

[106] Z Kuang YHao S Hwang et al ldquoThe Pseudomonas aeruginosaflagellum confers resistance to pulmonary surfactant protein-Aby impacting the production of exoproteases through quorum-sensingrdquo Molecular Microbiology vol 79 no 5 pp 1220ndash12352011

[107] S Zhang F X McCormack R C Levesque G A OrsquoTooleand G W Lau ldquoThe flagellum of Pseudomonas aeruginosa isrequired for resistance to clearance by surfactant protein ArdquoPLoS ONE vol 2 no 6 article e564 2007

[108] G W Lau D J Hassett H Ran and F Kong ldquoThe role ofpyocyanin in Pseudomonas aeruginosa infectionrdquo Trends inMolecular Medicine vol 10 no 12 pp 599ndash606 2004

[109] GW LauH Ran F Kong D J Hassett andDMavrodi ldquoPseu-domonas aeruginosa pyocyanin is critical for lung infection inmicerdquo Infection and Immunity vol 72 no 7 pp 4275ndash42782004

[110] D S Merrell M L Goodrich G Otto L S Tompkins and SFalkow ldquopH-regulated gene expression of the gastric pathogenHelicobacter pylorirdquo Infection and Immunity vol 71 no 6 pp3529ndash3539 2003

[111] K J Allen and M W Griffiths ldquoEffect of environmental andchemotactic stimuli on the activity of the Campylobacter jejuniflaA 12059028 promoterrdquo FEMS Microbiology Letters vol 205 no 1pp 43ndash48 2001

[112] Y Wen J Feng D R Scott E A Marcus and G SachsldquoThe pH-responsive regulon of HP0244 (FlgS) the cytoplasmichistidine kinase of Helicobacter pylorirdquo Journal of Bacteriologyvol 191 no 2 pp 449ndash460 2009

[113] D R Hendrixson ldquoA phase-variable mechanism controllingthe Campylobacter jejuni FlgR response regulator influencescommensalismrdquo Molecular Microbiology vol 61 no 6 pp1646ndash1659 2006

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

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International Journal of

Volume 2014

Zoology

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Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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BioinformaticsAdvances in

Marine BiologyJournal of

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Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Microbiology

6 Scientifica

[68] Nonmotile strains of P aeruginosamay have an advan-tage during chronic infection by evading stimulation of theinnate immune responses However a nonmotile flgKmutantis defective in surface attachment suggesting a role for flagellaandor motility in the initial cell-to-surface interaction [69]Mutants in fliM and cheY are also nonmotile and are unableto form biofilm cap structures on initial biofilm colonies [70]Other Pseudomonas species include Pseudomonas fluorescenswhich possesses multiple flagella and is found in the soil andwater Some P fluorescens strains have been shown to protectplant roots against fungal infections [71] P fluorescens doesnot typically cause disease in humans but it is an opportunis-tic pathogen in immunocompromised individuals [72 73]

Like the Epsilonproteobacteria and Vibrio P aeruginosapossesses a four-tiered transcriptional hierarchy for regulat-ing flagellar gene expression FleQ is the master regulatorfor flagellar synthesis and fleQ is regulated by global factorsoutside the flagellar regulons such as cyclic di-GMP asignaling molecule important in modulating the transitionbetween planktonic and biofilm lifestyles [74] The earlyflagellar genes are dependent on FleQ and RpoN for theirtranscription and encode components of the basal body aswell as the filament cap which is somewhat surprising sinceit is not needed until late in flagellar biogenesis [75] Theearly genes also encode regulatory proteins which includeflhF fleN fleS and fleR [75 76] FleN is an antiactivator ofFleQ that maintains the normal flagellum copy number ofone per cell by downregulating genes encoding early flagellarcomponents via a negative feedbackmechanism [77 78] FleSand FleR form a two-component system that is required forthe transcription of the middle RpoN-dependent genes [75]The signal(s) required for FleS activation is unknown Themiddle genes encode components of the basal body includingrod L ring hook hook cap and hook-filament junctionproteins [75] The late genes are FliA dependent and encodeFliC (flagellin) FleL (filament length control protein) FlgMFlgN (a protein required for initiation of filament assembly)and some chemotaxis proteins [75] Transcription of fliA isconstitutive [75] and FlgM (whose transcription is also FleQdependent but not RpoN dependent) represses the activityof FliA until completion of the hook-basal body structure atwhich point it is secreted from the cytoplasm via the exportapparatus which alleviates its inhibition of FliA

24 Caulobacter crescentus Caulobacter crescentus is anAlphaproteobacterium found in freshwater that is a modelorganism for cell cycle studies C crescentus divides asym-metrically to produce two cells with distinct morphologiesa swarmer cell and a stalked cell The nonmotile stalked cellcontains a polar stalk which secretes an exopolysaccharidethat facilitates adhesion to surfaces In addition the stalkedcell initiatesDNA replication upon the start of cell division Incontrast the swarmer cell is motile via a polar flagellum andDNA replication is repressed for a defined length of time untilthe cell differentiates into a stalked cell Flagellar biogenesis iscoordinated with the cell division cycle such that all progenyswarmer cells possess a functional flagellum

Flagellar genes inC crescentus are regulated by two sigmafactors The initiation of DNA replication in the swarmer cellresults in expression and activation of the global transcriptionfactor CtrA CtrA also acts as themaster regulator for flagellargene expression by activating transcription of the early genes(encode MS ring C ring and export apparatus FlbD FliX)Additionally CtrA controls expression of rpoD [79] and isneeded for transcription of the early flagellar genes [80]FlbD and FliX are transcriptional regulators that regulateexpression of the early and middle genes The middle andlate genes which encode the hook protein and flagellins aretranscribed only after the components encoded by the earlygenes have been assembled into the nascent flagellum FlbDis an RpoN-dependent transcriptional activator which bindsftr (flagella transcriptional regulation) sequence motifs toregulate gene expression Once the early genes are expressedFlbD binds the ftr sequence motifs in the promoter regionsof early flagellar genes to inhibit transcription of these genesFor example FlbD binds the ftr4 site located upstream ofthe early gene fliF to repress its transcription once the earlygenes have been expressed [81] In contrast FlbD stimulatestranscription of the middle genes by binding to the ftrsites of the middle and late genes [81 82] FlbD activity isregulated by FliX which acts to either inhibit or stimulateFlbD In the absence of early flagellar structures FliX inhibitsFlbD activity by binding FlbD preventing it from bindingftr sites [83] Once the early flagellar structures are formedFliX stimulates FlbD The mechanism for this stimulationis unknown but it may involve another factor such asan assembled component of the flagellum which convertsFliX into a positive regulator or it may involve covalentmodifications of FliX or FlbD [83] Although FlbD possessesan N-terminal response regulator domain FlbD activity doesnot appear to be regulated by phosphorylation via a sensorkinase The exact cellular cues that regulate FlbD are notknown but FlbD activity is linked to cell division [84]

Unlike the bacteria discussed thus far flagellar biogenesisin C crescentus does not involve FliA Rather the six flagellingenes found in C crescentus are RpoN dependent Theexpression of the flagellin genes is regulated by FlbD and FlbTwhich binds the 51015840 UTR of flagellin transcripts and repressestranslation until the hook-basal body structure is assembled[85] Though the mechanism for this temporal regulation isunknown one possibility is that FlbT represses translationof flagellin genes until a positive factor binds the 51015840 UTRregion of flagellins to promote translation upon assembly ofthe mature hook-basal body structure [85]

3 Coupling Flagellar Biosynthesis withOther Cellular Activities

Though flagellar biogenesis is a complex process in itselfit is further complicated by its coordination with othercellular processes Flagellar biosynthesis is timed with othercellular activities such as cell replication and quorum sensingEnvironmental stimuli also play a role in regulating flagellarbiogenesis as bacteria respond to changes in the environmentby altering behavior needed for survival Many of these

Scientifica 7

responses involve altering the state of flagellar biogenesiswithin these different contexts

31 Modulating Flagellar Gene Expression during the CellCycle Flagellar biosynthesis is linked to the cell cyclethrough various mechanisms and involves coordinating flag-ellar gene expression with growth phase and cell division InV cholerae chemotaxis and motility genes are upregulatedas the population enters stationary phase Of the 114 genesidentified with roles in chemotaxis and flagellar biogen-esis 72 genes were upregulated more than 2-fold duringstationary phase compared to mid-exponential phase [86]The upregulation of 60 of the 72 chemotaxis and flagellargenes is dependent on RpoS indicating a correlation betweenflagellar biogenesis and entry into stationary phase [86]The coordination between entry into stationary phase andflagellar biogenesis may aid the bacterium in exiting the hostwhen nutrients become depleted and prepare the bacteriumfor colonization of other niches or survival in environmentalreservoirs

C crescentus is a particularly good organism for studyinghow flagellar biogenesis is integrated with the cell cyclebecause of its two distinct cell morphologies The flagellarfilament is assembled just prior to cell division and allprogeny swarmer cells possess a fully functional flagellumThe synthesis of the flagellum is intimately linked to the cellcycle through regulatory proteins such as CtrA FlbD andFliX that are involved in both the cell cycle and flagellarbiogenesis This dual functionality may exist to ensure thateach swarmer cell is flagellated The master regulator forflagellar biogenesis CtrA regulates at least 95 genes [87]including the cell-division genes ftsZ [88] and ftsQA [89]Cells of a fliX mutant become filamentous as they enterlate log phase [90] demonstrating a role for this flagellarregulatory protein in cell division Flagellar biogenesis is alsolinked to the cell cycle via FlbT and FlaF FlaF is required forflagellar biogenesis and motility [91] FlbT protein levels areconstant throughout the cell cycle while FlaF levels correlateto flagellin levels both of which peak just prior to cell division[91] Since FlbT is a negative regulator for flagellin translationand peaks in flagellin levels correlate with FlaF levels thisobservation suggests that FlaF may temporally modulateFlbT activity [91]

Expression of Vibrio vulnificus FlhF which is needed forpolar localization of the flagellum is regulated by the quorumsensing master regulator SmcR flhF transcript levels in asmcRmutant are higher than wild-type levels indicating thatSmcR represses flhF expression [92] In wild-type cells flhFtranscript levels are highest during exponential phase anddecrease upon entry into stationary phase [92] In contrasttranscript levels of smcR increase as cultures enter stationaryphase suggesting that SmcR plays a major role for growthphase-dependent variation of flhF expression [92] Thisgrowth phase-dependent gene expression may be importantin host colonization as during the initial infection bacterialcell densities are low but at later stages of colonizationwhen bacterial cell densities are higher motility may not benecessary

Expression of flagellar genes is regulated in P fluorescensthroughout the growth phases by the Gac (GacAGasS) two-component system which limits flagellar biosynthesis duringexponential growth by downregulating transcription of fleQ(encodes the master regulator) [93] The Gac system posi-tively regulates production of virulence factors and quorumsensing molecules and is required for full virulence in animaland plant hosts [94] Levels of the Gac system componentsare regulated in response to growth phase as gacA and gacStranscript levels are highest at mid-exponential growth phase[95] The concomitant upregulation of virulence factors andthe downregulation of flagellar genes by the Gac system maybe a way to coordinate virulence and motility with growthphase so that virulence factors are expressed at high celldensities during host colonization and flagellar genes areturned off to facilitate attachment to the host

32 Connections between Quorum Sensing Host Colonizationand Flagellar Gene Expression In many pathogenic bacte-ria flagella aid in surface attachment and colonization Insuch cases successful colonization may require cooperationbetween quorum sensing and flagellar synthesis to integratesurface attachment and virulence gene expression at highcell densities in pathogens Surface colonization can leadto biofilm formation which is generally more resistant tohost defenses and antimicrobials Pathogens use differentmechanisms to couple expression of virulence genes withflagellar gene expression in the context of the host Thisincludes modulating quorum sensing and host colonizationbased on the status of the flagellum or modulating flagellarbiogenesis based on quorum sensing signals

Quorum sensing is used to regulate gene expression inresponse to cell densityThis is achieved through the secretionof a signaling molecule called an autoinducer (AI) AIs areproduced by bacteria and travel across the cell membrane andaccumulate in the environment Upon reaching a critical con-centration the AIs stimulate transcription of genes requiredfor group behavior luxS is responsible for the productionof AI-2 [96] and an H pylori mutant in luxS exhibitsdecreasedmotility compared towild type [97]flhA transcriptlevels are decreased in the luxS mutant but addition of 45-dihydroxy-23-pentanedione (functions as AI-2) into thegrowth medium restores flhA transcript levels to wild-typelevels [97] Like the H pylori luxS mutant a C jejuni luxSmutant is less motile than wild type [98] and the mutantdisplays reduced transcript levels of flaA [99] Using DNAmicroarrays He and coworkers found that a majority of theflagellar genes are downregulated in a C jejuni luxS mutant[98] Expression of chemotaxis genes however is unchangedin the luxS mutant indicating that the motility defect of theluxS mutant is due to defects in flagellar biogenesis and notchemotaxis [98] A rationale for the link between cell densityand flagellar biogenesis is that it may aid in colonization ofhost tissue by ensuring that a sufficient number of flagellatedbacteria are present to establish a successful infection

Regulatory proteins involved in flagellar biogenesis canregulate quorum sensing and colonization or they themselvescan be regulated by quorum sensing and colonization factorsFor example the RpoN-dependent activator FlrC not only

8 Scientifica

is required for flagellar biogenesis but also appears to play arole in host colonization by V cholerae Mutants expressinga FlrC variant incapable of being phosphorylated fail toproduce flagella and are therefore defective in colonization[54] Interestingly a mutant expressing a constitutively activeform of FlrC is also deficient in colonization even though thecells are motile and produce flagella [54] This observationdemonstrates the need for coordinating flagellar biogenesisand pathogenesis for successful colonization ConverselyVfr a major quorum sensing and virulence regulator inP aeruginosa downregulates expression of flagellar genesby repressing transcription from the fleQ promoter [100]Inhibition of flagellar biogenesis by Vfr may aid P aeruginosain persisting at the site of infection to achieve high celldensities and virulence gene expression

HapR is a transcriptional regulator involved in quorumsensing in V cholerae which also regulates expression ofvirulence genes HapR represses expression of several vir-ulence genes [101] and hapR expression is regulated by anunknown mechanism involving FliA [102] hapR expres-sion is depressed in flgM and flgD mutants (both of thesemutations alleviate FlgM repression on FliA activity) whiledeletion of fliA results in elevated hapR expression [102] Theauthors of this study speculated that expression of HapR isregulated by a regulator or small noncoding RNA (sRNA)that is part of the FliA regulon FliA-mediated regulation ofhapR appears to be linked to the shearing of flagella frombacteria that penetrate the mucin layer to infect the hostThis shearing of the flagella leads to secretion of FlgM viathe flagellar protein export apparatus and results in increasedexpression of FliA-dependent genes and repression of hapRexpression [102] The outcome of such a mechanism is thatvirulence genes which are negatively regulated by HapRare expressed specifically when V cholerae penetrates theintestinal mucous layer hapR production is also regulatedby the quorum sensing protein LuxO further linking viru-lence to cell density LuxO is an RpoN-dependent activator[103] which along with HapR regulates motility proteaseproduction and biofilm formation [101] High cell densitiesare common during late stages of infection and the HapR-mediated repression of virulence genes may aidV cholerae indetaching and finding new colonization sites [86 101]

V parahaemolyticus uses its polar flagellum to detectsurfaces by sensing impedance to the polar flagellum rotationrate [104] Once a surface is encountered expression of thelateral flagellar genes is induced Lateral flagella are importantfor host colonization as mutants that do not produce lateralflagella are deficient in adherence to HeLa cells and biofilmformation [61] Gode-Potratz and coworkers identified sim70genes that are surface responsive most of which are positivelyregulated [64] Some of the surface-regulated genes that areupregulated are virulence genes such as a putative GbpAhomolog and components of a type III secretion system [64]GbpA from V cholerae promotes adherence to chitinoussurfaces of zooplankton and human epithelia cells [105]which is consistent with the observed upregulation of gbpAin V parahaemolyticus when it encounters a surface

The P aeruginosa flagellum is an important determinantin the susceptibility of the bacterium to host defenses

P aeruginosa fliCmutants are unable to upregulate transcrip-tion of lasI and rhlI which encode enzymes that synthesizequorum sensing homoserine lactones [106] These quo-rum sensing molecules regulate production of exoproteaseswhich degrade surfactant protein-A (SP-A) an antimicro-bial that opsonizes and permeabilizes membranes of lungpathogens [106] In the absence of these exoproteases theP aeruginosa fliC mutants are cleared from the lung [106]Though it is not known why fliC mutants are unable toupregulate transcription of lasI and rhlI it is not due tothe inability to sense the environment [106] P aeruginosafliE flgE fliC and fliD mutants are also attenuated in LPSbiosynthesis which compromises the integrity of their outermembranes making them more susceptible to SP-A [107]Additionally flgE and fliC mutants make less pyocyanin aredox-active toxic secondary metabolite that is crucial forlung infection [108 109]

33 Reacting to pH Changes by Gastrointestinal PathogensGastrointestinal pathogens encounter varying environmentalconditions as they travel from the mouth to the stomach andthen to the intestines The gastric pathogen H pylori has theadded burden of long-term survival in the acidic environ-ment of the stomach Pathogens that colonize the intestinaltract must also survive passage through the stomach in highenough numbers to be able to colonize their target organsThus it is not surprising that gastrointestinal pathogens oftenmodulate gene expression in response to acidic conditions

Merrell and coworkers found that expression of approxi-mately 7 of theH pylori genes is altered by a shift to low pH[110] Many of these genes are involved in flagellar synthesiswith the majority of those being RpoN dependent [110] FliA-dependent genes such as flgM in H pylori [110] and flaAin C jejuni [111] are also upregulated at low pH conditionsThe upregulation of flagellar genes is critical for survival ofH pylori as the bacterium is not able to survive at low pHbut must swim across the mucous layer to the underlyinggastric epithelium to establish a successful infection Inaddition to enhancing transcription of flagellar genes acidicenvironments also stimulate motility and swimming speedspotentially in response to increased concentration of protonsin the stomach [110]

The basis for upregulation of the RpoN-dependent flag-ellar genes in H pylori likely involves a stimulation of FlgSactivity at low pHWen and coworkers reported thatH pylorirequires FlgS to survive acid stress (30-minute exposure topH25) [112]These researchers found that besides the knownRpoN-dependent flagellar genes FlgS was required for theupregulation of 86 genes in response to acid stress many ofwhich were previously known to be required for acid stresssurvival [112] It is not known if FlgS responds directly toacid stress or if the stress signal is mediated through anotherfactor

34 Altering Flagellar Biogenesis by Phase Variation In somebacteria flagellar biogenesis is subject to phase variationevents that affect expression of specific flagellar genes Phasevariation causes changes in phenotypes at frequencies that

Scientifica 9

are much higher than random mutations and contribute toheterogeneity within a population The ability of H pylori topersist in the host may involve the phase-variable dependentexpression of flagella that can serve as an advantage foradaptation to the host and for evading the host immuneresponse C jejuni utilizes phase variation to turn on andoff production of flagella a strategy that increases com-mensal colonization in poultry [113] Many phase variationevents occur by random reversible changes in the length ofshort DNA sequence repeats resulting from slipped-strandmispairing These events can take place within the codingsequence or in the promoter of a gene C jejuni phasevariants in which expression of flgR is in the OFF stateresult from the addition or removal of a nucleotide withinone of two homopolymeric nucleotide tracts consisting ofadenine and thymine These nucleotide tracts are locatedwithin the coding sequence of flgR causing the sequence toshift out of frame [113] Revertants to the ON state ariseupon the addition or removal of a nucleotide within theoriginal mutated homopolymeric nucleotide tract to restorethe wild-type sequence Alternatively pseudorevertants canarise in which nucleotides are removed or added close to theoriginal mutated homopolymeric nucleotide tract to restorethe correct reading frame but which result in changes in theamino acid sequence of the protein [113] flgS is also regulatedby phase variation in C jejuni providing multiple levels ofphase-variable control for theRpoN regulon [114 115] Similarmechanisms of phase variation also exist for flhA in C coli[116] and fliP in H pylori [117] which leads to the formationof truncated proteins that prevent flagellar biogenesis

A second type of phase variation in flagellar biogen-esis is mediated through alterations in DNA methylationwhich impact the expression of specific flagellar genesHost-adapted bacterial pathogens such as H pylori oftenpossess DNA methyltransferases and type III restriction-modification (R-M) systems that contain simple tandemDNA repeats which undergo slipped-strand mispairing dur-ing DNA replication that causes frameshift mutations andphase variation [118] R-M systems widespread among manybacteria confer protection from invasion by foreign DNAType III R-M systems are composed of a methyltransferase(mod) and an endonuclease (res) gene whose gene prod-ucts typically function together The switching between ONand OFF states of a DNA methyltransferase can regulateexpression of a ldquophasevarionrdquo via differential methylationof the genome in ON and OFF states [119] Many of thesephase-variable methyltransferase genes are associated withan inactive res gene resulting from frameshift mutationor are orphans that are not associated with a res genesuggesting that DNA restriction is not the primary roleof these methyltransferases (reviewed by Fox et al [120])For example H pylori modH encodes a phase-variableDNA methyltransferase and the cognate res gene containsa nonsense mutation frameshift mutation or is absent invarious H pylori strains [119] modH OFF strains havedecreased expression of flaA and fliK compared to themodHON strain [119] FlaA has a low ability to activate innateimmunity via the Toll-like receptor 5 [121] and modulatingexpression of the flagellin may be advantageous in evading

the host response Another H pylori methyltransferase withan inactive res gene is hpyAVIBM [122 123] hpyAVIBMencodes a C5 cytosine methyltransferase and contains AGrepeats in its open reading frame making it susceptible tophase variation via frame shift mutations [124] For instancehpyAVIBM from strain 26695 and hpyAVIBM from strainHPAG1 have five AG repeats whereas the four AG repeats instrain San 74 result in the translation of a truncated protein[124] Deletion of hpyAVIBM results in both the upregulationand downregulation of rpoN fliR fliD fliS motA fliK andflgK depending on the strain used [124] indicating that phasevariation of hpyAVIBM can alter flagellar biogenesis

P aeruginosa populations can possess two phenotypicvariants one which forms small rough colonies (S) andanother which forms large flat (L) colonies S variants formbiofilms in nonagitated liquid cultures whereas the L variantsdo not [125] S variants possess defects in flagellum-mediatedswimming flagellum-mediated swarming and type IV-pilus-mediated twitching but are able to revert back to the Lphenotype at relatively high frequencies suggesting that theshift between phenotypes is regulated by phase variation[125] Phase variation also occurs in P fluorescens whichcolonizes the alfalfa rhizosphere Phenotypic variants ariseduring rhizosphere colonization and are dependent on theactivity of a site-specific recombinase [126] C variants havewild-type colony morphology while F and S variants havea translucent and diffuse colony morphology [126] F andS variants which colonize distal parts of the roots andswim faster than the C variants overproduce fliC (flagellin)transcripts and synthesize flagellin filaments that aresim3 timeslonger than those of the C variant [126]

4 Conclusions and Future Directions

Flagellar biogenesis is a carefully choreographed processin which many different components of the flagellum aremade as they are needed for assembly into the nascentflagellum Bacteria have evolved elaborate and sundry reg-ulatory networks to couple flagellar gene expression withassembly A driving force for the evolution of such regulatorynetworks is undoubtedly the environmental and ecologicalconstraints imposed by a particular bacteriumrsquos niche Inaddition the degree to which a given bacterial species needsto couple flagellar biogenesis to other cellular processes suchas cell division has also likely shaped the evolution of theseregulatory networks We focused here on bacteria whichutilize RpoN for flagellar biogenesis yet even among thesebacteria the mechanisms used to regulate expression of theRpoN-dependent flagellar genes vary greatly For most of thesystems described in this review we have only scratched thesurface in our understanding of how they operate For each ofthese systems fundamental questions remain to be answeredsuch as the following are all of the flagellar genes of a givensystem expressed as part of a transcription hierarchy or aresome genes expressed constitutively Are there regulatorymechanisms within RpoN flagellar regulons that fine-tunegene expression so that components of the flagellum aremadeprecisely when needed What are the cellular cues sensed by

10 Scientifica

the regulatory proteins that control expression of the RpoN-dependent flagellar genes How does the flagellar exportapparatus control expression of RpoN-dependent flagellargenes in various bacteria Does FlgZ have a regulatoryrole in flagellar biogenesis in H pylori and other bacteriaAs our understanding of the molecular genetics of diversebacterial species expands novel regulatory mechanisms thatcontrol expression of flagellar genes within these bacteria willundoubtedly come to light For example it was only recentlythat sRNAs were identified in H pylori some of whichare potentially FliA dependent [127] and may have roles inflagellar biogenesis Finally with increasingly sophisticatedmethods in bioinformatics genomics and proteomics fur-ther characterization of flagellar regulatory networks willprovide important insights into the mechanisms governinggene expression and identify new regulatory proteins andmechanisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by award MCB-1244242 from theNational Science Foundation

References

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[2] J L McMurry J W Murphy and B Gonzalez-Pedrajo ldquoTheFliN-FliH interaction mediates localization of flagellar exportATPase FliI to the C ring complexrdquo Biochemistry vol 45 no39 pp 11790ndash11798 2006

[3] R M Macnab Ed Flagella and Motility ASM Press 1996[4] T Minamino K Imada and K Namba ldquoMolecular motors of

the bacterial flagellardquoCurrent Opinion in Structural Biology vol18 no 6 pp 693ndash701 2008

[5] T G Smith and T R Hoover ldquoDeciphering bacterial flagellargene regulatory networks in the genomic erardquo Advances inApplied Microbiology vol 67 pp 257ndash295 2009

[6] J K Anderson T G Smith and T R Hoover ldquoSense andsensibility flagellum-mediated gene regulationrdquo Trends inMicrobiology vol 18 no 1 pp 30ndash37 2010

[7] J D Helmann ldquoAlternative sigma factors and the regulation offlagellar gene expressionrdquoMolecular Microbiology vol 5 no 12pp 2875ndash2882 1991

[8] D L Popham D Szeto J Keener and S Kustu ldquoFunctionof a bacterial activator protein that binds to transcriptionalenhancersrdquo Science vol 243 no 4891 pp 629ndash635 1989

[9] S Sasse-Dwight and J D Gralla ldquoProbing the EscherichiacoliglnALG upstream activation mechanism in vivordquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8934ndash8938 1988

[10] M Kihara T Minamino S Yamaguchi and R M MacnabldquoIntergenic suppression between the flagellar MS ring protein

FliF of Salmonella and FlhA a membrane component of itsexport apparatusrdquo Journal of Bacteriology vol 183 no 5 pp1655ndash1662 2001

[11] J S van Arnam J L McMurry M Kihara and R M MacnabldquoAnalysis of an engineered Salmonella flagellar fusion proteinFliR-FlhBrdquo Journal of Bacteriology vol 186 no 8 pp 2495ndash2498 2004

[12] C S Barker I V Meshcheryakova A S Kostyukova and FA Samatey ldquoFliO regulation of FliP in the formation of theSalmonella enterica flagellumrdquo PLoS Genetics vol 6 no 9Article ID e1001143 2010

[13] A C Lowenthal M Hill L K Sycuro K Mehmood N RSalama and K M Ottemann ldquoFunctional analysis of the Heli-cobacter pylori flagellar switch proteinsrdquo Journal of Bacteriologyvol 191 no 23 pp 7147ndash7156 2009

[14] R M Macnab ldquoType III flagellar protein export and flagellarassemblyrdquo Biochimica et Biophysica Acta vol 1694 no 1ndash3 pp207ndash217 2004

[15] K A Eaton D R Morgan and S Krakowka ldquoCampylobacterpylori virulence factors in gnotobiotic pigletsrdquo Infection andImmunity vol 57 no 4 pp 1119ndash1125 1989

[16] K A Eaton D R Morgan and S Krakowka ldquoMotility as afactor in the colonisation of gnotobiotic piglets by Helicobacterpylorirdquo Journal of Medical Microbiology vol 37 no 2 pp 123ndash127 1992

[17] D G Newell H McBride and J M Dolby ldquoInvestigationson the role of flagella in the colonization of infant mice withCampylobacter jejuni and attachment of Campylobacter jejunito human epithelial cell linesrdquo Journal of Hygiene vol 95 no 2pp 217ndash227 1985

[18] TMorooka A Umeda andK Amako ldquoMotility as an intestinalcolonization factor forCampylobacter jejunirdquo Journal of GeneralMicrobiology vol 131 no 8 pp 1973ndash1980 1985

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[23] A Kusumoto K Kamisaka T Yakushi H Terashima AShinohara and M Homma ldquoRegulation of polar flagellarnumber by the flhF and flhG genes in Vibrio alginolyticusrdquoJournal of Biochemistry vol 139 no 1 pp 113ndash121 2006

[24] B M Pruszlig and P Matsumura ldquoA regulator of the flagellarregulon of Escherichia coli flhD also affects cell divisionrdquoJournal of Bacteriology vol 178 no 3 pp 668ndash674 1996

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Scientifica 11

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[28] D Beier and R Frank ldquoMolecular characterization of two-component systems of Helicobacter pylorirdquo Journal of Bacteri-ology vol 182 no 8 pp 2068ndash2076 2000

[29] P Brahmachary M G Dashti J W Olson and T R HooverldquoHelicobacter pylori FlgR is an enhancer-independent activatorof 12059054-RNA polymerase holoenzymerdquo Journal of Bacteriologyvol 186 no 14 pp 4535ndash4542 2004

[30] M M S M Wosten J A Wagenaar and J P M van PuttenldquoThe FlgSFlgR two-component signal transduction systemregulates the fla regulon in Campylobacter jejunirdquo The Journalof Biological Chemistry vol 279 no 16 pp 16214ndash16222 2004

[31] M Bush and R Dixon ldquoThe role of bacterial enhancer bindingproteins as specialized activators of 12059054-dependent transcrip-tionrdquo MicroBiology and Molecular Biology Reviews vol 76 no3 pp 497ndash529 2012

[32] J Schumacher N Joly M Rappas X Zhang and M BuckldquoStructures and organisation of AAA+ enhancer binding pro-teins in transcriptional activationrdquo Journal of Structural Biologyvol 156 no 1 pp 190ndash199 2006

[33] S N Joslin and D R Hendrixson ldquoAnalysis of the Campy-lobacter jejuni FlgR response regulator suggests integration ofdiverse mechanisms to activate an NtrC-like proteinrdquo Journalof Bacteriology vol 190 no 7 pp 2422ndash2433 2008

[34] J M Boll and D R Hendrixson ldquoA specificity determinant forphosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphaterdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 50 pp 20160ndash20165 2011

[35] S Porwollik B Noonan and P W OrsquoToole ldquoMolecular char-acterization of a flagellar export locus of Helicobacter pylorirdquoInfection and Immunity vol 67 no 5 pp 2060ndash2070 1999

[36] E Allan N Dorrell S Foynes M Anyim and B W WrenldquoMutational analysis of genes encoding the early flagellarcomponents of Helicobacter pylori evidence for transcriptionalregulation of flagellin A biosynthesisrdquo Journal of Bacteriologyvol 182 no 18 pp 5274ndash5277 2000

[37] T G Smith L Pereira and T R Hoover ldquoHelicobacter pyloriFlhB processing-deficient variants affect flagellar assembly butnot flagellar gene expressionrdquo Microbiology vol 155 no 4 pp1170ndash1180 2009

[38] D R Hendrixson B J Akerley and V J DiRita ldquoTranspo-son mutagenesis of Campylobacter jejuni identifies a bipartiteenergy taxis system required for motilityrdquoMolecular Microbiol-ogy vol 40 no 1 pp 214ndash224 2001

[39] J Tsang T G Smith L E Pereira and T R Hoover ldquoInsertionmutations inHelicobacter pylori flhA reveal strain differences inRpoN-dependent gene expressionrdquo Microbiology vol 159 part1 pp 58ndash67 2013

[40] J M Boll and D R Hendrixson ldquoA regulatory checkpointduring flagellar biogenesis in Campylobacter jejuni initiatessignal transduction to activate transcription of flagellar genesrdquoMBio vol 4 no 5 Article ID e00432-13 2013

[41] T Minamino H Doi and K Kutsukake ldquoSubstrate specificityswitching of the flagellum-specific export apparatus during flag-ellar morphogenesis in Salmonella typhimuriumrdquo Bioscience

Biotechnology and Biochemistry vol 63 no 7 pp 1301ndash13031999

[42] M Erhardt H M Singer D H Wee J P Keener and K THughes ldquoAn infrequent molecular ruler controls flagellar hooklength in Salmonella entericardquo EMBO Journal vol 30 no 14 pp2948ndash2961 2011

[43] K T Hughes K L Gillen M J Semon and J E KarlinseyldquoSensing structural intermediates in bacterial flagellar assemblyby export of a negative regulatorrdquo Science vol 262 no 5137 pp1277ndash1280 1993

[44] K A Ryan N Karim M Worku S A Moore C W Pennand P W OrsquoToole ldquoHP0958 is an essential motility gene inHelicobacter pylorirdquo FEMS Microbiology Letters vol 248 no 1pp 47ndash55 2005

[45] L Pereira and T R Hoover ldquoStable accumulation of 12059054 inHelicobacter pylori requires the novel protein HP0958rdquo Journalof Bacteriology vol 187 no 13 pp 4463ndash4469 2005

[46] J C Rain L Selig H de Reuse et al ldquoThe protein-proteininteraction map of Helicobacter pylorirdquo Nature vol 409 no6817 pp 211ndash215 2001

[47] F P Douillard K A Ryan D L Caly et al ldquoPosttranscriptionalregulation of flagellin synthesis in Helicobacter pylori by theRpoN chaperone HP0958rdquo Journal of Bacteriology vol 190 no24 pp 7975ndash7984 2008

[48] C Josenhans E Niehus S Amersbach et al ldquoFunctionalcharacterization of the antagonistic flagellar late regulators FliAand FlgM ofHelicobacter pylori and their effects on theH pyloritranscriptomerdquoMolecular Microbiology vol 43 no 2 pp 307ndash322 2002

[49] M Rust S Borchert E Niehus et al ldquoThe Helicobacter pylorianti-sigma factor FlgM is predominantly cytoplasmic andcooperates with the flagellar basal body protein FlhArdquo Journalof Bacteriology vol 191 no 15 pp 4824ndash4834 2009

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[51] M M S M Wosten L van Dijk A K J Veenendaal MR de Zoete N M C Bleumink-Pluijm and J P M vanPutten ldquoTemperature-dependent FlgMFliA complex forma-tion regulates Campylobacter jejuni flagella lengthrdquo MolecularMicrobiology vol 75 no 6 pp 1577ndash1591 2010

[52] M G Prouty N E Correa and K E Klose ldquoThe novel 12059054- and12059028-dependent flagellar gene transcription hierarchy of Vibrio

choleraerdquo Molecular Microbiology vol 39 no 6 pp 1595ndash16092001

[53] D Srivastava M L Hsieh A Khataokar M B Neiditch and CM Waters ldquoCyclic di-GMP inhibits Vibrio choleraemotility byrepressing induction of transcription and inducing extracellularpolysaccharide productionrdquo Molecular Microbiology vol 90no 6 pp 1262ndash1276 2013

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12 Scientifica

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Scientifica 13

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[92] S M Kim D H Lee and S H Choi ldquoEvidence that the Vibriovulnificus flagellar regulator FlhF is regulated by a quorumsensing master regulator SmcRrdquo Microbiology vol 158 part 8pp 2017ndash2025 2012

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[97] B A Rader S R Campagna M F Semmelhack B L Basslerand K Guillemin ldquoThe quorum-sensing molecule autoinducer2 regulates motility and flagellar morphogenesis inHelicobacterpylorirdquo Journal of Bacteriology vol 189 no 17 pp 6109ndash61172007

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[99] B Jeon K Itoh N Misawa and S Ryu ldquoEffects of quorumsensing on flaA transcription and autoagglutination in Campy-lobacter jejunirdquoMicrobiology and Immunology vol 47 no 11 pp833ndash839 2003

[100] N Dasgupta E P Ferrell K J Kanack S E H West andR Ramphal ldquofleQ the gene encoding the major flagellarregulator of Pseudomonas aeruginosa is 12059070 dependent and isdownregulated by Vfr a homolog of Escherichia coli cyclic AMPreceptor proteinrdquo Journal of Bacteriology vol 184 no 19 pp5240ndash5250 2002

[101] J Zhu M B Miller R E Vance M Dziejman B L Bassler andJ J Mekalanos ldquoQuorum-sensing regulators control virulencegene expression in Vibrio choleraerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no5 pp 3129ndash3134 2002

[102] Z Liu T Miyashiro A Tsou A Hsiao M Goulian and JZhu ldquoMucosal penetration primes Vibrio cholerae for hostcolonization by repressing quorum sensingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 28 pp 9769ndash9774 2008

[103] K E Klose V Novik and J J Mekalanos ldquoIdentificationof multiple 12059054-dependent transcriptional activators in Vibriocholeraerdquo Journal of Bacteriology vol 180 no 19 pp 5256ndash52591998

[104] I Kawagishi M Imagawa Y Imae L McCarter and MHomma ldquoThe sodium-driven polar flagellar motor of marineVibrio as the mechanosensor that regulates lateral flagellarexpressionrdquoMolecular Microbiology vol 20 no 4 pp 693ndash6991996

[105] T J Kirn B A Jude and R K Taylor ldquoA colonizationfactor links Vibrio cholerae environmental survival and humaninfectionrdquo Nature vol 438 no 7069 pp 863ndash866 2005

[106] Z Kuang YHao S Hwang et al ldquoThe Pseudomonas aeruginosaflagellum confers resistance to pulmonary surfactant protein-Aby impacting the production of exoproteases through quorum-sensingrdquo Molecular Microbiology vol 79 no 5 pp 1220ndash12352011

[107] S Zhang F X McCormack R C Levesque G A OrsquoTooleand G W Lau ldquoThe flagellum of Pseudomonas aeruginosa isrequired for resistance to clearance by surfactant protein ArdquoPLoS ONE vol 2 no 6 article e564 2007

[108] G W Lau D J Hassett H Ran and F Kong ldquoThe role ofpyocyanin in Pseudomonas aeruginosa infectionrdquo Trends inMolecular Medicine vol 10 no 12 pp 599ndash606 2004

[109] GW LauH Ran F Kong D J Hassett andDMavrodi ldquoPseu-domonas aeruginosa pyocyanin is critical for lung infection inmicerdquo Infection and Immunity vol 72 no 7 pp 4275ndash42782004

[110] D S Merrell M L Goodrich G Otto L S Tompkins and SFalkow ldquopH-regulated gene expression of the gastric pathogenHelicobacter pylorirdquo Infection and Immunity vol 71 no 6 pp3529ndash3539 2003

[111] K J Allen and M W Griffiths ldquoEffect of environmental andchemotactic stimuli on the activity of the Campylobacter jejuniflaA 12059028 promoterrdquo FEMS Microbiology Letters vol 205 no 1pp 43ndash48 2001

[112] Y Wen J Feng D R Scott E A Marcus and G SachsldquoThe pH-responsive regulon of HP0244 (FlgS) the cytoplasmichistidine kinase of Helicobacter pylorirdquo Journal of Bacteriologyvol 191 no 2 pp 449ndash460 2009

[113] D R Hendrixson ldquoA phase-variable mechanism controllingthe Campylobacter jejuni FlgR response regulator influencescommensalismrdquo Molecular Microbiology vol 61 no 6 pp1646ndash1659 2006

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

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Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Signal TransductionJournal of

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Microbiology

Scientifica 7

responses involve altering the state of flagellar biogenesiswithin these different contexts

31 Modulating Flagellar Gene Expression during the CellCycle Flagellar biosynthesis is linked to the cell cyclethrough various mechanisms and involves coordinating flag-ellar gene expression with growth phase and cell division InV cholerae chemotaxis and motility genes are upregulatedas the population enters stationary phase Of the 114 genesidentified with roles in chemotaxis and flagellar biogen-esis 72 genes were upregulated more than 2-fold duringstationary phase compared to mid-exponential phase [86]The upregulation of 60 of the 72 chemotaxis and flagellargenes is dependent on RpoS indicating a correlation betweenflagellar biogenesis and entry into stationary phase [86]The coordination between entry into stationary phase andflagellar biogenesis may aid the bacterium in exiting the hostwhen nutrients become depleted and prepare the bacteriumfor colonization of other niches or survival in environmentalreservoirs

C crescentus is a particularly good organism for studyinghow flagellar biogenesis is integrated with the cell cyclebecause of its two distinct cell morphologies The flagellarfilament is assembled just prior to cell division and allprogeny swarmer cells possess a fully functional flagellumThe synthesis of the flagellum is intimately linked to the cellcycle through regulatory proteins such as CtrA FlbD andFliX that are involved in both the cell cycle and flagellarbiogenesis This dual functionality may exist to ensure thateach swarmer cell is flagellated The master regulator forflagellar biogenesis CtrA regulates at least 95 genes [87]including the cell-division genes ftsZ [88] and ftsQA [89]Cells of a fliX mutant become filamentous as they enterlate log phase [90] demonstrating a role for this flagellarregulatory protein in cell division Flagellar biogenesis is alsolinked to the cell cycle via FlbT and FlaF FlaF is required forflagellar biogenesis and motility [91] FlbT protein levels areconstant throughout the cell cycle while FlaF levels correlateto flagellin levels both of which peak just prior to cell division[91] Since FlbT is a negative regulator for flagellin translationand peaks in flagellin levels correlate with FlaF levels thisobservation suggests that FlaF may temporally modulateFlbT activity [91]

Expression of Vibrio vulnificus FlhF which is needed forpolar localization of the flagellum is regulated by the quorumsensing master regulator SmcR flhF transcript levels in asmcRmutant are higher than wild-type levels indicating thatSmcR represses flhF expression [92] In wild-type cells flhFtranscript levels are highest during exponential phase anddecrease upon entry into stationary phase [92] In contrasttranscript levels of smcR increase as cultures enter stationaryphase suggesting that SmcR plays a major role for growthphase-dependent variation of flhF expression [92] Thisgrowth phase-dependent gene expression may be importantin host colonization as during the initial infection bacterialcell densities are low but at later stages of colonizationwhen bacterial cell densities are higher motility may not benecessary

Expression of flagellar genes is regulated in P fluorescensthroughout the growth phases by the Gac (GacAGasS) two-component system which limits flagellar biosynthesis duringexponential growth by downregulating transcription of fleQ(encodes the master regulator) [93] The Gac system posi-tively regulates production of virulence factors and quorumsensing molecules and is required for full virulence in animaland plant hosts [94] Levels of the Gac system componentsare regulated in response to growth phase as gacA and gacStranscript levels are highest at mid-exponential growth phase[95] The concomitant upregulation of virulence factors andthe downregulation of flagellar genes by the Gac system maybe a way to coordinate virulence and motility with growthphase so that virulence factors are expressed at high celldensities during host colonization and flagellar genes areturned off to facilitate attachment to the host

32 Connections between Quorum Sensing Host Colonizationand Flagellar Gene Expression In many pathogenic bacte-ria flagella aid in surface attachment and colonization Insuch cases successful colonization may require cooperationbetween quorum sensing and flagellar synthesis to integratesurface attachment and virulence gene expression at highcell densities in pathogens Surface colonization can leadto biofilm formation which is generally more resistant tohost defenses and antimicrobials Pathogens use differentmechanisms to couple expression of virulence genes withflagellar gene expression in the context of the host Thisincludes modulating quorum sensing and host colonizationbased on the status of the flagellum or modulating flagellarbiogenesis based on quorum sensing signals

Quorum sensing is used to regulate gene expression inresponse to cell densityThis is achieved through the secretionof a signaling molecule called an autoinducer (AI) AIs areproduced by bacteria and travel across the cell membrane andaccumulate in the environment Upon reaching a critical con-centration the AIs stimulate transcription of genes requiredfor group behavior luxS is responsible for the productionof AI-2 [96] and an H pylori mutant in luxS exhibitsdecreasedmotility compared towild type [97]flhA transcriptlevels are decreased in the luxS mutant but addition of 45-dihydroxy-23-pentanedione (functions as AI-2) into thegrowth medium restores flhA transcript levels to wild-typelevels [97] Like the H pylori luxS mutant a C jejuni luxSmutant is less motile than wild type [98] and the mutantdisplays reduced transcript levels of flaA [99] Using DNAmicroarrays He and coworkers found that a majority of theflagellar genes are downregulated in a C jejuni luxS mutant[98] Expression of chemotaxis genes however is unchangedin the luxS mutant indicating that the motility defect of theluxS mutant is due to defects in flagellar biogenesis and notchemotaxis [98] A rationale for the link between cell densityand flagellar biogenesis is that it may aid in colonization ofhost tissue by ensuring that a sufficient number of flagellatedbacteria are present to establish a successful infection

Regulatory proteins involved in flagellar biogenesis canregulate quorum sensing and colonization or they themselvescan be regulated by quorum sensing and colonization factorsFor example the RpoN-dependent activator FlrC not only

8 Scientifica

is required for flagellar biogenesis but also appears to play arole in host colonization by V cholerae Mutants expressinga FlrC variant incapable of being phosphorylated fail toproduce flagella and are therefore defective in colonization[54] Interestingly a mutant expressing a constitutively activeform of FlrC is also deficient in colonization even though thecells are motile and produce flagella [54] This observationdemonstrates the need for coordinating flagellar biogenesisand pathogenesis for successful colonization ConverselyVfr a major quorum sensing and virulence regulator inP aeruginosa downregulates expression of flagellar genesby repressing transcription from the fleQ promoter [100]Inhibition of flagellar biogenesis by Vfr may aid P aeruginosain persisting at the site of infection to achieve high celldensities and virulence gene expression

HapR is a transcriptional regulator involved in quorumsensing in V cholerae which also regulates expression ofvirulence genes HapR represses expression of several vir-ulence genes [101] and hapR expression is regulated by anunknown mechanism involving FliA [102] hapR expres-sion is depressed in flgM and flgD mutants (both of thesemutations alleviate FlgM repression on FliA activity) whiledeletion of fliA results in elevated hapR expression [102] Theauthors of this study speculated that expression of HapR isregulated by a regulator or small noncoding RNA (sRNA)that is part of the FliA regulon FliA-mediated regulation ofhapR appears to be linked to the shearing of flagella frombacteria that penetrate the mucin layer to infect the hostThis shearing of the flagella leads to secretion of FlgM viathe flagellar protein export apparatus and results in increasedexpression of FliA-dependent genes and repression of hapRexpression [102] The outcome of such a mechanism is thatvirulence genes which are negatively regulated by HapRare expressed specifically when V cholerae penetrates theintestinal mucous layer hapR production is also regulatedby the quorum sensing protein LuxO further linking viru-lence to cell density LuxO is an RpoN-dependent activator[103] which along with HapR regulates motility proteaseproduction and biofilm formation [101] High cell densitiesare common during late stages of infection and the HapR-mediated repression of virulence genes may aidV cholerae indetaching and finding new colonization sites [86 101]

V parahaemolyticus uses its polar flagellum to detectsurfaces by sensing impedance to the polar flagellum rotationrate [104] Once a surface is encountered expression of thelateral flagellar genes is induced Lateral flagella are importantfor host colonization as mutants that do not produce lateralflagella are deficient in adherence to HeLa cells and biofilmformation [61] Gode-Potratz and coworkers identified sim70genes that are surface responsive most of which are positivelyregulated [64] Some of the surface-regulated genes that areupregulated are virulence genes such as a putative GbpAhomolog and components of a type III secretion system [64]GbpA from V cholerae promotes adherence to chitinoussurfaces of zooplankton and human epithelia cells [105]which is consistent with the observed upregulation of gbpAin V parahaemolyticus when it encounters a surface

The P aeruginosa flagellum is an important determinantin the susceptibility of the bacterium to host defenses

P aeruginosa fliCmutants are unable to upregulate transcrip-tion of lasI and rhlI which encode enzymes that synthesizequorum sensing homoserine lactones [106] These quo-rum sensing molecules regulate production of exoproteaseswhich degrade surfactant protein-A (SP-A) an antimicro-bial that opsonizes and permeabilizes membranes of lungpathogens [106] In the absence of these exoproteases theP aeruginosa fliC mutants are cleared from the lung [106]Though it is not known why fliC mutants are unable toupregulate transcription of lasI and rhlI it is not due tothe inability to sense the environment [106] P aeruginosafliE flgE fliC and fliD mutants are also attenuated in LPSbiosynthesis which compromises the integrity of their outermembranes making them more susceptible to SP-A [107]Additionally flgE and fliC mutants make less pyocyanin aredox-active toxic secondary metabolite that is crucial forlung infection [108 109]

33 Reacting to pH Changes by Gastrointestinal PathogensGastrointestinal pathogens encounter varying environmentalconditions as they travel from the mouth to the stomach andthen to the intestines The gastric pathogen H pylori has theadded burden of long-term survival in the acidic environ-ment of the stomach Pathogens that colonize the intestinaltract must also survive passage through the stomach in highenough numbers to be able to colonize their target organsThus it is not surprising that gastrointestinal pathogens oftenmodulate gene expression in response to acidic conditions

Merrell and coworkers found that expression of approxi-mately 7 of theH pylori genes is altered by a shift to low pH[110] Many of these genes are involved in flagellar synthesiswith the majority of those being RpoN dependent [110] FliA-dependent genes such as flgM in H pylori [110] and flaAin C jejuni [111] are also upregulated at low pH conditionsThe upregulation of flagellar genes is critical for survival ofH pylori as the bacterium is not able to survive at low pHbut must swim across the mucous layer to the underlyinggastric epithelium to establish a successful infection Inaddition to enhancing transcription of flagellar genes acidicenvironments also stimulate motility and swimming speedspotentially in response to increased concentration of protonsin the stomach [110]

The basis for upregulation of the RpoN-dependent flag-ellar genes in H pylori likely involves a stimulation of FlgSactivity at low pHWen and coworkers reported thatH pylorirequires FlgS to survive acid stress (30-minute exposure topH25) [112]These researchers found that besides the knownRpoN-dependent flagellar genes FlgS was required for theupregulation of 86 genes in response to acid stress many ofwhich were previously known to be required for acid stresssurvival [112] It is not known if FlgS responds directly toacid stress or if the stress signal is mediated through anotherfactor

34 Altering Flagellar Biogenesis by Phase Variation In somebacteria flagellar biogenesis is subject to phase variationevents that affect expression of specific flagellar genes Phasevariation causes changes in phenotypes at frequencies that

Scientifica 9

are much higher than random mutations and contribute toheterogeneity within a population The ability of H pylori topersist in the host may involve the phase-variable dependentexpression of flagella that can serve as an advantage foradaptation to the host and for evading the host immuneresponse C jejuni utilizes phase variation to turn on andoff production of flagella a strategy that increases com-mensal colonization in poultry [113] Many phase variationevents occur by random reversible changes in the length ofshort DNA sequence repeats resulting from slipped-strandmispairing These events can take place within the codingsequence or in the promoter of a gene C jejuni phasevariants in which expression of flgR is in the OFF stateresult from the addition or removal of a nucleotide withinone of two homopolymeric nucleotide tracts consisting ofadenine and thymine These nucleotide tracts are locatedwithin the coding sequence of flgR causing the sequence toshift out of frame [113] Revertants to the ON state ariseupon the addition or removal of a nucleotide within theoriginal mutated homopolymeric nucleotide tract to restorethe wild-type sequence Alternatively pseudorevertants canarise in which nucleotides are removed or added close to theoriginal mutated homopolymeric nucleotide tract to restorethe correct reading frame but which result in changes in theamino acid sequence of the protein [113] flgS is also regulatedby phase variation in C jejuni providing multiple levels ofphase-variable control for theRpoN regulon [114 115] Similarmechanisms of phase variation also exist for flhA in C coli[116] and fliP in H pylori [117] which leads to the formationof truncated proteins that prevent flagellar biogenesis

A second type of phase variation in flagellar biogen-esis is mediated through alterations in DNA methylationwhich impact the expression of specific flagellar genesHost-adapted bacterial pathogens such as H pylori oftenpossess DNA methyltransferases and type III restriction-modification (R-M) systems that contain simple tandemDNA repeats which undergo slipped-strand mispairing dur-ing DNA replication that causes frameshift mutations andphase variation [118] R-M systems widespread among manybacteria confer protection from invasion by foreign DNAType III R-M systems are composed of a methyltransferase(mod) and an endonuclease (res) gene whose gene prod-ucts typically function together The switching between ONand OFF states of a DNA methyltransferase can regulateexpression of a ldquophasevarionrdquo via differential methylationof the genome in ON and OFF states [119] Many of thesephase-variable methyltransferase genes are associated withan inactive res gene resulting from frameshift mutationor are orphans that are not associated with a res genesuggesting that DNA restriction is not the primary roleof these methyltransferases (reviewed by Fox et al [120])For example H pylori modH encodes a phase-variableDNA methyltransferase and the cognate res gene containsa nonsense mutation frameshift mutation or is absent invarious H pylori strains [119] modH OFF strains havedecreased expression of flaA and fliK compared to themodHON strain [119] FlaA has a low ability to activate innateimmunity via the Toll-like receptor 5 [121] and modulatingexpression of the flagellin may be advantageous in evading

the host response Another H pylori methyltransferase withan inactive res gene is hpyAVIBM [122 123] hpyAVIBMencodes a C5 cytosine methyltransferase and contains AGrepeats in its open reading frame making it susceptible tophase variation via frame shift mutations [124] For instancehpyAVIBM from strain 26695 and hpyAVIBM from strainHPAG1 have five AG repeats whereas the four AG repeats instrain San 74 result in the translation of a truncated protein[124] Deletion of hpyAVIBM results in both the upregulationand downregulation of rpoN fliR fliD fliS motA fliK andflgK depending on the strain used [124] indicating that phasevariation of hpyAVIBM can alter flagellar biogenesis

P aeruginosa populations can possess two phenotypicvariants one which forms small rough colonies (S) andanother which forms large flat (L) colonies S variants formbiofilms in nonagitated liquid cultures whereas the L variantsdo not [125] S variants possess defects in flagellum-mediatedswimming flagellum-mediated swarming and type IV-pilus-mediated twitching but are able to revert back to the Lphenotype at relatively high frequencies suggesting that theshift between phenotypes is regulated by phase variation[125] Phase variation also occurs in P fluorescens whichcolonizes the alfalfa rhizosphere Phenotypic variants ariseduring rhizosphere colonization and are dependent on theactivity of a site-specific recombinase [126] C variants havewild-type colony morphology while F and S variants havea translucent and diffuse colony morphology [126] F andS variants which colonize distal parts of the roots andswim faster than the C variants overproduce fliC (flagellin)transcripts and synthesize flagellin filaments that aresim3 timeslonger than those of the C variant [126]

4 Conclusions and Future Directions

Flagellar biogenesis is a carefully choreographed processin which many different components of the flagellum aremade as they are needed for assembly into the nascentflagellum Bacteria have evolved elaborate and sundry reg-ulatory networks to couple flagellar gene expression withassembly A driving force for the evolution of such regulatorynetworks is undoubtedly the environmental and ecologicalconstraints imposed by a particular bacteriumrsquos niche Inaddition the degree to which a given bacterial species needsto couple flagellar biogenesis to other cellular processes suchas cell division has also likely shaped the evolution of theseregulatory networks We focused here on bacteria whichutilize RpoN for flagellar biogenesis yet even among thesebacteria the mechanisms used to regulate expression of theRpoN-dependent flagellar genes vary greatly For most of thesystems described in this review we have only scratched thesurface in our understanding of how they operate For each ofthese systems fundamental questions remain to be answeredsuch as the following are all of the flagellar genes of a givensystem expressed as part of a transcription hierarchy or aresome genes expressed constitutively Are there regulatorymechanisms within RpoN flagellar regulons that fine-tunegene expression so that components of the flagellum aremadeprecisely when needed What are the cellular cues sensed by

10 Scientifica

the regulatory proteins that control expression of the RpoN-dependent flagellar genes How does the flagellar exportapparatus control expression of RpoN-dependent flagellargenes in various bacteria Does FlgZ have a regulatoryrole in flagellar biogenesis in H pylori and other bacteriaAs our understanding of the molecular genetics of diversebacterial species expands novel regulatory mechanisms thatcontrol expression of flagellar genes within these bacteria willundoubtedly come to light For example it was only recentlythat sRNAs were identified in H pylori some of whichare potentially FliA dependent [127] and may have roles inflagellar biogenesis Finally with increasingly sophisticatedmethods in bioinformatics genomics and proteomics fur-ther characterization of flagellar regulatory networks willprovide important insights into the mechanisms governinggene expression and identify new regulatory proteins andmechanisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by award MCB-1244242 from theNational Science Foundation

References

[1] T Minamino K Imada and K Namba ldquoMechanisms of typeIII protein export for bacterial flagellar assemblyrdquo MolecularBioSystems vol 4 no 11 pp 1105ndash1115 2008

[2] J L McMurry J W Murphy and B Gonzalez-Pedrajo ldquoTheFliN-FliH interaction mediates localization of flagellar exportATPase FliI to the C ring complexrdquo Biochemistry vol 45 no39 pp 11790ndash11798 2006

[3] R M Macnab Ed Flagella and Motility ASM Press 1996[4] T Minamino K Imada and K Namba ldquoMolecular motors of

the bacterial flagellardquoCurrent Opinion in Structural Biology vol18 no 6 pp 693ndash701 2008

[5] T G Smith and T R Hoover ldquoDeciphering bacterial flagellargene regulatory networks in the genomic erardquo Advances inApplied Microbiology vol 67 pp 257ndash295 2009

[6] J K Anderson T G Smith and T R Hoover ldquoSense andsensibility flagellum-mediated gene regulationrdquo Trends inMicrobiology vol 18 no 1 pp 30ndash37 2010

[7] J D Helmann ldquoAlternative sigma factors and the regulation offlagellar gene expressionrdquoMolecular Microbiology vol 5 no 12pp 2875ndash2882 1991

[8] D L Popham D Szeto J Keener and S Kustu ldquoFunctionof a bacterial activator protein that binds to transcriptionalenhancersrdquo Science vol 243 no 4891 pp 629ndash635 1989

[9] S Sasse-Dwight and J D Gralla ldquoProbing the EscherichiacoliglnALG upstream activation mechanism in vivordquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8934ndash8938 1988

[10] M Kihara T Minamino S Yamaguchi and R M MacnabldquoIntergenic suppression between the flagellar MS ring protein

FliF of Salmonella and FlhA a membrane component of itsexport apparatusrdquo Journal of Bacteriology vol 183 no 5 pp1655ndash1662 2001

[11] J S van Arnam J L McMurry M Kihara and R M MacnabldquoAnalysis of an engineered Salmonella flagellar fusion proteinFliR-FlhBrdquo Journal of Bacteriology vol 186 no 8 pp 2495ndash2498 2004

[12] C S Barker I V Meshcheryakova A S Kostyukova and FA Samatey ldquoFliO regulation of FliP in the formation of theSalmonella enterica flagellumrdquo PLoS Genetics vol 6 no 9Article ID e1001143 2010

[13] A C Lowenthal M Hill L K Sycuro K Mehmood N RSalama and K M Ottemann ldquoFunctional analysis of the Heli-cobacter pylori flagellar switch proteinsrdquo Journal of Bacteriologyvol 191 no 23 pp 7147ndash7156 2009

[14] R M Macnab ldquoType III flagellar protein export and flagellarassemblyrdquo Biochimica et Biophysica Acta vol 1694 no 1ndash3 pp207ndash217 2004

[15] K A Eaton D R Morgan and S Krakowka ldquoCampylobacterpylori virulence factors in gnotobiotic pigletsrdquo Infection andImmunity vol 57 no 4 pp 1119ndash1125 1989

[16] K A Eaton D R Morgan and S Krakowka ldquoMotility as afactor in the colonisation of gnotobiotic piglets by Helicobacterpylorirdquo Journal of Medical Microbiology vol 37 no 2 pp 123ndash127 1992

[17] D G Newell H McBride and J M Dolby ldquoInvestigationson the role of flagella in the colonization of infant mice withCampylobacter jejuni and attachment of Campylobacter jejunito human epithelial cell linesrdquo Journal of Hygiene vol 95 no 2pp 217ndash227 1985

[18] TMorooka A Umeda andK Amako ldquoMotility as an intestinalcolonization factor forCampylobacter jejunirdquo Journal of GeneralMicrobiology vol 131 no 8 pp 1973ndash1980 1985

[19] C Y Kao B S Sheu SM Sheu et al ldquoHighermotility enhancesbacterial density and inflammatory response in dyspepticpatients infected with Helicobacter pylorirdquo Helicobacter vol 17no 6 pp 411ndash416 2012

[20] E Niehus H Gressmann F Ye et al ldquoGenome-wide analysisof transcriptional hierarchy and feedback regulation in theflagellar system of Helicobacter pylorirdquoMolecular Microbiologyvol 52 no 4 pp 947ndash961 2004

[21] M Balaban S N Joslin and D R Hendrixson ldquoFlhF and itsGTPase activity are required for distinct processes in flagellargene regulation and biosynthesis in Campylobacter jejunirdquoJournal of Bacteriology vol 191 no 21 pp 6602ndash6611 2009

[22] A Kusumoto A Shinohara H Terashima S Kojima TYakushi and M Homma ldquoCollaboration of FlhF and FlhGto regulate polarflagella number and localization in VibrioalginolyticusrdquoMicrobiology vol 154 no 5 pp 1390ndash1399 2008

[23] A Kusumoto K Kamisaka T Yakushi H Terashima AShinohara and M Homma ldquoRegulation of polar flagellarnumber by the flhF and flhG genes in Vibrio alginolyticusrdquoJournal of Biochemistry vol 139 no 1 pp 113ndash121 2006

[24] B M Pruszlig and P Matsumura ldquoA regulator of the flagellarregulon of Escherichia coli flhD also affects cell divisionrdquoJournal of Bacteriology vol 178 no 3 pp 668ndash674 1996

[25] B M Pruszlig and P Matsumura ldquoCell cycle regulation of flagellargenesrdquo Journal of Bacteriology vol 179 no 17 pp 5602ndash56041997

[26] G Spohn and V Scarlato ldquoMotility of Helicobacter pylori iscoordinately regulated by the transcriptional activator FlgR an

Scientifica 11

NtrC homologrdquo Journal of Bacteriology vol 181 no 2 pp 593ndash599 1999

[27] M Schirm E C Soo A J Aubry J Austin P Thibault and SM Logan ldquoStructural genetic and functional characterizationof the flagellin glycosylation process in Helicobacter pylorirdquoMolecular Microbiology vol 48 no 6 pp 1579ndash1592 2003

[28] D Beier and R Frank ldquoMolecular characterization of two-component systems of Helicobacter pylorirdquo Journal of Bacteri-ology vol 182 no 8 pp 2068ndash2076 2000

[29] P Brahmachary M G Dashti J W Olson and T R HooverldquoHelicobacter pylori FlgR is an enhancer-independent activatorof 12059054-RNA polymerase holoenzymerdquo Journal of Bacteriologyvol 186 no 14 pp 4535ndash4542 2004

[30] M M S M Wosten J A Wagenaar and J P M van PuttenldquoThe FlgSFlgR two-component signal transduction systemregulates the fla regulon in Campylobacter jejunirdquo The Journalof Biological Chemistry vol 279 no 16 pp 16214ndash16222 2004

[31] M Bush and R Dixon ldquoThe role of bacterial enhancer bindingproteins as specialized activators of 12059054-dependent transcrip-tionrdquo MicroBiology and Molecular Biology Reviews vol 76 no3 pp 497ndash529 2012

[32] J Schumacher N Joly M Rappas X Zhang and M BuckldquoStructures and organisation of AAA+ enhancer binding pro-teins in transcriptional activationrdquo Journal of Structural Biologyvol 156 no 1 pp 190ndash199 2006

[33] S N Joslin and D R Hendrixson ldquoAnalysis of the Campy-lobacter jejuni FlgR response regulator suggests integration ofdiverse mechanisms to activate an NtrC-like proteinrdquo Journalof Bacteriology vol 190 no 7 pp 2422ndash2433 2008

[34] J M Boll and D R Hendrixson ldquoA specificity determinant forphosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphaterdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 50 pp 20160ndash20165 2011

[35] S Porwollik B Noonan and P W OrsquoToole ldquoMolecular char-acterization of a flagellar export locus of Helicobacter pylorirdquoInfection and Immunity vol 67 no 5 pp 2060ndash2070 1999

[36] E Allan N Dorrell S Foynes M Anyim and B W WrenldquoMutational analysis of genes encoding the early flagellarcomponents of Helicobacter pylori evidence for transcriptionalregulation of flagellin A biosynthesisrdquo Journal of Bacteriologyvol 182 no 18 pp 5274ndash5277 2000

[37] T G Smith L Pereira and T R Hoover ldquoHelicobacter pyloriFlhB processing-deficient variants affect flagellar assembly butnot flagellar gene expressionrdquo Microbiology vol 155 no 4 pp1170ndash1180 2009

[38] D R Hendrixson B J Akerley and V J DiRita ldquoTranspo-son mutagenesis of Campylobacter jejuni identifies a bipartiteenergy taxis system required for motilityrdquoMolecular Microbiol-ogy vol 40 no 1 pp 214ndash224 2001

[39] J Tsang T G Smith L E Pereira and T R Hoover ldquoInsertionmutations inHelicobacter pylori flhA reveal strain differences inRpoN-dependent gene expressionrdquo Microbiology vol 159 part1 pp 58ndash67 2013

[40] J M Boll and D R Hendrixson ldquoA regulatory checkpointduring flagellar biogenesis in Campylobacter jejuni initiatessignal transduction to activate transcription of flagellar genesrdquoMBio vol 4 no 5 Article ID e00432-13 2013

[41] T Minamino H Doi and K Kutsukake ldquoSubstrate specificityswitching of the flagellum-specific export apparatus during flag-ellar morphogenesis in Salmonella typhimuriumrdquo Bioscience

Biotechnology and Biochemistry vol 63 no 7 pp 1301ndash13031999

[42] M Erhardt H M Singer D H Wee J P Keener and K THughes ldquoAn infrequent molecular ruler controls flagellar hooklength in Salmonella entericardquo EMBO Journal vol 30 no 14 pp2948ndash2961 2011

[43] K T Hughes K L Gillen M J Semon and J E KarlinseyldquoSensing structural intermediates in bacterial flagellar assemblyby export of a negative regulatorrdquo Science vol 262 no 5137 pp1277ndash1280 1993

[44] K A Ryan N Karim M Worku S A Moore C W Pennand P W OrsquoToole ldquoHP0958 is an essential motility gene inHelicobacter pylorirdquo FEMS Microbiology Letters vol 248 no 1pp 47ndash55 2005

[45] L Pereira and T R Hoover ldquoStable accumulation of 12059054 inHelicobacter pylori requires the novel protein HP0958rdquo Journalof Bacteriology vol 187 no 13 pp 4463ndash4469 2005

[46] J C Rain L Selig H de Reuse et al ldquoThe protein-proteininteraction map of Helicobacter pylorirdquo Nature vol 409 no6817 pp 211ndash215 2001

[47] F P Douillard K A Ryan D L Caly et al ldquoPosttranscriptionalregulation of flagellin synthesis in Helicobacter pylori by theRpoN chaperone HP0958rdquo Journal of Bacteriology vol 190 no24 pp 7975ndash7984 2008

[48] C Josenhans E Niehus S Amersbach et al ldquoFunctionalcharacterization of the antagonistic flagellar late regulators FliAand FlgM ofHelicobacter pylori and their effects on theH pyloritranscriptomerdquoMolecular Microbiology vol 43 no 2 pp 307ndash322 2002

[49] M Rust S Borchert E Niehus et al ldquoThe Helicobacter pylorianti-sigma factor FlgM is predominantly cytoplasmic andcooperates with the flagellar basal body protein FlhArdquo Journalof Bacteriology vol 191 no 15 pp 4824ndash4834 2009

[50] D R Hendrixson and V J DiRita ldquoTranscription of 12059054-dependent but not 12059028-dependent flagellar genes in Campy-lobacter jejuni is associated with formation of the flagellarsecretory apparatusrdquoMolecular Microbiology vol 50 no 2 pp687ndash702 2003

[51] M M S M Wosten L van Dijk A K J Veenendaal MR de Zoete N M C Bleumink-Pluijm and J P M vanPutten ldquoTemperature-dependent FlgMFliA complex forma-tion regulates Campylobacter jejuni flagella lengthrdquo MolecularMicrobiology vol 75 no 6 pp 1577ndash1591 2010

[52] M G Prouty N E Correa and K E Klose ldquoThe novel 12059054- and12059028-dependent flagellar gene transcription hierarchy of Vibrio

choleraerdquo Molecular Microbiology vol 39 no 6 pp 1595ndash16092001

[53] D Srivastava M L Hsieh A Khataokar M B Neiditch and CM Waters ldquoCyclic di-GMP inhibits Vibrio choleraemotility byrepressing induction of transcription and inducing extracellularpolysaccharide productionrdquo Molecular Microbiology vol 90no 6 pp 1262ndash1276 2013

[54] N E Correa C M Lauriano R McGee and K E KloseldquoPhosphorylation of the flagellar regulatory protein FlrC is nec-essary for Vibrio cholerae motility and enhanced colonizationrdquoMolecular Microbiology vol 35 no 4 pp 743ndash755 2000

[55] N E Correa J R Barker and K E Klose ldquoThe Vibrio choleraeFlgM homologue is an anti-12059028 factor that is secreted throughthe sheathed polar flagellumrdquo Journal of Bacteriology vol 186no 14 pp 4613ndash4619 2004

12 Scientifica

[56] MMoisi C Jenul SM Butler et al ldquoA novel regulatory proteininvolved in motility of Vibrio choleraerdquo Journal of Bacteriologyvol 191 no 22 pp 7027ndash7038 2009

[57] L Aravind and C P Ponting ldquoThe cytoplasmic helical linkerdomain of receptor histidine kinase and methyl-acceptingproteins is common to many prokaryotic signalling proteinsrdquoFEMS Microbiology Letters vol 176 no 1 pp 111ndash116 1999

[58] SMerino J G Shaw and JM Tomas ldquoBacterial lateral flagellaan inducible flagella systemrdquo FEMS Microbiology Letters vol263 no 2 pp 127ndash135 2006

[59] L L McCarter ldquoDual flagellar systems enable motility underdifferent circumstancesrdquo Journal of Molecular Microbiology andBiotechnology vol 7 no 1-2 pp 18ndash29 2004

[60] S Ulitzur ldquoInduction of swarming in Vibrio parahaemolyticusrdquoArchives of Microbiology vol 101 no 4 pp 357ndash363 1974

[61] K S Park M Arita T Iida and T Honda ldquovpaH a geneencoding a novel histone-like nucleoid structure-like proteinthat was possibly horizontally acquired regulates the biogen-esis of lateral flagella in trh-positive Vibrio parahaemolyticusTH3996rdquo Infection and Immunity vol 73 no 9 pp 5754ndash57612005

[62] LMcCarter andM Silverman ldquoIron regulation of swarmer celldifferentiation ofVibrio parahaemolyticusrdquo Journal of Bacteriol-ogy vol 171 no 2 pp 731ndash736 1989

[63] B J Stewart and L L McCarter ldquoLateral flagellar gene systemofVibrio parahaemolyticusrdquo Journal of Bacteriology vol 185 no15 pp 4508ndash4518 2003

[64] C J Gode-Potratz R J Kustusch P J Breheny D S Weissand L LMcCarter ldquoSurface sensing inVibrio parahaemolyticustriggers a programme of gene expression that promotes colo-nization and virulencerdquo Molecular Microbiology vol 79 no 1pp 240ndash263 2011

[65] J L Veesenmeyer A R Hauser T Lisboa and J Rello ldquoPseu-domonas aeruginosa virulence and therapy evolving transla-tional strategiesrdquo Critical Care Medicine vol 37 no 5 pp 1777ndash1786 2009

[66] D Balasubramanian L Schneper H Kumari and K MatheeldquoA dynamic and intricate regulatory network determines Pseu-domonas aeruginosa virulencerdquo Nucleic Acids Research vol 41no 1 pp 1ndash20 2013

[67] T C Montie D D Huntzinger R C Craven and I A HolderldquoLoss of virulence associated with absence of flagellum in anisogenic mutant of Pseudomonas aeruginosa in the burned-mouse modelrdquo Infection and Immunity vol 38 no 3 pp 1296ndash1298 1982

[68] Y R Patankar R R Lovewell M E Poynter J Jyot BI Kazmierczak and B Berwin ldquoFlagellar motility is a keydeterminant of themagnitude of the inflammasome response toPseudomonas aeruginosardquo Infection and Immunity vol 81 no 6pp 2043ndash2052 2013

[69] G A OrsquoToole and R Kolter ldquoFlagellar and twitching motilityare necessary for Pseudomonas aeruginosa biofilm develop-mentrdquoMolecular Microbiology vol 30 no 2 pp 295ndash304 1998

[70] K B Barken S J Pamp L Yang et al ldquoRoles of type IVpili flagellum-mediated motility and extracellular DNA in theformation of mature multicellular structures in Pseudomonasaeruginosa biofilmsrdquo Environmental Microbiology vol 10 no 9pp 2331ndash2343 2008

[71] M Leeman J A van Pelt F M den Ouden M Heinsbroek PA Bakker and B Schippers ldquoInduction of systemic resistance

against fusarium wilt of radish by lipopolysaccharides of Pseu-domonas fluorescensrdquo Phytopathology vol 85 no 9 pp 1021ndash1027 1995

[72] P Hsueh L Teng H Pan et al ldquoOutbreak of Pseudomonasfluorescens bacteremia among oncology patientsrdquo Journal ofClinical Microbiology vol 36 no 10 pp 2914ndash2917 1998

[73] VWongK Levi B Baddal J Turton andTC Boswell ldquoSpreadof Pseudomonas fluorescens due to contaminated drinkingwater in a bone marrow transplant unitrdquo Journal of ClinicalMicrobiology vol 49 no 6 pp 2093ndash2096 2011

[74] J W Hickman and C S Harwood ldquoIdentification of FleQ fromPseudomonas aeruginosa as a c-di-GMP-responsive transcrip-tion factorrdquoMolecular Microbiology vol 69 no 2 pp 376ndash3892008

[75] N Dasgupta M C Wolfgang A L Goodman et al ldquoA four-tiered transcriptional regulatory circuit controls flagellar bio-genesis in Pseudomonas aeruginosardquo Molecular Microbiologyvol 50 no 3 pp 809ndash824 2003

[76] S K Arora B W Ritchings E C Almira S Lory and RRamphal ldquoA transcriptional activator FleQ regulates mucinadhesion and flagellar gene expression in Pseudomonas aerugi-nosa in a cascade mannerrdquo Journal of Bacteriology vol 179 no17 pp 5574ndash5581 1997

[77] N Dasgupta and R Ramphal ldquoInteraction of the antiactivatorFleN with the transcriptional activator FleQ regulates flagellarnumber in Pseudomonas aeruginosardquo Journal of Bacteriologyvol 183 no 22 pp 6636ndash6644 2001

[78] N Dasgupta S K Arora and R Ramphal ldquofleN a gene thatregulates flagellar number in Pseudomonas aeruginosardquo Journalof Bacteriology vol 182 no 2 pp 357ndash364 2000

[79] J Malakooti and B Ely ldquoPrincipal sigma subunit of theCaulobacter crescentus RNA polymeraserdquo Journal of Bacteriol-ogy vol 177 no 23 pp 6854ndash6860 1995

[80] M T Laub H H McAdams T Feldblyum C M Fraser andL Shapiro ldquoGlobal analysis of the genetic network controllinga bacterial cell cyclerdquo Science vol 290 no 5499 pp 2144ndash21482000

[81] A K Benson G Ramakrishnan N Ohta J Feng A J Ninfaand A Newton ldquoThe Caulobacter crescentus FlbD proteinacts at ftr sequence elements both to activate and to represstranscription of cell cycle-regulated flagellar genesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 91 no 11 pp 4989ndash4993 1994

[82] D A Mullin S M van Way C A Blankenship and A HMullin ldquoFlbD has a DNA-binding activity near its carboxyterminus that recognizes ftr sequences involved in positive andnegative regulation of flagellar gene transcription in Caulobac-ter crescentusrdquo Journal of Bacteriology vol 176 no 19 pp 5971ndash5981 1994

[83] R J Dutton Z Xu and J W Gober ldquoLinking structural assem-bly to gene expression a novel mechanism for regulating theactivity of a 12059054 transcription factorrdquo Molecular Microbiologyvol 58 no 3 pp 743ndash757 2005

[84] R E Muir J Easter and J W Gober ldquoThe trans-acting flagellarregulatory proteins FliX and FlbD play a central role in linkingflagellar biogenesis and cytokinesis in Caulobacter crescentusrdquoMicrobiology vol 151 no 11 pp 3699ndash3711 2005

[85] P E Anderson and J W Gober ldquoFlbT the post-transcriptionalregulator of flagellin synthesis in Caulobacter crescentus inter-acts with the 51015840 untranslated region of flagellin mRNArdquoMolec-ular Microbiology vol 38 no 1 pp 41ndash52 2000

Scientifica 13

[86] A T Nielsen N A Dolganov G Otto M C Miller C Y WuandGK Schoolnik ldquoRpoS controls theVibrio choleraemucosalescape responserdquo PLoS Pathogens vol 2 no 10 article e1092006

[87] M T Laub S L Chen L Shapiro and H H McAdamsldquoGenes directly controlled by CtrA a master regulator of theCaulobacter cell cyclerdquo Proceedings of the National Academy ofSciences of the United States of America vol 99 no 7 pp 4632ndash4637 2002

[88] A J Kelly M J Sackett N Din E Quardokus and Y VBrun ldquoCell cycle-dependent transcriptional and proteolyticregulation of FtsZ in Caulobacterrdquo Genes and Development vol12 no 6 pp 880ndash893 1998

[89] M Wortinger M J Sackett and Y V Brun ldquoCtrA mediatesa DNA replication checkpoint that prevents cell division inCaulobacter crescentusrdquo EMBO Journal vol 19 no 17 pp 4503ndash4512 2000

[90] R E Muir T M OrsquoBrien and J W Gober ldquoThe Caulobactercrescentus flagellar gene fliX encodes a novel trans-acting fac-tor that couples flagellar assembly to transcriptionrdquo MolecularMicrobiology vol 39 no 6 pp 1623ndash1637 2001

[91] M Llewellyn R J Dutton J Easter D OrsquoDonnol and J WGober ldquoThe conserved flaF gene has a critical role in couplingflagellin translation and assembly in Caulobacter crescentusrdquoMolecular Microbiology vol 57 no 4 pp 1127ndash1142 2005

[92] S M Kim D H Lee and S H Choi ldquoEvidence that the Vibriovulnificus flagellar regulator FlhF is regulated by a quorumsensing master regulator SmcRrdquo Microbiology vol 158 part 8pp 2017ndash2025 2012

[93] F Martınez-Granero A Navazo E Barahona M Redondo-Nieto R Rivilla andMMartın ldquoTheGac-Rsm and SadB signaltransduction pathways converge on Algu to downregulatemotility in Pseudomonas fluorescensrdquo PLoS ONE vol 7 no 2Article ID e31765 2012

[94] C Reimmann M Beyeler A Latifi et al ldquoThe global activatorGacA of Pseudomonas aeruginosa PAO positively controls theproduction of the autoinducer N-butyryl-homoserine lactoneand the formation of the virulence factors pyocyanin cyanideand lipaserdquo Molecular Microbiology vol 24 no 2 pp 309ndash3191997

[95] J Dubern and G V Bloemberg ldquoInfluence of environmentalconditions on putisolvins I and II production in Pseudomonasputida strain PCL1445rdquo FEMSMicrobiology Letters vol 263 no2 pp 169ndash175 2006

[96] M G Surette M B Miller and B L Bassler ldquoQuorum sensingin Escherichia coli Salmonella typhimurium andVibrio harveyia new family of genes responsible for autoinducer productionrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 96 no 4 pp 1639ndash1644 1999

[97] B A Rader S R Campagna M F Semmelhack B L Basslerand K Guillemin ldquoThe quorum-sensing molecule autoinducer2 regulates motility and flagellar morphogenesis inHelicobacterpylorirdquo Journal of Bacteriology vol 189 no 17 pp 6109ndash61172007

[98] YHe J G Frye T P Strobaugh Jr andCChen ldquoAnalysis of AI-2LuxS-dependent transcription in Campylobacter jejuni strain81-176rdquo Foodborne Pathogens and Disease vol 5 no 4 pp 399ndash415 2008

[99] B Jeon K Itoh N Misawa and S Ryu ldquoEffects of quorumsensing on flaA transcription and autoagglutination in Campy-lobacter jejunirdquoMicrobiology and Immunology vol 47 no 11 pp833ndash839 2003

[100] N Dasgupta E P Ferrell K J Kanack S E H West andR Ramphal ldquofleQ the gene encoding the major flagellarregulator of Pseudomonas aeruginosa is 12059070 dependent and isdownregulated by Vfr a homolog of Escherichia coli cyclic AMPreceptor proteinrdquo Journal of Bacteriology vol 184 no 19 pp5240ndash5250 2002

[101] J Zhu M B Miller R E Vance M Dziejman B L Bassler andJ J Mekalanos ldquoQuorum-sensing regulators control virulencegene expression in Vibrio choleraerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no5 pp 3129ndash3134 2002

[102] Z Liu T Miyashiro A Tsou A Hsiao M Goulian and JZhu ldquoMucosal penetration primes Vibrio cholerae for hostcolonization by repressing quorum sensingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 28 pp 9769ndash9774 2008

[103] K E Klose V Novik and J J Mekalanos ldquoIdentificationof multiple 12059054-dependent transcriptional activators in Vibriocholeraerdquo Journal of Bacteriology vol 180 no 19 pp 5256ndash52591998

[104] I Kawagishi M Imagawa Y Imae L McCarter and MHomma ldquoThe sodium-driven polar flagellar motor of marineVibrio as the mechanosensor that regulates lateral flagellarexpressionrdquoMolecular Microbiology vol 20 no 4 pp 693ndash6991996

[105] T J Kirn B A Jude and R K Taylor ldquoA colonizationfactor links Vibrio cholerae environmental survival and humaninfectionrdquo Nature vol 438 no 7069 pp 863ndash866 2005

[106] Z Kuang YHao S Hwang et al ldquoThe Pseudomonas aeruginosaflagellum confers resistance to pulmonary surfactant protein-Aby impacting the production of exoproteases through quorum-sensingrdquo Molecular Microbiology vol 79 no 5 pp 1220ndash12352011

[107] S Zhang F X McCormack R C Levesque G A OrsquoTooleand G W Lau ldquoThe flagellum of Pseudomonas aeruginosa isrequired for resistance to clearance by surfactant protein ArdquoPLoS ONE vol 2 no 6 article e564 2007

[108] G W Lau D J Hassett H Ran and F Kong ldquoThe role ofpyocyanin in Pseudomonas aeruginosa infectionrdquo Trends inMolecular Medicine vol 10 no 12 pp 599ndash606 2004

[109] GW LauH Ran F Kong D J Hassett andDMavrodi ldquoPseu-domonas aeruginosa pyocyanin is critical for lung infection inmicerdquo Infection and Immunity vol 72 no 7 pp 4275ndash42782004

[110] D S Merrell M L Goodrich G Otto L S Tompkins and SFalkow ldquopH-regulated gene expression of the gastric pathogenHelicobacter pylorirdquo Infection and Immunity vol 71 no 6 pp3529ndash3539 2003

[111] K J Allen and M W Griffiths ldquoEffect of environmental andchemotactic stimuli on the activity of the Campylobacter jejuniflaA 12059028 promoterrdquo FEMS Microbiology Letters vol 205 no 1pp 43ndash48 2001

[112] Y Wen J Feng D R Scott E A Marcus and G SachsldquoThe pH-responsive regulon of HP0244 (FlgS) the cytoplasmichistidine kinase of Helicobacter pylorirdquo Journal of Bacteriologyvol 191 no 2 pp 449ndash460 2009

[113] D R Hendrixson ldquoA phase-variable mechanism controllingthe Campylobacter jejuni FlgR response regulator influencescommensalismrdquo Molecular Microbiology vol 61 no 6 pp1646ndash1659 2006

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Advances in

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Nucleic AcidsJournal of

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Enzyme Research

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International Journal of

Microbiology

8 Scientifica

is required for flagellar biogenesis but also appears to play arole in host colonization by V cholerae Mutants expressinga FlrC variant incapable of being phosphorylated fail toproduce flagella and are therefore defective in colonization[54] Interestingly a mutant expressing a constitutively activeform of FlrC is also deficient in colonization even though thecells are motile and produce flagella [54] This observationdemonstrates the need for coordinating flagellar biogenesisand pathogenesis for successful colonization ConverselyVfr a major quorum sensing and virulence regulator inP aeruginosa downregulates expression of flagellar genesby repressing transcription from the fleQ promoter [100]Inhibition of flagellar biogenesis by Vfr may aid P aeruginosain persisting at the site of infection to achieve high celldensities and virulence gene expression

HapR is a transcriptional regulator involved in quorumsensing in V cholerae which also regulates expression ofvirulence genes HapR represses expression of several vir-ulence genes [101] and hapR expression is regulated by anunknown mechanism involving FliA [102] hapR expres-sion is depressed in flgM and flgD mutants (both of thesemutations alleviate FlgM repression on FliA activity) whiledeletion of fliA results in elevated hapR expression [102] Theauthors of this study speculated that expression of HapR isregulated by a regulator or small noncoding RNA (sRNA)that is part of the FliA regulon FliA-mediated regulation ofhapR appears to be linked to the shearing of flagella frombacteria that penetrate the mucin layer to infect the hostThis shearing of the flagella leads to secretion of FlgM viathe flagellar protein export apparatus and results in increasedexpression of FliA-dependent genes and repression of hapRexpression [102] The outcome of such a mechanism is thatvirulence genes which are negatively regulated by HapRare expressed specifically when V cholerae penetrates theintestinal mucous layer hapR production is also regulatedby the quorum sensing protein LuxO further linking viru-lence to cell density LuxO is an RpoN-dependent activator[103] which along with HapR regulates motility proteaseproduction and biofilm formation [101] High cell densitiesare common during late stages of infection and the HapR-mediated repression of virulence genes may aidV cholerae indetaching and finding new colonization sites [86 101]

V parahaemolyticus uses its polar flagellum to detectsurfaces by sensing impedance to the polar flagellum rotationrate [104] Once a surface is encountered expression of thelateral flagellar genes is induced Lateral flagella are importantfor host colonization as mutants that do not produce lateralflagella are deficient in adherence to HeLa cells and biofilmformation [61] Gode-Potratz and coworkers identified sim70genes that are surface responsive most of which are positivelyregulated [64] Some of the surface-regulated genes that areupregulated are virulence genes such as a putative GbpAhomolog and components of a type III secretion system [64]GbpA from V cholerae promotes adherence to chitinoussurfaces of zooplankton and human epithelia cells [105]which is consistent with the observed upregulation of gbpAin V parahaemolyticus when it encounters a surface

The P aeruginosa flagellum is an important determinantin the susceptibility of the bacterium to host defenses

P aeruginosa fliCmutants are unable to upregulate transcrip-tion of lasI and rhlI which encode enzymes that synthesizequorum sensing homoserine lactones [106] These quo-rum sensing molecules regulate production of exoproteaseswhich degrade surfactant protein-A (SP-A) an antimicro-bial that opsonizes and permeabilizes membranes of lungpathogens [106] In the absence of these exoproteases theP aeruginosa fliC mutants are cleared from the lung [106]Though it is not known why fliC mutants are unable toupregulate transcription of lasI and rhlI it is not due tothe inability to sense the environment [106] P aeruginosafliE flgE fliC and fliD mutants are also attenuated in LPSbiosynthesis which compromises the integrity of their outermembranes making them more susceptible to SP-A [107]Additionally flgE and fliC mutants make less pyocyanin aredox-active toxic secondary metabolite that is crucial forlung infection [108 109]

33 Reacting to pH Changes by Gastrointestinal PathogensGastrointestinal pathogens encounter varying environmentalconditions as they travel from the mouth to the stomach andthen to the intestines The gastric pathogen H pylori has theadded burden of long-term survival in the acidic environ-ment of the stomach Pathogens that colonize the intestinaltract must also survive passage through the stomach in highenough numbers to be able to colonize their target organsThus it is not surprising that gastrointestinal pathogens oftenmodulate gene expression in response to acidic conditions

Merrell and coworkers found that expression of approxi-mately 7 of theH pylori genes is altered by a shift to low pH[110] Many of these genes are involved in flagellar synthesiswith the majority of those being RpoN dependent [110] FliA-dependent genes such as flgM in H pylori [110] and flaAin C jejuni [111] are also upregulated at low pH conditionsThe upregulation of flagellar genes is critical for survival ofH pylori as the bacterium is not able to survive at low pHbut must swim across the mucous layer to the underlyinggastric epithelium to establish a successful infection Inaddition to enhancing transcription of flagellar genes acidicenvironments also stimulate motility and swimming speedspotentially in response to increased concentration of protonsin the stomach [110]

The basis for upregulation of the RpoN-dependent flag-ellar genes in H pylori likely involves a stimulation of FlgSactivity at low pHWen and coworkers reported thatH pylorirequires FlgS to survive acid stress (30-minute exposure topH25) [112]These researchers found that besides the knownRpoN-dependent flagellar genes FlgS was required for theupregulation of 86 genes in response to acid stress many ofwhich were previously known to be required for acid stresssurvival [112] It is not known if FlgS responds directly toacid stress or if the stress signal is mediated through anotherfactor

34 Altering Flagellar Biogenesis by Phase Variation In somebacteria flagellar biogenesis is subject to phase variationevents that affect expression of specific flagellar genes Phasevariation causes changes in phenotypes at frequencies that

Scientifica 9

are much higher than random mutations and contribute toheterogeneity within a population The ability of H pylori topersist in the host may involve the phase-variable dependentexpression of flagella that can serve as an advantage foradaptation to the host and for evading the host immuneresponse C jejuni utilizes phase variation to turn on andoff production of flagella a strategy that increases com-mensal colonization in poultry [113] Many phase variationevents occur by random reversible changes in the length ofshort DNA sequence repeats resulting from slipped-strandmispairing These events can take place within the codingsequence or in the promoter of a gene C jejuni phasevariants in which expression of flgR is in the OFF stateresult from the addition or removal of a nucleotide withinone of two homopolymeric nucleotide tracts consisting ofadenine and thymine These nucleotide tracts are locatedwithin the coding sequence of flgR causing the sequence toshift out of frame [113] Revertants to the ON state ariseupon the addition or removal of a nucleotide within theoriginal mutated homopolymeric nucleotide tract to restorethe wild-type sequence Alternatively pseudorevertants canarise in which nucleotides are removed or added close to theoriginal mutated homopolymeric nucleotide tract to restorethe correct reading frame but which result in changes in theamino acid sequence of the protein [113] flgS is also regulatedby phase variation in C jejuni providing multiple levels ofphase-variable control for theRpoN regulon [114 115] Similarmechanisms of phase variation also exist for flhA in C coli[116] and fliP in H pylori [117] which leads to the formationof truncated proteins that prevent flagellar biogenesis

A second type of phase variation in flagellar biogen-esis is mediated through alterations in DNA methylationwhich impact the expression of specific flagellar genesHost-adapted bacterial pathogens such as H pylori oftenpossess DNA methyltransferases and type III restriction-modification (R-M) systems that contain simple tandemDNA repeats which undergo slipped-strand mispairing dur-ing DNA replication that causes frameshift mutations andphase variation [118] R-M systems widespread among manybacteria confer protection from invasion by foreign DNAType III R-M systems are composed of a methyltransferase(mod) and an endonuclease (res) gene whose gene prod-ucts typically function together The switching between ONand OFF states of a DNA methyltransferase can regulateexpression of a ldquophasevarionrdquo via differential methylationof the genome in ON and OFF states [119] Many of thesephase-variable methyltransferase genes are associated withan inactive res gene resulting from frameshift mutationor are orphans that are not associated with a res genesuggesting that DNA restriction is not the primary roleof these methyltransferases (reviewed by Fox et al [120])For example H pylori modH encodes a phase-variableDNA methyltransferase and the cognate res gene containsa nonsense mutation frameshift mutation or is absent invarious H pylori strains [119] modH OFF strains havedecreased expression of flaA and fliK compared to themodHON strain [119] FlaA has a low ability to activate innateimmunity via the Toll-like receptor 5 [121] and modulatingexpression of the flagellin may be advantageous in evading

the host response Another H pylori methyltransferase withan inactive res gene is hpyAVIBM [122 123] hpyAVIBMencodes a C5 cytosine methyltransferase and contains AGrepeats in its open reading frame making it susceptible tophase variation via frame shift mutations [124] For instancehpyAVIBM from strain 26695 and hpyAVIBM from strainHPAG1 have five AG repeats whereas the four AG repeats instrain San 74 result in the translation of a truncated protein[124] Deletion of hpyAVIBM results in both the upregulationand downregulation of rpoN fliR fliD fliS motA fliK andflgK depending on the strain used [124] indicating that phasevariation of hpyAVIBM can alter flagellar biogenesis

P aeruginosa populations can possess two phenotypicvariants one which forms small rough colonies (S) andanother which forms large flat (L) colonies S variants formbiofilms in nonagitated liquid cultures whereas the L variantsdo not [125] S variants possess defects in flagellum-mediatedswimming flagellum-mediated swarming and type IV-pilus-mediated twitching but are able to revert back to the Lphenotype at relatively high frequencies suggesting that theshift between phenotypes is regulated by phase variation[125] Phase variation also occurs in P fluorescens whichcolonizes the alfalfa rhizosphere Phenotypic variants ariseduring rhizosphere colonization and are dependent on theactivity of a site-specific recombinase [126] C variants havewild-type colony morphology while F and S variants havea translucent and diffuse colony morphology [126] F andS variants which colonize distal parts of the roots andswim faster than the C variants overproduce fliC (flagellin)transcripts and synthesize flagellin filaments that aresim3 timeslonger than those of the C variant [126]

4 Conclusions and Future Directions

Flagellar biogenesis is a carefully choreographed processin which many different components of the flagellum aremade as they are needed for assembly into the nascentflagellum Bacteria have evolved elaborate and sundry reg-ulatory networks to couple flagellar gene expression withassembly A driving force for the evolution of such regulatorynetworks is undoubtedly the environmental and ecologicalconstraints imposed by a particular bacteriumrsquos niche Inaddition the degree to which a given bacterial species needsto couple flagellar biogenesis to other cellular processes suchas cell division has also likely shaped the evolution of theseregulatory networks We focused here on bacteria whichutilize RpoN for flagellar biogenesis yet even among thesebacteria the mechanisms used to regulate expression of theRpoN-dependent flagellar genes vary greatly For most of thesystems described in this review we have only scratched thesurface in our understanding of how they operate For each ofthese systems fundamental questions remain to be answeredsuch as the following are all of the flagellar genes of a givensystem expressed as part of a transcription hierarchy or aresome genes expressed constitutively Are there regulatorymechanisms within RpoN flagellar regulons that fine-tunegene expression so that components of the flagellum aremadeprecisely when needed What are the cellular cues sensed by

10 Scientifica

the regulatory proteins that control expression of the RpoN-dependent flagellar genes How does the flagellar exportapparatus control expression of RpoN-dependent flagellargenes in various bacteria Does FlgZ have a regulatoryrole in flagellar biogenesis in H pylori and other bacteriaAs our understanding of the molecular genetics of diversebacterial species expands novel regulatory mechanisms thatcontrol expression of flagellar genes within these bacteria willundoubtedly come to light For example it was only recentlythat sRNAs were identified in H pylori some of whichare potentially FliA dependent [127] and may have roles inflagellar biogenesis Finally with increasingly sophisticatedmethods in bioinformatics genomics and proteomics fur-ther characterization of flagellar regulatory networks willprovide important insights into the mechanisms governinggene expression and identify new regulatory proteins andmechanisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by award MCB-1244242 from theNational Science Foundation

References

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[2] J L McMurry J W Murphy and B Gonzalez-Pedrajo ldquoTheFliN-FliH interaction mediates localization of flagellar exportATPase FliI to the C ring complexrdquo Biochemistry vol 45 no39 pp 11790ndash11798 2006

[3] R M Macnab Ed Flagella and Motility ASM Press 1996[4] T Minamino K Imada and K Namba ldquoMolecular motors of

the bacterial flagellardquoCurrent Opinion in Structural Biology vol18 no 6 pp 693ndash701 2008

[5] T G Smith and T R Hoover ldquoDeciphering bacterial flagellargene regulatory networks in the genomic erardquo Advances inApplied Microbiology vol 67 pp 257ndash295 2009

[6] J K Anderson T G Smith and T R Hoover ldquoSense andsensibility flagellum-mediated gene regulationrdquo Trends inMicrobiology vol 18 no 1 pp 30ndash37 2010

[7] J D Helmann ldquoAlternative sigma factors and the regulation offlagellar gene expressionrdquoMolecular Microbiology vol 5 no 12pp 2875ndash2882 1991

[8] D L Popham D Szeto J Keener and S Kustu ldquoFunctionof a bacterial activator protein that binds to transcriptionalenhancersrdquo Science vol 243 no 4891 pp 629ndash635 1989

[9] S Sasse-Dwight and J D Gralla ldquoProbing the EscherichiacoliglnALG upstream activation mechanism in vivordquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8934ndash8938 1988

[10] M Kihara T Minamino S Yamaguchi and R M MacnabldquoIntergenic suppression between the flagellar MS ring protein

FliF of Salmonella and FlhA a membrane component of itsexport apparatusrdquo Journal of Bacteriology vol 183 no 5 pp1655ndash1662 2001

[11] J S van Arnam J L McMurry M Kihara and R M MacnabldquoAnalysis of an engineered Salmonella flagellar fusion proteinFliR-FlhBrdquo Journal of Bacteriology vol 186 no 8 pp 2495ndash2498 2004

[12] C S Barker I V Meshcheryakova A S Kostyukova and FA Samatey ldquoFliO regulation of FliP in the formation of theSalmonella enterica flagellumrdquo PLoS Genetics vol 6 no 9Article ID e1001143 2010

[13] A C Lowenthal M Hill L K Sycuro K Mehmood N RSalama and K M Ottemann ldquoFunctional analysis of the Heli-cobacter pylori flagellar switch proteinsrdquo Journal of Bacteriologyvol 191 no 23 pp 7147ndash7156 2009

[14] R M Macnab ldquoType III flagellar protein export and flagellarassemblyrdquo Biochimica et Biophysica Acta vol 1694 no 1ndash3 pp207ndash217 2004

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[16] K A Eaton D R Morgan and S Krakowka ldquoMotility as afactor in the colonisation of gnotobiotic piglets by Helicobacterpylorirdquo Journal of Medical Microbiology vol 37 no 2 pp 123ndash127 1992

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[18] TMorooka A Umeda andK Amako ldquoMotility as an intestinalcolonization factor forCampylobacter jejunirdquo Journal of GeneralMicrobiology vol 131 no 8 pp 1973ndash1980 1985

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[22] A Kusumoto A Shinohara H Terashima S Kojima TYakushi and M Homma ldquoCollaboration of FlhF and FlhGto regulate polarflagella number and localization in VibrioalginolyticusrdquoMicrobiology vol 154 no 5 pp 1390ndash1399 2008

[23] A Kusumoto K Kamisaka T Yakushi H Terashima AShinohara and M Homma ldquoRegulation of polar flagellarnumber by the flhF and flhG genes in Vibrio alginolyticusrdquoJournal of Biochemistry vol 139 no 1 pp 113ndash121 2006

[24] B M Pruszlig and P Matsumura ldquoA regulator of the flagellarregulon of Escherichia coli flhD also affects cell divisionrdquoJournal of Bacteriology vol 178 no 3 pp 668ndash674 1996

[25] B M Pruszlig and P Matsumura ldquoCell cycle regulation of flagellargenesrdquo Journal of Bacteriology vol 179 no 17 pp 5602ndash56041997

[26] G Spohn and V Scarlato ldquoMotility of Helicobacter pylori iscoordinately regulated by the transcriptional activator FlgR an

Scientifica 11

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[28] D Beier and R Frank ldquoMolecular characterization of two-component systems of Helicobacter pylorirdquo Journal of Bacteri-ology vol 182 no 8 pp 2068ndash2076 2000

[29] P Brahmachary M G Dashti J W Olson and T R HooverldquoHelicobacter pylori FlgR is an enhancer-independent activatorof 12059054-RNA polymerase holoenzymerdquo Journal of Bacteriologyvol 186 no 14 pp 4535ndash4542 2004

[30] M M S M Wosten J A Wagenaar and J P M van PuttenldquoThe FlgSFlgR two-component signal transduction systemregulates the fla regulon in Campylobacter jejunirdquo The Journalof Biological Chemistry vol 279 no 16 pp 16214ndash16222 2004

[31] M Bush and R Dixon ldquoThe role of bacterial enhancer bindingproteins as specialized activators of 12059054-dependent transcrip-tionrdquo MicroBiology and Molecular Biology Reviews vol 76 no3 pp 497ndash529 2012

[32] J Schumacher N Joly M Rappas X Zhang and M BuckldquoStructures and organisation of AAA+ enhancer binding pro-teins in transcriptional activationrdquo Journal of Structural Biologyvol 156 no 1 pp 190ndash199 2006

[33] S N Joslin and D R Hendrixson ldquoAnalysis of the Campy-lobacter jejuni FlgR response regulator suggests integration ofdiverse mechanisms to activate an NtrC-like proteinrdquo Journalof Bacteriology vol 190 no 7 pp 2422ndash2433 2008

[34] J M Boll and D R Hendrixson ldquoA specificity determinant forphosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphaterdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 50 pp 20160ndash20165 2011

[35] S Porwollik B Noonan and P W OrsquoToole ldquoMolecular char-acterization of a flagellar export locus of Helicobacter pylorirdquoInfection and Immunity vol 67 no 5 pp 2060ndash2070 1999

[36] E Allan N Dorrell S Foynes M Anyim and B W WrenldquoMutational analysis of genes encoding the early flagellarcomponents of Helicobacter pylori evidence for transcriptionalregulation of flagellin A biosynthesisrdquo Journal of Bacteriologyvol 182 no 18 pp 5274ndash5277 2000

[37] T G Smith L Pereira and T R Hoover ldquoHelicobacter pyloriFlhB processing-deficient variants affect flagellar assembly butnot flagellar gene expressionrdquo Microbiology vol 155 no 4 pp1170ndash1180 2009

[38] D R Hendrixson B J Akerley and V J DiRita ldquoTranspo-son mutagenesis of Campylobacter jejuni identifies a bipartiteenergy taxis system required for motilityrdquoMolecular Microbiol-ogy vol 40 no 1 pp 214ndash224 2001

[39] J Tsang T G Smith L E Pereira and T R Hoover ldquoInsertionmutations inHelicobacter pylori flhA reveal strain differences inRpoN-dependent gene expressionrdquo Microbiology vol 159 part1 pp 58ndash67 2013

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[41] T Minamino H Doi and K Kutsukake ldquoSubstrate specificityswitching of the flagellum-specific export apparatus during flag-ellar morphogenesis in Salmonella typhimuriumrdquo Bioscience

Biotechnology and Biochemistry vol 63 no 7 pp 1301ndash13031999

[42] M Erhardt H M Singer D H Wee J P Keener and K THughes ldquoAn infrequent molecular ruler controls flagellar hooklength in Salmonella entericardquo EMBO Journal vol 30 no 14 pp2948ndash2961 2011

[43] K T Hughes K L Gillen M J Semon and J E KarlinseyldquoSensing structural intermediates in bacterial flagellar assemblyby export of a negative regulatorrdquo Science vol 262 no 5137 pp1277ndash1280 1993

[44] K A Ryan N Karim M Worku S A Moore C W Pennand P W OrsquoToole ldquoHP0958 is an essential motility gene inHelicobacter pylorirdquo FEMS Microbiology Letters vol 248 no 1pp 47ndash55 2005

[45] L Pereira and T R Hoover ldquoStable accumulation of 12059054 inHelicobacter pylori requires the novel protein HP0958rdquo Journalof Bacteriology vol 187 no 13 pp 4463ndash4469 2005

[46] J C Rain L Selig H de Reuse et al ldquoThe protein-proteininteraction map of Helicobacter pylorirdquo Nature vol 409 no6817 pp 211ndash215 2001

[47] F P Douillard K A Ryan D L Caly et al ldquoPosttranscriptionalregulation of flagellin synthesis in Helicobacter pylori by theRpoN chaperone HP0958rdquo Journal of Bacteriology vol 190 no24 pp 7975ndash7984 2008

[48] C Josenhans E Niehus S Amersbach et al ldquoFunctionalcharacterization of the antagonistic flagellar late regulators FliAand FlgM ofHelicobacter pylori and their effects on theH pyloritranscriptomerdquoMolecular Microbiology vol 43 no 2 pp 307ndash322 2002

[49] M Rust S Borchert E Niehus et al ldquoThe Helicobacter pylorianti-sigma factor FlgM is predominantly cytoplasmic andcooperates with the flagellar basal body protein FlhArdquo Journalof Bacteriology vol 191 no 15 pp 4824ndash4834 2009

[50] D R Hendrixson and V J DiRita ldquoTranscription of 12059054-dependent but not 12059028-dependent flagellar genes in Campy-lobacter jejuni is associated with formation of the flagellarsecretory apparatusrdquoMolecular Microbiology vol 50 no 2 pp687ndash702 2003

[51] M M S M Wosten L van Dijk A K J Veenendaal MR de Zoete N M C Bleumink-Pluijm and J P M vanPutten ldquoTemperature-dependent FlgMFliA complex forma-tion regulates Campylobacter jejuni flagella lengthrdquo MolecularMicrobiology vol 75 no 6 pp 1577ndash1591 2010

[52] M G Prouty N E Correa and K E Klose ldquoThe novel 12059054- and12059028-dependent flagellar gene transcription hierarchy of Vibrio

choleraerdquo Molecular Microbiology vol 39 no 6 pp 1595ndash16092001

[53] D Srivastava M L Hsieh A Khataokar M B Neiditch and CM Waters ldquoCyclic di-GMP inhibits Vibrio choleraemotility byrepressing induction of transcription and inducing extracellularpolysaccharide productionrdquo Molecular Microbiology vol 90no 6 pp 1262ndash1276 2013

[54] N E Correa C M Lauriano R McGee and K E KloseldquoPhosphorylation of the flagellar regulatory protein FlrC is nec-essary for Vibrio cholerae motility and enhanced colonizationrdquoMolecular Microbiology vol 35 no 4 pp 743ndash755 2000

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12 Scientifica

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against fusarium wilt of radish by lipopolysaccharides of Pseu-domonas fluorescensrdquo Phytopathology vol 85 no 9 pp 1021ndash1027 1995

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[81] A K Benson G Ramakrishnan N Ohta J Feng A J Ninfaand A Newton ldquoThe Caulobacter crescentus FlbD proteinacts at ftr sequence elements both to activate and to represstranscription of cell cycle-regulated flagellar genesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 91 no 11 pp 4989ndash4993 1994

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Scientifica 13

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[92] S M Kim D H Lee and S H Choi ldquoEvidence that the Vibriovulnificus flagellar regulator FlhF is regulated by a quorumsensing master regulator SmcRrdquo Microbiology vol 158 part 8pp 2017ndash2025 2012

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[97] B A Rader S R Campagna M F Semmelhack B L Basslerand K Guillemin ldquoThe quorum-sensing molecule autoinducer2 regulates motility and flagellar morphogenesis inHelicobacterpylorirdquo Journal of Bacteriology vol 189 no 17 pp 6109ndash61172007

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[99] B Jeon K Itoh N Misawa and S Ryu ldquoEffects of quorumsensing on flaA transcription and autoagglutination in Campy-lobacter jejunirdquoMicrobiology and Immunology vol 47 no 11 pp833ndash839 2003

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[101] J Zhu M B Miller R E Vance M Dziejman B L Bassler andJ J Mekalanos ldquoQuorum-sensing regulators control virulencegene expression in Vibrio choleraerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no5 pp 3129ndash3134 2002

[102] Z Liu T Miyashiro A Tsou A Hsiao M Goulian and JZhu ldquoMucosal penetration primes Vibrio cholerae for hostcolonization by repressing quorum sensingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 28 pp 9769ndash9774 2008

[103] K E Klose V Novik and J J Mekalanos ldquoIdentificationof multiple 12059054-dependent transcriptional activators in Vibriocholeraerdquo Journal of Bacteriology vol 180 no 19 pp 5256ndash52591998

[104] I Kawagishi M Imagawa Y Imae L McCarter and MHomma ldquoThe sodium-driven polar flagellar motor of marineVibrio as the mechanosensor that regulates lateral flagellarexpressionrdquoMolecular Microbiology vol 20 no 4 pp 693ndash6991996

[105] T J Kirn B A Jude and R K Taylor ldquoA colonizationfactor links Vibrio cholerae environmental survival and humaninfectionrdquo Nature vol 438 no 7069 pp 863ndash866 2005

[106] Z Kuang YHao S Hwang et al ldquoThe Pseudomonas aeruginosaflagellum confers resistance to pulmonary surfactant protein-Aby impacting the production of exoproteases through quorum-sensingrdquo Molecular Microbiology vol 79 no 5 pp 1220ndash12352011

[107] S Zhang F X McCormack R C Levesque G A OrsquoTooleand G W Lau ldquoThe flagellum of Pseudomonas aeruginosa isrequired for resistance to clearance by surfactant protein ArdquoPLoS ONE vol 2 no 6 article e564 2007

[108] G W Lau D J Hassett H Ran and F Kong ldquoThe role ofpyocyanin in Pseudomonas aeruginosa infectionrdquo Trends inMolecular Medicine vol 10 no 12 pp 599ndash606 2004

[109] GW LauH Ran F Kong D J Hassett andDMavrodi ldquoPseu-domonas aeruginosa pyocyanin is critical for lung infection inmicerdquo Infection and Immunity vol 72 no 7 pp 4275ndash42782004

[110] D S Merrell M L Goodrich G Otto L S Tompkins and SFalkow ldquopH-regulated gene expression of the gastric pathogenHelicobacter pylorirdquo Infection and Immunity vol 71 no 6 pp3529ndash3539 2003

[111] K J Allen and M W Griffiths ldquoEffect of environmental andchemotactic stimuli on the activity of the Campylobacter jejuniflaA 12059028 promoterrdquo FEMS Microbiology Letters vol 205 no 1pp 43ndash48 2001

[112] Y Wen J Feng D R Scott E A Marcus and G SachsldquoThe pH-responsive regulon of HP0244 (FlgS) the cytoplasmichistidine kinase of Helicobacter pylorirdquo Journal of Bacteriologyvol 191 no 2 pp 449ndash460 2009

[113] D R Hendrixson ldquoA phase-variable mechanism controllingthe Campylobacter jejuni FlgR response regulator influencescommensalismrdquo Molecular Microbiology vol 61 no 6 pp1646ndash1659 2006

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

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Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Advances in

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Nucleic AcidsJournal of

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Enzyme Research

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International Journal of

Microbiology

Scientifica 9

are much higher than random mutations and contribute toheterogeneity within a population The ability of H pylori topersist in the host may involve the phase-variable dependentexpression of flagella that can serve as an advantage foradaptation to the host and for evading the host immuneresponse C jejuni utilizes phase variation to turn on andoff production of flagella a strategy that increases com-mensal colonization in poultry [113] Many phase variationevents occur by random reversible changes in the length ofshort DNA sequence repeats resulting from slipped-strandmispairing These events can take place within the codingsequence or in the promoter of a gene C jejuni phasevariants in which expression of flgR is in the OFF stateresult from the addition or removal of a nucleotide withinone of two homopolymeric nucleotide tracts consisting ofadenine and thymine These nucleotide tracts are locatedwithin the coding sequence of flgR causing the sequence toshift out of frame [113] Revertants to the ON state ariseupon the addition or removal of a nucleotide within theoriginal mutated homopolymeric nucleotide tract to restorethe wild-type sequence Alternatively pseudorevertants canarise in which nucleotides are removed or added close to theoriginal mutated homopolymeric nucleotide tract to restorethe correct reading frame but which result in changes in theamino acid sequence of the protein [113] flgS is also regulatedby phase variation in C jejuni providing multiple levels ofphase-variable control for theRpoN regulon [114 115] Similarmechanisms of phase variation also exist for flhA in C coli[116] and fliP in H pylori [117] which leads to the formationof truncated proteins that prevent flagellar biogenesis

A second type of phase variation in flagellar biogen-esis is mediated through alterations in DNA methylationwhich impact the expression of specific flagellar genesHost-adapted bacterial pathogens such as H pylori oftenpossess DNA methyltransferases and type III restriction-modification (R-M) systems that contain simple tandemDNA repeats which undergo slipped-strand mispairing dur-ing DNA replication that causes frameshift mutations andphase variation [118] R-M systems widespread among manybacteria confer protection from invasion by foreign DNAType III R-M systems are composed of a methyltransferase(mod) and an endonuclease (res) gene whose gene prod-ucts typically function together The switching between ONand OFF states of a DNA methyltransferase can regulateexpression of a ldquophasevarionrdquo via differential methylationof the genome in ON and OFF states [119] Many of thesephase-variable methyltransferase genes are associated withan inactive res gene resulting from frameshift mutationor are orphans that are not associated with a res genesuggesting that DNA restriction is not the primary roleof these methyltransferases (reviewed by Fox et al [120])For example H pylori modH encodes a phase-variableDNA methyltransferase and the cognate res gene containsa nonsense mutation frameshift mutation or is absent invarious H pylori strains [119] modH OFF strains havedecreased expression of flaA and fliK compared to themodHON strain [119] FlaA has a low ability to activate innateimmunity via the Toll-like receptor 5 [121] and modulatingexpression of the flagellin may be advantageous in evading

the host response Another H pylori methyltransferase withan inactive res gene is hpyAVIBM [122 123] hpyAVIBMencodes a C5 cytosine methyltransferase and contains AGrepeats in its open reading frame making it susceptible tophase variation via frame shift mutations [124] For instancehpyAVIBM from strain 26695 and hpyAVIBM from strainHPAG1 have five AG repeats whereas the four AG repeats instrain San 74 result in the translation of a truncated protein[124] Deletion of hpyAVIBM results in both the upregulationand downregulation of rpoN fliR fliD fliS motA fliK andflgK depending on the strain used [124] indicating that phasevariation of hpyAVIBM can alter flagellar biogenesis

P aeruginosa populations can possess two phenotypicvariants one which forms small rough colonies (S) andanother which forms large flat (L) colonies S variants formbiofilms in nonagitated liquid cultures whereas the L variantsdo not [125] S variants possess defects in flagellum-mediatedswimming flagellum-mediated swarming and type IV-pilus-mediated twitching but are able to revert back to the Lphenotype at relatively high frequencies suggesting that theshift between phenotypes is regulated by phase variation[125] Phase variation also occurs in P fluorescens whichcolonizes the alfalfa rhizosphere Phenotypic variants ariseduring rhizosphere colonization and are dependent on theactivity of a site-specific recombinase [126] C variants havewild-type colony morphology while F and S variants havea translucent and diffuse colony morphology [126] F andS variants which colonize distal parts of the roots andswim faster than the C variants overproduce fliC (flagellin)transcripts and synthesize flagellin filaments that aresim3 timeslonger than those of the C variant [126]

4 Conclusions and Future Directions

Flagellar biogenesis is a carefully choreographed processin which many different components of the flagellum aremade as they are needed for assembly into the nascentflagellum Bacteria have evolved elaborate and sundry reg-ulatory networks to couple flagellar gene expression withassembly A driving force for the evolution of such regulatorynetworks is undoubtedly the environmental and ecologicalconstraints imposed by a particular bacteriumrsquos niche Inaddition the degree to which a given bacterial species needsto couple flagellar biogenesis to other cellular processes suchas cell division has also likely shaped the evolution of theseregulatory networks We focused here on bacteria whichutilize RpoN for flagellar biogenesis yet even among thesebacteria the mechanisms used to regulate expression of theRpoN-dependent flagellar genes vary greatly For most of thesystems described in this review we have only scratched thesurface in our understanding of how they operate For each ofthese systems fundamental questions remain to be answeredsuch as the following are all of the flagellar genes of a givensystem expressed as part of a transcription hierarchy or aresome genes expressed constitutively Are there regulatorymechanisms within RpoN flagellar regulons that fine-tunegene expression so that components of the flagellum aremadeprecisely when needed What are the cellular cues sensed by

10 Scientifica

the regulatory proteins that control expression of the RpoN-dependent flagellar genes How does the flagellar exportapparatus control expression of RpoN-dependent flagellargenes in various bacteria Does FlgZ have a regulatoryrole in flagellar biogenesis in H pylori and other bacteriaAs our understanding of the molecular genetics of diversebacterial species expands novel regulatory mechanisms thatcontrol expression of flagellar genes within these bacteria willundoubtedly come to light For example it was only recentlythat sRNAs were identified in H pylori some of whichare potentially FliA dependent [127] and may have roles inflagellar biogenesis Finally with increasingly sophisticatedmethods in bioinformatics genomics and proteomics fur-ther characterization of flagellar regulatory networks willprovide important insights into the mechanisms governinggene expression and identify new regulatory proteins andmechanisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by award MCB-1244242 from theNational Science Foundation

References

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[2] J L McMurry J W Murphy and B Gonzalez-Pedrajo ldquoTheFliN-FliH interaction mediates localization of flagellar exportATPase FliI to the C ring complexrdquo Biochemistry vol 45 no39 pp 11790ndash11798 2006

[3] R M Macnab Ed Flagella and Motility ASM Press 1996[4] T Minamino K Imada and K Namba ldquoMolecular motors of

the bacterial flagellardquoCurrent Opinion in Structural Biology vol18 no 6 pp 693ndash701 2008

[5] T G Smith and T R Hoover ldquoDeciphering bacterial flagellargene regulatory networks in the genomic erardquo Advances inApplied Microbiology vol 67 pp 257ndash295 2009

[6] J K Anderson T G Smith and T R Hoover ldquoSense andsensibility flagellum-mediated gene regulationrdquo Trends inMicrobiology vol 18 no 1 pp 30ndash37 2010

[7] J D Helmann ldquoAlternative sigma factors and the regulation offlagellar gene expressionrdquoMolecular Microbiology vol 5 no 12pp 2875ndash2882 1991

[8] D L Popham D Szeto J Keener and S Kustu ldquoFunctionof a bacterial activator protein that binds to transcriptionalenhancersrdquo Science vol 243 no 4891 pp 629ndash635 1989

[9] S Sasse-Dwight and J D Gralla ldquoProbing the EscherichiacoliglnALG upstream activation mechanism in vivordquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8934ndash8938 1988

[10] M Kihara T Minamino S Yamaguchi and R M MacnabldquoIntergenic suppression between the flagellar MS ring protein

FliF of Salmonella and FlhA a membrane component of itsexport apparatusrdquo Journal of Bacteriology vol 183 no 5 pp1655ndash1662 2001

[11] J S van Arnam J L McMurry M Kihara and R M MacnabldquoAnalysis of an engineered Salmonella flagellar fusion proteinFliR-FlhBrdquo Journal of Bacteriology vol 186 no 8 pp 2495ndash2498 2004

[12] C S Barker I V Meshcheryakova A S Kostyukova and FA Samatey ldquoFliO regulation of FliP in the formation of theSalmonella enterica flagellumrdquo PLoS Genetics vol 6 no 9Article ID e1001143 2010

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[14] R M Macnab ldquoType III flagellar protein export and flagellarassemblyrdquo Biochimica et Biophysica Acta vol 1694 no 1ndash3 pp207ndash217 2004

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[18] TMorooka A Umeda andK Amako ldquoMotility as an intestinalcolonization factor forCampylobacter jejunirdquo Journal of GeneralMicrobiology vol 131 no 8 pp 1973ndash1980 1985

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[23] A Kusumoto K Kamisaka T Yakushi H Terashima AShinohara and M Homma ldquoRegulation of polar flagellarnumber by the flhF and flhG genes in Vibrio alginolyticusrdquoJournal of Biochemistry vol 139 no 1 pp 113ndash121 2006

[24] B M Pruszlig and P Matsumura ldquoA regulator of the flagellarregulon of Escherichia coli flhD also affects cell divisionrdquoJournal of Bacteriology vol 178 no 3 pp 668ndash674 1996

[25] B M Pruszlig and P Matsumura ldquoCell cycle regulation of flagellargenesrdquo Journal of Bacteriology vol 179 no 17 pp 5602ndash56041997

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Scientifica 11

NtrC homologrdquo Journal of Bacteriology vol 181 no 2 pp 593ndash599 1999

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[28] D Beier and R Frank ldquoMolecular characterization of two-component systems of Helicobacter pylorirdquo Journal of Bacteri-ology vol 182 no 8 pp 2068ndash2076 2000

[29] P Brahmachary M G Dashti J W Olson and T R HooverldquoHelicobacter pylori FlgR is an enhancer-independent activatorof 12059054-RNA polymerase holoenzymerdquo Journal of Bacteriologyvol 186 no 14 pp 4535ndash4542 2004

[30] M M S M Wosten J A Wagenaar and J P M van PuttenldquoThe FlgSFlgR two-component signal transduction systemregulates the fla regulon in Campylobacter jejunirdquo The Journalof Biological Chemistry vol 279 no 16 pp 16214ndash16222 2004

[31] M Bush and R Dixon ldquoThe role of bacterial enhancer bindingproteins as specialized activators of 12059054-dependent transcrip-tionrdquo MicroBiology and Molecular Biology Reviews vol 76 no3 pp 497ndash529 2012

[32] J Schumacher N Joly M Rappas X Zhang and M BuckldquoStructures and organisation of AAA+ enhancer binding pro-teins in transcriptional activationrdquo Journal of Structural Biologyvol 156 no 1 pp 190ndash199 2006

[33] S N Joslin and D R Hendrixson ldquoAnalysis of the Campy-lobacter jejuni FlgR response regulator suggests integration ofdiverse mechanisms to activate an NtrC-like proteinrdquo Journalof Bacteriology vol 190 no 7 pp 2422ndash2433 2008

[34] J M Boll and D R Hendrixson ldquoA specificity determinant forphosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphaterdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 50 pp 20160ndash20165 2011

[35] S Porwollik B Noonan and P W OrsquoToole ldquoMolecular char-acterization of a flagellar export locus of Helicobacter pylorirdquoInfection and Immunity vol 67 no 5 pp 2060ndash2070 1999

[36] E Allan N Dorrell S Foynes M Anyim and B W WrenldquoMutational analysis of genes encoding the early flagellarcomponents of Helicobacter pylori evidence for transcriptionalregulation of flagellin A biosynthesisrdquo Journal of Bacteriologyvol 182 no 18 pp 5274ndash5277 2000

[37] T G Smith L Pereira and T R Hoover ldquoHelicobacter pyloriFlhB processing-deficient variants affect flagellar assembly butnot flagellar gene expressionrdquo Microbiology vol 155 no 4 pp1170ndash1180 2009

[38] D R Hendrixson B J Akerley and V J DiRita ldquoTranspo-son mutagenesis of Campylobacter jejuni identifies a bipartiteenergy taxis system required for motilityrdquoMolecular Microbiol-ogy vol 40 no 1 pp 214ndash224 2001

[39] J Tsang T G Smith L E Pereira and T R Hoover ldquoInsertionmutations inHelicobacter pylori flhA reveal strain differences inRpoN-dependent gene expressionrdquo Microbiology vol 159 part1 pp 58ndash67 2013

[40] J M Boll and D R Hendrixson ldquoA regulatory checkpointduring flagellar biogenesis in Campylobacter jejuni initiatessignal transduction to activate transcription of flagellar genesrdquoMBio vol 4 no 5 Article ID e00432-13 2013

[41] T Minamino H Doi and K Kutsukake ldquoSubstrate specificityswitching of the flagellum-specific export apparatus during flag-ellar morphogenesis in Salmonella typhimuriumrdquo Bioscience

Biotechnology and Biochemistry vol 63 no 7 pp 1301ndash13031999

[42] M Erhardt H M Singer D H Wee J P Keener and K THughes ldquoAn infrequent molecular ruler controls flagellar hooklength in Salmonella entericardquo EMBO Journal vol 30 no 14 pp2948ndash2961 2011

[43] K T Hughes K L Gillen M J Semon and J E KarlinseyldquoSensing structural intermediates in bacterial flagellar assemblyby export of a negative regulatorrdquo Science vol 262 no 5137 pp1277ndash1280 1993

[44] K A Ryan N Karim M Worku S A Moore C W Pennand P W OrsquoToole ldquoHP0958 is an essential motility gene inHelicobacter pylorirdquo FEMS Microbiology Letters vol 248 no 1pp 47ndash55 2005

[45] L Pereira and T R Hoover ldquoStable accumulation of 12059054 inHelicobacter pylori requires the novel protein HP0958rdquo Journalof Bacteriology vol 187 no 13 pp 4463ndash4469 2005

[46] J C Rain L Selig H de Reuse et al ldquoThe protein-proteininteraction map of Helicobacter pylorirdquo Nature vol 409 no6817 pp 211ndash215 2001

[47] F P Douillard K A Ryan D L Caly et al ldquoPosttranscriptionalregulation of flagellin synthesis in Helicobacter pylori by theRpoN chaperone HP0958rdquo Journal of Bacteriology vol 190 no24 pp 7975ndash7984 2008

[48] C Josenhans E Niehus S Amersbach et al ldquoFunctionalcharacterization of the antagonistic flagellar late regulators FliAand FlgM ofHelicobacter pylori and their effects on theH pyloritranscriptomerdquoMolecular Microbiology vol 43 no 2 pp 307ndash322 2002

[49] M Rust S Borchert E Niehus et al ldquoThe Helicobacter pylorianti-sigma factor FlgM is predominantly cytoplasmic andcooperates with the flagellar basal body protein FlhArdquo Journalof Bacteriology vol 191 no 15 pp 4824ndash4834 2009

[50] D R Hendrixson and V J DiRita ldquoTranscription of 12059054-dependent but not 12059028-dependent flagellar genes in Campy-lobacter jejuni is associated with formation of the flagellarsecretory apparatusrdquoMolecular Microbiology vol 50 no 2 pp687ndash702 2003

[51] M M S M Wosten L van Dijk A K J Veenendaal MR de Zoete N M C Bleumink-Pluijm and J P M vanPutten ldquoTemperature-dependent FlgMFliA complex forma-tion regulates Campylobacter jejuni flagella lengthrdquo MolecularMicrobiology vol 75 no 6 pp 1577ndash1591 2010

[52] M G Prouty N E Correa and K E Klose ldquoThe novel 12059054- and12059028-dependent flagellar gene transcription hierarchy of Vibrio

choleraerdquo Molecular Microbiology vol 39 no 6 pp 1595ndash16092001

[53] D Srivastava M L Hsieh A Khataokar M B Neiditch and CM Waters ldquoCyclic di-GMP inhibits Vibrio choleraemotility byrepressing induction of transcription and inducing extracellularpolysaccharide productionrdquo Molecular Microbiology vol 90no 6 pp 1262ndash1276 2013

[54] N E Correa C M Lauriano R McGee and K E KloseldquoPhosphorylation of the flagellar regulatory protein FlrC is nec-essary for Vibrio cholerae motility and enhanced colonizationrdquoMolecular Microbiology vol 35 no 4 pp 743ndash755 2000

[55] N E Correa J R Barker and K E Klose ldquoThe Vibrio choleraeFlgM homologue is an anti-12059028 factor that is secreted throughthe sheathed polar flagellumrdquo Journal of Bacteriology vol 186no 14 pp 4613ndash4619 2004

12 Scientifica

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[60] S Ulitzur ldquoInduction of swarming in Vibrio parahaemolyticusrdquoArchives of Microbiology vol 101 no 4 pp 357ndash363 1974

[61] K S Park M Arita T Iida and T Honda ldquovpaH a geneencoding a novel histone-like nucleoid structure-like proteinthat was possibly horizontally acquired regulates the biogen-esis of lateral flagella in trh-positive Vibrio parahaemolyticusTH3996rdquo Infection and Immunity vol 73 no 9 pp 5754ndash57612005

[62] LMcCarter andM Silverman ldquoIron regulation of swarmer celldifferentiation ofVibrio parahaemolyticusrdquo Journal of Bacteriol-ogy vol 171 no 2 pp 731ndash736 1989

[63] B J Stewart and L L McCarter ldquoLateral flagellar gene systemofVibrio parahaemolyticusrdquo Journal of Bacteriology vol 185 no15 pp 4508ndash4518 2003

[64] C J Gode-Potratz R J Kustusch P J Breheny D S Weissand L LMcCarter ldquoSurface sensing inVibrio parahaemolyticustriggers a programme of gene expression that promotes colo-nization and virulencerdquo Molecular Microbiology vol 79 no 1pp 240ndash263 2011

[65] J L Veesenmeyer A R Hauser T Lisboa and J Rello ldquoPseu-domonas aeruginosa virulence and therapy evolving transla-tional strategiesrdquo Critical Care Medicine vol 37 no 5 pp 1777ndash1786 2009

[66] D Balasubramanian L Schneper H Kumari and K MatheeldquoA dynamic and intricate regulatory network determines Pseu-domonas aeruginosa virulencerdquo Nucleic Acids Research vol 41no 1 pp 1ndash20 2013

[67] T C Montie D D Huntzinger R C Craven and I A HolderldquoLoss of virulence associated with absence of flagellum in anisogenic mutant of Pseudomonas aeruginosa in the burned-mouse modelrdquo Infection and Immunity vol 38 no 3 pp 1296ndash1298 1982

[68] Y R Patankar R R Lovewell M E Poynter J Jyot BI Kazmierczak and B Berwin ldquoFlagellar motility is a keydeterminant of themagnitude of the inflammasome response toPseudomonas aeruginosardquo Infection and Immunity vol 81 no 6pp 2043ndash2052 2013

[69] G A OrsquoToole and R Kolter ldquoFlagellar and twitching motilityare necessary for Pseudomonas aeruginosa biofilm develop-mentrdquoMolecular Microbiology vol 30 no 2 pp 295ndash304 1998

[70] K B Barken S J Pamp L Yang et al ldquoRoles of type IVpili flagellum-mediated motility and extracellular DNA in theformation of mature multicellular structures in Pseudomonasaeruginosa biofilmsrdquo Environmental Microbiology vol 10 no 9pp 2331ndash2343 2008

[71] M Leeman J A van Pelt F M den Ouden M Heinsbroek PA Bakker and B Schippers ldquoInduction of systemic resistance

against fusarium wilt of radish by lipopolysaccharides of Pseu-domonas fluorescensrdquo Phytopathology vol 85 no 9 pp 1021ndash1027 1995

[72] P Hsueh L Teng H Pan et al ldquoOutbreak of Pseudomonasfluorescens bacteremia among oncology patientsrdquo Journal ofClinical Microbiology vol 36 no 10 pp 2914ndash2917 1998

[73] VWongK Levi B Baddal J Turton andTC Boswell ldquoSpreadof Pseudomonas fluorescens due to contaminated drinkingwater in a bone marrow transplant unitrdquo Journal of ClinicalMicrobiology vol 49 no 6 pp 2093ndash2096 2011

[74] J W Hickman and C S Harwood ldquoIdentification of FleQ fromPseudomonas aeruginosa as a c-di-GMP-responsive transcrip-tion factorrdquoMolecular Microbiology vol 69 no 2 pp 376ndash3892008

[75] N Dasgupta M C Wolfgang A L Goodman et al ldquoA four-tiered transcriptional regulatory circuit controls flagellar bio-genesis in Pseudomonas aeruginosardquo Molecular Microbiologyvol 50 no 3 pp 809ndash824 2003

[76] S K Arora B W Ritchings E C Almira S Lory and RRamphal ldquoA transcriptional activator FleQ regulates mucinadhesion and flagellar gene expression in Pseudomonas aerugi-nosa in a cascade mannerrdquo Journal of Bacteriology vol 179 no17 pp 5574ndash5581 1997

[77] N Dasgupta and R Ramphal ldquoInteraction of the antiactivatorFleN with the transcriptional activator FleQ regulates flagellarnumber in Pseudomonas aeruginosardquo Journal of Bacteriologyvol 183 no 22 pp 6636ndash6644 2001

[78] N Dasgupta S K Arora and R Ramphal ldquofleN a gene thatregulates flagellar number in Pseudomonas aeruginosardquo Journalof Bacteriology vol 182 no 2 pp 357ndash364 2000

[79] J Malakooti and B Ely ldquoPrincipal sigma subunit of theCaulobacter crescentus RNA polymeraserdquo Journal of Bacteriol-ogy vol 177 no 23 pp 6854ndash6860 1995

[80] M T Laub H H McAdams T Feldblyum C M Fraser andL Shapiro ldquoGlobal analysis of the genetic network controllinga bacterial cell cyclerdquo Science vol 290 no 5499 pp 2144ndash21482000

[81] A K Benson G Ramakrishnan N Ohta J Feng A J Ninfaand A Newton ldquoThe Caulobacter crescentus FlbD proteinacts at ftr sequence elements both to activate and to represstranscription of cell cycle-regulated flagellar genesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 91 no 11 pp 4989ndash4993 1994

[82] D A Mullin S M van Way C A Blankenship and A HMullin ldquoFlbD has a DNA-binding activity near its carboxyterminus that recognizes ftr sequences involved in positive andnegative regulation of flagellar gene transcription in Caulobac-ter crescentusrdquo Journal of Bacteriology vol 176 no 19 pp 5971ndash5981 1994

[83] R J Dutton Z Xu and J W Gober ldquoLinking structural assem-bly to gene expression a novel mechanism for regulating theactivity of a 12059054 transcription factorrdquo Molecular Microbiologyvol 58 no 3 pp 743ndash757 2005

[84] R E Muir J Easter and J W Gober ldquoThe trans-acting flagellarregulatory proteins FliX and FlbD play a central role in linkingflagellar biogenesis and cytokinesis in Caulobacter crescentusrdquoMicrobiology vol 151 no 11 pp 3699ndash3711 2005

[85] P E Anderson and J W Gober ldquoFlbT the post-transcriptionalregulator of flagellin synthesis in Caulobacter crescentus inter-acts with the 51015840 untranslated region of flagellin mRNArdquoMolec-ular Microbiology vol 38 no 1 pp 41ndash52 2000

Scientifica 13

[86] A T Nielsen N A Dolganov G Otto M C Miller C Y WuandGK Schoolnik ldquoRpoS controls theVibrio choleraemucosalescape responserdquo PLoS Pathogens vol 2 no 10 article e1092006

[87] M T Laub S L Chen L Shapiro and H H McAdamsldquoGenes directly controlled by CtrA a master regulator of theCaulobacter cell cyclerdquo Proceedings of the National Academy ofSciences of the United States of America vol 99 no 7 pp 4632ndash4637 2002

[88] A J Kelly M J Sackett N Din E Quardokus and Y VBrun ldquoCell cycle-dependent transcriptional and proteolyticregulation of FtsZ in Caulobacterrdquo Genes and Development vol12 no 6 pp 880ndash893 1998

[89] M Wortinger M J Sackett and Y V Brun ldquoCtrA mediatesa DNA replication checkpoint that prevents cell division inCaulobacter crescentusrdquo EMBO Journal vol 19 no 17 pp 4503ndash4512 2000

[90] R E Muir T M OrsquoBrien and J W Gober ldquoThe Caulobactercrescentus flagellar gene fliX encodes a novel trans-acting fac-tor that couples flagellar assembly to transcriptionrdquo MolecularMicrobiology vol 39 no 6 pp 1623ndash1637 2001

[91] M Llewellyn R J Dutton J Easter D OrsquoDonnol and J WGober ldquoThe conserved flaF gene has a critical role in couplingflagellin translation and assembly in Caulobacter crescentusrdquoMolecular Microbiology vol 57 no 4 pp 1127ndash1142 2005

[92] S M Kim D H Lee and S H Choi ldquoEvidence that the Vibriovulnificus flagellar regulator FlhF is regulated by a quorumsensing master regulator SmcRrdquo Microbiology vol 158 part 8pp 2017ndash2025 2012

[93] F Martınez-Granero A Navazo E Barahona M Redondo-Nieto R Rivilla andMMartın ldquoTheGac-Rsm and SadB signaltransduction pathways converge on Algu to downregulatemotility in Pseudomonas fluorescensrdquo PLoS ONE vol 7 no 2Article ID e31765 2012

[94] C Reimmann M Beyeler A Latifi et al ldquoThe global activatorGacA of Pseudomonas aeruginosa PAO positively controls theproduction of the autoinducer N-butyryl-homoserine lactoneand the formation of the virulence factors pyocyanin cyanideand lipaserdquo Molecular Microbiology vol 24 no 2 pp 309ndash3191997

[95] J Dubern and G V Bloemberg ldquoInfluence of environmentalconditions on putisolvins I and II production in Pseudomonasputida strain PCL1445rdquo FEMSMicrobiology Letters vol 263 no2 pp 169ndash175 2006

[96] M G Surette M B Miller and B L Bassler ldquoQuorum sensingin Escherichia coli Salmonella typhimurium andVibrio harveyia new family of genes responsible for autoinducer productionrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 96 no 4 pp 1639ndash1644 1999

[97] B A Rader S R Campagna M F Semmelhack B L Basslerand K Guillemin ldquoThe quorum-sensing molecule autoinducer2 regulates motility and flagellar morphogenesis inHelicobacterpylorirdquo Journal of Bacteriology vol 189 no 17 pp 6109ndash61172007

[98] YHe J G Frye T P Strobaugh Jr andCChen ldquoAnalysis of AI-2LuxS-dependent transcription in Campylobacter jejuni strain81-176rdquo Foodborne Pathogens and Disease vol 5 no 4 pp 399ndash415 2008

[99] B Jeon K Itoh N Misawa and S Ryu ldquoEffects of quorumsensing on flaA transcription and autoagglutination in Campy-lobacter jejunirdquoMicrobiology and Immunology vol 47 no 11 pp833ndash839 2003

[100] N Dasgupta E P Ferrell K J Kanack S E H West andR Ramphal ldquofleQ the gene encoding the major flagellarregulator of Pseudomonas aeruginosa is 12059070 dependent and isdownregulated by Vfr a homolog of Escherichia coli cyclic AMPreceptor proteinrdquo Journal of Bacteriology vol 184 no 19 pp5240ndash5250 2002

[101] J Zhu M B Miller R E Vance M Dziejman B L Bassler andJ J Mekalanos ldquoQuorum-sensing regulators control virulencegene expression in Vibrio choleraerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no5 pp 3129ndash3134 2002

[102] Z Liu T Miyashiro A Tsou A Hsiao M Goulian and JZhu ldquoMucosal penetration primes Vibrio cholerae for hostcolonization by repressing quorum sensingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 28 pp 9769ndash9774 2008

[103] K E Klose V Novik and J J Mekalanos ldquoIdentificationof multiple 12059054-dependent transcriptional activators in Vibriocholeraerdquo Journal of Bacteriology vol 180 no 19 pp 5256ndash52591998

[104] I Kawagishi M Imagawa Y Imae L McCarter and MHomma ldquoThe sodium-driven polar flagellar motor of marineVibrio as the mechanosensor that regulates lateral flagellarexpressionrdquoMolecular Microbiology vol 20 no 4 pp 693ndash6991996

[105] T J Kirn B A Jude and R K Taylor ldquoA colonizationfactor links Vibrio cholerae environmental survival and humaninfectionrdquo Nature vol 438 no 7069 pp 863ndash866 2005

[106] Z Kuang YHao S Hwang et al ldquoThe Pseudomonas aeruginosaflagellum confers resistance to pulmonary surfactant protein-Aby impacting the production of exoproteases through quorum-sensingrdquo Molecular Microbiology vol 79 no 5 pp 1220ndash12352011

[107] S Zhang F X McCormack R C Levesque G A OrsquoTooleand G W Lau ldquoThe flagellum of Pseudomonas aeruginosa isrequired for resistance to clearance by surfactant protein ArdquoPLoS ONE vol 2 no 6 article e564 2007

[108] G W Lau D J Hassett H Ran and F Kong ldquoThe role ofpyocyanin in Pseudomonas aeruginosa infectionrdquo Trends inMolecular Medicine vol 10 no 12 pp 599ndash606 2004

[109] GW LauH Ran F Kong D J Hassett andDMavrodi ldquoPseu-domonas aeruginosa pyocyanin is critical for lung infection inmicerdquo Infection and Immunity vol 72 no 7 pp 4275ndash42782004

[110] D S Merrell M L Goodrich G Otto L S Tompkins and SFalkow ldquopH-regulated gene expression of the gastric pathogenHelicobacter pylorirdquo Infection and Immunity vol 71 no 6 pp3529ndash3539 2003

[111] K J Allen and M W Griffiths ldquoEffect of environmental andchemotactic stimuli on the activity of the Campylobacter jejuniflaA 12059028 promoterrdquo FEMS Microbiology Letters vol 205 no 1pp 43ndash48 2001

[112] Y Wen J Feng D R Scott E A Marcus and G SachsldquoThe pH-responsive regulon of HP0244 (FlgS) the cytoplasmichistidine kinase of Helicobacter pylorirdquo Journal of Bacteriologyvol 191 no 2 pp 449ndash460 2009

[113] D R Hendrixson ldquoA phase-variable mechanism controllingthe Campylobacter jejuni FlgR response regulator influencescommensalismrdquo Molecular Microbiology vol 61 no 6 pp1646ndash1659 2006

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 Scientifica

the regulatory proteins that control expression of the RpoN-dependent flagellar genes How does the flagellar exportapparatus control expression of RpoN-dependent flagellargenes in various bacteria Does FlgZ have a regulatoryrole in flagellar biogenesis in H pylori and other bacteriaAs our understanding of the molecular genetics of diversebacterial species expands novel regulatory mechanisms thatcontrol expression of flagellar genes within these bacteria willundoubtedly come to light For example it was only recentlythat sRNAs were identified in H pylori some of whichare potentially FliA dependent [127] and may have roles inflagellar biogenesis Finally with increasingly sophisticatedmethods in bioinformatics genomics and proteomics fur-ther characterization of flagellar regulatory networks willprovide important insights into the mechanisms governinggene expression and identify new regulatory proteins andmechanisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by award MCB-1244242 from theNational Science Foundation

References

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Scientifica 11

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Biotechnology and Biochemistry vol 63 no 7 pp 1301ndash13031999

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[50] D R Hendrixson and V J DiRita ldquoTranscription of 12059054-dependent but not 12059028-dependent flagellar genes in Campy-lobacter jejuni is associated with formation of the flagellarsecretory apparatusrdquoMolecular Microbiology vol 50 no 2 pp687ndash702 2003

[51] M M S M Wosten L van Dijk A K J Veenendaal MR de Zoete N M C Bleumink-Pluijm and J P M vanPutten ldquoTemperature-dependent FlgMFliA complex forma-tion regulates Campylobacter jejuni flagella lengthrdquo MolecularMicrobiology vol 75 no 6 pp 1577ndash1591 2010

[52] M G Prouty N E Correa and K E Klose ldquoThe novel 12059054- and12059028-dependent flagellar gene transcription hierarchy of Vibrio

choleraerdquo Molecular Microbiology vol 39 no 6 pp 1595ndash16092001

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12 Scientifica

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[69] G A OrsquoToole and R Kolter ldquoFlagellar and twitching motilityare necessary for Pseudomonas aeruginosa biofilm develop-mentrdquoMolecular Microbiology vol 30 no 2 pp 295ndash304 1998

[70] K B Barken S J Pamp L Yang et al ldquoRoles of type IVpili flagellum-mediated motility and extracellular DNA in theformation of mature multicellular structures in Pseudomonasaeruginosa biofilmsrdquo Environmental Microbiology vol 10 no 9pp 2331ndash2343 2008

[71] M Leeman J A van Pelt F M den Ouden M Heinsbroek PA Bakker and B Schippers ldquoInduction of systemic resistance

against fusarium wilt of radish by lipopolysaccharides of Pseu-domonas fluorescensrdquo Phytopathology vol 85 no 9 pp 1021ndash1027 1995

[72] P Hsueh L Teng H Pan et al ldquoOutbreak of Pseudomonasfluorescens bacteremia among oncology patientsrdquo Journal ofClinical Microbiology vol 36 no 10 pp 2914ndash2917 1998

[73] VWongK Levi B Baddal J Turton andTC Boswell ldquoSpreadof Pseudomonas fluorescens due to contaminated drinkingwater in a bone marrow transplant unitrdquo Journal of ClinicalMicrobiology vol 49 no 6 pp 2093ndash2096 2011

[74] J W Hickman and C S Harwood ldquoIdentification of FleQ fromPseudomonas aeruginosa as a c-di-GMP-responsive transcrip-tion factorrdquoMolecular Microbiology vol 69 no 2 pp 376ndash3892008

[75] N Dasgupta M C Wolfgang A L Goodman et al ldquoA four-tiered transcriptional regulatory circuit controls flagellar bio-genesis in Pseudomonas aeruginosardquo Molecular Microbiologyvol 50 no 3 pp 809ndash824 2003

[76] S K Arora B W Ritchings E C Almira S Lory and RRamphal ldquoA transcriptional activator FleQ regulates mucinadhesion and flagellar gene expression in Pseudomonas aerugi-nosa in a cascade mannerrdquo Journal of Bacteriology vol 179 no17 pp 5574ndash5581 1997

[77] N Dasgupta and R Ramphal ldquoInteraction of the antiactivatorFleN with the transcriptional activator FleQ regulates flagellarnumber in Pseudomonas aeruginosardquo Journal of Bacteriologyvol 183 no 22 pp 6636ndash6644 2001

[78] N Dasgupta S K Arora and R Ramphal ldquofleN a gene thatregulates flagellar number in Pseudomonas aeruginosardquo Journalof Bacteriology vol 182 no 2 pp 357ndash364 2000

[79] J Malakooti and B Ely ldquoPrincipal sigma subunit of theCaulobacter crescentus RNA polymeraserdquo Journal of Bacteriol-ogy vol 177 no 23 pp 6854ndash6860 1995

[80] M T Laub H H McAdams T Feldblyum C M Fraser andL Shapiro ldquoGlobal analysis of the genetic network controllinga bacterial cell cyclerdquo Science vol 290 no 5499 pp 2144ndash21482000

[81] A K Benson G Ramakrishnan N Ohta J Feng A J Ninfaand A Newton ldquoThe Caulobacter crescentus FlbD proteinacts at ftr sequence elements both to activate and to represstranscription of cell cycle-regulated flagellar genesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 91 no 11 pp 4989ndash4993 1994

[82] D A Mullin S M van Way C A Blankenship and A HMullin ldquoFlbD has a DNA-binding activity near its carboxyterminus that recognizes ftr sequences involved in positive andnegative regulation of flagellar gene transcription in Caulobac-ter crescentusrdquo Journal of Bacteriology vol 176 no 19 pp 5971ndash5981 1994

[83] R J Dutton Z Xu and J W Gober ldquoLinking structural assem-bly to gene expression a novel mechanism for regulating theactivity of a 12059054 transcription factorrdquo Molecular Microbiologyvol 58 no 3 pp 743ndash757 2005

[84] R E Muir J Easter and J W Gober ldquoThe trans-acting flagellarregulatory proteins FliX and FlbD play a central role in linkingflagellar biogenesis and cytokinesis in Caulobacter crescentusrdquoMicrobiology vol 151 no 11 pp 3699ndash3711 2005

[85] P E Anderson and J W Gober ldquoFlbT the post-transcriptionalregulator of flagellin synthesis in Caulobacter crescentus inter-acts with the 51015840 untranslated region of flagellin mRNArdquoMolec-ular Microbiology vol 38 no 1 pp 41ndash52 2000

Scientifica 13

[86] A T Nielsen N A Dolganov G Otto M C Miller C Y WuandGK Schoolnik ldquoRpoS controls theVibrio choleraemucosalescape responserdquo PLoS Pathogens vol 2 no 10 article e1092006

[87] M T Laub S L Chen L Shapiro and H H McAdamsldquoGenes directly controlled by CtrA a master regulator of theCaulobacter cell cyclerdquo Proceedings of the National Academy ofSciences of the United States of America vol 99 no 7 pp 4632ndash4637 2002

[88] A J Kelly M J Sackett N Din E Quardokus and Y VBrun ldquoCell cycle-dependent transcriptional and proteolyticregulation of FtsZ in Caulobacterrdquo Genes and Development vol12 no 6 pp 880ndash893 1998

[89] M Wortinger M J Sackett and Y V Brun ldquoCtrA mediatesa DNA replication checkpoint that prevents cell division inCaulobacter crescentusrdquo EMBO Journal vol 19 no 17 pp 4503ndash4512 2000

[90] R E Muir T M OrsquoBrien and J W Gober ldquoThe Caulobactercrescentus flagellar gene fliX encodes a novel trans-acting fac-tor that couples flagellar assembly to transcriptionrdquo MolecularMicrobiology vol 39 no 6 pp 1623ndash1637 2001

[91] M Llewellyn R J Dutton J Easter D OrsquoDonnol and J WGober ldquoThe conserved flaF gene has a critical role in couplingflagellin translation and assembly in Caulobacter crescentusrdquoMolecular Microbiology vol 57 no 4 pp 1127ndash1142 2005

[92] S M Kim D H Lee and S H Choi ldquoEvidence that the Vibriovulnificus flagellar regulator FlhF is regulated by a quorumsensing master regulator SmcRrdquo Microbiology vol 158 part 8pp 2017ndash2025 2012

[93] F Martınez-Granero A Navazo E Barahona M Redondo-Nieto R Rivilla andMMartın ldquoTheGac-Rsm and SadB signaltransduction pathways converge on Algu to downregulatemotility in Pseudomonas fluorescensrdquo PLoS ONE vol 7 no 2Article ID e31765 2012

[94] C Reimmann M Beyeler A Latifi et al ldquoThe global activatorGacA of Pseudomonas aeruginosa PAO positively controls theproduction of the autoinducer N-butyryl-homoserine lactoneand the formation of the virulence factors pyocyanin cyanideand lipaserdquo Molecular Microbiology vol 24 no 2 pp 309ndash3191997

[95] J Dubern and G V Bloemberg ldquoInfluence of environmentalconditions on putisolvins I and II production in Pseudomonasputida strain PCL1445rdquo FEMSMicrobiology Letters vol 263 no2 pp 169ndash175 2006

[96] M G Surette M B Miller and B L Bassler ldquoQuorum sensingin Escherichia coli Salmonella typhimurium andVibrio harveyia new family of genes responsible for autoinducer productionrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 96 no 4 pp 1639ndash1644 1999

[97] B A Rader S R Campagna M F Semmelhack B L Basslerand K Guillemin ldquoThe quorum-sensing molecule autoinducer2 regulates motility and flagellar morphogenesis inHelicobacterpylorirdquo Journal of Bacteriology vol 189 no 17 pp 6109ndash61172007

[98] YHe J G Frye T P Strobaugh Jr andCChen ldquoAnalysis of AI-2LuxS-dependent transcription in Campylobacter jejuni strain81-176rdquo Foodborne Pathogens and Disease vol 5 no 4 pp 399ndash415 2008

[99] B Jeon K Itoh N Misawa and S Ryu ldquoEffects of quorumsensing on flaA transcription and autoagglutination in Campy-lobacter jejunirdquoMicrobiology and Immunology vol 47 no 11 pp833ndash839 2003

[100] N Dasgupta E P Ferrell K J Kanack S E H West andR Ramphal ldquofleQ the gene encoding the major flagellarregulator of Pseudomonas aeruginosa is 12059070 dependent and isdownregulated by Vfr a homolog of Escherichia coli cyclic AMPreceptor proteinrdquo Journal of Bacteriology vol 184 no 19 pp5240ndash5250 2002

[101] J Zhu M B Miller R E Vance M Dziejman B L Bassler andJ J Mekalanos ldquoQuorum-sensing regulators control virulencegene expression in Vibrio choleraerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no5 pp 3129ndash3134 2002

[102] Z Liu T Miyashiro A Tsou A Hsiao M Goulian and JZhu ldquoMucosal penetration primes Vibrio cholerae for hostcolonization by repressing quorum sensingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 28 pp 9769ndash9774 2008

[103] K E Klose V Novik and J J Mekalanos ldquoIdentificationof multiple 12059054-dependent transcriptional activators in Vibriocholeraerdquo Journal of Bacteriology vol 180 no 19 pp 5256ndash52591998

[104] I Kawagishi M Imagawa Y Imae L McCarter and MHomma ldquoThe sodium-driven polar flagellar motor of marineVibrio as the mechanosensor that regulates lateral flagellarexpressionrdquoMolecular Microbiology vol 20 no 4 pp 693ndash6991996

[105] T J Kirn B A Jude and R K Taylor ldquoA colonizationfactor links Vibrio cholerae environmental survival and humaninfectionrdquo Nature vol 438 no 7069 pp 863ndash866 2005

[106] Z Kuang YHao S Hwang et al ldquoThe Pseudomonas aeruginosaflagellum confers resistance to pulmonary surfactant protein-Aby impacting the production of exoproteases through quorum-sensingrdquo Molecular Microbiology vol 79 no 5 pp 1220ndash12352011

[107] S Zhang F X McCormack R C Levesque G A OrsquoTooleand G W Lau ldquoThe flagellum of Pseudomonas aeruginosa isrequired for resistance to clearance by surfactant protein ArdquoPLoS ONE vol 2 no 6 article e564 2007

[108] G W Lau D J Hassett H Ran and F Kong ldquoThe role ofpyocyanin in Pseudomonas aeruginosa infectionrdquo Trends inMolecular Medicine vol 10 no 12 pp 599ndash606 2004

[109] GW LauH Ran F Kong D J Hassett andDMavrodi ldquoPseu-domonas aeruginosa pyocyanin is critical for lung infection inmicerdquo Infection and Immunity vol 72 no 7 pp 4275ndash42782004

[110] D S Merrell M L Goodrich G Otto L S Tompkins and SFalkow ldquopH-regulated gene expression of the gastric pathogenHelicobacter pylorirdquo Infection and Immunity vol 71 no 6 pp3529ndash3539 2003

[111] K J Allen and M W Griffiths ldquoEffect of environmental andchemotactic stimuli on the activity of the Campylobacter jejuniflaA 12059028 promoterrdquo FEMS Microbiology Letters vol 205 no 1pp 43ndash48 2001

[112] Y Wen J Feng D R Scott E A Marcus and G SachsldquoThe pH-responsive regulon of HP0244 (FlgS) the cytoplasmichistidine kinase of Helicobacter pylorirdquo Journal of Bacteriologyvol 191 no 2 pp 449ndash460 2009

[113] D R Hendrixson ldquoA phase-variable mechanism controllingthe Campylobacter jejuni FlgR response regulator influencescommensalismrdquo Molecular Microbiology vol 61 no 6 pp1646ndash1659 2006

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Scientifica 11

NtrC homologrdquo Journal of Bacteriology vol 181 no 2 pp 593ndash599 1999

[27] M Schirm E C Soo A J Aubry J Austin P Thibault and SM Logan ldquoStructural genetic and functional characterizationof the flagellin glycosylation process in Helicobacter pylorirdquoMolecular Microbiology vol 48 no 6 pp 1579ndash1592 2003

[28] D Beier and R Frank ldquoMolecular characterization of two-component systems of Helicobacter pylorirdquo Journal of Bacteri-ology vol 182 no 8 pp 2068ndash2076 2000

[29] P Brahmachary M G Dashti J W Olson and T R HooverldquoHelicobacter pylori FlgR is an enhancer-independent activatorof 12059054-RNA polymerase holoenzymerdquo Journal of Bacteriologyvol 186 no 14 pp 4535ndash4542 2004

[30] M M S M Wosten J A Wagenaar and J P M van PuttenldquoThe FlgSFlgR two-component signal transduction systemregulates the fla regulon in Campylobacter jejunirdquo The Journalof Biological Chemistry vol 279 no 16 pp 16214ndash16222 2004

[31] M Bush and R Dixon ldquoThe role of bacterial enhancer bindingproteins as specialized activators of 12059054-dependent transcrip-tionrdquo MicroBiology and Molecular Biology Reviews vol 76 no3 pp 497ndash529 2012

[32] J Schumacher N Joly M Rappas X Zhang and M BuckldquoStructures and organisation of AAA+ enhancer binding pro-teins in transcriptional activationrdquo Journal of Structural Biologyvol 156 no 1 pp 190ndash199 2006

[33] S N Joslin and D R Hendrixson ldquoAnalysis of the Campy-lobacter jejuni FlgR response regulator suggests integration ofdiverse mechanisms to activate an NtrC-like proteinrdquo Journalof Bacteriology vol 190 no 7 pp 2422ndash2433 2008

[34] J M Boll and D R Hendrixson ldquoA specificity determinant forphosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphaterdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 50 pp 20160ndash20165 2011

[35] S Porwollik B Noonan and P W OrsquoToole ldquoMolecular char-acterization of a flagellar export locus of Helicobacter pylorirdquoInfection and Immunity vol 67 no 5 pp 2060ndash2070 1999

[36] E Allan N Dorrell S Foynes M Anyim and B W WrenldquoMutational analysis of genes encoding the early flagellarcomponents of Helicobacter pylori evidence for transcriptionalregulation of flagellin A biosynthesisrdquo Journal of Bacteriologyvol 182 no 18 pp 5274ndash5277 2000

[37] T G Smith L Pereira and T R Hoover ldquoHelicobacter pyloriFlhB processing-deficient variants affect flagellar assembly butnot flagellar gene expressionrdquo Microbiology vol 155 no 4 pp1170ndash1180 2009

[38] D R Hendrixson B J Akerley and V J DiRita ldquoTranspo-son mutagenesis of Campylobacter jejuni identifies a bipartiteenergy taxis system required for motilityrdquoMolecular Microbiol-ogy vol 40 no 1 pp 214ndash224 2001

[39] J Tsang T G Smith L E Pereira and T R Hoover ldquoInsertionmutations inHelicobacter pylori flhA reveal strain differences inRpoN-dependent gene expressionrdquo Microbiology vol 159 part1 pp 58ndash67 2013

[40] J M Boll and D R Hendrixson ldquoA regulatory checkpointduring flagellar biogenesis in Campylobacter jejuni initiatessignal transduction to activate transcription of flagellar genesrdquoMBio vol 4 no 5 Article ID e00432-13 2013

[41] T Minamino H Doi and K Kutsukake ldquoSubstrate specificityswitching of the flagellum-specific export apparatus during flag-ellar morphogenesis in Salmonella typhimuriumrdquo Bioscience

Biotechnology and Biochemistry vol 63 no 7 pp 1301ndash13031999

[42] M Erhardt H M Singer D H Wee J P Keener and K THughes ldquoAn infrequent molecular ruler controls flagellar hooklength in Salmonella entericardquo EMBO Journal vol 30 no 14 pp2948ndash2961 2011

[43] K T Hughes K L Gillen M J Semon and J E KarlinseyldquoSensing structural intermediates in bacterial flagellar assemblyby export of a negative regulatorrdquo Science vol 262 no 5137 pp1277ndash1280 1993

[44] K A Ryan N Karim M Worku S A Moore C W Pennand P W OrsquoToole ldquoHP0958 is an essential motility gene inHelicobacter pylorirdquo FEMS Microbiology Letters vol 248 no 1pp 47ndash55 2005

[45] L Pereira and T R Hoover ldquoStable accumulation of 12059054 inHelicobacter pylori requires the novel protein HP0958rdquo Journalof Bacteriology vol 187 no 13 pp 4463ndash4469 2005

[46] J C Rain L Selig H de Reuse et al ldquoThe protein-proteininteraction map of Helicobacter pylorirdquo Nature vol 409 no6817 pp 211ndash215 2001

[47] F P Douillard K A Ryan D L Caly et al ldquoPosttranscriptionalregulation of flagellin synthesis in Helicobacter pylori by theRpoN chaperone HP0958rdquo Journal of Bacteriology vol 190 no24 pp 7975ndash7984 2008

[48] C Josenhans E Niehus S Amersbach et al ldquoFunctionalcharacterization of the antagonistic flagellar late regulators FliAand FlgM ofHelicobacter pylori and their effects on theH pyloritranscriptomerdquoMolecular Microbiology vol 43 no 2 pp 307ndash322 2002

[49] M Rust S Borchert E Niehus et al ldquoThe Helicobacter pylorianti-sigma factor FlgM is predominantly cytoplasmic andcooperates with the flagellar basal body protein FlhArdquo Journalof Bacteriology vol 191 no 15 pp 4824ndash4834 2009

[50] D R Hendrixson and V J DiRita ldquoTranscription of 12059054-dependent but not 12059028-dependent flagellar genes in Campy-lobacter jejuni is associated with formation of the flagellarsecretory apparatusrdquoMolecular Microbiology vol 50 no 2 pp687ndash702 2003

[51] M M S M Wosten L van Dijk A K J Veenendaal MR de Zoete N M C Bleumink-Pluijm and J P M vanPutten ldquoTemperature-dependent FlgMFliA complex forma-tion regulates Campylobacter jejuni flagella lengthrdquo MolecularMicrobiology vol 75 no 6 pp 1577ndash1591 2010

[52] M G Prouty N E Correa and K E Klose ldquoThe novel 12059054- and12059028-dependent flagellar gene transcription hierarchy of Vibrio

choleraerdquo Molecular Microbiology vol 39 no 6 pp 1595ndash16092001

[53] D Srivastava M L Hsieh A Khataokar M B Neiditch and CM Waters ldquoCyclic di-GMP inhibits Vibrio choleraemotility byrepressing induction of transcription and inducing extracellularpolysaccharide productionrdquo Molecular Microbiology vol 90no 6 pp 1262ndash1276 2013

[54] N E Correa C M Lauriano R McGee and K E KloseldquoPhosphorylation of the flagellar regulatory protein FlrC is nec-essary for Vibrio cholerae motility and enhanced colonizationrdquoMolecular Microbiology vol 35 no 4 pp 743ndash755 2000

[55] N E Correa J R Barker and K E Klose ldquoThe Vibrio choleraeFlgM homologue is an anti-12059028 factor that is secreted throughthe sheathed polar flagellumrdquo Journal of Bacteriology vol 186no 14 pp 4613ndash4619 2004

12 Scientifica

[56] MMoisi C Jenul SM Butler et al ldquoA novel regulatory proteininvolved in motility of Vibrio choleraerdquo Journal of Bacteriologyvol 191 no 22 pp 7027ndash7038 2009

[57] L Aravind and C P Ponting ldquoThe cytoplasmic helical linkerdomain of receptor histidine kinase and methyl-acceptingproteins is common to many prokaryotic signalling proteinsrdquoFEMS Microbiology Letters vol 176 no 1 pp 111ndash116 1999

[58] SMerino J G Shaw and JM Tomas ldquoBacterial lateral flagellaan inducible flagella systemrdquo FEMS Microbiology Letters vol263 no 2 pp 127ndash135 2006

[59] L L McCarter ldquoDual flagellar systems enable motility underdifferent circumstancesrdquo Journal of Molecular Microbiology andBiotechnology vol 7 no 1-2 pp 18ndash29 2004

[60] S Ulitzur ldquoInduction of swarming in Vibrio parahaemolyticusrdquoArchives of Microbiology vol 101 no 4 pp 357ndash363 1974

[61] K S Park M Arita T Iida and T Honda ldquovpaH a geneencoding a novel histone-like nucleoid structure-like proteinthat was possibly horizontally acquired regulates the biogen-esis of lateral flagella in trh-positive Vibrio parahaemolyticusTH3996rdquo Infection and Immunity vol 73 no 9 pp 5754ndash57612005

[62] LMcCarter andM Silverman ldquoIron regulation of swarmer celldifferentiation ofVibrio parahaemolyticusrdquo Journal of Bacteriol-ogy vol 171 no 2 pp 731ndash736 1989

[63] B J Stewart and L L McCarter ldquoLateral flagellar gene systemofVibrio parahaemolyticusrdquo Journal of Bacteriology vol 185 no15 pp 4508ndash4518 2003

[64] C J Gode-Potratz R J Kustusch P J Breheny D S Weissand L LMcCarter ldquoSurface sensing inVibrio parahaemolyticustriggers a programme of gene expression that promotes colo-nization and virulencerdquo Molecular Microbiology vol 79 no 1pp 240ndash263 2011

[65] J L Veesenmeyer A R Hauser T Lisboa and J Rello ldquoPseu-domonas aeruginosa virulence and therapy evolving transla-tional strategiesrdquo Critical Care Medicine vol 37 no 5 pp 1777ndash1786 2009

[66] D Balasubramanian L Schneper H Kumari and K MatheeldquoA dynamic and intricate regulatory network determines Pseu-domonas aeruginosa virulencerdquo Nucleic Acids Research vol 41no 1 pp 1ndash20 2013

[67] T C Montie D D Huntzinger R C Craven and I A HolderldquoLoss of virulence associated with absence of flagellum in anisogenic mutant of Pseudomonas aeruginosa in the burned-mouse modelrdquo Infection and Immunity vol 38 no 3 pp 1296ndash1298 1982

[68] Y R Patankar R R Lovewell M E Poynter J Jyot BI Kazmierczak and B Berwin ldquoFlagellar motility is a keydeterminant of themagnitude of the inflammasome response toPseudomonas aeruginosardquo Infection and Immunity vol 81 no 6pp 2043ndash2052 2013

[69] G A OrsquoToole and R Kolter ldquoFlagellar and twitching motilityare necessary for Pseudomonas aeruginosa biofilm develop-mentrdquoMolecular Microbiology vol 30 no 2 pp 295ndash304 1998

[70] K B Barken S J Pamp L Yang et al ldquoRoles of type IVpili flagellum-mediated motility and extracellular DNA in theformation of mature multicellular structures in Pseudomonasaeruginosa biofilmsrdquo Environmental Microbiology vol 10 no 9pp 2331ndash2343 2008

[71] M Leeman J A van Pelt F M den Ouden M Heinsbroek PA Bakker and B Schippers ldquoInduction of systemic resistance

against fusarium wilt of radish by lipopolysaccharides of Pseu-domonas fluorescensrdquo Phytopathology vol 85 no 9 pp 1021ndash1027 1995

[72] P Hsueh L Teng H Pan et al ldquoOutbreak of Pseudomonasfluorescens bacteremia among oncology patientsrdquo Journal ofClinical Microbiology vol 36 no 10 pp 2914ndash2917 1998

[73] VWongK Levi B Baddal J Turton andTC Boswell ldquoSpreadof Pseudomonas fluorescens due to contaminated drinkingwater in a bone marrow transplant unitrdquo Journal of ClinicalMicrobiology vol 49 no 6 pp 2093ndash2096 2011

[74] J W Hickman and C S Harwood ldquoIdentification of FleQ fromPseudomonas aeruginosa as a c-di-GMP-responsive transcrip-tion factorrdquoMolecular Microbiology vol 69 no 2 pp 376ndash3892008

[75] N Dasgupta M C Wolfgang A L Goodman et al ldquoA four-tiered transcriptional regulatory circuit controls flagellar bio-genesis in Pseudomonas aeruginosardquo Molecular Microbiologyvol 50 no 3 pp 809ndash824 2003

[76] S K Arora B W Ritchings E C Almira S Lory and RRamphal ldquoA transcriptional activator FleQ regulates mucinadhesion and flagellar gene expression in Pseudomonas aerugi-nosa in a cascade mannerrdquo Journal of Bacteriology vol 179 no17 pp 5574ndash5581 1997

[77] N Dasgupta and R Ramphal ldquoInteraction of the antiactivatorFleN with the transcriptional activator FleQ regulates flagellarnumber in Pseudomonas aeruginosardquo Journal of Bacteriologyvol 183 no 22 pp 6636ndash6644 2001

[78] N Dasgupta S K Arora and R Ramphal ldquofleN a gene thatregulates flagellar number in Pseudomonas aeruginosardquo Journalof Bacteriology vol 182 no 2 pp 357ndash364 2000

[79] J Malakooti and B Ely ldquoPrincipal sigma subunit of theCaulobacter crescentus RNA polymeraserdquo Journal of Bacteriol-ogy vol 177 no 23 pp 6854ndash6860 1995

[80] M T Laub H H McAdams T Feldblyum C M Fraser andL Shapiro ldquoGlobal analysis of the genetic network controllinga bacterial cell cyclerdquo Science vol 290 no 5499 pp 2144ndash21482000

[81] A K Benson G Ramakrishnan N Ohta J Feng A J Ninfaand A Newton ldquoThe Caulobacter crescentus FlbD proteinacts at ftr sequence elements both to activate and to represstranscription of cell cycle-regulated flagellar genesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 91 no 11 pp 4989ndash4993 1994

[82] D A Mullin S M van Way C A Blankenship and A HMullin ldquoFlbD has a DNA-binding activity near its carboxyterminus that recognizes ftr sequences involved in positive andnegative regulation of flagellar gene transcription in Caulobac-ter crescentusrdquo Journal of Bacteriology vol 176 no 19 pp 5971ndash5981 1994

[83] R J Dutton Z Xu and J W Gober ldquoLinking structural assem-bly to gene expression a novel mechanism for regulating theactivity of a 12059054 transcription factorrdquo Molecular Microbiologyvol 58 no 3 pp 743ndash757 2005

[84] R E Muir J Easter and J W Gober ldquoThe trans-acting flagellarregulatory proteins FliX and FlbD play a central role in linkingflagellar biogenesis and cytokinesis in Caulobacter crescentusrdquoMicrobiology vol 151 no 11 pp 3699ndash3711 2005

[85] P E Anderson and J W Gober ldquoFlbT the post-transcriptionalregulator of flagellin synthesis in Caulobacter crescentus inter-acts with the 51015840 untranslated region of flagellin mRNArdquoMolec-ular Microbiology vol 38 no 1 pp 41ndash52 2000

Scientifica 13

[86] A T Nielsen N A Dolganov G Otto M C Miller C Y WuandGK Schoolnik ldquoRpoS controls theVibrio choleraemucosalescape responserdquo PLoS Pathogens vol 2 no 10 article e1092006

[87] M T Laub S L Chen L Shapiro and H H McAdamsldquoGenes directly controlled by CtrA a master regulator of theCaulobacter cell cyclerdquo Proceedings of the National Academy ofSciences of the United States of America vol 99 no 7 pp 4632ndash4637 2002

[88] A J Kelly M J Sackett N Din E Quardokus and Y VBrun ldquoCell cycle-dependent transcriptional and proteolyticregulation of FtsZ in Caulobacterrdquo Genes and Development vol12 no 6 pp 880ndash893 1998

[89] M Wortinger M J Sackett and Y V Brun ldquoCtrA mediatesa DNA replication checkpoint that prevents cell division inCaulobacter crescentusrdquo EMBO Journal vol 19 no 17 pp 4503ndash4512 2000

[90] R E Muir T M OrsquoBrien and J W Gober ldquoThe Caulobactercrescentus flagellar gene fliX encodes a novel trans-acting fac-tor that couples flagellar assembly to transcriptionrdquo MolecularMicrobiology vol 39 no 6 pp 1623ndash1637 2001

[91] M Llewellyn R J Dutton J Easter D OrsquoDonnol and J WGober ldquoThe conserved flaF gene has a critical role in couplingflagellin translation and assembly in Caulobacter crescentusrdquoMolecular Microbiology vol 57 no 4 pp 1127ndash1142 2005

[92] S M Kim D H Lee and S H Choi ldquoEvidence that the Vibriovulnificus flagellar regulator FlhF is regulated by a quorumsensing master regulator SmcRrdquo Microbiology vol 158 part 8pp 2017ndash2025 2012

[93] F Martınez-Granero A Navazo E Barahona M Redondo-Nieto R Rivilla andMMartın ldquoTheGac-Rsm and SadB signaltransduction pathways converge on Algu to downregulatemotility in Pseudomonas fluorescensrdquo PLoS ONE vol 7 no 2Article ID e31765 2012

[94] C Reimmann M Beyeler A Latifi et al ldquoThe global activatorGacA of Pseudomonas aeruginosa PAO positively controls theproduction of the autoinducer N-butyryl-homoserine lactoneand the formation of the virulence factors pyocyanin cyanideand lipaserdquo Molecular Microbiology vol 24 no 2 pp 309ndash3191997

[95] J Dubern and G V Bloemberg ldquoInfluence of environmentalconditions on putisolvins I and II production in Pseudomonasputida strain PCL1445rdquo FEMSMicrobiology Letters vol 263 no2 pp 169ndash175 2006

[96] M G Surette M B Miller and B L Bassler ldquoQuorum sensingin Escherichia coli Salmonella typhimurium andVibrio harveyia new family of genes responsible for autoinducer productionrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 96 no 4 pp 1639ndash1644 1999

[97] B A Rader S R Campagna M F Semmelhack B L Basslerand K Guillemin ldquoThe quorum-sensing molecule autoinducer2 regulates motility and flagellar morphogenesis inHelicobacterpylorirdquo Journal of Bacteriology vol 189 no 17 pp 6109ndash61172007

[98] YHe J G Frye T P Strobaugh Jr andCChen ldquoAnalysis of AI-2LuxS-dependent transcription in Campylobacter jejuni strain81-176rdquo Foodborne Pathogens and Disease vol 5 no 4 pp 399ndash415 2008

[99] B Jeon K Itoh N Misawa and S Ryu ldquoEffects of quorumsensing on flaA transcription and autoagglutination in Campy-lobacter jejunirdquoMicrobiology and Immunology vol 47 no 11 pp833ndash839 2003

[100] N Dasgupta E P Ferrell K J Kanack S E H West andR Ramphal ldquofleQ the gene encoding the major flagellarregulator of Pseudomonas aeruginosa is 12059070 dependent and isdownregulated by Vfr a homolog of Escherichia coli cyclic AMPreceptor proteinrdquo Journal of Bacteriology vol 184 no 19 pp5240ndash5250 2002

[101] J Zhu M B Miller R E Vance M Dziejman B L Bassler andJ J Mekalanos ldquoQuorum-sensing regulators control virulencegene expression in Vibrio choleraerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no5 pp 3129ndash3134 2002

[102] Z Liu T Miyashiro A Tsou A Hsiao M Goulian and JZhu ldquoMucosal penetration primes Vibrio cholerae for hostcolonization by repressing quorum sensingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 28 pp 9769ndash9774 2008

[103] K E Klose V Novik and J J Mekalanos ldquoIdentificationof multiple 12059054-dependent transcriptional activators in Vibriocholeraerdquo Journal of Bacteriology vol 180 no 19 pp 5256ndash52591998

[104] I Kawagishi M Imagawa Y Imae L McCarter and MHomma ldquoThe sodium-driven polar flagellar motor of marineVibrio as the mechanosensor that regulates lateral flagellarexpressionrdquoMolecular Microbiology vol 20 no 4 pp 693ndash6991996

[105] T J Kirn B A Jude and R K Taylor ldquoA colonizationfactor links Vibrio cholerae environmental survival and humaninfectionrdquo Nature vol 438 no 7069 pp 863ndash866 2005

[106] Z Kuang YHao S Hwang et al ldquoThe Pseudomonas aeruginosaflagellum confers resistance to pulmonary surfactant protein-Aby impacting the production of exoproteases through quorum-sensingrdquo Molecular Microbiology vol 79 no 5 pp 1220ndash12352011

[107] S Zhang F X McCormack R C Levesque G A OrsquoTooleand G W Lau ldquoThe flagellum of Pseudomonas aeruginosa isrequired for resistance to clearance by surfactant protein ArdquoPLoS ONE vol 2 no 6 article e564 2007

[108] G W Lau D J Hassett H Ran and F Kong ldquoThe role ofpyocyanin in Pseudomonas aeruginosa infectionrdquo Trends inMolecular Medicine vol 10 no 12 pp 599ndash606 2004

[109] GW LauH Ran F Kong D J Hassett andDMavrodi ldquoPseu-domonas aeruginosa pyocyanin is critical for lung infection inmicerdquo Infection and Immunity vol 72 no 7 pp 4275ndash42782004

[110] D S Merrell M L Goodrich G Otto L S Tompkins and SFalkow ldquopH-regulated gene expression of the gastric pathogenHelicobacter pylorirdquo Infection and Immunity vol 71 no 6 pp3529ndash3539 2003

[111] K J Allen and M W Griffiths ldquoEffect of environmental andchemotactic stimuli on the activity of the Campylobacter jejuniflaA 12059028 promoterrdquo FEMS Microbiology Letters vol 205 no 1pp 43ndash48 2001

[112] Y Wen J Feng D R Scott E A Marcus and G SachsldquoThe pH-responsive regulon of HP0244 (FlgS) the cytoplasmichistidine kinase of Helicobacter pylorirdquo Journal of Bacteriologyvol 191 no 2 pp 449ndash460 2009

[113] D R Hendrixson ldquoA phase-variable mechanism controllingthe Campylobacter jejuni FlgR response regulator influencescommensalismrdquo Molecular Microbiology vol 61 no 6 pp1646ndash1659 2006

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 Scientifica

[56] MMoisi C Jenul SM Butler et al ldquoA novel regulatory proteininvolved in motility of Vibrio choleraerdquo Journal of Bacteriologyvol 191 no 22 pp 7027ndash7038 2009

[57] L Aravind and C P Ponting ldquoThe cytoplasmic helical linkerdomain of receptor histidine kinase and methyl-acceptingproteins is common to many prokaryotic signalling proteinsrdquoFEMS Microbiology Letters vol 176 no 1 pp 111ndash116 1999

[58] SMerino J G Shaw and JM Tomas ldquoBacterial lateral flagellaan inducible flagella systemrdquo FEMS Microbiology Letters vol263 no 2 pp 127ndash135 2006

[59] L L McCarter ldquoDual flagellar systems enable motility underdifferent circumstancesrdquo Journal of Molecular Microbiology andBiotechnology vol 7 no 1-2 pp 18ndash29 2004

[60] S Ulitzur ldquoInduction of swarming in Vibrio parahaemolyticusrdquoArchives of Microbiology vol 101 no 4 pp 357ndash363 1974

[61] K S Park M Arita T Iida and T Honda ldquovpaH a geneencoding a novel histone-like nucleoid structure-like proteinthat was possibly horizontally acquired regulates the biogen-esis of lateral flagella in trh-positive Vibrio parahaemolyticusTH3996rdquo Infection and Immunity vol 73 no 9 pp 5754ndash57612005

[62] LMcCarter andM Silverman ldquoIron regulation of swarmer celldifferentiation ofVibrio parahaemolyticusrdquo Journal of Bacteriol-ogy vol 171 no 2 pp 731ndash736 1989

[63] B J Stewart and L L McCarter ldquoLateral flagellar gene systemofVibrio parahaemolyticusrdquo Journal of Bacteriology vol 185 no15 pp 4508ndash4518 2003

[64] C J Gode-Potratz R J Kustusch P J Breheny D S Weissand L LMcCarter ldquoSurface sensing inVibrio parahaemolyticustriggers a programme of gene expression that promotes colo-nization and virulencerdquo Molecular Microbiology vol 79 no 1pp 240ndash263 2011

[65] J L Veesenmeyer A R Hauser T Lisboa and J Rello ldquoPseu-domonas aeruginosa virulence and therapy evolving transla-tional strategiesrdquo Critical Care Medicine vol 37 no 5 pp 1777ndash1786 2009

[66] D Balasubramanian L Schneper H Kumari and K MatheeldquoA dynamic and intricate regulatory network determines Pseu-domonas aeruginosa virulencerdquo Nucleic Acids Research vol 41no 1 pp 1ndash20 2013

[67] T C Montie D D Huntzinger R C Craven and I A HolderldquoLoss of virulence associated with absence of flagellum in anisogenic mutant of Pseudomonas aeruginosa in the burned-mouse modelrdquo Infection and Immunity vol 38 no 3 pp 1296ndash1298 1982

[68] Y R Patankar R R Lovewell M E Poynter J Jyot BI Kazmierczak and B Berwin ldquoFlagellar motility is a keydeterminant of themagnitude of the inflammasome response toPseudomonas aeruginosardquo Infection and Immunity vol 81 no 6pp 2043ndash2052 2013

[69] G A OrsquoToole and R Kolter ldquoFlagellar and twitching motilityare necessary for Pseudomonas aeruginosa biofilm develop-mentrdquoMolecular Microbiology vol 30 no 2 pp 295ndash304 1998

[70] K B Barken S J Pamp L Yang et al ldquoRoles of type IVpili flagellum-mediated motility and extracellular DNA in theformation of mature multicellular structures in Pseudomonasaeruginosa biofilmsrdquo Environmental Microbiology vol 10 no 9pp 2331ndash2343 2008

[71] M Leeman J A van Pelt F M den Ouden M Heinsbroek PA Bakker and B Schippers ldquoInduction of systemic resistance

against fusarium wilt of radish by lipopolysaccharides of Pseu-domonas fluorescensrdquo Phytopathology vol 85 no 9 pp 1021ndash1027 1995

[72] P Hsueh L Teng H Pan et al ldquoOutbreak of Pseudomonasfluorescens bacteremia among oncology patientsrdquo Journal ofClinical Microbiology vol 36 no 10 pp 2914ndash2917 1998

[73] VWongK Levi B Baddal J Turton andTC Boswell ldquoSpreadof Pseudomonas fluorescens due to contaminated drinkingwater in a bone marrow transplant unitrdquo Journal of ClinicalMicrobiology vol 49 no 6 pp 2093ndash2096 2011

[74] J W Hickman and C S Harwood ldquoIdentification of FleQ fromPseudomonas aeruginosa as a c-di-GMP-responsive transcrip-tion factorrdquoMolecular Microbiology vol 69 no 2 pp 376ndash3892008

[75] N Dasgupta M C Wolfgang A L Goodman et al ldquoA four-tiered transcriptional regulatory circuit controls flagellar bio-genesis in Pseudomonas aeruginosardquo Molecular Microbiologyvol 50 no 3 pp 809ndash824 2003

[76] S K Arora B W Ritchings E C Almira S Lory and RRamphal ldquoA transcriptional activator FleQ regulates mucinadhesion and flagellar gene expression in Pseudomonas aerugi-nosa in a cascade mannerrdquo Journal of Bacteriology vol 179 no17 pp 5574ndash5581 1997

[77] N Dasgupta and R Ramphal ldquoInteraction of the antiactivatorFleN with the transcriptional activator FleQ regulates flagellarnumber in Pseudomonas aeruginosardquo Journal of Bacteriologyvol 183 no 22 pp 6636ndash6644 2001

[78] N Dasgupta S K Arora and R Ramphal ldquofleN a gene thatregulates flagellar number in Pseudomonas aeruginosardquo Journalof Bacteriology vol 182 no 2 pp 357ndash364 2000

[79] J Malakooti and B Ely ldquoPrincipal sigma subunit of theCaulobacter crescentus RNA polymeraserdquo Journal of Bacteriol-ogy vol 177 no 23 pp 6854ndash6860 1995

[80] M T Laub H H McAdams T Feldblyum C M Fraser andL Shapiro ldquoGlobal analysis of the genetic network controllinga bacterial cell cyclerdquo Science vol 290 no 5499 pp 2144ndash21482000

[81] A K Benson G Ramakrishnan N Ohta J Feng A J Ninfaand A Newton ldquoThe Caulobacter crescentus FlbD proteinacts at ftr sequence elements both to activate and to represstranscription of cell cycle-regulated flagellar genesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 91 no 11 pp 4989ndash4993 1994

[82] D A Mullin S M van Way C A Blankenship and A HMullin ldquoFlbD has a DNA-binding activity near its carboxyterminus that recognizes ftr sequences involved in positive andnegative regulation of flagellar gene transcription in Caulobac-ter crescentusrdquo Journal of Bacteriology vol 176 no 19 pp 5971ndash5981 1994

[83] R J Dutton Z Xu and J W Gober ldquoLinking structural assem-bly to gene expression a novel mechanism for regulating theactivity of a 12059054 transcription factorrdquo Molecular Microbiologyvol 58 no 3 pp 743ndash757 2005

[84] R E Muir J Easter and J W Gober ldquoThe trans-acting flagellarregulatory proteins FliX and FlbD play a central role in linkingflagellar biogenesis and cytokinesis in Caulobacter crescentusrdquoMicrobiology vol 151 no 11 pp 3699ndash3711 2005

[85] P E Anderson and J W Gober ldquoFlbT the post-transcriptionalregulator of flagellin synthesis in Caulobacter crescentus inter-acts with the 51015840 untranslated region of flagellin mRNArdquoMolec-ular Microbiology vol 38 no 1 pp 41ndash52 2000

Scientifica 13

[86] A T Nielsen N A Dolganov G Otto M C Miller C Y WuandGK Schoolnik ldquoRpoS controls theVibrio choleraemucosalescape responserdquo PLoS Pathogens vol 2 no 10 article e1092006

[87] M T Laub S L Chen L Shapiro and H H McAdamsldquoGenes directly controlled by CtrA a master regulator of theCaulobacter cell cyclerdquo Proceedings of the National Academy ofSciences of the United States of America vol 99 no 7 pp 4632ndash4637 2002

[88] A J Kelly M J Sackett N Din E Quardokus and Y VBrun ldquoCell cycle-dependent transcriptional and proteolyticregulation of FtsZ in Caulobacterrdquo Genes and Development vol12 no 6 pp 880ndash893 1998

[89] M Wortinger M J Sackett and Y V Brun ldquoCtrA mediatesa DNA replication checkpoint that prevents cell division inCaulobacter crescentusrdquo EMBO Journal vol 19 no 17 pp 4503ndash4512 2000

[90] R E Muir T M OrsquoBrien and J W Gober ldquoThe Caulobactercrescentus flagellar gene fliX encodes a novel trans-acting fac-tor that couples flagellar assembly to transcriptionrdquo MolecularMicrobiology vol 39 no 6 pp 1623ndash1637 2001

[91] M Llewellyn R J Dutton J Easter D OrsquoDonnol and J WGober ldquoThe conserved flaF gene has a critical role in couplingflagellin translation and assembly in Caulobacter crescentusrdquoMolecular Microbiology vol 57 no 4 pp 1127ndash1142 2005

[92] S M Kim D H Lee and S H Choi ldquoEvidence that the Vibriovulnificus flagellar regulator FlhF is regulated by a quorumsensing master regulator SmcRrdquo Microbiology vol 158 part 8pp 2017ndash2025 2012

[93] F Martınez-Granero A Navazo E Barahona M Redondo-Nieto R Rivilla andMMartın ldquoTheGac-Rsm and SadB signaltransduction pathways converge on Algu to downregulatemotility in Pseudomonas fluorescensrdquo PLoS ONE vol 7 no 2Article ID e31765 2012

[94] C Reimmann M Beyeler A Latifi et al ldquoThe global activatorGacA of Pseudomonas aeruginosa PAO positively controls theproduction of the autoinducer N-butyryl-homoserine lactoneand the formation of the virulence factors pyocyanin cyanideand lipaserdquo Molecular Microbiology vol 24 no 2 pp 309ndash3191997

[95] J Dubern and G V Bloemberg ldquoInfluence of environmentalconditions on putisolvins I and II production in Pseudomonasputida strain PCL1445rdquo FEMSMicrobiology Letters vol 263 no2 pp 169ndash175 2006

[96] M G Surette M B Miller and B L Bassler ldquoQuorum sensingin Escherichia coli Salmonella typhimurium andVibrio harveyia new family of genes responsible for autoinducer productionrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 96 no 4 pp 1639ndash1644 1999

[97] B A Rader S R Campagna M F Semmelhack B L Basslerand K Guillemin ldquoThe quorum-sensing molecule autoinducer2 regulates motility and flagellar morphogenesis inHelicobacterpylorirdquo Journal of Bacteriology vol 189 no 17 pp 6109ndash61172007

[98] YHe J G Frye T P Strobaugh Jr andCChen ldquoAnalysis of AI-2LuxS-dependent transcription in Campylobacter jejuni strain81-176rdquo Foodborne Pathogens and Disease vol 5 no 4 pp 399ndash415 2008

[99] B Jeon K Itoh N Misawa and S Ryu ldquoEffects of quorumsensing on flaA transcription and autoagglutination in Campy-lobacter jejunirdquoMicrobiology and Immunology vol 47 no 11 pp833ndash839 2003

[100] N Dasgupta E P Ferrell K J Kanack S E H West andR Ramphal ldquofleQ the gene encoding the major flagellarregulator of Pseudomonas aeruginosa is 12059070 dependent and isdownregulated by Vfr a homolog of Escherichia coli cyclic AMPreceptor proteinrdquo Journal of Bacteriology vol 184 no 19 pp5240ndash5250 2002

[101] J Zhu M B Miller R E Vance M Dziejman B L Bassler andJ J Mekalanos ldquoQuorum-sensing regulators control virulencegene expression in Vibrio choleraerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no5 pp 3129ndash3134 2002

[102] Z Liu T Miyashiro A Tsou A Hsiao M Goulian and JZhu ldquoMucosal penetration primes Vibrio cholerae for hostcolonization by repressing quorum sensingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 28 pp 9769ndash9774 2008

[103] K E Klose V Novik and J J Mekalanos ldquoIdentificationof multiple 12059054-dependent transcriptional activators in Vibriocholeraerdquo Journal of Bacteriology vol 180 no 19 pp 5256ndash52591998

[104] I Kawagishi M Imagawa Y Imae L McCarter and MHomma ldquoThe sodium-driven polar flagellar motor of marineVibrio as the mechanosensor that regulates lateral flagellarexpressionrdquoMolecular Microbiology vol 20 no 4 pp 693ndash6991996

[105] T J Kirn B A Jude and R K Taylor ldquoA colonizationfactor links Vibrio cholerae environmental survival and humaninfectionrdquo Nature vol 438 no 7069 pp 863ndash866 2005

[106] Z Kuang YHao S Hwang et al ldquoThe Pseudomonas aeruginosaflagellum confers resistance to pulmonary surfactant protein-Aby impacting the production of exoproteases through quorum-sensingrdquo Molecular Microbiology vol 79 no 5 pp 1220ndash12352011

[107] S Zhang F X McCormack R C Levesque G A OrsquoTooleand G W Lau ldquoThe flagellum of Pseudomonas aeruginosa isrequired for resistance to clearance by surfactant protein ArdquoPLoS ONE vol 2 no 6 article e564 2007

[108] G W Lau D J Hassett H Ran and F Kong ldquoThe role ofpyocyanin in Pseudomonas aeruginosa infectionrdquo Trends inMolecular Medicine vol 10 no 12 pp 599ndash606 2004

[109] GW LauH Ran F Kong D J Hassett andDMavrodi ldquoPseu-domonas aeruginosa pyocyanin is critical for lung infection inmicerdquo Infection and Immunity vol 72 no 7 pp 4275ndash42782004

[110] D S Merrell M L Goodrich G Otto L S Tompkins and SFalkow ldquopH-regulated gene expression of the gastric pathogenHelicobacter pylorirdquo Infection and Immunity vol 71 no 6 pp3529ndash3539 2003

[111] K J Allen and M W Griffiths ldquoEffect of environmental andchemotactic stimuli on the activity of the Campylobacter jejuniflaA 12059028 promoterrdquo FEMS Microbiology Letters vol 205 no 1pp 43ndash48 2001

[112] Y Wen J Feng D R Scott E A Marcus and G SachsldquoThe pH-responsive regulon of HP0244 (FlgS) the cytoplasmichistidine kinase of Helicobacter pylorirdquo Journal of Bacteriologyvol 191 no 2 pp 449ndash460 2009

[113] D R Hendrixson ldquoA phase-variable mechanism controllingthe Campylobacter jejuni FlgR response regulator influencescommensalismrdquo Molecular Microbiology vol 61 no 6 pp1646ndash1659 2006

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Scientifica 13

[86] A T Nielsen N A Dolganov G Otto M C Miller C Y WuandGK Schoolnik ldquoRpoS controls theVibrio choleraemucosalescape responserdquo PLoS Pathogens vol 2 no 10 article e1092006

[87] M T Laub S L Chen L Shapiro and H H McAdamsldquoGenes directly controlled by CtrA a master regulator of theCaulobacter cell cyclerdquo Proceedings of the National Academy ofSciences of the United States of America vol 99 no 7 pp 4632ndash4637 2002

[88] A J Kelly M J Sackett N Din E Quardokus and Y VBrun ldquoCell cycle-dependent transcriptional and proteolyticregulation of FtsZ in Caulobacterrdquo Genes and Development vol12 no 6 pp 880ndash893 1998

[89] M Wortinger M J Sackett and Y V Brun ldquoCtrA mediatesa DNA replication checkpoint that prevents cell division inCaulobacter crescentusrdquo EMBO Journal vol 19 no 17 pp 4503ndash4512 2000

[90] R E Muir T M OrsquoBrien and J W Gober ldquoThe Caulobactercrescentus flagellar gene fliX encodes a novel trans-acting fac-tor that couples flagellar assembly to transcriptionrdquo MolecularMicrobiology vol 39 no 6 pp 1623ndash1637 2001

[91] M Llewellyn R J Dutton J Easter D OrsquoDonnol and J WGober ldquoThe conserved flaF gene has a critical role in couplingflagellin translation and assembly in Caulobacter crescentusrdquoMolecular Microbiology vol 57 no 4 pp 1127ndash1142 2005

[92] S M Kim D H Lee and S H Choi ldquoEvidence that the Vibriovulnificus flagellar regulator FlhF is regulated by a quorumsensing master regulator SmcRrdquo Microbiology vol 158 part 8pp 2017ndash2025 2012

[93] F Martınez-Granero A Navazo E Barahona M Redondo-Nieto R Rivilla andMMartın ldquoTheGac-Rsm and SadB signaltransduction pathways converge on Algu to downregulatemotility in Pseudomonas fluorescensrdquo PLoS ONE vol 7 no 2Article ID e31765 2012

[94] C Reimmann M Beyeler A Latifi et al ldquoThe global activatorGacA of Pseudomonas aeruginosa PAO positively controls theproduction of the autoinducer N-butyryl-homoserine lactoneand the formation of the virulence factors pyocyanin cyanideand lipaserdquo Molecular Microbiology vol 24 no 2 pp 309ndash3191997

[95] J Dubern and G V Bloemberg ldquoInfluence of environmentalconditions on putisolvins I and II production in Pseudomonasputida strain PCL1445rdquo FEMSMicrobiology Letters vol 263 no2 pp 169ndash175 2006

[96] M G Surette M B Miller and B L Bassler ldquoQuorum sensingin Escherichia coli Salmonella typhimurium andVibrio harveyia new family of genes responsible for autoinducer productionrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 96 no 4 pp 1639ndash1644 1999

[97] B A Rader S R Campagna M F Semmelhack B L Basslerand K Guillemin ldquoThe quorum-sensing molecule autoinducer2 regulates motility and flagellar morphogenesis inHelicobacterpylorirdquo Journal of Bacteriology vol 189 no 17 pp 6109ndash61172007

[98] YHe J G Frye T P Strobaugh Jr andCChen ldquoAnalysis of AI-2LuxS-dependent transcription in Campylobacter jejuni strain81-176rdquo Foodborne Pathogens and Disease vol 5 no 4 pp 399ndash415 2008

[99] B Jeon K Itoh N Misawa and S Ryu ldquoEffects of quorumsensing on flaA transcription and autoagglutination in Campy-lobacter jejunirdquoMicrobiology and Immunology vol 47 no 11 pp833ndash839 2003

[100] N Dasgupta E P Ferrell K J Kanack S E H West andR Ramphal ldquofleQ the gene encoding the major flagellarregulator of Pseudomonas aeruginosa is 12059070 dependent and isdownregulated by Vfr a homolog of Escherichia coli cyclic AMPreceptor proteinrdquo Journal of Bacteriology vol 184 no 19 pp5240ndash5250 2002

[101] J Zhu M B Miller R E Vance M Dziejman B L Bassler andJ J Mekalanos ldquoQuorum-sensing regulators control virulencegene expression in Vibrio choleraerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no5 pp 3129ndash3134 2002

[102] Z Liu T Miyashiro A Tsou A Hsiao M Goulian and JZhu ldquoMucosal penetration primes Vibrio cholerae for hostcolonization by repressing quorum sensingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 28 pp 9769ndash9774 2008

[103] K E Klose V Novik and J J Mekalanos ldquoIdentificationof multiple 12059054-dependent transcriptional activators in Vibriocholeraerdquo Journal of Bacteriology vol 180 no 19 pp 5256ndash52591998

[104] I Kawagishi M Imagawa Y Imae L McCarter and MHomma ldquoThe sodium-driven polar flagellar motor of marineVibrio as the mechanosensor that regulates lateral flagellarexpressionrdquoMolecular Microbiology vol 20 no 4 pp 693ndash6991996

[105] T J Kirn B A Jude and R K Taylor ldquoA colonizationfactor links Vibrio cholerae environmental survival and humaninfectionrdquo Nature vol 438 no 7069 pp 863ndash866 2005

[106] Z Kuang YHao S Hwang et al ldquoThe Pseudomonas aeruginosaflagellum confers resistance to pulmonary surfactant protein-Aby impacting the production of exoproteases through quorum-sensingrdquo Molecular Microbiology vol 79 no 5 pp 1220ndash12352011

[107] S Zhang F X McCormack R C Levesque G A OrsquoTooleand G W Lau ldquoThe flagellum of Pseudomonas aeruginosa isrequired for resistance to clearance by surfactant protein ArdquoPLoS ONE vol 2 no 6 article e564 2007

[108] G W Lau D J Hassett H Ran and F Kong ldquoThe role ofpyocyanin in Pseudomonas aeruginosa infectionrdquo Trends inMolecular Medicine vol 10 no 12 pp 599ndash606 2004

[109] GW LauH Ran F Kong D J Hassett andDMavrodi ldquoPseu-domonas aeruginosa pyocyanin is critical for lung infection inmicerdquo Infection and Immunity vol 72 no 7 pp 4275ndash42782004

[110] D S Merrell M L Goodrich G Otto L S Tompkins and SFalkow ldquopH-regulated gene expression of the gastric pathogenHelicobacter pylorirdquo Infection and Immunity vol 71 no 6 pp3529ndash3539 2003

[111] K J Allen and M W Griffiths ldquoEffect of environmental andchemotactic stimuli on the activity of the Campylobacter jejuniflaA 12059028 promoterrdquo FEMS Microbiology Letters vol 205 no 1pp 43ndash48 2001

[112] Y Wen J Feng D R Scott E A Marcus and G SachsldquoThe pH-responsive regulon of HP0244 (FlgS) the cytoplasmichistidine kinase of Helicobacter pylorirdquo Journal of Bacteriologyvol 191 no 2 pp 449ndash460 2009

[113] D R Hendrixson ldquoA phase-variable mechanism controllingthe Campylobacter jejuni FlgR response regulator influencescommensalismrdquo Molecular Microbiology vol 61 no 6 pp1646ndash1659 2006

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

14 Scientifica

[114] D R Hendrixson ldquoRestoration of flagellar biosynthesis byvaried mutational events in Campylobacter jejunirdquo MolecularMicrobiology vol 70 no 2 pp 519ndash536 2008

[115] P Lertsethtakarn K M Ottemann and D R HendrixsonldquoMotility and chemotaxis in Campylobacter and HelicobacterrdquoAnnual Review of Microbiology vol 65 pp 389ndash410 2011

[116] S F Park D Purdy and S Leach ldquoLocalized reversibleframeshift mutation in the flhA gene confers phase variabilityto flagellin gene expression in Campylobacter colirdquo Journal ofBacteriology vol 182 no 1 pp 207ndash210 2000

[117] C Josenhans K A Eaton T Thevenot and S SuerbaumldquoSwitching of flagellar motility in Helicobacter pylori byreversible length variation of a short homopolymeric sequencerepeat in fliP a gene encoding a basal body proteinrdquo Infectionand Immunity vol 68 no 8 pp 4598ndash4603 2000

[118] N deVries DDuinsbergen E J Kuipers et al ldquoTranscriptionalphase variation of a type III restriction-modification system inHelicobacter pylorirdquo Journal of Bacteriology vol 184 no 23 pp6615ndash6623 2002

[119] Y N Srikhanta R J Gorrell J A Steen et al ldquoPhasevarionmediated epigenetic gene regulation in Helicobacter pylorirdquoPLoS ONE vol 6 no 12 Article ID e27569 2011

[120] K L Fox Y N Srikhanta and M P Jennings ldquoPhase variabletype III restriction-modification systems of host-adapted bacte-rial pathogensrdquoMolecular Microbiology vol 65 no 6 pp 1375ndash1379 2007

[121] S K Lee A Stack E Katzowitsch S I Aizawa S Suerbaumand C Josenhans ldquoHelicobacter pylori flagellins have very lowintrinsic activity to stimulate human gastric epithelial cells viaTLR5rdquoMicrobes and Infection vol 5 no 15 pp 1345ndash1356 2003

[122] L F Lin J Posfai R J Roberts and H Kong ldquoComparativegenomics of the restriction-modification systems in Helicobac-ter pylorirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2740ndash2745 2001

[123] Y Zheng J Posfai R D Morgan T Vincze and R J RobertsldquoUsing shotgun sequence data to find active restriction enzymegenesrdquo Nucleic Acids Research vol 37 no 1 article e1 2009

[124] R Kumar A K Mukhopadhyay P Ghosh and D N RaoldquoComparative transcriptomics ofH pylori strains AM5 SS1 andtheir hpyAVIBM deletion mutants possible roles of cytosinemethylationrdquo PLoS ONE vol 7 no 8 Article ID e42303 2012

[125] E Deziel Y Comeau and R Villemur ldquoInitiation of biofilmformation by Pseudomonas aeruginosa 57RP correlates withemergence of hyperpiliated and highly adherent phenotypicvariants deficient in swimming swarming and twitchingmotil-itiesrdquo Journal of Bacteriology vol 183 no 4 pp 1195ndash1204 2001

[126] M Sanchez-Contreras M Martın M Villacieros F OrsquoGaraI Bonilla and R Rivilla ldquoPhenotypic selection and phasevariation occur during alfalfa root colonization byPseudomonasfluorescensF113rdquo Journal of Bacteriology vol 184 no 6 pp 1587ndash1596 2002

[127] C M Sharma S Hoffmann F Darfeuille et al ldquoThe pri-mary transcriptome of the major human pathogenHelicobacterpylorirdquo Nature vol 464 no 7286 pp 250ndash255 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology