Annual Joint Meeting of the Missouri and Missouri Valley Branches of the American Society of Microbiology

March 9-10, 2018

Hosted by

MissouriValleyBranch

Missouri Branch Officers

President(through6/30/19)

Lea M. Daniels Email: drleadaniel@gmail.com President-Elect: TBD Councilor (through 6/30/19) Anna Oller University of Central Missouri Email: oller@ucmo.edu Meeting Coordinator Curtis V. Smith Ph.D. Professor of Biological Sciences Interim Dean of Mathematics, Science, Business, and Technology Kansas City Kansas Community College 7250 State Avenue Kansas City, Kansas 66110 Office Phone 913-288-7314 Fax 913-288-7677 Administrative Assistant's 913-288-7518; or .7627

MissouriValleyBranchOfficers

President(through5/31/19)

JulieShafferUniversityofNebraskaatKearneyDepartmentofBiologyBHS335b240111thAveKearney,NE68849308-865-8661shafferjj@unk.edu

President-Elect(through5/31/19)

TravisBourretMedMicro,CrissII,Rm514CreightonUniversity2500CaliforniaPlazaOmaha,NE,68178402-280-3750TravisBourret@creighton.edu

Secretary-Treasurer(through5/31/19)

DonaldRowenUniversityofNebraska,OmahaDepartmentofBiology6001DodgeStreetOmaha,NE68164(402)554-2143FAX:(402)554-3532drowen@unomaha.edu

Councilor

FranklinR.Champlin,Ph.D.OklahomaStateUniversityCenterforHealthSciencesDepartmentofBiochemistryandMicrobiology1111West17thStreetTulsa,OK74107918-561-8490franklin.champlin@okstate.edu

A virtual map can be found at http://www.kckcc.edu/explore-kckcc/campus/maps-directions/map

Take State Ave to Campus Blvd (just west of 72nd and State). Follow to Quindaro Ln. Parking between Argentine Ln and Armourdale Ln. Enter the Henry Louis Social Science Building. Go downstairs and to the back which takes you into the lower level of Jewell.

Keynote Speakers

Dr.MelanieR.Mormile,AssociateProvostatMissouriUniversityofScienceandTechnology,ASMDistinguishedLecturerDr.Mormile’sspecialtyiswiththemetaboliccapabilitiesofthemembersoftheOrderHalanaerobialesandtheirpotentialbiotechnologicalapplications.Shehasresearched:1)theabilityofahaloalkaliphilicbacteriumisolatedfromSoapLake,WashingtontogenerateelectricityatpH11.0and7%salinity:2)AstreamlinedstrategyforbiohydrogenproductionwithHalanaerobiumhydrogeniformans,analkaliphilicbacterium;3)activity-basedmetagenomicscreeningandbiochemicalcharacterizationofbovinerumenprotozoanglycosylhydrolases;4)activity-basedmetagenomicsscreeningandbiochemicalcharacterizationofbovineruminalprotozoanglycosidehydrolases;and5)characterizationofamoderatelyhalo-acidophilicbacteriumisolatedfromLakeBrown,WesternAustralia;and6)theimpactofelevatedCO2concentrationsoncarbonatemineralprecipitationabilityofsulfate-reducingbacteriaandimplicationsforCO2sequestration.Shehas3patents:1)conversionofglycerolto1,3propanediolunderhaloalkalineconditions;2)acombinedfossilfreeprocessoflignocellulosispretreatmentwithbiologicalproduction;and3)fossilfuel-freeprocessofLignocellulosicPretreatmentwithBiologicalHydrogenProduction.

Dr.KarlKlose,PhD,Professor,UniveristyofTexasatSanAntonio,ASMDistinguishedLecturerDr.KarlKlosereceivedhisPh.D.inMicrobiologyatUCBerkeley,andperformedpostdoctoralstudiesatHarvardMedicalSchool.HewasthenhiredasafacultymemberattheUniversityofTexasHealthScienceCenterinSanAntonio(UTHSCSA),andlatermovedtotheUniversityofTexasatSanAntonio(UTSA),andisthefounderoftheSouthTexasCenterforEmergingInfectiousDiseases.Klose’sresearchfocusesonunderstandingbacterialpathogenesis.HislaboratorystudiesVibriocholeraeandthepotentialbioweaponFrancisellatularensis.Kloseisanauthoronmorethan90publications,andhasreceivedfundingfromnumeroussources.HehasmentoredmanyPh.D.,Masters,andundergraduatestudents.HeservedasthePresidentoftheTexasBranchoftheAmericanSocietyforMicrobiology(2001-2003),andhasbeenanorganizerofmultiplenationalandinternationalmeetings.HehastwicebeenarecipientofASMVisitingProfessorships,inKolkata,IndiaandinValparaiso,Chile.Hereceivedthe2002PresidentialJuniorResearchScholarawardatUTHSCSAandthe2009President’sDistinguishedResearchAchievementAwardatUTSA.KlosehasgivenaTEDxtalkonantibiotic-resistantbacteriathatisavailableon

YouTubeandthathasreceivedover40,000views(https://www.youtube.com/results?search_query=karl+klose).KlosewasrecentlyelectedafellowoftheAmericanAcademyofMicrobiology.FeaturedBranchSpeakers

Dr.HeatherSeitz,Ph.D.AssociateProfessorofScience,JohnsonCountyCommunityCollegePULSELeadershipFellow-MidwestGreatPlainsRegionalNetworkDr.SeitzisthefirstauthorandleaderofthetaskforcethatcreatedtheMicrobiologyforHealthSciencesConceptInventory(MHSCI)andhasworkedcloselytoanalyzedthedataonstudentmisconceptions.Shehasdatatosharefromover3,000studentsat15institutionsintheUnitedStates.Helpingtouncoverstudentmisconceptionsisincrediblyimportantinimprovingunderstanding.Validatedandreliableconceptinventoriesaredesignedtouncoverstudentmisconceptions.Recently,theAmericanSocietyofMicrobiologysupportedthedevelopmentoftwoconceptinventoriesthatwillbediscussed:theMicrobiologyConceptInventoryandtheMicrobiologyforHealthSciencesConceptInventory.TheconceptinventoriesthathaverecentlybeenpublishedarealignedwiththeASMCurriculumGuidelines.Thedatafromthedevelopmentoftheseconceptinventorieswasusedtohelpuncoverthemostcommonmisconceptionsinundergraduatemicrobiology.Theprocessusedtocreatetheconceptinventoriesandnationaltrendsinthedatacollectedwillbehighlighted.Examplesofhowtheconceptinventoriescanbeimplementedintocoursedesign,assessment,andfacultydevelopmentwillalsobeshared.

Dr.MarkHoffman,Ph.D.,servesastheChiefResearchInformationofficerforChildren’sMercyandtheChildren’sResearchInstitute.Dr.Hoffman’sroleistoaccelerateandimprovealltypesofresearchattheChildren’sResearchInstitutethroughsuchasdata,applicationsandtechnology.In2013,hejoinedthefacultyattheUniversityofMissouriatKansasCity(UMKC)intheDepartmentsofBiomedicalandHealthInformaticsandPediatrics.AtUMKChelaunchedheCenterforHealthInsightsandbroughtnewInformaticscapabilitiestotheUniversity,IncludingREDCap,i2b2andtheCernerHealthFactsdata.Duringhis16yearsatCernerand3yearsatUMKChehadtheopportunitytomeetawidevarietyofclinicalresearchorganizationsaroundtheworld,learningabouttheirsuccessesandchallenges.In2016Dr.HoffmanjoinedChildren’sMercywhereheistheprimaryinvestigatoronaCDCgranttoutilizeclinicalandlaboratorydatawarehousestoinformqualityimprovementandhecontinuestoserveonthefacultyatUMKC.HehasdeliveredaTEDtalkonthe“Envrome”andwonthe

iThermometercategoryintheGooglewearabledevicesinhealthcarechallengein2015.Heisaninventorof19issuedpatentsandamemberoftheAmericanAcademyofInventors.

Dr.ErikaLutter,PhD,AssistantProfessor,OklahomaStateUniversityMylabisinterestedinmechanismsofhost-cellexit,transmissionandmolecularpathogenesisoftheobligate-intracellularpathogenC.trachomatis.C.trachomatiscausesinfectionsthathavesignificantglobalmedicalandeconomicimpact.Itistheleadingcauseofinfectiousblindness(trachoma)worldwideandthemostreportedbacterialsexuallytransmittedinfectionintheUnitedStates.Asanobligateintracellularpathogenwithacomplexbi-phasicdevelopmentallifecycle,thewaysinwhichC.trachomatisutilizeshostsignallingprocessesforsurvivalandtransmissionarepoorlyunderstood.Untilrecently,C.trachomatiswasgeneticallyintractablebutthelatestadventoftransformationmethodshasenabledthedevelopmentofgenetictoolstomutate,complementandexpressgeneticconstructswithinC.trachomatis.OurresearchusesacombinationofthesenewlydevelopedgenetictoolsandcellularbiologytoaddressmygoalofdecipheringtheinteractionofChlamydialproteinsandmyosinphosphataseandtheirroleinhost-cellexitandthetransmissionofC.trachomatis.

Dr.AnnaSelmecki,PhD,AssistantProfessor,CreightonUniversityMyresearchisfocusedonunderstandingthemechanismsthatcontrolgenomestability.MylabatCreightonUniversityMedicalSchoolusesexperimentalevolutionandcomparativegenomicstoidentifymutationsthataltergenomestabilityandgeneexpressioninbothpathogenicandnon-pathogenicmicrobes.Weusemolecularbiology,biochemistryandfluorescentmicroscopytodeterminethemechanisticroleofnovelgenomestabilizingmutations.Ourexperimentsaddressthegeneticandenvironmentalcausesofincreasedratesofchromosomeaneuploidyinyeast.Finally,wedevelopmathematicalmodelstounderstandhowgenomestabilityimpactstheadaptationatthecellularandpopulationleveloverdifferenttimescales.Myvisionisthattheseapproacheswillgenerateamorecomprehensiveandmechanisticunderstandingofgenomestabilityanditsroleinadaptationthanhasbeenpossiblewithtraditionalgeneticapproaches.

ScheduleMarch95:00-5:15p.m. Welcome LowerLevelJewell5:15-5:45p.m. HeatherSeitz,JCCC—evaluatingnationaldataon

studentmisconceptionsinmicrobiology

5:45-6:15p.m. MarkHoffman,—Medicaldatawarehousestoinformqualityimprovements

6:30-7:30p.m. Dinner Adjacentdiningroom7:30-8:45p.m. Keynotespeaker:MelanieMormile—SoapLake:

MicrobialStudiesfromaSaltyAlkalineLake

8:45-9:00p.m. Closingannouncements March108:00-9:30a.m. PosterSessionI(Undergraduates) LowerLevelJewell9:30-11:00a.m. OralPresentationsI

3roomswithpresentationsGeneralSession1,Room2325Medical,Room2326UndergraduateSession1,Room2335

11:00a.m.-12:00p.m.

OralSessionII3roomswithpresentations

GeneralSession2,Room2325UndergraduateSession2,Room2326UndergraduateSession3,Room2335

12:00-1:30p.m. LunchandKeynotespeaker:KarlKlose—KillerBunniesandBioweapons:TularemiaandBiodefense

LowerLevelDiningRoom

1:30-2:00p.m. StudentsmeetwithKeynotespeakers Room23252:00-3:00p.m. PostersessionII

(GraduateStudents)LowerLevelJewell

3:00-3:30p.m. ErikaLutter,OklahomaStateUniversity--Chlamydia

3:30-4:00p.m. AnnaSelmecki,CreightonUniversityMedicalCenter—yeastevolution

4:00-4:15p.m. Break 4:15-4:45p.m. BranchBusiness Missouri—Room2325

MissouriValley—Room23264:45-5:00p.m. AwardsandClosingremarks LowerLevelJewell

GeneralMicrobiologyGraduateStudentOralPresentationsSession1:Jewell23259:30a.m. NirakarAdhikari D-motifMutationincAMPPhosphodiesterase,RegA,Leadsto

DevelopmentalPhenotypeDefectinDictyosteliumdiscoideum9:45a.m. BirajKayastha TheRoleofβ-carbonicanhydrasesinCalciumDepositionby

Pseudomonasaeruginosa10:00a.m. JorgeLightfoot MetabolicallyEngineeringAspergillusnidulansusingRNA

Interference10:15a.m. NickKuburich InvestigatingaPhosphodiesteraseInvolvedinAmoeboid

MulticellularDevelopmentandSignalingSession2:Jewell232511:00a.m. MarielDegadoCruz ExploringthePotentialRoleofVps1asaFusionProtein11:15a.m. M.M.King CarPisaCa2+RegulatedPutativePhytasethatisUniqueto

PseudomonadsandisHighlyConservedinClinicalIsolates11:30a.m. JaredSmothers MyosinmediatesproteinrecyclingtowardtheGolgi11:45a.m. WesShort SearchingforRecruitmentDomainsandResiduesofVps1toTarget

MembraneMedicalMicrobiology/ImmunologyGraduateStudentOralPresentationsSession1:Jewell2326

9:30a.m. AbbyRigsbee EffectoftheOuterMembranePermeabilizerCompound48/80onIntrinsicResistancetotheHydrophobicBiocideTriclosaninOpportunisticSerratiaSpecies

9:45a.m. JeffShaw ThreeTransketolasesinSalmonellaentericaContributetoDefendingAgainstOxidativeandNitrosativeStresses

10:00a.m. RobertTodd SegmentalAneuploidiesFlankedbyInvertedRepeatsCauseAzoleResistanceintheFungalPathogenCandidaalbicans

10:15a.m. AmandaZalud ROS/RNSInducedChangesinBorreliaburgdorferiOuterSurfaceProteins

10:30a.m. MacyOlson DeterminationoftheRoleofChlamydialInclusionMembraneProteinsinInclusionGrowthandDevelopmentbyProximityLabelingofInteractingPartners

10:45a.m. WilliamBoyle TheBorreliaburgdorferibb0168-EncodedDnaKSuppressorEnhancespHDependentLipoproteinExpression

UndergraduateorHighSchoolOralPresentationsSession1:Jewell2335

9:30a.m. LeaKafer UtilizingGalleriamellonellatoDeterminetheRoleofCalciuminPseudomonasaeruginosaVirulence

9:45a.m. AlisonGuyer RoleofcentromereheterogeneityinclinicalisolatesofCandidaalbicans

10:00a.m. NathanielHigdon AnalyzingProteinInteractionsofTheHerpesSimplexType1UL34Protein

10:15a.m. KeeganMcGill Determining the Localization of the Hypothetical Membrane ProteinCBU_1651fromCoxiellaburnetii

10:30a.m. SamKoshy Characterizationoftheroleofredox-activecysteinesintheregulatoryfunctionofDksAintheLymediseasespirocheteBorreliaburgdorferi

Session2:Jewell232611:00a.m. AmburD.Robertson IdentifyingIntracellularTick-borneIllnessesinBison,Bisonbison,via

BloodCellStainingandPCR11:15a.m. DanielMcLeod

GeneratingDeletionandPointMutationstoStudyaNovelPhytase-likeProtein,CarP,ThatProtectsPseudomonasaeruginosafromElevatedCalcium

11:30a.m. GeorgieO.Tauber SoilMicrobeIsolationSurroundingNativeandInvasiveGrassestoTestforAntimicrobialPropertiesagainstE.S.K.A.P.E.relatives

11:45a.m. MakaylaNemecek LongevityAnalysisofGerm-freeDrosophilamelanogasterSession3:Jewell2335

11:00a.m. CullenHorstmann SilverNanoparticlesonYeastViabilitywithBioinformaticsAnalysis11:15a.m. RyanWindish SiteDirectedMutagenesisofKnownVps1UbiquitinationSites11:30a.m. NicholasWood InitialCharacterizationoftheTwoClpPIsoformsofChlamydia

trachomatisSuggestsIndependentFunctionalityforEach11:45a.m. ChristopherJohnson

Abstracts PosterSession1(UndergraduateStudents)1. ImpactofFluconazoleonGenomeCopyNumberChangesinCandidaalbicans.AnnBraverman

(Undergraduate)*,RobertTodd,AnnaSelmecki.CreightonUniversityMedicalSchool,Omaha,Nebraska

Candidaalbicansisanopportunisticfungalpathogenthatcausesdebilitatinginfectionsinimmune-compromisedindividuals.Fluconazole,afungistaticanti-fungaldrug,isthemostcommonlyprescribedtreatmentforCandidiasisduetoitsrapidbio-availabilityandlowoccurrenceofsideeffects.However,duetothefungistaticnatureandcommon,widespreaduse,fluconazoleresistancehasbecomeamajorthreattopublichealth.Onemechanismassociatedwithantifungaldrugresistanceistheamplificationofimportantdrug-responsegenes.Thisincreaseingenecopynumbercanariseduetoabnormalgenomereplicationorsegregation,andmaycausechangesinwholegenomecopynumber(ploidy).Infungi,significantfitnessincreasescanbeattributedtoploidyincreases(Selmeckietal.2015;Gersteinetal.,2009;Adamsetal.1974).Here,wehaveoptimizedaflowcytometrybasedassaytorapidlydetectploidychangesinyeast-formfungiwhenexposedtofluconazole.Ourploidyassaycoupledwithanin-vitroevolutionexperimentalsystemallowsustoaddresshowfluconazoleexposurealtersgenomestability.2. SegmentalAneuploidiesFlankedbyInvertedRepeatsCauseAzoleResistanceintheFungalPathogen

Candidaalbicans.RobertTodd,TylerWikoff(undergraduate)*,ShilpaNair(undergraduate)*,CurtisFocht,RobertThomas,AnnaSelmecki.CreightonUniversitySchoolofMedicine,Omaha,Nebraska.

Candidaalbicansisthemostprevalentfungalpathogeninimmunocompromisedindividuals.Currently,treatmentofCandidainfectionsreliesheavilyonazoles,afamilyoffungistaticdrugsthatdisruptthebiosynthesisofthefungal-specificsterol,ergosterol.Aneuploidy,amplificationorlossofachromosome,isacommongenomicfeaturefoundin50%ofazole-resistantC.albicans.PreviousworkfromtheSelmeckiLabhasshownthataspecificsegmentalaneuploidy,anamplificationoftheleftarmofchromosome5,canconferazoleresistanceduetotheamplificationoftwodrug-responsegenes.Currently,littleisknownabouthowsegmentalaneuploidiesform.Usingnext-generationsequencingtechnology,chromosomekaryotyping,andlong-rangeDNAsequencingwedescribeseveralnovelsegmentalaneuploidiesfoundinazoleresistantCandidastrainsfromdiversegeneticbackgrounds.Thesesegmentalaneuploidiesconsistofamplifiedregionsofthegenome,someofwhichcanundergocopynumberincreasesofgreaterthan12copies.Theseamplificationscontainimportantdrugresistancegenesandcorrelatewithasignificantincreaseinazoleminimalinhibitoryconcentration.Here,wedescribethesenovelsegmentalaneuploidiesthatconferazole-resistanceandidentifygeneticfeaturesthatelucidateacommonmechanismofformation.3. TheEffectofGarliconEscherichiacoliandStaphylococcusaureus.AmberMason(undergraduate)*Kansas

CityKansasCommunityCollege,KansasCity,Kansas.Theactiveingredientingarlicthatinhibitsbacteriahasbeenshowntobeallicin.Theonlywaythesesubstanceworkisintherawuncookedformofgarlic,orgarlicextractasusedintheseexperiments.Concentrationsof1ml,1.5ml,and2mlwereusedtoinvestigatetheinhibitoryeffectsonE.coliandStaphaureus.Bothmicrobeswerestreakedonseparateplatesforconfluentgrowthandinhibitoryeffectsrecordedafter48hoursincubation.TheresultsdemonstratedthatE.coliwasinhibitedmoreeffectivelywithincreasingconcentrationsthanStaphaureuswhereafewresistantcolonieswereevident.

4. TheRoleofApigenininTreatingLeukemia.RemingtonWilkerson(undergraduate)*,KansasCityKansasCommunityCollege,KansasCity,Kansas.

Apigenin-4’,5,6trihydroxyflavone,aninitiatorofcaspase-9,hasbeenshowntoinduceleukemiacellapoptosis.Itisfoundinrelativelylargequantitiesincelery,parsley,cilantro,oranges,onion,andchamomiletea.Chemicallyengineeredsupplementshavebeendevelopedfromthechamomileplant.Apigenindemonstratesantioxidantbenefitsthathelpmetabolizecelldamagingfreeradicalsanddisruptionofmetastasizingtumorcellsingeneral.Apigeninhasalsobeenshowntohaveanti-inflammatorypropertiesthathelpreduceinflammationandautoimmunediseases.ThisisareviewoftheliteratureposterthatfindsevidencefromtheNationalCancerInstituteandelsewhereshowingthatapigenincanprolongthelifeofleukemiapatients.5. GeneticKnockdownSystemforChlamydiatrachomatis.EmilyGietzen,PrakashSahandErikaLutter,

OklahomaStateUniversity,StillwaterOKChlamydiatrachomatisisanobligateintracellularpathogenthatiscommonlysexuallytransmittedamonghumans.UntilrecentlyChlamydiahasbeengeneticallyintractable,therebylimitinggeneticapproaches.However,recentdevelopmentshaveallowedforthedevelopmentofnovelgenetictoolswhichcanbeusedtomutatespecificgenes.Unfortunately,geneinactivationbytargetronorantibioticcassetteinsertioncanresultinpolareffectsofneighboringgenesmakingitdifficulttostudythegeneswithinoperons.ThisstudyfocusesondevelopinganovelknockdownstrategybyexpressingthereversecomplementspecificChlamydiagenesonashuttleplasmid.Oncecloned,theplasmidsaretransformedbackintoChlamydiaandthegenesexpressedintranswillbetranscribedandbindtheRNAofthecorrespondinggeneproducingdoublestrandedRNAwhichisdegraded.Thismethodwillallowustolookatindividualgenesinanoperonwithoutthepolareffectsofmutations.Thisstrategyisbeingusedonanoperoncontaining6genes.Afterthereversecomplementofeachgeneisexpressed,thedecreasedexpressionofthetargetgenewillbeassessedbyreversetranscriptionPCR.TheseexperimentswillbethefirsttoutilizeagenespecificknockdownstrategyinChlamydia.6. CharacterizationElizabethkingiaursingiiMutantsExpressingElevatedVancomycinResistance.BradenLanier

(undergraduate)*,WilliamL.Johnson,andJohnE.Gustafson.OklahomaStateUniversityDepartmentofBiochemistryandMolecularBiology,Stillwater,Oklahoma.

TheGram-negativeElizabethkingiabacterialgenusexhibitclinically-relevantintrinsicmultipleantibioticresistance.ClinicalreportssuggestthatthepeptidoglycanbiosynthesistargetingantibioticvancomycinwhichiscommonlyusedtotreatGram-positiveinfections,canalsobeusedtotreatElizabethkingiainfections.Inthisstudy,weinitiallydeterminedthevancomycinMICsofsixgenomically-characterizedElizabethkingiaspeciesfollowingCLSIguidelines,whichrangedfrom2to64mg/L.TheseMICssuggestthatallElizabethkingiaspeciesarevancomycin-resistant,andtherefore,refractorytothebactericidalandgrowthinhibitoryactionsofthisdrug.ElizabethkingiaursingiidemonstratedthelowestMIC(2mg/L)whileElizabethkingiameningosepticademonstratedthehighestMIC(64mg/L).WenextisolatedsingleE.ursingiicoloniesthatsurvivedonheartinfusionagarsupplementedwithvancomycin(12,14,16,18and20mg/L)andnotedthatthenumberofsurvivingcolonieswasreducedasthevancomycinconcentrationwasincreased.ThefiveisolatesselectedoffthevancomycincontainingHIAplatesdisplayedMICsrangingfrom32to256mg/L,whicharemuchhigherMICsthanthatexhibitedbytheparentstrain.TheseresultssuggestthatElizabethkingiacanacquireelevatedvancomycinresistancefollowingbriefvancomycinexposure.ThesefindingsdonotsupportstudiesthatsuggestvancomycintherapyiseffectiveagainstElizabethkingiainfections.

7. DrosophilamelanogasterNoravirusORF1ProteinisLocalizedtotheNucleus.LarissaK.Attema(undergraduate)*,AlexisM.Page,BrandonE.Luedtke,BradL.EricsonandKimberlyA.Carlson.DepartmentofBiology,UniversityofNebraskaatKearney,Kearney,Nebraska.

Noravirusisapicorna-likevirusthatistransmittedviathefecal-oralroute.Thegenomeoftheviruscontainsfouropenreadingframes(ORFs)knownasORF1,ORF2,ORF3,andORF4. ORF1wasthefocus inthisstudy,and it isbelievedtoencodeanRNAiinhibitor.WhenweperformedasequenceanalysisofORF1,wediscoveredaputativebipartitenuclearlocalizationsignal(NLS),whichisasequenceofaminoacidsthatdirectsthetransportofproteinsintothenucleiofcells.ThegoalofthisprojectwastoverifythatthisNLSwastransportingORF1intothenucleusofthe cell. We created anORF1-GFP construct, as well as a mutant construct that deleted the NLS from ORF,transfectedtheseintoS2cells,andobservedusingflorescentmicroscopy.Theresultssuggestnuclearlocalization,astheORF1-GFPstainingoverlapswithDAPIstaininginnucleioftheS2cells,andthemutantconstructshowedGFPstaininginthecytoplasmwithnooverlapwithDAPIinthenuclei.Toourknowledge,thisisthefirstexampleofanRNAvirusthatspecifiesanRNAiinhibitorthattranslocatestothenucleus.8. THEMASSEMERGENCEOF17-YEARCICADAACCELERATESLITTERDECOMPOSITION.Ismert,K.,Reazin,C.,

Morris,S.,Jumpponen,A.,Sikes,B.,Zeglin,L.1KansasStateUniversity,Manhattan,KS(KPBS)2UniversityofKansas,Lawrence,KS(KU)

Magicicadaseptendecimaccumulatenutrientsduringtheirfeedingandprovideanutrientpulseastheyemergetomate.Tounderstandhowthematingandsubsequentmassivedepositofcicadaimpactlitterdecompositioninsoil,weconductedalitterbagexperimentintwofieldsitesinthestateofKansas.Wetargetedburoak(QuercusmacrocarpaMichx.)andhackberry(CeltisoccidentalisL.)leaflitter,withandwithoutcicadacarcasses.Atbothsites,wecomparedlitterbagswith10gofhackberryorburoaklitterwithorwithoutanadditional2gofcicada.The30µmmeshlitterbagswereincubatedinsituandsampledatthetimeofdeployment(T0),onemonth(T1),twomonths(T2),fourmonths(T3),orsix(KPBS)andtwelve(KU)monthslater(T4).Ourdatashowthat(i)bothlittertypesdecomposedfasterwithcicadaand(ii)althoughtherecalcitrantlitterdecomposedfasterinthecicada-amendedtreatment,itstillretainedgreaterproportionoftheoriginalmassatT4thantheeasilydecomposablelitter.Theseresultshighlightnutrientpulsesfrominsectpopulationdynamicsmayalternutrientcyclinganddecomposition.Analysesoftheseexperimentscontinueandadditionaleffortsaimtodissectbacterialandfungalcommunitiesandtheirdynamicsovertime.9. UsingInversePCRandSequencingtoIdentifyGenesinPseudomonasaeruginosathatContributeto

SusceptibilitytotheAntimicrobialPeptideDASamp2.MatthewM.Froid(Undergraduate)*,DonaldRowen,PhD.UniversityofNebraska-Omaha,DepartmentofBiology,OmahaNebraska

Increasedresistancetoconventionalantibioticsismakingthetreatmentofbacterialinfectionsmoredifficult.Duetothisdilemma,newalternativestotraditionalantibioticsarecurrentlybeingsought.DASamp2isapromisingnewantimicrobialpeptidethathasbeenexperimentallyshowntobeeffectiveagainstbothGrampositiveandnegativebacteria.InanefforttodeterminethetargetofDASamp2andtodeterminetheeaseatwhichbacteriacandevelopresistancetothiscompound,wepreviouslyisolatedmutantPsuedomonasaeruginosastrainswithshowedincreasedresistancetoDASamp2.Ideterminedthatmutationinonemutant,(RMB1)waslocatedinthegenemexTbyusinginversePCRandsequencing.Thegene,mexT,isbelievedtoencodeatranscriptionalregulatorinvolvedintheproductionofextracellulareffluxpumps.However,theregulationoftheproductionofeffluxpumpsinP.aeruginosaiscomplexandpoorlyunderstood.FurthercharacterizationofthisgeneregulatorynetworkisvaluableduetothelargeincreaseinresistancetoDASamp2(8foldorhigher)seenintheRMB1strain.SuchasignificantincreaseinresistanceishighlysuggestivethattheeffluxpumpsregulatedbymexTplayaroleindeterminingthesensitivityofP.aeruginosacellstoDASamp2.

10. IsolationandCharacterizationofHalo-AcidophilicMicroorganismsfromLakeGneiss,WesternAustralia.AshleySegobiano(Undergraduate)*,MelanieR.Mormile,andSarahStewartJohnson.MissouriUniversityofScienceandTechnology,Rolla,Mo.

WesternAustralialakes,locatedintheYilgronCraton,maybethebestterrestrialanaloguetopreviousMartianconditionsastherearemanygeochemicalsimilarities.Wehypothesizethathaloacidophilicbacteriaresideintheselakes.LakeGneissisamongthemostextremeoftheselakes,withapHofaround1.4andsaturatedsaltconditions.Thepurposeofthepresentstudywastodevelopenrichmentsfortheisolationandcharacterizationofhaloacidophilestogainabetterunderstandingoftheorganismsthatresideintheseharshenvironments.Mediumdesignedtosimulatelakeconditions,microscopicobservation,andmolecularanalyseswereemployedtoachievetheaforementionedgoals.Aftersixmonthsofgrowth,thefirstenrichmentofLakeGneisshadturbidityanddevelopedapHof0.5.DNAextractionwasperformedonthefirstenrichmentofLakeGneissandagenomiclibrarywasgenerated.OurresultssupportourhypothesisthatbacteriacanexistatextremelowpHsandsaltmolaritiesandprovideshintsofthekindoflifethatcouldhavepreviouslyexistedonMars.11. Detectionof theNoravirusRegulatedProteins,Vir-1andVago, inDrosophilamelanogasterHemolymph.

IsaacJ.Lee(Undergraduate)*,AmandaMacke,DarbyJ.Carlson,andKimberlyA.Carlson.DepartmentofBiology,UniversityofNebraskaatKearney,KearneyNE68849

Research into the innate immune response ofDrosophilamelanogaster against virusesmay help identify theirfunctionsinhumans.Twoviralregulatedproteins,VirusinducedRNA-1(Vir-1)andVago,arecandidatesforanalysisbecausetheyarebiosyntheticproductsoftheinnateimmunesystem.Asofyet,thefunctionoftheseproteinsisuncharacterizedinNoravirusinfection.ThepathologyofNoravirusisunknown,butacognitivelocomotordefecthasbeenidentifiedinourlab.OurhypothesisisthatifNoravirusinfectioniscausingthelocomotordefect,thenthemostlikelyrouteoftransmissionfromtheguttothebrainwouldbethroughthehemolymph.WesternblotanalysisofNoravirusinfectedfliesdemonstratesthepresenceofNoravirus,Vir-1,andVagowithinthehemolymph.SinceNoraviruswaspreviouslythoughttoonlybelocatedwithinthegutofD.melanogaster,thisisanewfindingthatmayindicateinfectioninothertissues.MoreresearchmustbeconductiononVir-1andVago,butitisnowpossiblyidentifiedaspartoftheproteomecomprisingthehemolymphofNoraviruspositiveflies.12. IsolationofSoilMicrobestoTestagainstE.S.K.A.P.E.RelativesforAntimicrobialProperties.SaraNansel

(Undergraduate)*andClaudiaCarvalho.FortHaysStateUniversity,Hays,Kansas.TheE.S.K.A.P.E.pathogens,(Enterococcusfaecium,Staphylococcusaureus,Klebsiellapneumoniae,Acinetobacterbaumannii,Pseudomonasaeruginosa,andEnterobacterspecies)areagroupofbacteriathathavedevelopedmultipleresistancestoantibiotics(Boucheretal.).Antibioticresistanceoccurswhenbacteriadevelopthecapabilitytoadaptandmultiplyinthepresenceofantibiotics.Thispresentsaworld-widehealthconcernasantibioticsthatwerecommonlyusedtotreattheseinfectionsarenolongereffective.BytestingbacteriaobtainedfromsoilsamplescollectedaroundtheHays,KSareaagainstrelativesoftheE.S.K.A.P.E.pathogens,thegoalistofindnewantimicrobialpropertiesthatwillactagainsttherelativesandthen,potentially,theE.S.K.A.P.E.pathogens.Fromeachsoilsample,16isolateswereselectedbasedonmorphologicaldifferencestotestagainsttheE.S.K.A.P.E.relativestoobserveinhibitoryeffects.Afterselectingsixpureisolatesexhibitingthisproperty,acombinationofstainingtechniques,biochemicaltesting,aswellasgeneticanalysiswasperformedforbacterialidentificationandcharacterization.TheisolateswerefoundtobebothGramnegativeandGrampositiveandoftheBacillusandPseudomonasgenera.Organicextractioniscurrentlybeingperformedtoisolateandpurifytheinhibitorycomponentofeachisolate.

13. ChlamydiatrachomatisManipulationofHostKinases.NickNelson(undergraduate)*,PrakashSah,andErikaLutter,OklahomaStateUniversity,StillwaterOK

Chlamydiatrachomatisisanintracellularpathogenthatcausesanestimated3millioninfectionseachyearintheUnitedStates.Infectionscanleadtofurthercomplicationssuchasinfertilityandectopicpregnancy.Duringinfection,C.trachomatislivesinaparasitophorousvacuoletermedaninclusion.Fromwithintheinclusionthebacteriamanipulatehost-cellfunctionsinordertoaidinitssurvival.Manyproteins,includingkinases,arerecruitedtotheinclusionmembrane.Withinthisstudywefocusedonhostkinasesandphosphorylatedkinasesubstrates.Recruitmentofkinasesandkinasesubstratestotheinclusionduringinfectionwasmonitoredbyimmunofluorescenceusingkinasespecificantibodies.TheoverallproteinlevelsandlevelsofphosphorylatedproteinsubstrateswereanalyzedbySDS-PAGEandwesternblot.TheanalysisofphosphorylatedsubstratesforMAPK,CDKandPKAshowedalteredphosphorylationastheC.trachomatisinfectionprogressedover48hours.BoththeMAPK-CDKandPKApathwayscontrolandplayasignificantroleincellproliferationandapoptosiswhichwouldaidC.trachomatisinitssurvivability.ManipulationofhostkinaseactivitymanipulationbyC.trachomatisisessentialtodecipheringhost-pathogeninteractionsandmayleadtofuturenoveltherapeutictargets.14. RelationshipBetween Locomotor Function andNoraVirus Infection inDrosophilamelanogaster.Amanda

McCown(undergraduate)*,AbigailBenz,&KimberlyA.Carlson,DepartmentofBiology,UniversityofNebraskaatKearney,Kearney,Nebraska.

NoravirusisamemberofthepicornavirusfamilythatinfectsDrosophilamelanogasterwithnopublishedpathogeniceffects.ApreviouslyunstudiedpathogeniceffectofNoravirusislocomotorability.Inthisstudy,theeffectNoravirushasonlongevityandlocomotorabilityisbeingexamined.Locomotionisexaminedusinggeotaxis,whereflieshaveoneminutetocrossathresholdoneinchfromthetopofthecage.TreatmentgroupsincludeNoravirusinfected,uninfectedandDrosophilaCVirus(DCV)infectedflies.LongevitycurvescreatedusingaStudent’st-testdemonstratethatNoravirusinfectedfliesaresignificantlyslowerintheirclimbingabilitiescomparedtouninfectedflies,whichsupportsarelationshipbetweengeotaxisandlocomotordysfunctionininfectedflies.NosignificantdifferencewasseeninlocomotorabilitybetweenDCVinfectedandNoravirusinfectedflies.NoravirusviralloadwasdeterminedutilizingqRT-PCRwithresultsthatdemonstrateabimodalcurveforNoravirusinfection.ThisdatasuggeststhatNoravirusdoeseffectlocomotorfunctionandcanbeclassifiedasapathogeniceffectofthevirus.15. InvestigatingtheroleofinflammatorycytokinesduringinfluenzaAvirusandAspergillusfumigatus

coinfectionsinvivo.MeaganD.Rippee-Brooks(Undergraduate)*,ChristopherR.Lupfer.MissouriStateUniversityDepartmentofBiology,Springfield,Missouri.

BacterialcoinfectionswithinfluenzaAvirus(IAV)areextremelyseriousandlife-threatening.However,thereexistslimiteddatagatheredabouttheimportanceoffungalinfectionswithIAV.Clinicalcasereportsindicatethatfungalcoinfectionsdohappenandsuggestthepandemic2009IAVhasapropensitytopredisposepatientstosecondaryfungalinfectionsmorethanpreviousIAVstrains.However,thefactorsinvolvedremaintobedetermined.Wehavedeveloped an in vitromodel using primary mouse bone marrow derived macrophages infected with IAV andcoinfectedwiththeopportunisticfungalpathogenAspergillusfumigatus.OurresultsindicatethatIAVandfungalcoinfectionssynergisticallyenhancecellsignalingandcytokineproduction.Weproposethisexacerbatedimmuneresponse during IAV andA. fumigatus predisposes clinical patients tomore severe pneumonia, andwe seek toidentifythepathwaysresponsiblefortheheightenedcytokineresponsesandtheirsignificanceinvivo.

16. PrevalenceofCulturableEndophyticBacteriainaVarietyofSeedSpecies.FaithHiggins*(Undergraduate),GiannaMorrie*(Undergraduate),AshtenGrabill*(Undergraduate),HeatherM.Seitz.JohnsonCountyCommunityCollege,OverlandPark,Kansas.

Antibioticresistanceisaseriousthreattotheworld,anduntreatableinfectionsarenolongerapredictionforthefuture,theyarehappeningrightnow.Unfortunately,thepaceofantibioticdiscoveryisnotkeepingupwiththerapidevolutionofresistancetomicrobes.Fewnewantibioticshavebeendiscoveredinthepast30years(McIntosh,2016).Endophyticbacteriacanbedefinedasthosebacteriathatcolonizetheinternaltissueoftheplantshowingnoexternalsignofinfectionornegativeeffectontheirhost(Holliday,1989&Boyle,2006).Endophyticbacteriaareabletolessenorpreventthedeleteriouseffectofcertainpathogenicorganismsinplants(Ryan,2008).Theinvestigationofendophyticstrainsfornovelantimicrobialmetabolitesisanuntappedsourcewithminimalpreviousresearch.Wearefocusingonfindingandclassifyingendophyticbacteriathatproducenovelantibioticsthatcantreatpathogens.Inthisresearchwewilldemonstrateourapproachtoidentifyingendophyticbacteriaandsharetheprevalenceofculturablestrainsofbacteriafoundonavarietyofspecies.Furtherwehavedocumentedtheantimicrobialpropertiesoftheendophyticbacteriafrommultiplespecies.

17. TranslesionSynthesisProteinAbundanceinProliferatingCells.JazmineSnow(Undergraduate)*,SebastianWendel,NicholasWallace.DivisionofBiology,KansasStateUniversity,Manhattan,KS

HumanPapillomavirus(HPV)causesnearlyeverycervicalcancer,withapproximately14millioninfectionsannuallyintheUnitedStates.ThesecancersaretheresultofHPVoncogenes(HPVE6andE7)manipulatingthehostcells.Thesechangeswerecharacterizedbytranscriptomeanalysisoftumorandcontrolcervicalsamplesandfoundincreasedexpressionofgenesneededfortranslesionsynthesis(TLS).ThisledtothehypothesisthathighlyproliferatingcellsencountermoreDNAdamageresultinginaninductionofTLS,aDNAdamagetolerancepathway.Totestthishypothesis,wedeterminedwhethertheproliferationofhumanforeskinfibroblasts(HFFs)couldbemodulated.WegrewHFFsinagradientoffetalbovineserum(FBS)concentrations.Whenthecellsnearedconfluence,theywereharvestedandcountedonahemocytometer.ThisdatashowedanincreaseincellproliferationcorrelatingwithincreasingFBSconcentration(R2=0.87).HarvestingproteinsfromparalleltreatedcellsdeterminedthatTLSproteinlevelsmirroredproliferationrates.OurinvitrodatashowthatHPVoncogenespreventcervicalcancercellsfrominducingexpressionofPolƞ,aTLSpolymerase.IwilldetermineifHPVoncoproteinspreventproliferation-inducedincreasesinTLSproteinlevelsbyrepeatingtheseexperimentsinHFFsexpressingHPVoncogenes.18. IdentificationofCandidateCellDivisionProteinsinPlanctomyceslimnophilusUsingtheBACTHTwo-Hybrid

System.BriannaSteiert(Undergraduate)*,Dr.LilahRahn-Lee.WilliamJewellCollege,Liberty,Missouri.Understandingcelldivisionmechanismsinbacteriaisimportantbecausecelldivisionisfundamentalforbacterialreproductionandsurvival.MostbacteriadividebybinaryfissionandareassistedbyproteinssuchasFtsZthatassemblesintoaringatmid-cellduringdivision.Thisresearchusesplanctomycetes,agroupofbacteriathatareunusualbecausetheydonotuseFtsZfordivision.Planctomyceteshavemanydistinctivephenotypicfeaturesincludingthecompartmentalizationofcellswithinternalmembranesandamembrane-boundnucleoid.ThepurposeofthisresearchistostudythedivisionmechanismbyscreeningaPlanctomyceslimnophilusgenomicDNApreylibraryagainstseveralcelldivisionproteinbaitsusingtheBACTHtwo-hybridsystem.Throughbioinformaticswork,planctomycetesgeneswerecomparedtogenesinotherbacterialgenomesknowntobeinvolvedincelldivisiontoidentifythebaitsofFtsKandFtsI.Baitandpreyinteractionswillbescreenedthroughco-transformedintoDHM1cells,andpreythatinteractwithbaitwillbecharacterizedtodeterminethePlanctomyceslimnophilussequenceintheprey.Thesegeneswillbecandidatesformembersoftheplanctomyceteuniquecelldivisionmechanism.

19. Theconstructionofageneticdevicetoinvestigatetheregulationofploidyincyanobacteria.SekiK.Anderson(Undergraduate)*andDr.LilahRahn-Lee.WilliamJewellCollege,Liberty,Missouri.

Cyanobacteriaisabacterialphylumknowntoexhibitpolyploidy;differentcyanobacteriaspecieshavedifferentcopynumbers,anddependingonthestrain,ploidyrangesfromaslowasthreecopiestoashighas218genomecopies.Bacterialstrainsaredefinedbytheirdifferencesingeneticmakeup,sowehypothesizethereisageneticfactorthatplaysaroleindeterminingthenumberofchromosomecopieswithinacyanobacteriaspecies.Weareinterestedinstudyingtwostrainswithinthesamegenusthataredifferentingenomecopynumber;Anabaenacylindricahasanaverageof25genomecopiesandAnabaenavariabilishasanaverageof8genomecopies.Tolookforgeneticdeterminantsofcopynumber,weareconstructingabistablesensorthatdetectsthenumberofchromosomecopiesastrainofcyanobacteriahas.Oncethegeneticdeviceismadeandhasbeentuned,weplantotransformagenomiclibraryfromonespeciestotheotheranduseourbistablegeneticdevicetoscreenforindividualswithchangedcopynumber.Thisresearchwillexpanduponourunderstandingofcelldivisionandbacterialdiversity,asweaimtodiscoverthemechanismscyanobacteriausetocountandregulatetheirchromosomecopynumber.20. Prevalenceof antibiotic resistant strains ofEnterococcus spp. andAcinetobacter spp. in community household

environment. Rebekah Elliott (Undergraduate)*, Jada Caplinger, and Anuradha Ghosh. Department of Biology,PittsburgStateUniversity(Pittsburg,KS)

Withincreasingprevalenceofantibioticresistancethreats,thereisanupsurgeintheoccurrenceofcommunity-acquiredinfections.ThepurposeofthisstudyistoassesstheecologyandprevalenceofEnterococcusspp.andAcinetobacterspp.(thatarewell-knownantibioticresistantnosocomialpathogens)inthehouseholdenvironment.Eachhouseholdsamplingkitcontained5swabsforeachofshoebottom,restroom,cleaningsupply,kitchentop,anddoorstep/handleaswellasademographicdatasheettobefilledup.Atotalof30suchkits(n=150)havebeenprocessed.Theswabsweresubjectedtoenrichmentusingselectivemediafortestbacterialspecies.TotalnumberkitspositiveforgrowthofsuspectedenterococciandAcinetobacterspp.weredetermined.Onlyfewcleaningsuppliesshowedgrowthforenterococciwhereasthekitchentopshowedmorefrequententerococcalcontamination.AlthoughmajorityofthelocationsswabbedwerecontaminatedwithsuspectedAcinetobacterspp.,doorstep/handleswerefreeofanyselectedmicrobe.Overall,mostoftheswabbedlocationswerecontaminatedwithbiochemicallyconfirmedAcinetobacterspp. incontrasttofewerwithenterococci.Apanelofantibioticsweretestedfortheirsusceptibility.FurtherPCRamplificationofselectivegeneswillbecarriedouttoconfirmatthespecieslevel.Theantibiotic-resistantisolateswillbegenotypedandcomparedtotheirrelativenosocomialstrains. The community will be outreached with recommended cleaning protocol and stewardship in antibioticconsumptionandresistance.Theoutcomeofthisstudymayhelpfacilitateeffectiveandappropriateantibiotictreatmentofcommunity-acquiredinfections.GeneralMicrobiologyGraduateStudentOralPresentations1. D-motifMutationincAMPPhosphodiesterase,RegA,LeadstoDevelopmentalPhenotypeDefectinDictyostelium

discoideum.NirakarAdhikari(doctoralstudent)*,NickKuburich,JeffHadwiger.OklahomaStateUniversity,Stillwater,Oklahoma.

CyclicnucleotidephosphodiesterasemoleculeshaveclinicalsignificancebecausetheydownregulatesecondmessengercAMPorcGMPincell.InDictyosteliumdiscoideumtheMAPkinase(MAPK),ERK2,down-regulatesthephosphodiesterase,RegA.RegAlowersthelevelofcAMP,animportantregulatorofcellaggregationanddifferentiation.RegAhasaMAPKdockingsite(D-motif)thatmightbeimportantininteractionswithMAPKs.CharacterizingthefunctionofthisD-motifinRegAwill likely provide insights on the interactionsbetweenRegAandMAPKsduring thedevelopmental life cycle ofDictyostelium.Thesefindingscanleadtoabetterunderstandinginothereukaryotesincludingmammals.Intheabsence

ofD-motif,littleornointeractionisexpectedtooccurbetweenRegAD-

andErk2.Thismightpreventthedown-regulation

ofRegAD-

andcausereducedlevelsofcAMP.LowlevelsofcAMPareexpectedtodelaytheaggregationanddifferentiation

ofDictyosteliumcells.RegAD-motifwasmutagenizedandthecorrespondingmutantgene(regAD-

)hasbeenexpressed

intoregA-

mutantcells.ThecloneswithstablephenotypesshowedthattheseregAD-

cloneswereslowerinoverallrateofdevelopment and have defect in their developmental stage. Western blot analysis will be used to determine if any

interactionsoccurbetweenRegAD-

andErk2.

2. TheRoleofβ-carbonicanhydrasesinCalciumDepositionbyPseudomonasaeruginosa.BirajB.Kayastha(Doctoral)*1,ShalakaLotlikar1,ErinGallaway1,ClaudiuT.Supuran2,andMariannaPatrauchan1,1OklahomaStateUniversity,Stillwater,OK,2UniveristyofFlorence,Florence,Italy

Pseudomonasaeruginosaisanopportunistichumanpathogencausinglife-threateninginfectionsinpatientswithcysticfibrosisandinfectiveendocarditis,whichcanbeassociatedwithcalcificationatlaterstages.EarlierresultsshowedthatP.aeruginosaproducesextracellulardepositsofcalcium(Ca2+)whengrownatelevatedCa2+levels.Weidentifiedandcharacterizedthreeβ-carbonicanhydrases(CAs):psCA1,psCA2,andpsCA3.WhilethedeletionofallthreeencodinggenesdelayedP.aeruginosagrowthatambientairby4hours,itcausednoimpactonstaticgrowthat5%CO2.MeasuringbothdepositedandfreeCa2+inliquidmediumforthewildtypeshowedthatstaticgrowthconditionsand5%CO2favorCa

2+deposition.AnalysesofCAmutantsdeterminedthatpsCA1isthemajorcontributortocalciumdeposition.DeletionofpsCA1alonecaused~2folddecreaseinCa2+depositionandalmostabolishedfreeCa2+removalfromthemediumatboth5and10mMCa2+.TheresultsshowthatpsCA2alsocontributetoCa2+depositionat10mMCa2+,butpsCA3playsnoroleinthisprocess.CurrentlywearetestingtheeffectofCAinhibitorsontheβ-CA-inducedCa2+depositionandtheroleofβ-CA-dependentcalcificationinbiofilmformationandvirulenceofP.aeruginosa.3. MetabolicallyEngineeringAspergillusnidulansusingRNAInterference.JorgeLightfoot(Doctoral)*,Rolf

Prade.OklahomaStateUniversity,Stillwater,OklahomaThefilamentousfungi,A.nidulans,canproducenearly100gramsperliterofindustriallyrelevantproteinsunderoptimalconditions.However,manyoftheseproteinsaredegradedorproducedalongsideotherproteins,whichdrasticallyreducetheirefficacyinacellulosefermentationreaction.Weproposeanovelmechanism,utilizingRNAinterference,tocombinatoriallysilencegenes,whichdegradeorcontaminateclientproteins.Usingdualpromoters,wewillflankasequencecontaining30or40bpcomplementarysequencesformultipleclientgenes.ThiswillinducedoublestrandedRNAproduction,inturnloadingtheseindividualcomplementarysequencesintotheArgonautecomplex,silencingthemessengerRNAforeachtargetgene.WehavealsoutilizedLC-MS/MStoexaminechangesintheproteomeofoursilencedstrains.Wehaveseenmarkeddecreasesinourtargetgenesequencesaswellastheinductionofnewproteins,actingasacompensationmechanismforthefungus.Oursilencedstrains,whentransformedtoproduceclientproteins,havealsohadamarkedchangeintheamountofproteinproduced,aswellashowlongitlastsinthemediaduringproduction.Wehavecontinuedthisworkbysilencinggenesresponsibleforunwantedamylolyticactivityinclientproteinproduction.

4. Investigating a Phosphodiesterase Involved in Amoeboid Multicellular Development and Signaling. NickKuburich(Doctoral)*,NirakarAdhikari,JeffHadwiger.OklahomaStateUniversity,Stillwater,Oklahoma

ManyeukaryoticsignalingpathwaysusecAMPasasecondarymessengertoevokespecificresponsestodifferentexternal stimuli. Here, localized levels of cAMP can be controlled by phosphodiesterases,which are sometimesregulatedbyphosphorylation.Dictyosteliumdiscoideumoffersanexcellentmodelsystemtostudytheregulationofphosphodiesterases as it contains relatively few cAMP-specific phosphodiesterases compared tomammals. ThecAMP-specificphosphodiesterase,RegA,regulatesimportantstepsinDictyosteliumdevelopmentandisnegativelyregulated by theMAP kinase, ERK2. This inactivation occurs periodically by external cAMP pulse where a cell-signalingpathwayactivatesERK2.Mammalian studieshave suggested that thecAMP-dependentproteinkinase,PKA,canalsoregulatethephosphodiesteraseactivity.ThisputativeregulationofPKAontheactivityofRegAhasnotbeenfullyinvestigatedinDictyostelium.MassspectrometrywasusedtodetectpotentialphosphorylationsitesonRegA. Two sites of interest have been identified, including a PKA phosphorylation site. Phosphomimic andphosphoablative mutations for the three sites have been constructed. The phenotypes of cells carrying thesemutationshavebeenanalyzedfortheirimpactondevelopmentandcellfate.ThesedatasupportthehypothesisthatRegAisregulatedbymultiplephosphorylationtoregulatesignalingduringmulticellulardevelopment.

5. ExploringthePotentialRoleofVps1asaFusionProtein.MarielDelgadoCruz(Masters)*,Dr.Kim,Kyoungtae.MissouriStateUniversity,Springfield,MO

Proteintraffickingwithinthecellismorethanjusttheseriesofstepsnecessarytogetcargofromoneendofthecelltoanotherlocation,italsoinvolves,theeasilyoverlooked,fusionstep.Fusionistheverylaststepintraffickingwhenthetransportedcargoispassedonfromthecarriervesicleontothetargetmembrane.ThisstepinintracellulartraffickinghasoftenbeencharacterizedbytheuseofSNAREproteins,however,thereareamultitudeofproteinsinvolvedinthisstep.AmongthemareRabs,Golgins,andMultitetheringproteins.Vps1wassuspectedtoplayaroleinhomotypicGolgifusionafternoticingthatadepletioninthisGTPaseresultedinanincreaseofLateGolginumbers.ThisledtothehypothesisthatVps1isfunctioningasafusionproteinactingtobringtwolateGolgicompartmentstogetherinordertoformalessernumberoflargerGolgicompartments.ThisquestionwasexploredusingyeasttwohybridwhereithasbeenfoundthatVps1interactswithGolgilocalizedSNAREs:Tlg1,Tlg2,Vti1,andSnc1.TofurtherinvestigatethisquestionaGSTpulldownassayandaninvivoyeastmatingexperimentsareallunderway.6. CarPisaCa2+RegulatedPutativePhytasethatisUniquetoPseudomonadsandisHighlyConservedin

ClinicalIsolates.M.M.King(Doctoral)*,S.Mares,andM.Patrauchan.OklahomaStateUniversityStillwater,OK

Pseudomonasaeruginosaisaversatilepathogencausingvariousinfectionsinahumanbody.ItisimportanttoidentifythehostfactorsthatsignaltoP.aeruginosathepatient’simmunocompromisedstatus,triggeringthevirulenceofthepathogen.Ourearlierstudiessuggestedthatelevatedcalcium(Ca2+)isoneofsuchsignals,asitincreasesvirulenceinP.aeruginosa.WehaveidentifiedaproteinCarPthatisimportantforCa2+-regulatedinfectivityinP.aeruginosa.SequenceanalysispredictedphytaseactivityandshowedthatcarPisuniquetoPseudomonads.High-resolutionRNAseqandsequenceanalysessuggestedcomplexregulationofcarPbymultiplehostfactors.PromoteractivityassaysconfirmedtheregulatoryroleofelevatedCa2+,H2O2,andCO2.TocharacterizetheroleofcarPduringinfectionsweaimtodeterminewhethertheencodingandregulatorysequencesofcarPareconservedinclinicalisolates(CI).Theinsilicoanalysisrevealedthesesequencesaremorethan98%identicalin~2,000CIcollectedfrompatientswithburnwounds,upperrespiratoryandurinarytractinfections,andkeratitis.Theidentifiedmutationsdonotalterproteinsequences.ThisworkshowsthatcarPanditsregulatoryelementsarepreservedduringinfectionsandhighlyresponsivetohostenvironment,suggestingtheirimportanceinP.aeruginosapathogenicity.7. MyosinmediatesproteinrecyclingtowardtheGolgi.JaredSmothers(Master’s)*,PaulBallhorn*,Vy

Nguyen*,Dr.KyoungtaeKim*MissouriStateUniversity,Springfield,MissouriMyosinfamilyproteinsareATP-dependentmotorsthatsharethesamebasicpropertiesofactinbinding.ThechemicalenergyprovidedbyATP-hydrolysisintheheaddomaingeneratesthe“powerstroke”necessaryto“walk”alongactinfilaments.Inthisstudy,usingconfocalmicroscopyweassessedthepotentialrolesofallfivemyosin’sinSaccharomycescerevisiae,andtheireffectontherecyclingofSnc1andVps10.MYO1knockoutstrainshadnosignificantdefectinSnc1recycling,butdisplayedseveredefectsinthetraffickingofVps10towardtheGolgi,manifestedbyabnormallocalizationtothelumenofthevacuole.Myo3andMyo5areparalogsthathavelittletonosignificanttrafficdefectwhenknockedoutindividually,butthedoubledeletionofbothMYO3and5ledtoseveredefectsinSnc1andVps10trafficking.TwotemperaturesensitivestrainsofMyo2,myo2-16andmyo2-66,demonstratedtraffickingdefectsatrestrictedtemperatureconditions.Amongallfivemembers,onlyMYO4deletiondidnotaffectproteinrecyclingpathways.Together,ourdataprovidesnovelinsightsintothefunctionofMyo-familyproteinsinproteinrecyclingtrafficsdestinedtowardsthetrans-GolgiNetwork.

8. SearchingforRecruitmentDomainsandResiduesofVps1toTargetMembrane.JohnC.(Wes)Short(Masters)*,ShivaKumarGoudGadila,KyoungtaeKim.MissouriStateUniversity,Springfield,Missouri.

Intracellularmembranetraffickingrequiresclassicaldynaminsanddynamin-likeproteins(DLPs),ubiquitousthroughouttheeukaryoticdomain.DysfunctionofclassicaldynaminsinhumansislinkedtoAlzheimer’sandotherneuropathies.LossoforthologyeastDLPVps1disruptspathwaysofvesicletrafficking,whichweusetoinvestigategenefunction.InvitroassayshaveshownthatVps1interactsdirectlywithmembranetoexertitsmembrane-remodelingpower.However,themolecularsignalfortherecruitmentofVps1totargetmembraneshasnotbeendescribed.Toinvestigate,geneticvariantsofVps1wereN-terminallyfusedwithmRFPandoverexpressedinwildtype(WT)andvps1∆backgroundwithgenomically-taggedGFPmembrane-associatedproteinmarkers,andexaminedbyconfocalmicroscopyforcolocalization.TheVps1variantsweresetsofdomainfragmentsaloneandincombinations,seriallyshortenedC-terminaltruncations,andselectedpointmutationsatsuggestedbindingsites,whichwouldreestablishorabolishVps1recruitmentandfunction.Resultsshowedthatalltestedvariantsabolishedthenormalphenotype,andnotestedpointmutationscausedabnormality,suggestingthatthefully-formed3Dshapewithitsintactcatalyticandpolymerizationdomainsisessentialtoitsabilitytotargetmembranes.Furtherstructuralanalysiswillnarrowdownpossiblebindingpatchesforfurthertesting.

MedicalMicrobiology/ImmunologyGraduateStudentOralPresentations1. EffectoftheOuterMembranePermeabilizerCompound48/80onIntrinsicResistancetotheHydrophobicBiocide

TriclosaninOpportunisticSerratiaSpecies.1A.Rigsbee(Masters)*,2S.KatzAmburn,3B.King,4K.Boyina,4J.Yang,1W.Sprinkles,3A.A.McDonald,5V.M.Patel,1F.R.Champlin.1OklahomaStateUniversityCenterforHealthSciences,Tulsa,OK;2RogersStateUniversity,Claremore,OK;3NortheasternStateUniversity,BrokenArrow,OK;4OklahomaStateUniversity,Stillwater,OK;5UnionHighSchool,Tulsa,OK.

Unlikemosthydrophobicmolecules,thebiocidetriclosanisabletopenetratethegram-negativebacterialoutermembrane.AtypicalresistancetotriclosaninthenosocomialopportunistsPseudomonasaeruginosaandSerratiamarcescensislargelyduetooutermembranepropertiesthatresultinimpermeabilityforhydrophobiccompounds.ThepresentstudywasundertakentodetermineifthesecellenvelopeimpermeabilitypropertiesareconservedandunderlietriclosanresistanceinotheropportunisticSerratiaspecies.Generalintrinsicresistancetohydrophobiccompoundswasassessedusingthreedisparatebioassaysandonechemicalassay.Batchculturekineticsinthepresenceofcombinationsoftriclosanorthehydrophobicantibioticnovobiocinandtheoutermembranepermeabilizercompound48/80allowedanalysisofoutermembraneinvolvementinintrinsicresistance.Tenindividualspeciesrangedfromgenerallyrefractorytosensitization,asseenwithSerratialiquefaciens,toextremelysusceptive,asseenwithSerratiaodorifera.Moreover,thosespecieswhichexhibitedintrinsicresistancetobothnovobiocinandtriclosanwerereadilysensitizedtodifferentdegreesbychemicaldisruptionoftheiroutermembraneexclusionaryproperties.ThesedatasuggestthatdisparateopportunisticpathogenswithinthegenusSerratiadifferphenotypicallywithregardtothedegreetowhichoutermembraneexclusioncontributestointrinsicresistancetohydrophobicmoleculesingeneral,andtriclosanspecifically.2. ThreeTransketolasesinSalmonellaentericaContributetoDefendingAgainstOxidativeandNitrosativeStresses.

JeffA.Shaw(Doctoral)*andTravisJ.Bourret.CreightonUniversitySchoolofMedicine,Omaha,Nebraska.As a facultative intracellular pathogen, Salmonella enterica serovar Typhimuriummust combat host-derived reactiveoxygenspecies(ROS)andreactivenitrogenspecies(RNS)thatcandamagebacterialDNA,modifyproteinsandenzymes,anddisrupttheredoxstateofinvadingintracellularbacteria.Topowerantioxidantdefensesandovercomethesecytotoxiceffects,S.Typhimuriumutilizes theoxidativebranchof thepentosephosphatepathway (oxPPP) togenerate reducingpowerintheformofNADPH.Inthenon-oxidativebranch(non-oxPPP),enzymesgeneratemetabolicintermediatesthatcanbechanneledtoglycolysis,recycledbacktotheoxPPP,orutilizedasprecursorsorbiosyntheticreactions.Thisstudyinvestigatedthecontributionoftransketolasesinthenon-oxPPPtoresistingROSandRNS.TheS.Typhimuriumgenomecontains genes for three transketolases (TktA, TktB, and STM2340-41), each of which catalyzed the transketolaseenzymaticreaction. WhenexposedtoROSandRNS,mutantstrains lackingall threetransketolasesdemonstratedthegreatest sensitivity in vitro, most of which was attributable to loss of TktA. Furthermore, a ΔtktA ΔtktB mutant hadattenuatedvirulence following intraperitoneal infectionofC57BL/6mice,butvirulencewas restored in iNOS-deficientmice.ThisstudyrevealsanessentialrolefortransketolasesinresistingROSandRNS,andsuggeststhattheymaycontributetotheintracellularsurvivalofS.Typhimurium.

3. SegmentalAneuploidiesFlankedbyInvertedRepeatsCauseAzoleResistanceintheFungalPathogenCandidaalbicans.RobertTodd(graduate)*,TylerWikoff,ShilpaNair,CurtisFocht,RobertThomas,AnnaSelmecki.CreightonUniversitySchoolofMedicine,Omaha,Nebraska.

Candidaalbicansisthemostprevalentfungalpathogeninimmunocompromisedindividuals.Currently,treatmentofCandidainfectionsreliesheavilyonazoles,afamilyoffungistaticdrugsthatdisruptthebiosynthesisofthefungal-specificsterol,ergosterol.Aneuploidy,amplificationorlossofachromosome,isacommongenomicfeaturefoundin50%ofazole-resistantC.albicans.PreviousworkfromtheSelmeckiLabhasshownthataspecificsegmentalaneuploidy,anamplificationoftheleftarmofchromosome5,canconferazoleresistanceduetotheamplificationoftwodrug-responsegenes.Currently,littleisknownabouthowsegmentalaneuploidiesform.Usingnext-generationsequencingtechnology,chromosomekaryotyping,andlong-rangeDNAsequencingwedescribeseveralnovelsegmentalaneuploidiesfoundinazoleresistantCandidastrainsfromdiversegeneticbackgrounds.Thesesegmentalaneuploidiesconsistofamplifiedregionsofthegenome,someofwhichcanundergocopynumberincreasesofgreaterthan12copies.Theseamplificationscontainimportantdrugresistancegenesandcorrelatewithasignificantincreaseinazoleminimalinhibitoryconcentration.Here,wedescribethesenovelsegmentalaneuploidiesthatconferazoleresistanceandidentifygeneticfeaturesthatelucidateacommonmechanismofformation.4. ROS/RNSInducedChangesinBorreliaburgdorferiOuterSurfaceProteins.AmandaK.Zalud(doctoral)*,TravisJ.

Bourret.CreightonUniversity,Omaha,Nebraska.Borreliaburgdorferi,thecausativeagentofLymedisease,isnaturallymaintainedinvariousmammalianhostsanditstickvectorIxodesscapularis.TheabilityofB.burgdorferitosuccessfullycompleteitsinfectiouscycledependsontheregulationofalargerepertoireoflipoproteins.Lipoproteinexpressioniscontrolledbyalimitednumberoftranscriptionfactorsinresponsetodiverseenvironmentalchallengesencounteredduringinfection,includingchangesinpH,temperature,osmolarity,nutrientavailability,andhost-derivedreactiveoxygenspecies(ROS)andreactivenitrogenspecies(RNS).Inthefollowingstudy,wetestedthehypothesisthatROSandRNSserveasregulatorysignalsforlipoproteinexpressioninB.burgdorferi.Totestthishypothesis,wecomparedtheexpressionofimmunogeniclipoproteinsinB.burgdorferigrowninBSKIIandexposedtohydrogenperoxideorspermineNONOate.AshiftinpHfrom7.6to6.8leadtowidespreadchangesinlipoproteinexpression,whichhasbeenshownpreviously.Surprisingly,sublethalconcentrationsofhydrogenperoxideorthenitricoxidedonorspermineNONOateinhibitedthepH-dependentchangesinlipoproteinexpression.ThesedatasuggestROSandRNScontributetotheregulationoflipoproteinexpressioninB.burgdorferiandmayimpactitsabilitytocompleteitsnaturalinfectiouscycle.5. DeterminationoftheRoleofChlamydialInclusionMembraneProteinsinInclusionGrowthandDevelopmentby

ProximityLabelingofInteractingPartners.MacyG.Olson(doctoralstudent)*,ElizabethA.RucksandScotP.Ouellette.UniversityofNebraskaMedicalCenter,PathologyandMicrobiology,Omaha,Nebraska.

Chlamydiatrachomatis(Ctr),adevelopmentally-regulated,obligateintracellularpathogen,istheleadingcauseofbacterialsexuallytransmittedinfections.Chlamydiaegrowwithinapathogen-specifiedorganelle,termedtheinclusion.Theinclusionmembranemediatesinteractionsbetweenthehostandpathogen.Tosupportchlamydial-hostinteractions,theinclusionmembraneisheavilymodifiedthroughoutthedevelopmentalcyclebychlamydialproteinscalledIncswhichencodetwoormoretransmembranemotifs.WehypothesizethatCtrusestemporalcontrolofIncexpressiontofacilitateinclusiondevelopment,wherebyIncsexpressedearly(e.g.IncF)playanessentialroleinorganization/establishmentoftheinclusionandIncsexpressedlater(e.g.IncA)areinvolvedinnutrientacquisition.Here,wecompareIncFandIncAover-expressiontodeterminetheirrolesininclusiondevelopment,includinghowtheseIncsareinvolvedinprotein-proteininteractionsattheinclusionmembrane.Toexamineourhypothesis,wecreatedCtrL2transformantswithIncF-APEX2,IncATM(transmembranedomain)-APEX2,IncA-APEX2,orAPEX2only.APEX2isanascorbateperoxidasethatbiotinylatesproximalandinteractingproteinsinvivo.PreliminarystudiesshowthatbiotinylatedproteinsidentifiedbymassspectrometryanalysissupportcurrentlyknowndataforIncsandtheireukaryoticproteinbindingpartners.Thesestudiesareimportantforunderstandingmolecularmechanismsinvolvedinestablishmentofthisintracellularpathogenicniche.

6. TheBorreliaburgdorferibb0168-EncodedDnaKSuppressorEnhancespHDependentLipoproteinExpression.WilliamK.Boyle(doctoral)1*,JeffA.Shaw1,AshleyGroshong2,JonS.Blevins3,FrankC.Gherardini4andTravisJ.Bourret1.CreightonUniversitySchoolofMedicine,Omaha,Nebraska1,UConnHealth,Farmington,Connecticut2,UniversityofArkansasforMedicalSciences,LittleRock,Arkansas3,NationalInstituteofAllergyandInfectiousDiseases,Hamilton,Montana4.

Borreliaburgdorferi,thecausativeagentofLymedisease,mustsensetransmissionfavoringconditionswithintickvectorsandincreasetheexpressionofinfectivityassociatedlipoproteins.Wehypothesizedthataputativetranscriptionalregulator,theDnaKsuppressorprotein(DksA),playsaroleincoordinatingthetranscriptionalresponseofB.burgdorferitoenvironmentalsignalsencounteredduringinfection.Totestthishypothesis,wild-typeanddksA-deficientB.burgdorferistrainsweresubjectedtogrowthconditionsmimickingthetickmidgutduringbloodmealacquisitionbyshiftingmid-logcultures(5x107spirochetes/ml)growninBSKII(pH7.6)toBSKII(pH6.8),andallowingthemtoreachstationaryphase(2x108spirochetes/ml).ImmunoblotsofspirochetelysateswithserumfromB.burgdorferi-infectedmiceindicatethatwild-typespirochetesincreasedtheexpressionofimmunogenicproteinsinresponsetopHshift,whiledksA-deficientstrainsexhibitedaconstitutivelylowexpressionofthesepH-inducibleproteins.Further,weobservedthatdksAisrequiredfortheexpressionofthekeyinfectivityassociatedlipoproteinsDbpAandOspC,alongwiththealternativesigmafactorRpoSinresponsetopHshift.Together,thesedataindicateDksAisaregulatorthat,directlyorindirectly,controlstheRpoS-dependentexpressionofimmunogenicproteinsinresponsetochangesinpH.

UndergraduateorHighSchoolOralPresentations

1. UtilizingGalleriamellonellatoDeterminetheRoleofCalciuminPseudomonasaeruginosaVirulence.LeahKafer(Undergraduate)*1,MichelleKing1,MarietteBarbier2,MariannaPatrauchan1OklahomaStateUniversity,Stillwater,OK1,WestVirginiaUniversity,Morgantown,WV2

PseudomonasaeruginosaisanopportunisticpathogeninfectingthelungsofCysticFibrosispatients,knowntohaveabnormallyhighlevelsofcalcium(Ca2+).OurlabhasshownthatelevatedCa2+increasesplantinfectivityandtheproductionofseveralvirulencefactorsincludingpyocyanin,pyoverdine,andrhamnolipidinP.aeruginosa.Basedontheseobservations,wehypothesizedthatelevatedCa2+enhancesP.aeruginosavirulenceinananimalhostaswell.Totestthishypothesis,Ihaveoptimizedavirulencemodelusingwaxworms,Galleriamellonella.First,weaimedtodeterminethehalflethaldose(LD50)ofP.aeruginosa.Forthis,weinjectedthewormswithPAO1grownin0mMor5mMCa2+.ThelatterdiedingreaterquantitiesandfasterthanthoseinfectedwithPAO1grownwithoutCa2+.TheLD50ofPAO1in0mMCa2+istwo-foldhigherthanthatofPAO1grownin5mMCa2+.Currently,wearedeterminingtheroleinvirulenceofthreeproteins,EfhP,CarP,andCalC,previouslyshowntomediateCa2+regulationofvirulencefactorsproduction.ThisknowledgewillenableidentificationofthecomponentsofCa2+regulatorynetworkcontrollingP.aeruginosavirulence,whichisasteptowardslearningnovelwaystofightP.aeruginosainfections.2. RoleofcentromereheterogeneityinclinicalisolatesofCandidaalbicans.AlisonGuyer(undergraduate)*,

RobertTodd,AnnaSelmeckiCandidaalbicansisafungusnaturallyfoundwithinthehumanmicrobiome.Whenexposedtostress,C.albicanscanrapidlyevolvethrougheffectivemeanssuchasgainorlossofanentirechromosome(aneuploidy).Specifically,whenC.albicansisexposedtotheantifungaldrugfluconazoleitmayacquireasegmentalaneuploidycalledisochromosome5L[i(5L)].i(5L)arisesfromabreakpointinthechromosome5centromere(CEN5),generatingtwocopiesoftheleftarmofchr5.Previousresearchhasdemonstratedi(5L)tobeakeycomponentinfluconazoledrugresistance,butthemechanismdrivingi(5L)formationisunknown.AninterestingfeaturefoundwithinCEN5isaheterozygoussequenceencodingalongterminalrepeat(LTR).Thepurposeofthisstudyistoexplorethepotentialroleofchr5heterogeneityintheformationofi(5L)andacquisitionoffluconazoledrugresistance.WeconductedaninvitroevolutionexperimentinwhichC.albicanswereexposedtofluconazoleandscreenedforchangesinheterozygosityoftheLTRandanotherheterozygouslocusonchr5L,thematingtypelocus(MTL).WefoundthatmostC.albicansclinicalisolatesmaintainedbothheterozygouslociafter100generationsinfluconazole.

3. AnalyzingProteinInteractionsOfTheHerpesSimplexType1UL34Protein.NathanielB.A.Higdon(Undergraduate)*,SusanL.Bjerke.WashburnUniversity,Topeka,Kansas.

HerpesSimplexVirusType-1(HSV-1)iseasilycommunicableandinfectionscanoccurindifferentlocations.HSV-1proliferateswithinthehostcellnucleus.Oncereplicationiscompletethevirusexitsthenucleus.ViralproteinUL34isessentialfordeparturefromthenucleus.It’sunknownwhichnuclearproteinsUL34isinteractingwithduringthisphase.UL34isahighlyconservedproteininallhumanherpesvirusesandanidealcandidateforfuturedrugtreatments.BlockingUL34functioninHSV-1wouldpreventexitofthenucleusandspreadofinfection.TodetermineinteractionpartnersforUL34,pulldownassayswereperformed.Inourassay,purifiedUL34proteinwasmixedwithHEp-2celllysate;UL34andanybindingpartnerswerethenremovedfromthemixture.OurpastresultsshowedsomepotentialUL34bindingpartners,however,uponexperimentalreplications,inconsistentresultswereobtained.Toobtainsimilarresultsfromourassays,weexpressedanewGSTcontrolproteininE.coli.WewillusethiscontroltocomparetotheproteinsthatarebeingpulleddownbyaGST-taggedUL34protein.WehopeourresultswillagainshowpotentialUL34bindingpartners.Ifmoreconvincingproteininteractionsoccur,isolationexperimentswillbeperformedtoidentifythebindingprotein(s).

4. DeterminingtheLocalizationoftheHypotheticalMembraneProteinCBU_1651fromCoxiellaburnetii

KeeganMcGill(Undergraduate)*BrandonLuedkte,UniversityofNebraskaatKearneyDepartmentofBiology,Kearney,Nebraska

CoxiellaburnetiiisanobligateintracellularpathogenandtheetiologicalagentofQueryFever.Tocausedisease,C.burnetii uses the Type IVB secretion system (T4BSS) to establish a replicative compartment, termed theparasitophorousvacuole(PV),andfromheremanipulatehostcellfunctionsviathereleaseofeffectorproteins.Apotentialeffectorproteinencodedbythegenecbu_1651,whichisuniquetoC.burnetii,isofinterest.Usinginsilicoanalyses,cbu_1651ispredictedtohaveatransmembranedomainandislikelyco-regulatedwiththeT4BSSsinceitislocatedbetweentheT4BSSgenesicmWandicmXandsuggestsapossibleimportantfunctionforCBU_1651duringpathogenesis.TheoverallgoalofthisstudywastocharacterizethelocalizationofCBU_1651duringinvivoandinvitrogrowth.WehavesuccessfullydevelopedpolyclonalantiseraagainstCBU_1651andareusingitforsubsequentassays.WehavefoundbywesternblotthatCBU_1651issecretedinaT4BSSdependentandlipiddependentmannerintothegrowthmedium.Intissueculture,indirectfluorescentantibody(IFA)assaysindicatedCBU_1651tolocalizetothePVlumen.ThisdatasuggeststhatCBU_1651ismediatedbyamechanismthatsensesenvironmentalstimuli.

5. Characterizationoftheroleofredox-activecysteinesintheregulatoryfunctionofDksAintheLymedisease

spirocheteBorreliaburgdorferi.SamKoshy(Undergraduate)*,WilliamBoyle,andTravisJ.Bourret,CreightonUniversitySchoolofMedicine,Omaha,NE

BorreliaburgdorferiisthecausativeagentofLymedisease,andisestimatedtoinfectasmanyas300,000individualsperyearintheUnitedStates.B.burgdorferi’slifecycleincludesthewhite-footedmouse(Peromyscusleucopus),ticksofthegenusIxodes,andhumans.Asitistransmitted,itundergoesavarietyofenvironmentalchangesthatincludeshiftsinpH,temperature,andnutrientavailability.ThisprojectaimstoexploretherelevanceofcysteinesencodingthezincfingerdomaininthetranscriptionfactorDksA(DnaKsuppressorprotein)thatisknowntoplayakeyroleinthestringentresponse.Thezincfingerwillbeassessedbyperformingsite-directedmutagenesisofcysteineswithinaB.burgdorferidksAalleleencodedonplasmids.TheeffectsofthesepointmutationsonDksAfunctionwillbedeterminedbyintroducingthelibraryofmutantplasmidsintodksA-deficientB.burgdorferistrains.Theabilityofthesestrainstocontroltheexpressionofstringentresponsegenes,survivenutrientlimitationandoxidativestress,andinfectpotentialhostswillbeconducted,highlightingtheimportanceofthezincfingermotifwithinDksAinregulatingthestringentresponse,andtogaininsightintothehowB.burgdorferisurvivesandproliferatesinhostileenvironments.

6. IdentifyingIntracellularTick-borneIllnessesinBison,Bisonbison,viaBloodCellStainingandPCR.Robertson,A.D.(Undergraduate)*,andA.R.Oller.DepartmentofBiologyandAgriculture,UniversityofCentralMissouri.

PrevioustickvectorstudieshaveconfirmedBabesia,Anaplasma,andEhrlichiainCentralMissouriticks.Eachmicroorganismisknowntocausezoonoticcasesofdiseaseinmammalianhostslikeruminantsviaticks.Althoughsometickstudiesutilizedcattle,Bisonstudiesarelacking.Withbison,becominganincreasingfixtureinpasturesduetovariousdietarychangesinmeatconsumptionpractices,thepresenceoftick-borneillnessesincaptivebisonherdsisgreatlyneeded.Theveterinariancollectedapproximately40bloodsamplesviatailveinvenipunctureusingEDTAasananticoagulant,andwasrefrigerated.Samplenumberscontainedgender,age,andweight.Thisprojectlookedatbloodparasitemia(%)ratestoconfirmthepresenceofcirculatingbloodparasites,asitisimportanttoestablishexpectedparasitesandco-infectionswithinbisonherdsinMissouri.BloodsmearsweremadeandstainedwithafilteredWright-Geimsastainthatwouldalsoallowparasitevisualization.Outofatotalof38Bison,fivehavebeenthoroughlyexaminedforpathogens.4thusfarhavesuspectedcasesofAnaplasma,threeEhrlichia,andoneBabesia.Someofthebisonhavecoinfections;furtherexaminationofbloodsmears,alongwithPCRtestingwillyieldmoreconciseresults.IACUCapprovalofUCM#928wasalreadyobtainedandgrantedforthispathogensurvey.

7. GeneratingDeletionandPointMutationstoStudyaNovelPhytase-likeProtein,CarP,That

ProtectsPseudomonasaeruginosafromElevatedCalcium.DanielMcLeod(Undergraduate)*,MichelleKing,Dr.MariannaPatrauchan.OklahomaStateUniversity,Stillwater,OK.

Pseudomonasaeruginosaisanopportunisticpathogenthatinfectswoundsandthelungsofcysticfibrosispatients.Previously,weshowedthatseveralvirulencefactorsinP.aeruginosaareinducedbyelevatedcalcium(Ca2+).Weidentified a putative phytase, CarP, and determined its role in protecting cells against Ca2+ and regulating Ca2+-inducedvirulencefactors.Therefore,wehypothesizedthatthisproteinbindsCa2+andbelongstotheCa2+regulatorynetwork in P. aeruginosa. To study the role of carP in Ca2+ regulation, we used a transposon mutant and acomplementationstrain,wherecarPisclonedunderanarabinose-induciblepromoter.HereweaimtogenerateacleancarPdeletionstrainandacomplementedstrainwherecarPwillberegulatedbyitsnativepromoter.SequenceanalysesshowednoknownCa2+bindingmotifsinCarP,andweaimtoidentifytheresponsibleresidues,whichwilllikelypresentanovelCa2+-bindingmotif.Weareusing inversePCRto replaceE183andE291predicted tobindCa2+with a glutamine,whichwill preventbinding.Wewill thenmeasure theproteins’ ability tobindCa2+ usingisothermalchromatography.Obtainingthesemutantswillalsoenablefuturefunctionalstudies,characterizingtheroleofCarPinP.aeruginosavirulence.

8. SoilMicrobeIsolationSurroundingNativeandInvasiveGrassestoTestforAntimicrobialPropertiesagainstE.S.K.A.P.E.relatives.GeorgieO.Tauber(Undergraduate)*,ClaudiaM.Carvalho,andMitchellJ.GreerDepartmentofBiologicalSciences,FortHaysStateUniversity,Hays,Kansas

Asnon-nativespecies(suchasBothriochloaischaemumandB.blahdii)continuetoinvadenativelands,theyalterthelandscape.Bothriochloaspp.havebeenshowntohaveallelopathiceffects.Theseallelochemicalsneedmoreinvestigationastowhetherornotthesebiochemicalsalterthemicrobesinthesurroundingsoiland/orhaveantimicrobialproperties.Ifso,theseantimicrobialpropertiescouldleadtonewantibiotics.AntibioticandantimicrobialresistancesuchasintheE.S.K.A.P.Epathogens,hasbeguntorunrampantaroundtheglobeandhascausedgreatdilemmatomanyphysicians.TheE.S.K.A.P.E.pathogenspresentarealthreattooursocietytoday.SamplesweretakenfromsoilsurroundingBothriochloaischaemum,B.blahdii,andAndropogongerardii-anativegrass.Afterinitialserialdilutionandselectionofcolonies,theisolatedcoloniesweretestedagainstE.S.K.A.P.E.pathogenrelativesforantimicrobialproperties.Therewerezerozonesofinhibitionfromsamplestakenneartheinvasivespecies,whiletwocoloniesfromAndropogongerardiisamplesshowedzonesofinhibition(bothagainstStaphylococcusepidermidis).Thebacteriawerefoundtobeagram-postive.Furthertestingwillbeperformedtodeterminetheidentityofthebacteria.

9. LongevityAnalysisofGerm-freeDrosophilamelanogaster.MakaylaNemecek(Undergraduate)*,RebeccaBest,ShelbyPeters,CarliePrososki,LesleyTowery,BradL.Ericson,DarbyJ.Carlson,&Kimberly.A.Carlson,DepartmentofBiology,UniversityofNebraskaatKearney,Kearney,NE68849

Gastrointestinalmicrobiotaandvirusesareakeycomponentincharacterizingguthealthandlongevity.Noravirus,apersistentvirusthatreplicatesinthegutofDrosophilamelanogaster,showsnolethalitytotheorganism.VirusessimilarinnaturetoNoravirusinteractwithgutmicrobiota,andthehealthoftheorganismandlongevitymaybedependent on a persistent viral infection. Our hypothesis is that Nora virus may be needed within thegastrointestinaltractofD.melanogastertomaintainafavorableenvironmentforgutmicrobiotaandincreasethelifespan. GermfreeD.melanogasterweregeneratedwiththeuseofantibioticsanddividedintofourtreatmentgroups: Noraviruspositive/bacteriapositive,Noravirusnegative/bacteriapositive,Noravirusnegative/bacterianegative,andNoravirusnegative/bacterianegative.ThepresenceofNoraviruswasdetectedwiththeuseofRT-PCRandbacterialspecieswasdeterminedbyplatinghomogenizedfliesonLuriaBrothplates.Alongevitystudywasconductedoneachof the treatmentgroupsanddemonstrated thatNoravirusdoesnotenhance longevity,butmicrobiotaisneededwhenvirusispresenttolive.ThisdatasuggeststhatNoravirusinfectionisnotbeneficialtothemicrobialenvironmentwithinthegastrointestinaltract,butfurthertestingisneeded.

10. SilverNanoparticlesonYeastViabilitywithBioinformaticsAnalysis.CullenHorstmann(undergraduate)*,and

KyoungtaeKim.MissouriStateUniversityDepartmentofBiology,Springfield,Missouri.Nanoparticleshavebecomecommoninmanycommerciallyusedproductssuchaszincsunscreenandwaterresistantclothes.Theymayalsobeutilizedinthetargetedtreatmentofcancer,printablemonitoringsystems,andcosteffectivephonesinthefuture.Theeffectsnanoparticleshaveonbiologicalorganismsiscrucialfortheresponsibleuseofthesetechnologies.Weinvestigatedtheeffectsofsilver(Ag)nanoparticlesonbuddingyeast(Saccharomycescerevisiae)usinggrowthassays,FUN-1stainingformetabolicactivity,RNAseq,andRTPCR.OurgrowthassayshowedthatAghasaninhibitoryeffectwithitsconcentrationsabove5µg/ml.HundredsofgenesinAgtreatedcellsweredifferentiallyexpressedaccordingtoourtranscriptomeinvestigation.AlargefractionofupregulatedgenesareidentifiedtoregulateribosomalbiogenesisandRNAprocessing,whereasdownregulatedgenesareknowntoberesponsibleformitochondrialfunctionsbasedonouranalysisofgeneontologyterms.Furthermore,wevalidatedtheRNAseqresultsusinganRTPCRassay.TheresultingexpressionprofileleadsustosuspectthatAgnanoparticleexposurecreatesastressenvironmentinthecell.11. SiteDirectedMutagenesisofKnownVps1UbiquitinationSites.RyanWindish(Undergraduate)*,Kyoungtae

Kim.MissouriStateUniversity,Springfield,Missouri.Ubiquitinationisacellularprocessthatisimportantforproteindegradation.Itoccursthroughaseriesofenzymaticreactionsinwhichubiquitin,akeyfactorubiquitouslyexpressedineukaryoticorganisms,tagsthesubstrateproteinstobedegradedviaproteasomes.VacuolarProteinSorting1(Vps1),whichisyeast’shomologuetomammaliandynamin,hasfiveknownubiquitinationsites.ThisproteiniscrucialtotheretrievalofbothVps10andSnc1,whichplaysignificantrolesforintracellularvesiculartraffickingpathways.WehaveperformedexperimentsbymutatingtheknownubiquitinationsitesofVps1throughsitedirectedmutagenesis.AmutantstrainharboringVps1K561NmutationdisplayedseveredefectsinthetraffickingofSnc1andVps10,suggestingthattheVps1ubiquitinationsitesarepivotalfortheirtraffickingtowardtheGolgiandthatproperturnoverofVps1regulatesthesecargotraffickingprocesses.

12. InitialCharacterizationoftheTwoClpPIsoformsofChlamydiatrachomatisSuggestsIndependentFunctionalityforEach.NicholasA.Wood(Undergraduate)*1,2,KrystalChung3,NathaliaRodriguesdeAlmeida1,MartinConda-Sheridan1,DerekJ.Fisher3,ScotP.Ouellette11UniversityofNebraskaMedicalCenter,Omaha,Nebraska.2UniversityofSouthDakota,Vermillion,SouthDakota.3SouthernIllinoisUniversity,Carbondale,Illinois.

Chlamydia trachomatis is anobligate intracellular bacterium thatdifferentiatesbetween twodistinct formsduring itsdevelopmentalcycle:elementarybodies(EBs)andreticulatebodies(RBs).TheEBistheelectrondense,infectiousform.Withinthecell,theEBdifferentiatesintotheRB,whichreplicatesanddevelopswithinahostmembranederivedvesicle,termedaninclusion.RBsreplicatewithinthisinclusionandeventuallydifferentiatebackintoEBsbeforereleasefromthehostcell.GiventheuniquefunctionalandmorphologicalformsofChlamydia,weareinterestedinproteomicregulationandturnoverthroughproteindegradation.WehypothesizethattheClpproteasesystemplaysanintegralroleinproteomicturnoverbydegradingspecificproteinsfromonedevelopmentalformortheother.Chlamydiaencodesfiveclpgenes:clpX,clpC,twoclpPparalogs,andclpB.ClpCandClpXarechaperoneproteinsthatunfoldandfeedproteinsintotheClpPproteasetobedegraded,andClpBisadeaggregase.Our initialcharacterizationfocusesonthetwoClpPparalogs.Wedemonstrate theirdevelopmental expression,potential foroligomerization, importanceduring infectionbyantibioticstargeting ClpPs, and the effect of overexpression of inactive ClpPmutant proteins. Together, these data indicate animportantroleforClpPproteinsinchlamydialgrowthandpathogenesis.13. UsinginterpretableneuralnetworkmodelsofPseudomonasaeruginosageneexpressiontorevealpotential

targetsofanunstudiedtranscriptionfactorimplicatedinhighresistancetoanovelantimicrobialpeptide.ChristopherJohnson(Undergraduate)*andDr.DonaldRowen.UniversityofNebraskaatOmaha,Omaha,Nebraska

Antimicrobialpeptides(AMPs)areanintriguingalternativetocurrentlyavailableantibiotictherapies.DASamP2,anAMPdevelopedbytheWanglabattheUniversityofNebraskaMedicalCenter,iseffectiveagainstPseudomonasaeruginosaandStaphylococcusaureus,butthemechanismofactionisnotcurrentlyunderstood.TocharacterizethegeneticfactorsaffectingsusceptibilitytothisAMP,ourlabhaspreviouslyusedtransposonmutagenesistoconstructnumerousmutantstrainsofP.aeruginosademonstratingincreasedresistancetoDASamP2.AnalysisofoneofourmostresistantmutantsrevealedthatupregulationofthetranscriptionfactorPA5189mayberesponsibleforproducingaresistantphenotypecapableofsurvivingover10xMIC.Thoughthisgeneisunstudied,wecanextractbiologicallymeaningfulinsightsfrompubliclyavailablegeneexpressiondatasetsbyusingdenoisingautoencodersordisentangleddeepvariationalautoencoderstoproduceacompressedrepresentationofallgeneexpressiondatasets.Analyzingatrainedneuralnetworkallowsustoconnectrawexpressiondatatohigher-levelbiologicalfeatures.EarlyresultssuggestPA5189maybeinvolvedintheregulationofnumerouseffluxpumps,porins,andothergenes–mostofwhicharealsounstudied,potentiallyindicatingthatwearelookingatanewfacetofhowP.aeruginosaadaptstoantimicrobialassault.

PosterSession2(GraduateStudents)

1. SodiumPyruvateAlterstheImmuneResponsetoInfluenzaAVirusInfectioninMacrophagesHazarAbuSalamah(Graduate)*.Dr.ChristopherLupfer.DepartmentofBiology,MissouriStateUniversity,Springfield,MO65897

Pyruvateistheendproductofglycolysis.ItcaneitherbetransportedintothemitochondriaforuseintheTCAcycleorbeusedtoregenerateNAD+duringaerobicglycolysis.WerecentlydiscoveredthatadditionofsodiumpyruvatetotheculturemediumduringinfectionofmacrophageswithinfluenzaAvirusaffectstheproductionofcytokinesinvolvedinimmunesignaling.Thepurposeofthepresentstudywastodeterminewhethersodiumpyruvate’sroleinenergyproductioninthemacrophagesmayalter the immune response to the infection.While infectionofmacrophageswith influenzaAvirusresulted inhigh levelsofcytokines (IL-6, IL-1β,andTNF-α) in theabsenceofsodiumpyruvate, theadditionofsodiumpyruvatesignificantlyimpairedcytokineproduction.Furthermore,sodiumpyruvatedidnotaffectvirusgrowth,suggestingtheeffectofsodiumpyruvateisontheimmuneresponseproducedbythemacrophagesandnottheviabilityofthevirus.

2. CharacterizationoftheRoleofPA5189ofPseudomonasaeruginosainResistancetoanAntimicrobialPeptide.ZacharyScott(Masters)*,ChristopherJohnson,andDonaldRowenDepartmentofBiology,UniversityofNebraskaatOmaha,Omaha,Nebraska

Pseudomonasaeruginosaisagramnegativebacillusbacteriumknownforitshighdegreeofantimicrobialresistanceandpathogenicity.Antimicrobialpeptides(AMPs)arepeptides,usuallybetween12and50aminoacidsinlengththatpossessantimicrobialactivity.ThemechanismofactionofonlyafewAMPsisknown.IamworkingwiththesyntheticAMPDASamp2whichhasbeenshowntobeeffectiveagainstP.aeruginosainliquidculturesandinbiofilms.OurlabhasusedtransposonmutagenesisofP.aeruginosatotrytoelucidatethemechanismofactionofDASamp2.Weisolated12mutantswithalteredsusceptibilitytoDASamp2.Ofthoseisolated,theF2-1mutantwasverypromisingbecauseitsMICwasincreased8fold.WefoundthattheF2-1mutanthadatransposoninsertedintothepromoterregionofthegenePA5189.PA5189ispredictedtoencodeatranscriptionfactorofunknownfunction.WehypothesizedthatthetransposonwascausingoverexpressionofPA5189.UsingqRT-PCR,IhaveobservedthatthelevelofPA5189mRNAis7foldhigherintheF2-1mutantthaninwildtypePCR.ThissuggeststhatoverexpressionofthepredictedtranscriptionfactorPA5189canaffectsensitivityofP.aeruginosacellstoanAMP.3. CHARACTERIZATIONOFTWONOVELANTIMICROBIALSAGAINSTAcinetobacterbaumannii. InfenciaXavier

Raj(Masters)*1,AnuradhaRoy2,andIndranilBiswas1.1 Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center,KansasCity,KS66160.2HighThroughputScreeningLab,UniversityofKansas,Lawrence,KS66045

AcinetobacterbaumanniibelongstoagroupofESKAPEpathogens;whichisanopportunisticpathogenprevalentinnosocomialinfections.Thispathogencancauseawiderangeofinfectionsfromminorskinandsoft-tissueinfectionstomoresevereinvasivediseasessuchasbacteremia,meningitisandventilator-associatedpneumonia.A.baumanniihastheabilitytoacquireantibioticresistancecassettesfromtheenvironmentandthistraithasallowedtheorganismtopersistinhealthcaresettingsandalsofacilitatedglobalemergenceofmultidrugresistance(MDR).Theorganismisbecomingresistancetomostofthecommonantibioticsincludingaminoglycosides,β–lactamsandquinolones.TofightagainsttheincreasinginfectionscausedbyESKAPEpathogens,therehasbeenanincreasedeffortinthelastfiveyearstoidentifynovelsmallanti-infectivemoleculesortomodifyexistingcompoundstoincreasetheirpotency.Usingahighthroughputscreeningassay,werecentlyexaminedseveralsmall-moleculelibrariesattheIDADCoreFacility at KU, Lawrence. We identified several potential candidates among ~20000 compounds that eitherspecificallyinhibitedA.baumanniistrainsordisplayedbroad-spectruminhibitoryactivityagainstA.buamanniiandother gram-negative ESKAPE pathogens. The main goal of this study is to characterize two such anti-infectivemoleculestodeterminetheirinhibitoryspectra,modeofaction,andsynergisticactivitieswithotherantibiotics.4. TheHostProteinKinaseCismanipulatedbyChlamydiatrachomatisDuringInfection

PrakashSah1(Doctoral)*,TedHackstadt2,ErikaLutter11Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, USA;2LaboratoryofIntracellularParasites,NIAID,NIHRockyMountainLaboratories,Hamilton,USA

Chlamydia trachomatis is responsible for causing a range of diseases such as blinding trachoma and urogenitalinfectionsleadingtoseriouscomplications.Insideahostcell,C.trachomatislivesinaparasitophorousvacuolecalledaninclusionfromwhereitisabletosecretevariouseffectorstomanipulatehost-cellularfunctionstoitsbenefit.Currently,notmuchisknownaboutChlamydialmanipulationofhostkinasessuchasProteinKinaseC(PKC).PKCsaremembersofAGC familyof kinasesand involved in regulating various cellular functions suchas, growthandproliferation,migration,survivalandapoptosis.WehypothesizethatC.trachomatismanipulatesPKCpathwaystoregulateintracellulardevelopmentinsidethehost,asPKCsareimportant inregulatingvariouscellularfunctions.Indirect immunofluorescenceof infectedcells verified recruitmentofmultiplePKC isoenzymes tomicrodomains(Src-familykinasesrichregions)ontheinclusionofC.trachomatisL2/434.RecruitmentofPKCsubstrates,includingMarcks,wasalsoconfirmed.RecruitmentofPKCsandPKCsubstrateswasfoundtobespeciesspecific.InhibitionofPKCactivitywithStaurosporineandGo6983resulted indecreasedrecoverable infectiousprogeny.WesternblotanalysisrevealeddifferentialactivationofPKCatdifferenttimepointsduringinfection.TheseresultsconfirmPKCsareimportantforintracellulargrowthanddevelopmentofC.trachomatis.

5. AProkaryoticdCTPDeaminaseintheEukaryoteDictyosteliumdiscoideum.HengLiang(Doctoral)*,PI:CatherineP.Chia,SchoolofBiologicalSciences,UniversityofNebraska-Lincoln,Lincoln,NE,USA.

DNAsynthesisiscrucialinalllivingorganisms.Deoxycytidinetriphosphate(dCTP)deaminase(EC3.5.4.13)isanenzymeinthepathwaywhichconvertsdCTPintodeoxythymidinetriphosphate(dTTP),oneofthebuildingblocksofDNA.Althoughtypicallyfoundonlyingram-negativeprokaryoticcells,twogenesencodingpredicteddCTPdeaminases(dcd1:DDB_G0293580anddcd2:DDB_G0268194)areidentifiedintheannotatedgenomeofDictyosteliumdiscoideum,aeukaryoticsoilamoeba.D.discoideumalsohasgenesfordCMPdeaminaseandthymidylatesynthase,enzymesforpyrimidinebiosynthesisineukaryotes.Weareinvestigatingwhetherdcd1anddcd2areexpressed,andwhethertheactivitiesandlocationoftheirrespectiveproteinsareregulatedeitherbythecellcycleorduringdevelopment.PhylogeneticanalysesofthepredictedproteinsequenceswithprokaryoticdCTPdeaminasesindicatethatdcd1anddcd2havebacterialancestorsthatlikelyenteredtheD.discoideumgenomethroughtwoindependentgenetransferevents.Consistentwithitssuggestedbacterialorigin,theD.discoideumdcd1rescuedtheslowgrowthphenotypeofanE.colidCTPdeaminaseknockout,indicatingthatdcd1encodesanenzymecapableoffunctioningintheprokaryoticpyrimidinebiosynthesispathway.Constructionofadcd1knockoutinD.discoideumisinprogresstoexaminethespecificrolesofdCTPdeaminaseinD.discoideum.6. CharacterizationoftheIS3FamilyofInsertionSequencesintheGenomeofHalanaerobium

hydrogeniformans.RonaldL.Frank,KodyA.Bassett(Masters)*,MelanieR.Mormile.MissouriUniversityofScienceandTechnology,Rolla,Missouri.

Halanaerobiumhydrogeniformansisananaerobic,gram-negative,rod-shaped,haloalkaliphilicbacteriumisolatedfromSoapLakeinWashingtonState.Thegenomesequencewasdeterminedin2011aspartofanefforttorevealsomeoftheadaptationsthatenablethisbacteriumtogrowinahighsalt,highalkalineenvironment.Oneprongofthateffortisagenome-widesurveyofanunusuallyabundantnumberoftransposableelements.Theresultsreportedherearelimitedtooneofninefamiliesofbacterialinsertionsequencesfoundinthis2.6Mbgenome.TheH.hydrogeniformansgenomecontains29locithatharboreitherfunctional,defective,orfossilremnantsoftheIS3familyofbacterialinsertionsequences.Theelementshavebeendividedintofivegroups(ISHahy2,3,4,5,andasolopartialelement).EachofthefivegroupsappearstohaveoriginatedfromanindependentinvasionofthegenomebyauniqueIS3-relatedelement.ThetransposaseoftypicalIS3elementsisencodedbytwooverlappingout-of-frameorfsAandBbyaprogrammedtranslationalframeshiftataslipperysiteupstreamofthestopcodoninorfA.WedescribeherethecharacteristicsofthefivegroupsofIS3-relatedelementsinHalanaerobiumhydrogeniformansastheyrelatetothatmodel.7. TheFattyAcidKinaseFakAShapestheMetabolomeofStaphylococcusaureus.ZacharyDeMars(Doctoral)*

andJeffreyL.Bose.UniversityofKansasMedicalCenter,KansasCity,Kansas.Staphylococcusaureusiscapableofphosphorylatingexogenousfattyacidswhicharethenabletobeincorporatedintothebacteria’smembraneviathefattyacidkinaseFakA.Additionally,FakAplaysasignificantroleinvirulencefactorregulationandskindisease.WepreviouslyshowedthatafakAmutantdisplaysalteredgrowthkineticsinvitro,observedduringlate-exponentialphaseofgrowth.Here,weshowthatthisisbothglucoseandaeration-dependent,indicatinganalteredacetateswitch.Consistentwiththis,acetateproductionwasalteredandthegrowthbenefitwas eliminated with inactivation of the acetate-generating enzyme AckA. Using a mass spectrometry-basedapproach,weidentifiedalteredconcentrationsofTCAcycleintermediatesandbothintracellularandextracellularaminoacids.Together,thesedatademonstratearedirectionofcarbohydratecarbonflowandalteredaminoacidmetabolisminthefakAmutant.WhileATPlevelsweresimilar,inactivationoffakAincreasestheNAD/NADHratio,indicatingamoreoxidizedcellularenvironment.Finally,wesuggestthattheglobalmetabolicregulatoryproteinsCcpAandCodYasbeingimportantregulatorymechanismforthealteredgrowthinafakAmutant.Together,thisdataidentifiesapreviouslyunidentifiedroleforfakAintheglobalphysiologyofS.aureusthatlinksthefattyacidandcentralmetabolism.

8. CharacterizationoftheNitrogenAssimilationRegulator(glnG)RoleinEscherichiacoliColonizationoftheMammalianIntestines.JustinBowen(Master’s)*,TyrellConway,JerremeJackson.OklahomaStateUniversityMicrobiologyandMolecularGenetics,Stillwater,OK.

Nitrogen,oftenintheformofammonia,isusedbybacteriatogenerateaminoacids.TheglnGgeneinEscherichiacolicodesforthenitrogenassimilationregulatorprotein,NR1,whichactivatesthegenesresponsibleforsurvivalduringammonialimitation.Undernitrogen-limitedgrowthconditionsNR1activatestranscriptionofglutaminesynthetasewhich,alongwithadditionalproteins,facilitatestransportanddegradationofnitrogen-containingcompounds.WhiletheE.colitranscriptionalresponsetonitrogenstarvationinvitrohasbeenstudiedextensively,nitrogenstarvationduringE.colicolonizationofthemammalianintestinehasreceivedlittleattention.Inthisstudy,wewilluseaglnGknockout(DglnG)mutanttostudytheroleofnitrogenmetabolisminMG1655colonizationofthemouseintestine.TotalRNAfromMG1655recoveredfromthemucosalliningofthemouseintestinewillbepreparedandsequencedbydifferentialRNA-seqanalysis,whichwillallowmappingofallglnG-dependentpromoters.WeexpecttheglnGmutantE.colitofailtocolonizetheintestinesiftheglnGgeneisvitaltocolonizationofthemammalianintestine.

9. GENERATIONOFYEAST2-HYBRIDCLONESTOEXAMINETHEROLEOFNUCLEOTIDEOLIGOMERIZATIONAND

BINDINGDOMAIN(NOD)-LIKERECEPTORS.AbbigaleMabary(Masters)*,FacultyAdvisor:Dr.ChristopherLupfer.MissouriStateUniversity.Springfield,Missouri.

NOD-likereceptors(NLRs)areaclassofcytoplasmicproteinsessentialfortheinitiationandregulationofimmuneresponsestoinfectiousdisease,metabolicandcellulardamageandcancer.Thehumangenomeencodesfor22NLRproteins.However,onlyabouthalfofthe22NLRshaveknownfunctions,andthemechanismsbywhichtheyfunctionareevenmoreambiguous.PreviousresearchindicatesthatsomeNLRsactivateinflammation,whileothers,likeNLRP12,functionsasregulatorsofinflammation,thusservingasanegativefeedbackmechanism.NLRP12suppressesinflammationbyinhibitingthetranscriptionfactorNF-κB,whichactivatestranscriptionforcytokinesthatactivatetheimmuneresponsecascade.InhibitionofNF-κBbyNLRP12isimportantinthepreventionofahyper-inflammation,whichisinvolvedinsevereinfectionsaswellascancerdevelopment.AlthoughthegeneralfunctionofNLRP12isknown,howitisactivatedisnotknown.Weare,therefore,embarkingonajourneytofindnovelproteinsthatinteractwithNLRP12todecipherthemechanismsbywhichtheyfunction.Wearegeneratingayeast2-hybridsystemtoexaminetheinteractionofNLRP12withahumancDNAlibrary.Novelinteractionsdiscoveredthroughthis2-hybridscreenshouldprovideinsightintothefunctionofthisNLRproteinandhelpusunderstandtheimmuneresponsetoinfectiousandnon-infectiousdisease.10. Ecology and prevalence of ticks and tick-borne bacterial pathogens in a peri-urban landscape of the

MidwesternU.S. Abrar Alzahrani (Masters)*1, NicholasA.Burnett1, Ram Raghavan2, and Anuradha Ghosh1 1.

Dept.ofBiology,PittsburgStateUniversity(Pittsburg,KS),2.Dept.ofDiagnosticMedicine/Pathobiology,KansasStateUniversity(Manhattan,KS)

Ticks transmit a wide variety of pathogens including viruses, bacteria, protozoa, and helminthes to vertebrates. Their life cycle depends on blood meals from various hosts as well as on environmental conditions such as the temperature and habitat type. The present study proposed to assess the prevalence of various tick species and infection prevalence of bacterial pathogens causing various diseases within the tick community of southeast Kansas and adjacent area. Over 2000 ticks were collected during warmer months of 2016 and 2017 (May-August) from three types of tick habitats (woodland, open grassland and woodland/grassland ecotones) using the flag-drag method. All the ticks (adults and nymphs) were sexed and identified in the laboratory. Majority of these were identified as Amblyomma spp. followed by Dermacentor spp. and rarely Ixodes spp. The ticks were surface-sterilized and total genomic DNA is currently being extracted from the adult ticks; and will be subjected to PCR amplification using bacterial species-specific primers. Microclimate data as well as landscape fragmentation pattern will be analyzed using GIS-based monitoring method. It is comprehensible that a better understanding of the variations in tick-pathogen prevalence is crucial for implementing sound surveillance and management programs and to understand risk for human/animal diseases.