review article cell envelope of corynebacteria: structure...

Hindawi Publishing CorporationISRNMicrobiologyVolume 2013 Article ID 935736 11 pageshttpdxdoiorg1011552013935736

Review ArticleCell Envelope of Corynebacteria Structure andInfluence on Pathogenicity

Andreas Burkovski

Lehrstuhl fur Mikrobiologie Friedrich-Alexander-Universitat Erlangen-Nurnberg Staudtstr120573e 5 91058 Erlangen Germany

Correspondence should be addressed to Andreas Burkovski aburkovbiologieuni-erlangende

Received 2 December 2012 Accepted 31 December 2012

Academic Editors S H Flint G Koraimann and T Krishnan

Copyright copy 2013 Andreas Burkovski This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

To date the genus Corynebacterium comprises 88 species More than half of these are connected to human and animal infectionswith the most prominent member of the pathogenic species being Corynebacterium diphtheriae which is also the type species ofthe genus Corynebacterium species are characterized by a complex cell wall architecture the plasma membrane of these bacteriais followed by a peptidoglycan layer which itself is covalently linked to a polymer of arabinogalactan Bound to this an outerlayer of mycolic acids is found which is functionally equivalent to the outer membrane of Gram-negative bacteria As final layerfree polysaccharides glycolipids and proteins are foundThe composition of the different substructures of the corynebacterial cellenvelope and their influence on pathogenicity are discussed in this paper

1 The Genus Corynebacterium

The genus Corynebacterium belongs to the class of Acti-nobacteria (highG+CGram-positive bacteria) and comprisesa collection of morphologically similar irregular- or club-shaped nonsporulating (mico)aerobic microorganisms [1 2]To date 88 species were taxonomically classified [3] Morethan half of these that is 53 species are occasional or rarecauses of infections with the most prominent member ofthe pathogenic species being Corynebacterium diphtheriaewhich is also the type species of the whole genus Sev-eral pathogenic species are considered to be part of thehuman skin flora for example Corynebacterium amycola-tum or Corynebacterium jeikeium others are consideredas zoonotic agents for example Corynebacterium pseudo-tuberculosis Corynebacterium ulcerans or Corynebacteriumxerosis [3] Biotechnologically important species used for theindustrial production of nucleotides and amino acids areCorynebacterium ammoniagenes Corynebacterium efficiensand Corynebacterium glutamicum C glutamicum especiallyis dominating the field of white (bacterial) biotechnologywith a production of two million tons of L-glutamate and 18million tons of L-lysine per year [4] and an increasing appli-cation as platform organism for the industrial production ofvarious metabolites [5]

Based on their medical importance as etiological agentof diphtheria and their enormous biotechnological potentialC diphtheriae and C glutamicum respectively have beenthe best investigated Corynebacterium species for manyyears In fact these were also the first species for whichgenome sequences became available [6ndash8] With the devel-opment of high throughput sequencing techniques to datenumerous genome sequences are available including thoseofCorynebacteriumaurimucosum [9]Corynebacterium bovis[10] various C diphtheriae strains [11ndash13] C jeikeium [14]Corynebacterium kroppenstedtii [15] several C pseudotuber-culosis strains [16ndash24] Corynebacterium resistens [25] twoC ulcerans strains [26] Corynebacterium urealyticum [27]and Corynebacterium variabile [28] This wealth of genomeinformation now provides a basis for the characterization ofcorynebacterial cell envelope components and correspondinggenes both on the whole cell level by global analysis tech-niques (genomics transcriptomics and proteomics) and ona molecular level by specific approaches

2 General Cell Envelope Architecture

Almost all Corynebacterium species are characterized by acomplex cell wall architecture the plasmamembrane of these

2 ISRNMicrobiology

bacteria is covered by a peptidoglycan layer which itselfis covalently linked to arabinogalactan an additional het-eropolysaccharide meshwork Bound to this an outer layerof mycolic acids is found which is functionally equivalent tothe outer membrane of Gram-negative bacteria As top layerouter surface material composed of free polysaccharidesglycolipids and proteins (including S-layer proteins pili andother surface proteins) is found (reviewed in [29 30] see alsoFigure 1)

The corynebacterial cell envelope has been investigatedby different optical techniques Electron microscopy of thinsections after freeze substitution revealed a layered cellenvelope organization comprising a plasma membrane athick electron-dense layer an electron-transparent layerand a thin outer layer [31ndash33] This picture resembles thetypical mycobacterial cell envelope appearanceThe electron-dense layer is traditionally interpreted as peptidoglycanthe electron-transparent layer as mycolic acid layer Whencomparing the thickness determined from the electronmicroscopic pictures of corynebacteria andmycobacteria thequestion arose why their electron-transparent layer has asimilar thickness despite the fact that mycobacterial mycolicacids have about a threefold length compared to corynebac-terial ones [33] A solution of this problem was indicatedby cryoelectron tomography studies These revealed a typicalouter membrane with a bilayer structure in Mycobacteriumsmegmatis Mycobacterium bovis and C glutamicum Thethickness of mycobacterial outer layers was smaller thanexpected and based on this observation an alternativemodelfor mycolic acid distribution was proposed implicating afolding of mycolic acids [34 35]

The general structure and composition of the corynebac-terial cell envelope were earlier reviewed by [29 30] inrespect to biochemical and genetic properties especially in Cglutamicum The aim of this paper will be the presentationof cell envelope properties with a broader focus on differentcorynebacteria and the importance of cell envelope compo-nents for pathogen host interaction

3 Cytoplasmic Membrane andFatty Acid Synthesis

31 Plasma Membrane Composition The cytoplasmic orplasma membrane is the main diffusion barrier of cells andseparates cytoplasm and environment As in other bacteriathe corynebacterial plasma membrane is mainly composedof phospholipids assembled into a lipid bilayer which addi-tionally contains other polar lipids besides a great variety ofproteins crucial for transport processes and bioenergetics ofthe cell

The main phospholipid found in C glutamicum is phos-phatidylglycerol followed by diphosphatidylglycerol phos-phatidylinositol and minor amounts of phosphatidylinositoldimannosides (PIM

2) [33 36ndash38] Fatty acids dominating in

the plasma membrane are the saturated palmitic acid (160)and the desaturated decenoic acid (181) [39ndash42] Howeveras in other bacteria fatty acid composition might changesignificantly depending on environmental conditions such as

low or high temperature [43] or the carbon source available[42]

32 Fatty Acid Synthesis Fatty acids are synthesized by suc-cessive cycles of multistep reactions [44] Two distinct typesof these fatty acid synthases (FASs) are distinguished based ontheir general composition the FAS-II type characteristicallyfound in bacteria contains the minimum seven functionaldomains necessary for fatty acid synthesis organized in onepolypeptide whereas the FAS-I type typically found ineukaryotes is comprised of a large multifunctional proteincomplex Interestingly and as an exception from the rule FAS-I proteins are found in members of the CorynebacterineaeC glutamicum and C efficiens contain even two FAS-I-type complexes FAS-IA and FAS-IB which were functionallycharacterized in detail for C glutamicum [42] FAS-IA isessential in C glutamicum while FAS-IB is not howeverFAS-IB-devoid mutant strains exhibit an altered patternof fatty and mycolic acids showing that FAS-IB is activeand necessary to generate the typical wild-type fatty acidprofile [42] The regulatory mechanism allowing adaptationof FAS activity to environmental stimuli (see above) isunknown

C glutamicum does not only have genes coding for twoFAS-I but also genes coding for FAS-II [42] which areinvolved in elongation of mycolic acid chains in mycobacte-ria Since C glutamicum does not contain elongated mycolicacids and FAS-II is absent in other corynebacteria such as Cdiphtheriae it was speculated that these proteins might playa minor physiological role or even not be functional in Cglutamicum [42]

33 Lipomannan and Lipoarabinomannan As in other pro-and eukaryotes the corynebacterial lipid bilayer of thecytoplasmic membrane is not symmetric In corynebacte-ria the proposed reason for asymmetry is an insertionof glycoconjugates in the outer sheet of the cytoplasmicmembrane [29] Lipomannan (LM) and lipoarabinomannan(LAM) derivatives were found in different Corynebacteriumspecies [33] They might be inserted into the plasma mem-brane via covalently bound palmitic acid or decenoic acidmolecules besides their appearance in the outer surfacematerial of corynebacteria Distribution of LM- and LAM-like substances seems to be species-specific inC glutamicumLM-like molecules are dominating in C xerosis and Camycolatum LAM-like substances were preferentially foundwhile a C diphtheriae strain showed an almost equal distri-bution of LM and LAM derivatives [33] An excellent reviewdealing with the synthesis of PIM LM and LAM derivativeswas published recently [45]

34 Lipoarabinomannan as a Virulence Factor A 10 kDalipoarabinomannan polymer was identified in C diphtheriaeand designated CdiLAM The basic structure of CdiLAMshows similarity to mycobacterial LAM however in contrastto lipoarabinomannans in other Actinobacteria CdiLAMpresents an unusual substitution at position 4 of 120572 1rarr 6mannan backbone by 120572-D-Araf CdLAM might be part of

ISRNMicrobiology 3

Top layer

Mycomembrane

Arabinogalactan

Peptidoglycan

Plasma membrane

LM LAM

Porin

Figure 1 The corynebacterial cell envelope A schematic representation of the general envelope structure of corynebacteria is shown Thedifferent layers are found in almost all species and distribution of single components like lipomannan (LM) and lipoarabinomannan (LAM)might differ (for details see text) Porin proteins might also be present in top layer S-layer and pili proteins not shown (figure courtesy of SMorbach Friedrich-Alexander-Universitat Erlangen-Nurnberg)

the plasma membrane but is also located at the surface ofC diphtheriae (see the following) and facilitates binding toepithelial cells [46]

The role of lipoarabinomannan with respect to initi-ation of immune responses was addressed in C glutam-icum recently [47] Characterization of a C glutamicumstrain devoid of 120572(1rarr 2) arabinofuranosyltransferase AftErevealed that AftE is involved in the synthesis of arabinansof LAM Absence of AftE leads to a hypermannosylatedvariant of LAM designated hLM Both LAM and hLMwere able to modulate the initiation of immune response byinteracting with TLR2 As shown by a number of in vitroassays arabinose branching of lipoarabinomannan impactsT-helper-cell differentiation and LAMas well as hLM activatedendritic cells via TLR2 Interestingly alterations of lipoara-binomannan seem to be discriminated by TLR2 and signalpathway induction by hLM was shown to be broader Inaccordance with this observation hLM was shown to be astronger inducer of immune responses in mice

4 The Cell Wall HeteropolysaccharideMeshwork

In contrast to for example Escherichia coli or Bacillussubtilis the cell wall skeleton of corynebacteria and relatedtaxa is not exclusively composed of peptidoglycan but inaddition a layer of arabinogalactan is covalently bound to

the peptidoglycan which itself is linked to mycolic acids (forreview see [29 33 48])

41 Peptidoglycan As in other bacteria the glycan part ofthe murein sacculus is composed of alternating 120573-14-linkedN-acetylglucosamine and N-acetyl muramic acid unitswhich form the glycan part of the macromolecule Cross-linking between different glycan polymers occurs via peptideside chains attached to the carboxyl group of muramicacid via peptide bonds Corynebacterial peptidoglycan isdirectly cross-linked as shown for C bovis Corynebacteriumpseudodiphthericum C pseudotuberculosis C striatum Culcerans and C xerosis [49] Interpeptide bridges as foundin other Gram-positives are absent In summary the pepti-doglycan of Corynebacterium in sensu stricto is of the A1120574type [49] In C diphtheriae the major peptide units foundare the tetrapeptide L-Ala-D-Glu-meso-DAP-D-Ala and thetripeptide L-Ala-D-Glu-meso-DAP [50] Interestingly onlya portion of the peptide side chains are cross-linked via D-Ala-meso-DAP bridges while the others are supposed to beconnected by DAP-DAP bridges [29]

Due to the similar peptidoglycan structure as well asthe homology and synteny of genes involved in cell wallsynthesis peptidoglycan synthesis is assumed to be similarto that in E coli [34] (for a topical review on E coli cellwall synthesis see [51]) and can be separated into threedistinct parts First the building blocks of peptidoglycan

4 ISRNMicrobiology

have to be synthesized in the cytoplasm For this purposeUDP-N-acetylglucosamine is synthesized and partially con-verted to UDP-N-acetylmuramic acid by the murA andmurB gene products [30 52] Next UDP-N-acetylmuramyl-pentapeptide is formed a process in which the murE murFmurD andmurC gene products are involved

The second step of building block synthesis is locatedat the cytoplasmic membrane and involves transfer ofa phospho-N-acetylmuramyl-pentapeptide to polyprenolphosphate catalyzed by themraY gene product and resultingin lipid I As in many other bacteria undecaprenol (C

55)

might be the polyprenol used since at least in C glutamicumthis compound is used for polyprenyl monophosphoman-nose synthesis [53] Next N-acetylglucosamine is transferredfrom UDP-N-acetylglucosamine to lipid I This is catalyzedby murG and yields lipid II or N-acetylglucosamine-120573-(14)-N-acetylmuramyl(pentapeptide)-pyrophosphoryl-polypre-nol The generated lipid intermediate mediates the transportof the hydrophilic disaccharide pentapeptide precursor fromthe cytoplasm across the hydrophobic plasma membrane tothe peptidoglycan layer

As third step disaccharide pentapeptide precursors areintegrated into the growing peptidoglycan meshwork bytransglycosylation and transpeptidation reactions catalyzedby penicillin binding proteins [54] Typically several copiesof these can be found in different Corynebacterium speciessuch as C diphtheriae [52] and C glutamicum [55] (forreview see [30]) For C glutamicum five out of nine putativepenicillin binding proteins were shown to be functional inpeptidoglycan synthesis [55]

A strict coordination of the described steps of cell wallsynthesis is crucial for survival of bacteria since otherwisethe cells would be prone to disruption due to the highinternal turgor pressure The regulation of this process andcell division has been reviewed for C glutamicum [54] andother Actinobacteria [56] recently

42 Linker Unit As shown in mycobacteria arabinogalactanis covalently bound to the murein sacculus via a polysaccha-ride linker unit making up phosphodiester bonds to about10 of the muramic acid residues of the peptidoglycan [57]The mycobacterial linker unit consists of galactose rham-nose and N-acetylglucosamine linked as Galf -(1rarr 4)-Rhap-(1rarr 3)-GlcNAc via a 1-O-phosphoryl bond of GlcNAc to the6-OHposition ofmuramic acid [58] Studies ofC diphtheriaerevealed a very similar linkage profile of arabinogalactan tothat ofMycobacterium tuberculosis [30 33]

43 Arabinogalactan Arabinogalactan is a heteropolysac-charide consisting of D-arabinose and D-galactose present intheir furanose form (Araf Galf ) InC glutamicum alternating120573(1rarr 5) and 120573(1rarr 6)-linked Galf residues form a linearchain of up to 30 sugar moieties which is bound to pepti-doglycan by the linker unit The galactan chain is decoratedwith arabinan branched from the C5 of 120573(1rarr 6) linkedGalf In C glutamicum three branched arabinan domains arelinked per galactan domain at the 8th 10th and 12th Galfresidues Enzymes involved in arabinogalactan synthesis are

well characterized in C glutamicum [59ndash62] and have beenreviewed recently [30]

Whereas C glutamicum arabinogalactan consists exclu-sively of arabinose and galactoseC diphtheriae arabinogalac-tan contains significant amounts of mannose and C amy-colatum and C xerosis arabinogalactans are characterized byadditional glucose content [33] In any case arabinogalactanprovides a covalent connection not only to peptidoglycan butalso to the outer membrane layer

5 Mycolic Acids and Trehalosyl Mycolates

51 Composition and Biosynthesis A second permeabilitybarrier equivalent to the outer membrane of Gram-negativebacteria is a key feature of the CMN group (Corynebac-teriumMycobacterium andNocardia) of Actinobacteria Thefunctionality of this barrier is critically influenced by itsmycolic acid content [63] The inner half of the corynebac-terial mycolic acid layer is mainly formed by mycolic acidsesterified to the 5-OH group of the penultimate 120573(1rarr 2)-linked or ultimate Araf residue of arabinogalactan whilein the outer sheet trehalose and glycerol esterified mycolicacids are predominating Additionally minor amounts of freemycolic acids are found A recent biochemical disclosure ofthe outer membrane of C glutamicum showed that the lipidscomposing the mycomembrane consist almost exclusively ofmycolic acids derivatives whereas only minor amounts ifany of phospholipids and lipomannans were detected [64]

In corynebacteria fatty acids with around 30 carbonatoms (corynomycolates) in nocardia with about 50 carbonatoms (nocardomycolates) and in mycobacteria with about70 to ninety carbon atoms (eumycolates) are found [29]In contrast to the linear fatty acids of the phospholipidsmycolic acids are120572-branched120573-hydroxy fatty acids requiringcarboxylation and condensation of two fatty acids for theirsynthesis [65] The enzymes involved in these steps wereidentified by mutation analyses in C glutamicum and twocarboxylases were identified to be essential for mycolic(and fatty) acid synthesis AccD2 and AccD3 [66] Theseare conserved in Corynebacterineae and provide the crucialcarboxylated intermediate for condensation of merochainand 120572-branch [67] Further proteins involved in mycolic acidsynthesis in C glutamicum and related organisms are AccD1involved in malonyl-CoA synthesis Pks a ketoacyl synthaseinvolved in fatty acid elongation and FadD a fatty acid acyl-AMP ligase ([65 67] for a recent review see [30])

Three pathways for trehalose synthesis were describedin C glutamicum the OtsA-OtsB pathway synthesizingtrehalose from UDP-glucose and glucose-6-phosphate theTreY-TreZ pathway using malto-oligosaccharides or 120572-14-glucans as substrate and the TreS pathway using maltoseas an educt for trehalose synthesis [68 69] It is suggestedthat the transfer of mycolic acid moieties to trehalose occursoutside the cytoplasm [70] since in absence of internallysynthesized trehalose either trehalose or externally addedglucose maltose andmaltotriose can be used as substrate formycolic acidmodification and result in the corresponding di-and monocorynomycolates [70]

ISRNMicrobiology 5

The production of arabinogalactan-linkedmycolates tre-halosylmonocorynomycolates and trehalosyldicorynomyco-lates indicates the presence of mycolyltransferases In factproteins similar to the mycobacterial antigen 85 showingmycoloyltransferase activity also designated fibronectin-binding protein were identified in corynebacteria The firstmember was the PS1 protein fromC glutamicum [71] Later itwas shown that six of these proteins are present in this speciesfive in C efficiens and four in C diphtheriae [29 72 73] Theenzymes are fully redundant in C glutamicum in respect tomycoloylmoiety transfer to trehalose and partially redundantwith respect to transfer of arabinogalactan [29 72 74] Theproteins associated with mycolic acid transport across theplasmamembranewere characterized inM smegmatis andCglutamicum recently [75] InC glutamicum fourmmpL genesencode large membrane proteins associated with mycolatemetabolism and transport which have partially redundantfunction [75]

Besides the plasmamembrane lipids the outermembranefatty acid composition also has to be adapted to differenttemperatures a crucial process for function of the mycolicacid layer as a diffusion barrier [76] In fact one stress-induced protein designated ElrF was identified which isconserved in Corynebacterineae and plays a role in theregulation of outer membrane lipid composition in responseto heat stress [77]

52 Corynomycolates and Pathogenicity Almost all Coryne-bacterium and Mycobacterium species are characterized bya complex cell wall architecture comprising an outer layer ofmycolic acids which is functionally equivalent to the outermembrane of Gram-negative bacteria not only in respectto its physiological role as a permeability barrier but alsoas important component of host pathogen interaction Ithas long been known that constituents of the mycolic acidlayer may be immune stimulatory and effecting macrophagefunctionThese effects are best investigated inM tuberculosis[78] where trehalose dimycolate also designated as cordfactor inhibits fusion events inside the host macrophageand at the same time contributes to macrophage activationHowever limited data for corynebacteria are availableInvestigations of C pseudotuberculosis (formerly Corynebac-terium ovis) indicate a lethal effect of outer membrane lipidson caprine and murine macrophages Lipid extracts of Cpseudotuberculosis had negative effects on glycolytic activityviability and membrane integrity [79] When macrophageswere infected with C pseudotuberculosis uptake of the bacte-ria and lysosome fusion was functional but bacteria survivedinternalization However the macrophages were destroyed[80] For C glutamicum priming and activation of murinemacrophages by trehalose dimycolates were reported [81]

6 Top Layer

Cell surface molecules extracted from different corynebac-teria consist of over 90 carbohydrates and of a minorportion (less than 10) of proteins Analyses of surfacesaccharides ofC diphtheriae strains indicated the presence of

sugars such as N-acetylglucosamine N-acetylgalactosaminegalactose mannose and sialic acid [82] In C amycolatumand C xerosis a neutral glucan consisting mainly of glucosewith an apparent mass of 110 kDa was found in additionto arabinomannans consisting of arabinose and mannosein a 1 1 ratio of 13 and 17 kDa [29 33] Additionally theabove-mentioned lipoarabinomannan and lipomannan weredetected in the outer layer besides trehalose dicorynomy-colate trehalose monocorynomycolate and phospholipidsInterestingly the same lipid compositionwas found for wholebacteria indicating that all classes of lipid molecules wereexposed on the corynebacterial cell surface [33] in contrastto the mycobacterial situation [83] Also a more distinctcomposition of plasma membrane and mycomembrane wasreported for C glutamicum [64]

While Puech and coworkers could only detect a fewbands on coomassie-stained SDS polyacrylamide gels fordifferent corynebacteria proteome analyses supported theidea that a plethora of proteins is exposed on the surfaceof corynebacteria Corresponding studies carried out for Cdiphtheriae [84] C efficiens [85] C glutamicum [64 85 86]C jeikeium [87] and C pseudotuberculosis [88] revealeda significant number of proteins for every single speciesOften these proteins are uncharacterized some clearly havefunctions for nutrient uptake and growth while others areinvolved in host pathogen interactions

61 Corynebacterial Porins Since the permeability barrier ofthe mycolic acid layer most likely hinders nutrient uptakeby plasma membrane transporters the outer membrane hasto be selectively permeabilized to allow growth For thispurpose porin proteins are inserted into the membrane Ingeneral these are functionally equivalent to Gram-negativeporins but resemble a completely different structure mul-timers of 120572-helical subunits instead of the Gram-negativetrimeric 120573-barrels

Corynebacterial porins are best investigated in C glu-tamicum where several different channel-forming proteinswere identified namely PorA PorB PorC and PorH [89ndash92]Homologs of these were found in Corynebacterium callunaeC efficiens [93] and C diphtheria [94] Despite the factthat C amycolatum does not have corynemycolic acids andcontains only small amounts of extractable lipids [33 9596] a channel-forming protein was also isolated from thispathogenic species [97] Reconstitution experiments withpurified PorA and PorH from C glutamicum and homologystudies indicated that the major cell wall channels of Ccallunae C diphtheriae C efficiens and C glutamicum areformed of two porins one of the PorA and one of the PorHtype [98] Furthermore forC glutamicumPorA andPorH anO-mycolationwas shown an extremely unusualmodification[99]

Comparative studies of C glutamicum wild type and aPorA-lacking mutant strain revealed a drastically decreasedsusceptibility of the mutant towards ampicillin kanamycinstreptomycin tetracycline and gentamicin [92] Correspond-ing studies with pathogenic corynebacteria are missinghowever based on the structural and functional similarities

6 ISRNMicrobiology

depicted above differences in the porin repertoire might beone reason for different antibiotic susceptibility observed forthe different species

62 S-Layer Proteins In some Corynebacterium strains thecell surface is covered with a crystalline surface layer com-posed of single protein species which are anchored in thecorynebacterial outer membrane [100] In C glutamicumthe S-layer protein PS2 has an apparent molecular weightof 63 kDa and is anchored in the mycomembrane by a C-terminal hydrophobic domain [101] Electron and atomicforce microscopy applications revealed a highly orderedhexagonal S-layer [31 101ndash104] and a certain degree ofvariability in different C glutamicum isolates [103] The PS2encoding cspB gene is located on a genomic island [103] asituation often found for virulence genes however no distinctphenotype was found for S-layer mutant strains

63 Sialidase Sialidases also designated as neuraminidasesare glycosyl hydrolases that catalyze the removal of terminalsialic acid residues from a variety of glycoconjugates of thehost surface [105] The sugar is subsequently metabolizedor used to decorate the surface of the pathogen In factC diphtheriae exposes sialic acids on its outer surface [82]Sialidase activity was first identified in a crude preparationof diphtheria toxin [106] and sialidase production andcomposition of cell surface carbohydrates of C diphtheriaeseem to be directly depending on iron concentration inthe medium [107ndash109] A putative exosialidase designatedNanH was identified in C diphtheriae Biochemical stud-ies revealed trans-sialylation activity however it is stillunclear if NanH is involved in sialic acids decoration or not[107]

64 Pili Proteinaceous protrusions such as fimbriae and piliare pivotal players for the attachment of bacteria to abioticand biotic surfaces In corynebacteria pili were first reportedin Corynebacterium renale [110] Later detailed molecularbiological analyses were carried out in C diphtheriae (forreview see [111]) C diphtheriae type strain NCTC13129produces three distinct pilus structures SpaA- SpaD- andSpaH-type pili which are polymerized by specific class Csortases [112] and covalently linked to cell surface by sortase F[113] Each type of pilus is composed of the name-giving shaftproteins SpaA SpaD and SpaH and minor pili subunitsthat is SpaB SpaC SpaE SpaF SpaG and SpaI For SpaBand SpaC a function in cell line specificity of pathogenattachment was shown [114] Ott and coworkers observedthat different C diphtheriae isolates are characterized by adifferent pili repertoire [115] which is characterized by differ-ent biophysical properties [116] Interestingly pili formationand adhesion rate are not strictly coupled processes and baldstrains are also able to attach to host cells [115] indicating thepresence of adhesion factors besides pili

Besides in C diphtheriae pilus gene clusters werefound in several pathogenic corynebacteria including

Corynebacterium accolens C amycolatum C aurimucosumCorynebacterium glucuronolyticum C jeikeium Corynebac-terium pseudogenitalium C striatum Corynebacteriumtuberculostearicum and C urealyticum [111]

65 67-72p Hemagglutinin Besides pili C diphtheriaeexhibits nonfimbrial surface proteins 67-72p [117] Recentstudies indicated that 67-72p is encoded by a single geneDIP0733 [118] The corresponding gene product DIP0733enables C diphtheriae to recognize and bind specifically tohuman erythrocytes a process-designated hemagglutinationIn vitro experiments with protein-coated latex beads indi-cated that DIP0733 contributes to invasion and induction ofapoptosis in HEp-2 cells [118]

66 NlpCP60 Proteins Pathogen factors responsible foradhesion are at least partially characterized however themolecular background of invasion remains unclear Basedon a comprehensive analysis of proteins secreted by C diph-theriae [84] Ott and coworker [119] started to characterizethe surface-associated proteinDIP1281 annotated as invasionassociated protein DIP1281 I is a member of the NlpCP60family a large superfamily of several diverse groups ofproteins [120] including putative proteases and probablyinvasion-associated proteins They are found in bacteriabacteriophages RNA viruses and eukaryotes and variousmembers are highly conserved among nonpathogenic andpathogenic corynebacteria such asC diphtheriaeC efficiensC glutamicum and C jeikeium DIP1281 mutant cells com-pletely lacked the ability to adhere to host cells and con-sequently to invade these Based on proteome fluorescenceand atomic force microscopy it was concluded that DIP1281is a pleotropic effector of the C diphtheriae outer surfacerather than a specific virulence factor [119] an idea that wassupported by results obtained for corresponding mutants ofthe nonpathogenic C glutamicum R strain [121] Neverthe-less proteins of this family seem to play a major role incorynebacterial cell surface organization and cell separationFurthermore the results obtained with C diphtheriaemutantstrains [119] support the idea that the corynebacterial cellenvelope components are important determinants of hostpathogen interactions

7 Concluding Remarks

Corynebacteria show a fascinating complex cell wall synthe-sis and architecture (see also Figure 1) Enormous progresswas made in the characterization of fatty acids sugar moi-eties and cell wall polysaccharides however the proteincontent of the top envelope layer has only been character-ized partly despite its probable importance for intercellularcommunication and host pathogen interaction as targets fortherapy or its importance for biotechnological productionFurthermore porin and S-layer proteinsmay have interestingbiotechnological functions in respect to surface display ofproteins and setup of nanostructures [100 122]

ISRNMicrobiology 7

References

[1] M Ventura C Canchaya A Tauch et al ldquoGenomics ofActinobacteria tracing the evolutionary history of an ancientphylumrdquo Microbiology and Molecular Biology Reviews vol 71no 3 pp 495ndash548 2007

[2] X Y Zhi W J Li and E Stackebrandt ldquoAn update of thestructure and 16S rRNA gene sequence-based definition ofhigher ranks of the class Actinobacteria with the proposalof two new suborders and four new families and emendeddescriptions of the existing higher taxardquo International Journalof Systematic and Evolutionary Microbiology vol 59 no 3 pp589ndash608 2009

[3] K Bernard ldquoThe genus Corynebacterium and other medicallyrelevant coryneform-like bacteriardquo Journal of Clinical Microbi-ology vol 50 no 10 pp 3152ndash3158 2012

[4] J Becker and C Wittmann ldquoBio-based production of chem-icals materials and fuelsmdashCorynebacterium glutamicum asversatile cell factoryrdquo Current Opinion in Biotechnology vol 23no 4 pp 631ndash640 2012

[5] A A VertesM Inui andH Yukawa ldquoPostgenomic approachesto using corynebacteria as biocatalystsrdquo Annual Review ofMicrobiology vol 66 pp 521ndash550 2012

[6] A M Cerdeno-Tarraga A Efstratiou L G Dover et al ldquoThecomplete genome sequence and analysis of Corynebacteriumdiphtheriae NCTC13129rdquo Nucleic Acids Research vol 31 no 22pp 6516ndash6523 2003

[7] M Ikeda and S Nakagawa ldquoThe Corynebacterium glutamicumgenome features and impacts on biotechnological processesrdquoAppliedMicrobiology and Biotechnology vol 62 no 2-3 pp 99ndash109 2003

[8] J Kalinowski B Bathe D Bartels et al ldquoThe completeCorynebacterium glutamicum ATCC 13032 genome sequenceand its impact on the production of L-aspartate-derived aminoacids and vitaminsrdquo Journal of Biotechnology vol 104 no 1ndash3pp 5ndash25 2003

[9] E Trost S Gotker J Schneider et al ldquoComplete genomesequence and lifestyle of black-pigmented Corynebacteriumaurimucosum ATCC 700975 (formerly C nigricans CN-1)isolated from a vaginal swab of a woman with spontaneousabortionrdquo BMC Genomics vol 11 no 1 article 91 2010

[10] J Schroder A Glaub J Schneider E Trost andA Tauch ldquoDraftgenome sequence ofCorynebacterium bovisDSM 20582 whichcauses clinical mastitis in dairy cowsrdquo Journal of Bacteriologyvol 194 no 16 article 4437 2012

[11] E Trost J Blom C Soares Sde et al ldquoPangenomic studyof Corynebacterium diphtheriae that provides insights intothe genomic diversity of pathogenic isolates from cases ofclassical diphtheriardquo Endocarditis and Pneumonia Journal ofBacteriology vol 194 no 12 pp 3199ndash3215 2012

[12] V Sangal N P Tucker A Burkovski and P A HoskissonldquoDraft genome sequence ofCorynebacterium diphtheriae biovarintermedius NCTC 5011rdquo Journal of Bacteriology vol 194 no17 article 4738 2012

[13] V Sangal N P Tucker A Burkovski and P A Hoskisson ldquoThedraft genome sequence ofCorynebacteriumdiphtheriae bvmitisNCTC 3529 reveals significant diversity between the primarydisease-causing biovarsrdquo Journal of Bacteriology vol 194 no 12article 3269 2012

[14] A Tauch O Kaiser T Hain et al ldquoComplete genome sequenceand analysis of the multiresistant nosocomial pathogenCorynebacterium jeikeium K411 a lipid-requiring bacterium of

the human skin florardquo Journal of Bacteriology vol 187 no 13pp 4671ndash4682 2005

[15] A Tauch J Schneider R Szczepanowski et al ldquoUltrafastpyrosequencing of Corynebacterium kroppenstedtii DSM44385revealed insights into the physiology of a lipophilic Corynebac-terium that lacks mycolic acidsrdquo Journal of Biotechnology vol136 no 1-2 pp 22ndash30 2008

[16] L T Cerdeira A C Pinto M P Schneider et al ldquoWhole-genome sequence of Corynebacterium pseudotuberculosisPAT10 strain isolated from sheep in Patagonia ArgentinardquoJournal of Bacteriology vol 193 no 22 pp 6420ndash6421 2011

[17] L T Cerdeira M P Schneider A C Pinto et al ldquoCompletegenome sequence ofCorynebacterium pseudotuberculosis strainCIP 52 97 isolated from a horse in Kenyardquo Journal of Bacteriol-ogy vol 193 no 24 pp 7025ndash7026 2011

[18] T Lopes A Silva RThiago et al ldquoComplete genome sequenceof Corynebacterium pseudotuberculosis strain Cp267 isolatedfrom a llamardquo Journal of Bacteriology vol 194 no 13 pp 3567ndash3568 2012

[19] F E Pethick A F Lainson R Yaga et al ldquoComplete genomesequences of Corynebacterium pseudotuberculosis strains 399-5 and 4202-A isolated from sheep in scotland and Australiarespectivelyrdquo Journal of Bacteriology vol 194 no 17 pp 4736ndash4737 2012

[20] F E Pethick A F Lainson R Yaga et al ldquoComplete genomesequence of Corynebacterium pseudotuberculosis strain 106-Aisolated from a Horse in North Americardquo Journal of Bacteriol-ogy vol 194 no 16 pp 4476ndash4476 2012

[21] R T J Ramos A Silva A R Carneiro et al ldquoGenomesequence of the Corynebacterium pseudotuberculosis Cp316strain isolated from the abscess of a Californian horserdquo Journalof Bacteriology vol 194 no 23 pp 6620ndash6621 2012

[22] A Silva R T J Ramos A R Carneiro et al ldquoComplete genomesequence of Corynebacterium pseudotuberculosis Cp31 isolatedfrom an Egyptian buffalordquo Journal of Bacteriology vol 194 no23 pp 6663ndash6664 2012

[23] A Silva M P C Schneider L Cerdeira et al ldquoCompletegenome sequence of Corynebacterium pseudotuberculosis I19 astrain isolated from a cow in Israel with bovinemastitisrdquo Journalof Bacteriology vol 193 no 1 pp 323ndash324 2011

[24] E Trost L Ott J Schneider et al ldquoThe complete genomesequence of Corynebacterium pseudotuberculosis FRC41 iso-lated from a 12-year-old girl with necrotizing lymphadenitisreveals insights into gene-regulatory networks contributing tovirulencerdquo BMC Genomics vol 11 no 1 article 728 2010

[25] J Schroder I Maus K Meyer et al ldquoComplete genomesequence lifestyle and multi-drug resistance of the humanpathogen Corynebacterium resistens DSM 45100 isolated fromblood samples of a leukemia patientrdquo BMC Genomics vol 13no 1 article 141 2012

[26] E Trost A Al-Dilaimi P Papavasiliou et al ldquoComparativeanalysis of two complete Corynebacterium ulcerans genomesand detection of candidate virulence factorsrdquo BMC Genomicsvol 12 article 383 2011

[27] A Tauch E Trost A Tilker et al ldquoThe lifestyle of Corynebac-terium urealyticum derived from its complete genome sequenceestablished by pyrosequencingrdquo Journal of Biotechnology vol136 no 1-2 pp 11ndash21 2008

[28] J Schroder I Maus E Trost and A Tauch ldquoComplete genomesequence of Corynebacterium variabile DSM 44702 isolatedfrom the surface of smear-ripened cheeses and insights into

8 ISRNMicrobiology

cheese ripening and flavor generationrdquo BMC Genomics vol 12article 545 2011

[29] M Daffe ldquoThe cell envelope of corynebacteriardquo inHandbook ofCorynebacterium glutamicum L Eggeling andM Bott Eds pp121ndash148 Taylor amp Francis Boca Raton Fla USA 2005

[30] L Eggeling S B Gurdyal and L Alderwick ldquoStructure andsynthesis of the cell wallrdquo in Corynebacteria A Burkovski Edpp 267ndash294 Caister Academic Press Norfolk UK 2008

[31] M Chami N Bayan J C Dedieu G Leblon E Shechter andT Gulik-Krzywicki ldquoOrganization of the outer layers of the cellenvelope of Corynebacterium glutamicum a combined freeze-etch electron microscopy and biochemical studyrdquo Biology of theCell vol 83 no 2-3 pp 219ndash229 1995

[32] S Marienfeld E M Uhlemann R Schmid R Kramer and ABurkovski ldquoUltrastructure of the Corynebacterium glutamicumcell wallrdquo Antonie van Leeuwenhoek vol 72 no 4 pp 291ndash2971997

[33] V Puech M Chami A Lemassu et al ldquoStructure of the cellenvelope of corynebacteria importance of the non-covalentlybound lipids in the formation of the cell wall permeabilitybarrier and fracture planerdquo Microbiology vol 147 no 5 pp1365ndash1382 2001

[34] C Hoffmann A Leis M Niederweis J M Plitzko and HEngelhardt ldquoDisclosure of the mycobacterial outer membranecryo-electron tomography and vitreous sections reveal thelipid bilayer structurerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 105 no 10 pp 3963ndash3967 2008

[35] M Niederweis O Danilchanka J Huff C Hoffmann andH Engelhardt ldquoMycobacterial outer membranes in search ofproteinsrdquoTrends inMicrobiology vol 18 no 3 pp 109ndash116 2010

[36] C Hoischen and R Kramer ldquoMembrane alteration is necessarybut not sufficient for effective glutamate secretion inCorynebac-terium glutamicumrdquo Journal of Bacteriology vol 172 no 6 pp3409ndash3416 1990

[37] K Nampoothiri C Hoischen B Bathe et al ldquoExpression ofgenes of lipid synthesis and altered lipid compositionmodulatesL-glutamate efflux of Corynebacterium glutamicumrdquo AppliedMicrobiology and Biotechnology vol 58 no 1 pp 89ndash96 2002

[38] M Shibukawa M Kurima and S Oruchi ldquoL-Glutamic acidfermentation with molasses XII Relationship between thekind of phospholipids and their fatty acid composition inthe mechanism of extracellular accumulation of L-glutamaterdquoAgricultural and Biological Chemistry vol 34 no 8 pp 1136ndash1141 1970

[39] P J Brennan and D P Lehane ldquoThe phospholipids ofcorynebacteriardquo Lipids vol 6 no 6 pp 401ndash409 1971

[40] M D Collins M Goodfellow and D E Minnikin ldquoFattyacid composition of some mycolic acid-containing coryneformbacteriardquo Journal of General Microbiology vol 128 no 11 pp2503ndash2509 1982

[41] D E Minnikin M Goodfellow andM D Collins ldquoLipid com-position in the classification an identification of coryneformand relaxed taxardquo in Coryneform Bacteria I J Bousfield and GGalley Eds p 85 Academic Press London UK 1978

[42] E Radmacher L J AlderwickG S Besra et al ldquoTwo functionalFAS-I type fatty acid synthases in Corynebacterium glutam-icumrdquoMicrobiology vol 151 no 7 pp 2421ndash2427 2005

[43] N Ozcan C S Ejsing A Shevchenko A Lipski S Morbachand R Kramer ldquoOsmolality temperature and membrane lipidcomposition modulate the activity of betaine transporter BetP

in Corynebacterium glutamicumrdquo Journal of Bacteriology vol189 no 20 pp 7485ndash7496 2007

[44] C O Rock and J E Cronan ldquoEscherichia coli as a model forthe regulation of dissociable (type II) fatty acid biosynthesisrdquoBiochimica et Biophysica Acta vol 1302 no 1 pp 1ndash16 1996

[45] A K Mishra K Krumbach D Rittmann et al ldquoLipoarabi-nomannan biosynthesis in Corynebacterineae the interplay oftwo 120572(1rarr 2)-mannopyranosyltransferases MptC and MptD inmannan branchingrdquo Molecular Microbiology vol 80 no 5 pp1241ndash1259 2011

[46] L O Moreira A L Mattos-Guaraldi and A F B AndradeldquoNovel lipoarabinomannan-like lipoglycan (CdiLAM) con-tributes to the adherence of Corynebacterium diphtheriae toepithelial cellsrdquoArchives of Microbiology vol 190 no 5 pp 521ndash530 2008

[47] A K Mishra K Krumbach D Rittmann et al ldquoDeletion ofmanC in Corynebacterium glutamicum results in a phospho-myo-inositol mannoside- and lipoglycan-deficient mutantrdquoMicrobiology vol 158 no 7 pp 1908ndash1917 2012

[48] I C Sutcliffe ldquoMacroamphiphilic cell envelope componentsof Rhodococcus equi and closely related bacteriardquo VeterinaryMicrobiology vol 56 no 3-4 pp 287ndash299 1997

[49] KH Schleifer andOKandler ldquoPeptidoglycan types of bacterialcell walls and their taxonomic implicationsrdquo BacteriologicalReviews vol 36 no 4 pp 407ndash477 1972

[50] K Kato J L Strominger and S Kotani ldquoStructure of thecell wall of Corynebacterium diphtheriae I Mechanism ofhydrolysis by the L-3 enzyme and the structure of the peptiderdquoBiochemistry vol 7 no 8 pp 2762ndash2773 1968

[51] D J Scheffers and M G Pinho ldquoBacterial cell wall synthesisnew insights from localization studiesrdquoMicrobiology andMolec-ular Biology Reviews vol 69 no 4 pp 585ndash607 2005

[52] L G Dover A M Cerdeno-Tarraga M J Pallen J Parkhilland G S Besra ldquoComparative cell wall core biosynthesisin the mycolated pathogens Mycobacterium tuberculosis andCorynebacterium diphtheriaerdquo FEMSMicrobiology Reviews vol28 no 2 pp 225ndash250 2004

[53] K J C Gibson L Eggeling W N Maughan et al ldquoDisruptionofCg-Ppm1 a polyprenylmonophosphomannose synthase andthe generation of lipoglycan-less mutants in Corynebacteriumglutamicumrdquo The Journal of Biological Chemistry vol 278 no42 pp 40842ndash40850 2003

[54] M Letek M Fiuza E Ordonez et al ldquoCell growth andcell division in the rod-shaped actinomycete Corynebacteriumglutamicumrdquo Antonie van Leeuwenhoek vol 94 no 1 pp 99ndash109 2008

[55] N Valbuena M Letek E Ordonez et al ldquoCharacterizationof HMW-PBPs from the rod-shaped actinomycete Corynebac-terium glutamicum peptidoglycan synthesis in cells lackingactin-like cytoskeletal structuresrdquo Molecular Microbiology vol66 no 3 pp 643ndash657 2007

[56] M LetekM Fiuza A FVilladangos LMMateos and J AGilldquoCytoskeletal proteins of Actinobacteriardquo International Journalof Cell Biology vol 2012 Article ID 905832 10 pages 2012

[57] E Lederer A Adam R Ciorbaru J F Petit and J WietzerbinldquoCell walls of mycobacteria and related organisms chemistryand immunostimulant propertiesrdquoMolecular and Cellular Bio-chemistry vol 7 no 2 pp 87ndash104 1975

[58] MMcNeilM Daffe and P J Brennan ldquoEvidence for the natureof the link between the arabinogalactan and peptidoglycan ofmycobacterial cell wallsrdquo The Journal of Biological Chemistryvol 265 no 30 pp 18200ndash18206 1990

ISRNMicrobiology 9

[59] L J Alderwick L G DoverM Seidel et al ldquoArabinan-deficientmutants of Corynebacterium glutamicum and the consequentflux in decaprenylmonophosphoryl-D-arabinose metabolismrdquoGlycobiology vol 16 no 11 pp 1073ndash1081 2006

[60] L J Alderwick E Radmacher M Seidel et al ldquoDeletion of Cg-emb in Corynebacterianeae leads to a novel truncated cell wallarabinogalactan whereas inactivation of Cg-ubiA results in anArabinan-deficient mutant with a cell wall galactan corerdquo TheJournal of Biological Chemistry vol 280 no 37 pp 32362ndash323712005

[61] M Seidel L J Alderwick H L Birch H Sahm L Eggelingand G S Besra ldquoIdentification of a novel arabinofuranosyl-transferase AftB involved in a terminal step of cell wall arabinanbiosynthesis in Corynebacterianeae such as Corynebacteriumglutamicum and Mycobacterium tuberculosisrdquo The Journal ofBiological Chemistry vol 282 no 20 pp 14729ndash14740 2007

[62] M Seidel L J Alderwick H Sahm G S Besra and LEggeling ldquoTopology and mutational analysis of the single Embarabinofuranosyltransferase of Corynebacterium glutamicumas a model of Emb proteins of Mycobacterium tuberculosisrdquoGlycobiology vol 17 no 2 pp 210ndash219 2007

[63] H Gebhardt X Meniche M Tropis R Kramer M Daffeand S Morbach ldquoThe key role of the mycolic acid contentin the functionality of the cell wall permeability barrier inCorynebacterineaerdquoMicrobiology vol 153 no 5 pp 1424ndash14342007

[64] C H Marchand C Salmeron R B Raad et al ldquoBiochemicaldisclosure of themycolate outer membrane ofCorynebacteriumglutamicumrdquo Journal of Bacteriology vol 194 no 3 pp 587ndash5972012

[65] D Portevin C de Sousa-DrsquoAuria C Houssin et al ldquoA polyke-tide synthase catalyzes the last condensation step of mycolicacid biosynthesis in mycobacteria and related organismsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 1 pp 314ndash319 2004

[66] R Gande L G Dover K Krumbach et al ldquoThe two carboxy-lases ofCorynebacterium glutamicum essential for fatty acid andmycolic acid synthesisrdquo Journal of Bacteriology vol 189 no 14pp 5257ndash5264 2007

[67] D Portevin C de Sousa-DrsquoAuria H Montrozier et al ldquoTheacyl-AMP ligase FadD32 and AccD4-containing acyl-CoAcarboxylase are required for the synthesis of mycolic acidsand essential for mycobacterial growth identification of thecarboxylation product and determination of the acyl-CoAcarboxylase componentsrdquo The Journal of Biological Chemistryvol 280 no 10 pp 8862ndash8874 2005

[68] M Tzvetkov C Klopprogge O Zelder and W Liebl ldquoGeneticdissection of trehalose biosynthesis inCorynebacterium glutam-icum inactivation of trehalose production leads to impairedgrowth and an altered cell wall lipid compositionrdquoMicrobiologyvol 149 no 7 pp 1659ndash1673 2003

[69] A Wolf R Kramer and S Morbach ldquoThree pathwaysfor trehalose metabolism in Corynebacterium glutamicumATCC13032 and their significance in response to osmoticstressrdquo Molecular Microbiology vol 49 no 4 pp 1119ndash11342003

[70] M Tropis X Meniche A Wolf et al ldquoThe crucial role oftrehalose and structurally related oligosaccharides in thebiosynthesis and transfer of mycolic acids in Corynebacter-ineaerdquo The Journal of Biological Chemistry vol 280 no 28 pp26573ndash26585 2005

[71] G Joliff L Mathieu V Hahn et al ldquoCloning and nucleotidesequence of the csp1 gene encoding PS1 one of the two majorsecreted proteins of Corynebacterium glutamicum the deducedN-terminal region of PS1 is similar to the Mycobacteriumantigen 85 complexrdquo Molecular Microbiology vol 6 no 16 pp2349ndash2362 1992

[72] S Brand K Niehaus A Puhler and J Kalinowski ldquoIdentifica-tion and functional analysis of six mycolyltransferase genes ofCorynebacterium glutamicumATCC 13032 the genes cop1 cmt1and cmt2 can replace each other in the synthesis of trehalosedicorynomycolate a component of the mycolic acid layer of thecell enveloperdquoArchives ofMicrobiology vol 180 no 1 pp 33ndash442003

[73] C de Sousa-DrsquoAuria R Kacem V Puech et al ldquoNew insightsinto the biogenesis of the cell envelope of corynebacteriaidentification and functional characterization of five newmycoloyltransferase genes in Corynebacterium glutamicumrdquoFEMS Microbiology Letters vol 224 no 1 pp 35ndash44 2003

[74] R Kacem C de Sousa-DrsquoAuria M Tropis et al ldquoImportanceof mycoloyltransferases on the physiology of CorynebacteriumglutamicumrdquoMicrobiology vol 150 no 1 pp 73ndash84 2004

[75] C Varela D Rittmann A Singh et al ldquoMmpL genes areassociated with mycolic acid metabolism in mycobacteria andcorynebacteriardquo Chemistry and Biology vol 19 no 4 pp 498ndash506 2012

[76] Y Yang F Shi G Tao and XWang ldquoPurification and structureanalysis of mycolic acids in Corynebacterium glutamicumrdquoJournal of Microbiology vol 50 no 2 pp 235ndash240 2012

[77] X Meniche C Labarre C de Sousa-DrsquoAuria et al ldquoIdenti-fication of a stress-induced factor of Corynebacterineae thatis involved in the regulation of the outer membrane lipidcompositionrdquo Journal of Bacteriology vol 191 no 23 pp 7323ndash7332 2009

[78] J Indrigo R L Hunter Jr and J K Actor ldquoCord factortrehalose 661015840-dimycolate (TDM) mediates trafficking eventsduringmycobacterial infection ofmurinemacrophagesrdquoMicro-biology vol 149 no 8 pp 2049ndash2059 2003

[79] G C Hard ldquoComparative toxic effect of the surface lipid ofCorynebacterium ovis on peritoneal macrophagesrdquo Infectionand Immunity vol 12 no 6 pp 1439ndash1449 1975

[80] J J Tashjian and S G Campbell ldquoInteraction betweencaprine macrophages and Corynebacterium pseudotuberculosisan electron microscopic studyrdquo American Journal of VeterinaryResearch vol 44 no 4 pp 690ndash693 1983

[81] M Chami K Andreau A Lemassu et al ldquoPriming and activa-tion ofmousemacrophages by trehalose 661015840-dicorynomycolatevesicles from Corynebacterium glutamicumrdquo FEMS Immunol-ogy and Medical Microbiology vol 32 no 2 pp 141ndash147 2002

[82] A L Mattos-Guaraldi E A Cappelli J O Previato L C DFormiga and A F B Andrade ldquoCharacterization of surfacesaccharides in two Corynebacterium diphtheriae strainsrdquo FEMSMicrobiology Letters vol 170 no 1 pp 159ndash166 1999

[83] A Ortalo-Magne A Lemassu M A Laneelle et al ldquoIdenti-fication of the surface-exposed lipids on the cell envelopes ofMycobacterium tuberculosis and other mycobacterial speciesrdquoJournal of Bacteriology vol 178 no 2 pp 456ndash461 1996

[84] N Hansmeier T C Chao J Kalinowski A Puhler and ATauch ldquoMapping and comprehensive analysis of the extra-cellular and cell surface proteome of the human pathogenCorynebacterium diphtheriaerdquo Proteomics vol 6 no 8 pp2465ndash2476 2006

10 ISRNMicrobiology

[85] N Hansmeier T C Chao A Puhler A Tauch and J Kali-nowski ldquoThe cytosolic cell surface and extracellular proteomesof the biotechnologically important soil bacterium Corynebac-terium efficiens YS-314 in comparison to those of Corynebac-terium glutamicum ATCC 13032rdquo Proteomics vol 6 no 1 pp233ndash250 2006

[86] THermannM FinkemeierW Pfefferle GWersch R Kramerand A Burkovski ldquoTwo-dimensional electrophoretic analysisof Corynebacterium glutamicum membrane fraction and sur-face proteinsrdquo Electrophoresis vol 21 no 3 pp 654ndash659 2000

[87] N Hansmeier T C Chao S Daschkey et al ldquoA comprehensiveproteome map of the lipid-requiring nosocomial pathogenCorynebacterium jeikeium K411rdquo Proteomics vol 7 no 7 pp1076ndash1096 2007

[88] L G Pacheco S E Slade N Seyffert et al ldquoA combinedapproach for comparative exoproteome analysis of Corynebac-terium pseudotuberculosisrdquo BMCMicrobiology vol 11 article 122011

[89] T Lichtinger F G Rieszlig A Burkovski et al ldquoThe low-molecular-mass subunit of the cell wall channel of the gram-positive Corynebacterium glutamicum immunological local-ization cloning and sequencing of its gene porArdquo EuropeanJournal of Biochemistry vol 268 no 2 pp 462ndash469 2001

[90] P Hunten N Costa-Riu D Palm F Lottspeich and RBenz ldquoIdentification and characterization of PorH a new cellwall channel of Corynebacterium glutamicumrdquo Biochimica etBiophysicaActa - Biomembranes vol 1715 no 1 pp 25ndash36 2005

[91] N Costa-Riu E Maier A Burkovski R Kramer F Lottspeichand R Benz ldquoIdentification of an anion-specific channel inthe cell wall of the Gram-positive bacterium Corynebacteriumglutamicumrdquo Molecular Microbiology vol 50 no 4 pp 1295ndash1308 2003

[92] N Costa-Riu A Burkovski R Kramer and R Benz ldquoPorArepresents the major cell wall channel of the gram-positive bac-terium Corynebacterium glutamicumrdquo Journal of Bacteriologyvol 185 no 16 pp 4779ndash4786 2003

[93] P Hunten B Schiffler F Lottspeich and R Benz ldquoPorH a newchannel-forming protein present in the cell wall of Corynebac-terium efficiens and Corynebacterium callunaerdquo Microbiologyvol 151 no 7 pp 2429ndash2438 2005

[94] B Schiffler E Barth M Daffe and R Benz ldquoCorynebacteriumdiphtheriae identification and characterization of a channel-forming protein in the cell wallrdquo Journal of Bacteriology vol 189no 21 pp 7709ndash7719 2007

[95] M D Collins R A Burton and D Jones ldquoCorynebacteriumamycolatum sp nov a new mycolic acid-less Corynebacteriumspecies from human skinrdquo FEMS Microbiology Letters vol 49no 3 pp 349ndash352 1988

[96] C Barreau F Bimet M Kiredjian N Rouillon and C BizetldquoComparative chemotaxonomic studies of mycolic acid-freecoryneform bacteria of human originrdquo Journal of ClinicalMicrobiology vol 31 no 8 pp 2085ndash2090 1993

[97] U Dorner B Schiffler M A Laneelle M Daffe and R BenzldquoIdentification of a cell-wall channel in the corynemycolic acid-free Gram-positive bacterium Corynebacterium amycolatumrdquoInternational Microbiology vol 12 no 1 pp 29ndash38 2009

[98] E Barth M A Barcelo C Klackta and R Benz ldquoRecon-stitution experiments and gene deletions reveal the existenceof two-component major cell wall channels in the genusCorynebacteriumrdquo Journal of Bacteriology vol 192 no 3 pp786ndash800 2010

[99] E Huc X Meniche R Benz et al ldquoO-mycoloylated proteinsfrom Corynebacterium an unprecedented post-translationalmodification in bacteriardquo The Journal of Biological Chemistryvol 285 no 29 pp 21908ndash21912 2010

[100] N Bayan C Houssin M Chami and G Leblon ldquoMycomem-brane and S-layer two important structures ofCorynebacteriumglutamicum cell envelope with promising biotechnology appli-cationsrdquo Journal of Biotechnology vol 104 no 1ndash3 pp 55ndash672003

[101] M Chami N Bayan J L Peyret T Gulik-Krzywicki G Leblonand E Shechter ldquoThe S-layer protein of Corynebacteriumglutamicum is anchored to the cell wall by its C-terminalhydrophobic domainrdquoMolecularMicrobiology vol 23 no 3 pp483ndash492 1997

[102] S Scheuring H Stahlberg M Chami C Houssin J LRigaud and A Engel ldquoCharting and unzipping the surfacelayer of Corynebacterium glutamicum with the atomic forcemicroscoperdquo Molecular Microbiology vol 44 no 3 pp 675ndash684 2002

[103] N Hansmeier F W Bartels R Ros et al ldquoClassificationof hyper-variable Corynebacterium glutamicum surface-layerproteins by sequence analyses and atomic force microscopyrdquoJournal of Biotechnology vol 112 no 1-2 pp 177ndash193 2004

[104] V Dupres D Alsteens K Pauwels and Y F Dufrene ldquoInvivo imaging of S-layer nanoarrays onCorynebacterium glutam-icumrdquo Langmuir vol 25 no 17 pp 9653ndash9655 2009

[105] E R Vimr K A Kalivoda E L Deszo and S M SteenbergenldquoDiversity of microbial sialic acid metabolismrdquo Microbiologyand Molecular Biology Reviews vol 68 no 1 pp 132ndash153 2004

[106] B S Blumberg and I Warren ldquoThe effect of sialidase ontransferrins and other serum proteinsrdquoBiochimica et BiophysicaActa vol 50 no 1 pp 90ndash101 1961

[107] S Kim D B Oh O Kwon and H A Kang ldquoIdentification andfunctional characterization of the NanH extracellular sialidasefrom Corynebacterium diphtheriaerdquo Journal of Biochemistryvol 147 no 4 pp 523ndash533 2010

[108] L de Oliveira Moreira A F B Andrade M D D Valeet al ldquoEffects of iron limitation on adherence and cell surfacecarbohydrates of Corynebacterium diphtheriae strainsrdquo Appliedand Environmental Microbiology vol 69 no 10 pp 5907ndash59132003

[109] L Warren and C W Spearing ldquoSialidase (Neuraminidase) ofCorynebacterium diphtheriaerdquo Journal of Bacteriology vol 86pp 950ndash955 1963

[110] R Yanagawa K Otsuki and T Tokui ldquoElectron microscopy offine structure of Corynebacterium renale with special referenceto pilirdquo Japanese Journal of Veterinary Research vol 16 no 1 pp31ndash37 1968

[111] E A Rogers A Das andH Ton-That ldquoAdhesion by pathogeniccorynebacteriardquo Advances in Experimental Medicine and Biol-ogy vol 715 pp 91ndash103 2011

[112] H Ton-That and O Schneewind ldquoAssembly of pili in Gram-positive bacteriardquo Trends inMicrobiology vol 12 no 5 pp 228ndash234 2004

[113] S Dramsi P Trieu-Cuot and H Bierne ldquoSorting sortasesa nomenclature proposal for the various sortases of Gram-positive bacteriardquo Research in Microbiology vol 156 no 3 pp289ndash297 2005

[114] A Mandlik A Swierczynski A Das and H Ton-ThatldquoCorynebacterium diphtheriae employs specific minor pilins totarget human pharyngeal epithelial cellsrdquoMolecular Microbiol-ogy vol 64 no 1 pp 111ndash124 2007

ISRNMicrobiology 11

[115] L Ott M Holler J Rheinlaender T E Schaffer M Hensel andA Burkovski ldquoStrain-specific differences in pili formation andthe interaction of Corynebacterium diphtheriae with host cellsrdquoBMCMicrobiology vol 10 article 257 2010

[116] J Rheinlaender A Grabner L Ott A Burkovski and T ESchaffer ldquoContour and persistence length of Corynebacteriumdiphtheriae pili by atomic force microscopyrdquo European Bio-physics Journal vol 41 no 6 pp 561ndash570 2012

[117] A V Colombo R Hirata Jr C M R de Souza et alldquoCorynebacterium diphtheriae surface proteins as adhesins tohuman erythrocytesrdquo FEMS Microbiology Letters vol 197 no2 pp 235ndash239 2001

[118] P S Sabbadini M C Assis E Trost et al ldquoCorynebacteriumdiphtheriae 67-72p hemagglutinin characterized as the proteinDIP0733 contributes to invasion and induction of apoptosis inHEp-2 cellsrdquoMicrobial Pathogenesis vol 52 no 3 pp 165ndash1762012

[119] L Ott M Holler R G Gerlach et al ldquoCorynebacteriumdiphtheriae invasion-associated protein (DIP1281) is involvedin cell surface organization adhesion and internalization inepithelial cellsrdquo BMCMicrobiology vol 10 article 2 2010

[120] V Anantharaman and L Aravind ldquoEvolutionary history struc-tural features and biochemical diversity of the NlpCP60 super-family of enzymesrdquo Genome Biology vol 4 no 2 article R112003

[121] Y Tsuge H Ogino H Teramoto M Inui and H YukawaldquoDeletion of cgR 1596 and cgR 2070 encoding NlpCP60 pro-teins causes a defect in cell separation in Corynebacteriumglutamicum Rrdquo Journal of Bacteriology vol 190 no 24 pp8204ndash8214 2008

[122] T Tateno K Hatada T Tanaka H Fukuda and A KondoldquoDevelopment of novel cell surface display in Corynebacteriumglutamicum using porinrdquo Applied Microbiology and Biotechnol-ogy vol 84 no 4 pp 733ndash739 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of


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Enzyme Research



Microbiology

2 ISRNMicrobiology

bacteria is covered by a peptidoglycan layer which itselfis covalently linked to arabinogalactan an additional het-eropolysaccharide meshwork Bound to this an outer layerof mycolic acids is found which is functionally equivalent tothe outer membrane of Gram-negative bacteria As top layerouter surface material composed of free polysaccharidesglycolipids and proteins (including S-layer proteins pili andother surface proteins) is found (reviewed in [29 30] see alsoFigure 1)

The corynebacterial cell envelope has been investigatedby different optical techniques Electron microscopy of thinsections after freeze substitution revealed a layered cellenvelope organization comprising a plasma membrane athick electron-dense layer an electron-transparent layerand a thin outer layer [31ndash33] This picture resembles thetypical mycobacterial cell envelope appearanceThe electron-dense layer is traditionally interpreted as peptidoglycanthe electron-transparent layer as mycolic acid layer Whencomparing the thickness determined from the electronmicroscopic pictures of corynebacteria andmycobacteria thequestion arose why their electron-transparent layer has asimilar thickness despite the fact that mycobacterial mycolicacids have about a threefold length compared to corynebac-terial ones [33] A solution of this problem was indicatedby cryoelectron tomography studies These revealed a typicalouter membrane with a bilayer structure in Mycobacteriumsmegmatis Mycobacterium bovis and C glutamicum Thethickness of mycobacterial outer layers was smaller thanexpected and based on this observation an alternativemodelfor mycolic acid distribution was proposed implicating afolding of mycolic acids [34 35]

The general structure and composition of the corynebac-terial cell envelope were earlier reviewed by [29 30] inrespect to biochemical and genetic properties especially in Cglutamicum The aim of this paper will be the presentationof cell envelope properties with a broader focus on differentcorynebacteria and the importance of cell envelope compo-nents for pathogen host interaction

3 Cytoplasmic Membrane andFatty Acid Synthesis

31 Plasma Membrane Composition The cytoplasmic orplasma membrane is the main diffusion barrier of cells andseparates cytoplasm and environment As in other bacteriathe corynebacterial plasma membrane is mainly composedof phospholipids assembled into a lipid bilayer which addi-tionally contains other polar lipids besides a great variety ofproteins crucial for transport processes and bioenergetics ofthe cell

The main phospholipid found in C glutamicum is phos-phatidylglycerol followed by diphosphatidylglycerol phos-phatidylinositol and minor amounts of phosphatidylinositoldimannosides (PIM

2) [33 36ndash38] Fatty acids dominating in

the plasma membrane are the saturated palmitic acid (160)and the desaturated decenoic acid (181) [39ndash42] Howeveras in other bacteria fatty acid composition might changesignificantly depending on environmental conditions such as

low or high temperature [43] or the carbon source available[42]

32 Fatty Acid Synthesis Fatty acids are synthesized by suc-cessive cycles of multistep reactions [44] Two distinct typesof these fatty acid synthases (FASs) are distinguished based ontheir general composition the FAS-II type characteristicallyfound in bacteria contains the minimum seven functionaldomains necessary for fatty acid synthesis organized in onepolypeptide whereas the FAS-I type typically found ineukaryotes is comprised of a large multifunctional proteincomplex Interestingly and as an exception from the rule FAS-I proteins are found in members of the CorynebacterineaeC glutamicum and C efficiens contain even two FAS-I-type complexes FAS-IA and FAS-IB which were functionallycharacterized in detail for C glutamicum [42] FAS-IA isessential in C glutamicum while FAS-IB is not howeverFAS-IB-devoid mutant strains exhibit an altered patternof fatty and mycolic acids showing that FAS-IB is activeand necessary to generate the typical wild-type fatty acidprofile [42] The regulatory mechanism allowing adaptationof FAS activity to environmental stimuli (see above) isunknown

C glutamicum does not only have genes coding for twoFAS-I but also genes coding for FAS-II [42] which areinvolved in elongation of mycolic acid chains in mycobacte-ria Since C glutamicum does not contain elongated mycolicacids and FAS-II is absent in other corynebacteria such as Cdiphtheriae it was speculated that these proteins might playa minor physiological role or even not be functional in Cglutamicum [42]

33 Lipomannan and Lipoarabinomannan As in other pro-and eukaryotes the corynebacterial lipid bilayer of thecytoplasmic membrane is not symmetric In corynebacte-ria the proposed reason for asymmetry is an insertionof glycoconjugates in the outer sheet of the cytoplasmicmembrane [29] Lipomannan (LM) and lipoarabinomannan(LAM) derivatives were found in different Corynebacteriumspecies [33] They might be inserted into the plasma mem-brane via covalently bound palmitic acid or decenoic acidmolecules besides their appearance in the outer surfacematerial of corynebacteria Distribution of LM- and LAM-like substances seems to be species-specific inC glutamicumLM-like molecules are dominating in C xerosis and Camycolatum LAM-like substances were preferentially foundwhile a C diphtheriae strain showed an almost equal distri-bution of LM and LAM derivatives [33] An excellent reviewdealing with the synthesis of PIM LM and LAM derivativeswas published recently [45]

34 Lipoarabinomannan as a Virulence Factor A 10 kDalipoarabinomannan polymer was identified in C diphtheriaeand designated CdiLAM The basic structure of CdiLAMshows similarity to mycobacterial LAM however in contrastto lipoarabinomannans in other Actinobacteria CdiLAMpresents an unusual substitution at position 4 of 120572 1rarr 6mannan backbone by 120572-D-Araf CdLAM might be part of

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review article cell envelope of corynebacteria: structure...

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