YoungMicrobiologistsSymposium2016

Posters

PostersessionI·Wednesday29thJune2016

1. Assemblyandactivityof[NiFe]-hydrogenases

AlexanderFinneyAmultitudeofmicroorganismspossess theability tometabolisemolecularhydrogen (H2).The major enzyme family involved in hydrogen metabolism are Hydrogenases. Theseenzymescatalysethereversibleconversionofmolecularhydrogentoprotonsandelectrons(H2 ↔ 2H++ 2e-). Increasingly these enzymes are being utilised for biotechnologicalapplicationssuchashydrogenfuelcellsbuttheyalsorepresentpromisingdrugtargetsforinhibition of bacterial growth within the gastrointestinal tract. Therefore, furtherunderstanding and discoveries made in the hydrogenase field warrants progression intoappliedmedicalandbiotechnologicalresearchareas.Hydrogenasesarecategorisedbytheiractivesitearchitecture.Onewellstudiedgroupistermedthe[NiFe]-hydrogenases,whichallharbouracomplexNi-Fe-CO-2CNactivesiteinthe‘large’subunitandhavethreeIron-Sulfurclusterswithin a ‘small’ subunit. Due to the complexity of hydrogenase protein assemblythereare largenumbersofotherproteins involved inhydrogenasebiosynthesis.Muchofthe basic understanding of these complex enzymes derives from the study of nativeEscherichia coli hydrogenase enzymes. The first aim of my project concerns E. coliHydrogenase 1 (Hyd-1) and its specific export to the periplasm by the Twin-ArginineTranslocase (Tat) pathway. The Tat pathway allows export of folded, cofactor rich andmultimeric protein substrates in bacteria, archaea and plant chloroplasts. By structuralcharacterisations,Hyd-1isactiveasatetrameroftwo‘large’andtwo‘small’subunitswithintheperiplasm.However,thepresenceofthistetramerhasnotbeenconfirmedwithinthebiosyntheticpathwayofHyd-1,specificallyinregardtoexport.HereweshowdevelopmentofanovelE.colistrainwhichwillserveasanassayforTat-dependentexportoftheHyd-1tetramer.ThesecondaimofmyprojectlookstousevariedapproachestoactivateacryptichydrogenaseinE.coli–Hydrogenase4.PreviousworkhasnotshownanyactivityfromEcHyd-4buttheenzymecomplexispredictedtoresembleRespiratoryComplex1indicativeofprotonor ionpumpingcapability. Searching specifically forHyd-4orthologues ina closelyrelated bacteria has warranted discovery of a large hydrogenase-related gene cluster inPhytopathogenPectobacteriumatrosepticum thatencodesan intactHyd-4enzyme. InitialcharacterisationofthisbacteriahasshedlightonthefirstactiveHyd-4complex.

2. ProbioticpotentialoftwoantagonisticKluyveromycesstrainsisolatedfromatraditionalFrenchcheesenamedthe“Tommesd’Orchies”

CeugniezAlexandre,CoucheneyFrançoise,JacquesPhilippe,DriderDjamelUniversityofLille1.CharlesViolletteInstituteIn our previous study (Ceugniez et al., 2015), we reported the antagonistic properties ofKluyveromycesmarxianusS-2-05 and K.lactisS-3-05 against a panel of bacteria and evenagainstyeastCandidaalbicans. Inthepresentstudy,weunveil theprobioticpropertiesoftheaforementionedstrains.Thus,KluyveromycesmarxianusS-2-05andK.lactisS-3-05weretested for their capabilities to surviveunder thegastrointestinal tractand to colonise thewell-known intestinal Caco-2 cells. These assessments were conducted with bothantagonistic strains as well as with the probiotic Saccharomycesboulardii and thepathogenicCandidaalbicansascontrols.The antagonistic strains unveiled a good survival in the intestinal tract, with an averageadhesion toCaco-2 cells.Usually, these testswere sufficient to support theabilityof anystrain to colonise the colonic microbiota. However, the capacity to fix and colonise thecolonicmucuswaslittlestudiedsofar.Nevertheless,themajormicrobiotawaspresentontheout-layeroftheintestinalmucusandnotdirectlyonthecellsaccordingtoJohanssonetal. (2011). The MATS method allowed describing a high complementarity betweenKluyveromyces strainsandmucus,higherthanthatobservedfor theprobioticS.boulardii.ThisstudyunderscoredthehighpotentialofK.lactisS-3-05andK.marxianusS-2-05,usuallyassignedasQPSorGRAS,tobeusedasprobiotics.References:Ceugniez,A.etal.2015.FoodMicrobiol.52,177-184Johansson,M.E.V.etal.,2011.Cell.Mol.Life.Sci.68,3635-3641

3. TheroleofLXAlocusencodedproteinsinthepathogenesisofBurkholderiacepaciacomplex

AndrewO’Connor,LouiseCullen,SiobhánMcClean CentreforMicrobialHostInteractions,InstituteofTechnology,Tallaght,Dublin24,Ireland

Burkholderiacepaciacomplex(Bcc)isagroupof20closelyrelatedspeciesofGram-negativebacteria found in a variety of ecological niches ranging from agricultural crops to therespiratorytractsofhumans.Bccisofclinical importanceasanopportunisticpathogen,inparticularcausingproblematiclunginfectionsinpeoplewithcysticfibrosis(CF).Bccishighlyantibiotic resistant and infections caused by Bcc are rarely eradicated and are associatedwith poor clinical outcomes andmortality. Thehigh-level intrinsicmultidrug resistanceofBcc threatens the development of new treatments to improve and extending the life ofpatientswithchronicinfections.Bcc can survive and persist in host cells during chronic infection through a number ofmechanismsthatarenotfullyunderstood.WehaveexaminedtheadaptationsofaseriesofIrish clinical Bcc isolates during the chronic infection of two CF patients and found thesequential isolates increased theirattachment to lungepithelial cellsover timeof chronicinfection. In-depth proteomic analysis on our sequential Bcc isolates showed that 19proteins encoded within a cluster of genes on chromosome 2 were consistently up-regulated. These genes are encoded within a 50-gene cluster, which was previouslydesignated by Saas et al., (2013) as the low-oxygen-activated (lxa) locus due to theirupregulation in response to low oxygen conditions. The consistent up-regulation duringchronic infection suggests that this locus is not exclusively associated with low oxygenconditionsandtheymayplayanimportantroleinchronicinfection.Twoparticularproteinsof interestwithin the up-regulated cluster of genes of interestwere a putative universalstressprotein(pUSP,BCAM0276)andaphospholipidbindingprotein(PBP,BCAM0280).InordertoinvestigatetheroleoftheseproteinsinpathogenesisandchronicinfectionofBcc,wedevelopedtargeteddeletionmutantsoftheindividualpuspandpbpgenesusingtheBccstrain K56-2 (Flannagan et al., 2008). Attachment to cystic fibrosis lung epithelial cells(CFBE41o-)was reducedby90% inΔpusp andΔpbpmutantscomparedwith thewildtypeK56-2.VirulenceoftheΔpbpmutantinGalleriamellonellawasreducedover5-foldrelativeto K562 wild type, while the Δpusp mutant showed no impairment in virulence. TheseresultsdemonstratethatbothproteinsencodedwithintheLXAlocusmayplayaroleinthepathogenesisofBccandfurtherinvestigationswillidentifytheirroleinchroniccolonisation.References:FLANNAGAN,R.S.,LINN,T.&VALVANO,M.A.2008.AsystemfortheconstructionoftargetedunmarkedgenedeletionsinthegenusBurkholderia.EnvironMicrobiol,10,1652-60.SASS,A.M.,SCHMERK,C.,AGNOLI,K.,NORVILLE,P.J.,EBERL,L.,VALVANO,M.A.&MAHENTHIRALINGAM,E.2013.Theunexpecteddiscoveryofanovellow-oxygen-activatedlocusfortheanoxicpersistenceofBurkholderiacenocepacia.ISMEJ,7,1568-81

4. CharacterizationoftheyebEFGlocusinSalmonellaenterica

ÁngelaMéridaandJosepCasadesúsDepartamentodeGenética,UniversidaddeSevilla,Seville,SpainThegenomeofSalmonellaentericaserovarTyphimuriumcontainsmorethan19,000GATCsiteswhich serve as targets for the DNA adeninemethyltransferase (or Dammethylase).Some of these GATC sites are located within regulatory regions, affecting protein-DNAinteractions that control transcription ofmultiple genes. Previouswork of our laboratorydescribed176genes showingdifferential expression inDam+andDam–Salmonellahosts[1]. The gene cluster formed by yebG, yebF and yebE appeared among the upregulatedgenesinDam–mutants.Very little is known about the function and regulation of these genes. yebG has beendescribedasageneoftheSOSregulon[2],yebFencodesasecretionprotein[3],andYebEispredictedtobeasingle-passinnermembraneproteinregulatedbytheCpxstressresponseregulator[4].Interestingly,severalGATCsitesarefoundupstreamthepredictedtranscriptionstartsiteofyebG,openingthepossibilityof itsregulationviaDNAmethylation. Inthiscommunicationwepresentourpreliminarydataaboutregulationoftheyebcluster.TheexpressionofthethreegenesisinducedbynalidixicacidbutonlyyebGhasaputativeSOS-boxupstreamitstranscriptionstartsite,andshowsLexA-dependentexpression.Single-cellanalysisrevealsthatonly1.6%ofcellsexpressyebG instationaryphase.IncontrasttoothergenesthathaveGATCsitesupstreamtheirregulatoryregionsandpresentphenotypicheterogeneity[5],heterogeneousexpressionofyebG isDam-independent.Flowcytometryanalysisalsorevealsthatonlyafractionofcellssurviveswhennalidixicacidisaddedtotheculture, and the surviving subpopulation shows high levels of yebGexpression. However,ΔyebGmutantsarenotmoresensitivetonalidixicacidthanwildtypecells,suggestingthatthe YebG protein is not essential for nalidixic acid resistance. Despite this result, theheterogeneityinyebGexpressioncouldgeneratephenotypicheterogeneityinafractionofthebacterial culture,perhapsmaking this subpopulationmore resistant toenvironmentalchallenges.References1.BalbontínR.etal.DNAadeninemethylationregulatesvirulencegeneexpressioninSalmonellaentericserovarTyphimurium.2006.JournalofBacteriology188(23):8160-8168.2.Oh,T.J.,Kim,I.G.IdentificationofgeneticfactorsalteringtheSOSinductionofDNAdamage-inducibleyebGgeneinEscherichiacoli.1999.FEMSMicrobiologyLetters177:271-277.3.Zhang,G.,Brokx,S.,Weiner,J.H.ExtracellularaccumulationofrecombinantproteinsfusedtothecarrierproteinYebFinEscherichiacoli.2006.NatureBiotechnology24(1):100-104.4.Price,N.,Raivio,T.CharacterizationoftheCpxReguloninEscherichiacolistrainMC4100.2009.JournalofBacteriology191(6):1798-1815.5.Cota,I.etal.EpigeneticcontrolofSalmonellaentericaO-antigenchainlength:Atradeoffbetweenvirulenceandbacteriophageresistance.2015.PlosGenetics11(11)

5. UnderstandingthedefenceresponseofplantstoEscherichiacoliO157:H7AshleighHolmesEscherichia coli O157:H7 are well known food-borne pathogens that can spend some oftheir life cycle in theenvironmentanduseplants as secondaryhosts. This abilityposesafoodsafetythreatwhenthebacteriacolonisecropplantsthatareeatenraworminimallyprocessed,i.e.thatthepathogensarenotdestroyedbycooking.Plantsmountadefensive response tomicro-organisms initially throughtherecognitionofmicrobe-associated molecular patterns (MAMPs), such as flagellin (the monomer offlagella), and lipopolysaccharide (LPS). Binding of theMAMP to its cognate plant patternrecognition receptor (PRR) triggers a cascade reaction in the plant cell that ultimatelyinducesexpressionofgenes involved indefensivepathways. This initial response inducestheproductionofreactiveoxygenspecies(ROS)andthethickeningofplantcellwallsfromthedepositionofcallosetokillandrestrictcolonisationbythemicrobe.PreviousworkinourlabhasshownthatE.coliO157:H7adheretoplanttissuesviaflagella,E.coliCommonPilus(ECP)andF9fimbriae.Asitsnamesuggests,ECPisconservedamongstthe Enterobacteriaceae family, where homologues are found in not only in other E. colipathovars but inKlebsiella species. The F9 fimbriae are encoded on an operonwhich islimited tohumanpathogenic strainsofE. coli such asO157:H7anduropathogenicE. coli(UPEC).Inlightofthis,wewantedtoinvestigatewhethertheplantrecognisesthepresenceof these organelles to initiate a defence response. The transcriptomic response ofArabidopsisthalianaCol-0leavestoinfiltratedflagella,ECPandF9fimbriaewastestedusingmicroarrays. Flagellawere included toactasapositive controlas theMAMPresponse toflagellin iswell documented in the literature. No responsehas been reported, as yet, tobacterial fimbriae even though these organelles are involved in initial attachment andbiofilm formation. Genes involved in the specific response to fimbriae are identifiedthrough comparison with those induced by flagella and from other published datasets.These results expand the repertoire of MAMPs recognised by plants to control microbecolonisation.

6. InvestigationofthelipoproteintranslocationsysteminN.meningitidisB.Ricchettia,b,ElenaDelTordelloa,MariaGiuliania,VincenzoScarlatobandIsabelDelanyaaGSKbUniversityofBolognaLipoproteins(LPs)ofpathogenicGram-negativebacteriaareinvolvedindifferentbiologicalprocesses ranging from outer membrane (OM) stabilization to activation of the immuneresponse. For some Gram-negative bacteria, like E. coli, the lipoprotein translocationmachineryiswellcharacterizedand,forN.meningitidis,homologuesoftheseproteinscanbe identified from the genome. Unlike E. coli, N. meningitidis displays many LPs on itssurface suggesting the presence of one ormore unknown factors involved in the surfaceexposureof LPs [1].A recent studyhas identified an additional translocation component,Slam(SurfaceLipoproteinAssemblyModulator),involvedinthesurfaceexposureofspecificN.meningitidisLPs[2].WegeneratedtheSlamdeletionmutant indifferentN.meningitidisstrains.ThroughFACSanalysis, we analyzed the surface exposure of fHbp (factor H binding protein) as arepresentativemeningococcalsurfaceLP,whichisacomponentofanovelvaccineagainstN. meningitidis serogroup B. The deletion of Slam affects the surface exposure of theanalyzedLPintheselectedmenigococcalstrains.Inparticular,theabsenceofSlamresultsindecreasedfHbplevelsonsurfaceandtheselowlevelswereaconsequenceofageneralreductionoffHbpamountintheSlamKOascomparedtothewildtype.ThesedatasuggestthatSlamisnotessentialforthesurfaceexposureoffHbp.We also attempted the heterologous expression of N. meningitidis fHbp in the E. colibackground.Interestingly,co-expressionoffHbpwithafunctionalSlamresultsinhighlevelsof fHbp expression which is surface exposed. These data evidence the role of Slam inefficientexpressionandsurfacelocalizationoffHbpinE.coli.Taken together, these results confirm a role in the N. meningitidis OM-translocationmechanismfortheproposedoutermembraneproteinSlam.References:1.Bos,M.P.,V.Robert,andJ.Tommassen,Biogenesisofthegram-negativebacterialoutermembrane.AnnuRevMicrobiol,2007.61:p.191-214.2.YogeshHooda,C.C.-L.L.,AndrewJudd,CarolynM.Buckwalter,HyejinEstherShin,ScottD.Gray-OwenandTrevorF.Moraes,SlamisanoutermembraneproteinthatisrequiredforthesurfacedisplayoflipidatedvirulencefactorsinNeisseria.Naturemicrobiology,2016.1.

7. RiskassessmentfactorsinvolvedinthecontaminationoffreshproducewithverocytotoxigenicEscherichiacoli

BernhardMerget(1,4),NorvalStrachan(1),KenForbes(2),FionaBrennan(3),NicolaHolden(4)(1)TheUniversityofAberdeen,SchoolofBiologicalSciences,Aberdeen,AB243UU,UK(2)TheUniversityofAberdeen,SchoolofMedicineandDentistry,Foresterhill,Aberdeen,AB252ZD,UK(3)NationalUniversityofIreland,DepartmentofMicrobiology,SchoolofNaturalSciences,Galway,Ireland(4)TheJamesHuttonInstitute,Cell&MolecularSciences,Invergowrie,Dundee,DD25DA,UKAimsFood-bornepathogens,includingverocytotoxigenicEscherichiacoli(VTEC)poseamajorrisktohumanhealth,which is increasinglyassociatedwiththeconsumptionof freshproduce.Quantitativeriskassessments (QRA)aimtoevaluatetheriskofcontaminationthroughouttheproductionchain.However,previousQRAhavepoorlyexaminedtheriskofpre-harvestfactors, due to a lack of experimental data (1). The project aims to develop a novel riskassessment framework, which takes pre-harvest factors (2) into account andmodels thelevelofcontaminationfromfarm-to-fork.MethodsandresultsPre-harvest factors, such as bacterial growth in plants or the ability for colonisation andinternalisation have been evaluated and included in a QRA. This was facilitated byquantitative and qualitative metabolite analyses of host plants, growth rate data andphenotypicclassificationofpathogenicstrains.Growthrateswerefoundtobemorestronglydependentonhostplantspecies/tissuetypethan the bacterial strain. However, several pathogenic serotypes did show an increasedgrowthinplants,inparticularserotypesO157:H7andO145.ConclusionsThelackofpre-harvestdatainpreviousriskassessmentshasresultedinanunderestimationof factors such as the presence of internalised bacteria. Furthermore, the ability to growinternally and persist for several days within plants has not been taken into account inindustrialmeasurestoprotectfood,whichincreasestherisktoconsumers.SignificanceofstudyThe implementation of pre-harvest factors will increase robustness and accuracy of QRAsignificantly. It will help the development of guidelines for the industry and regulatoryauthoritiesandimprovefoodsafetyintheUKandEurope.References

(1) Danyluk,MD,&Schaffner,DW(2011).Quantitativeassessmentofthemicrobialriskofleafygreensfromfarmtoconsumption:preliminaryframework,data,andriskestimates.JournalofFoodProtection,74(5),700-708

(2) Wright,KM,Chapman,S,McGeachy,K,Humphris,S,Campbell,E,Toth,IK,andHolden,NJ(2013).TheendophyticlifestyleofEscherichiacoliO157:H7:quantificationandinternallocalizationinroots.Phytopathology,103(4),333-340

8. TheimpactofthemicrobiotaonClostridiumdifficilecolonisationanddiseaseseverity

CaitlinJukesUniversityofGlasgowClostridium difficile is the leading cause of antibiotic associated diarrhoea. Three mousemodels of infection in which disease severity is dependent on the antibiotics used prechallengewereused.Onemodelusesanantibioticcocktail(ABC)andclindamycintoinducesevere disease, one clindamycin only to induce colonisation and one just the ABC alonegivingtransientcolonisation.MicewerechallengedwithC.difficileandmicrobiomesamplescollectedthroughoutantibioticpre-treatmentandinfection.Colonisationresistanceseemsto be heavilymediated by bacteria that are involved in bilemetabolism. Loss of bile salthydrolase (BSH) producing members of the microbiota seems to be essential for loss ofcolonisationresistance.ThisisbecausebilesaltsplayanimportantroleinC.difficilesporegerminationwithmodificationofthesebilesaltsdecreasingpotencyasgerminants.PCRsforBSHgenesshowedlossofBSHafterclindamycinindicatingalossofBSHproducingbacteriaandbilemetabolism.ThiswassupportedbyboththemicrobiomedatawhichshowedlossofBSH andmass spectrometer analysis which showed a significant increase in primary bilesalts compared to controls in both regimes resulting in an environment suited forproliferationandoutgrowth.LossofBSHproducingbacteriadidnotcorrelatewithincreasedseverityofdisease,howeverfurtheranalysisofthemicrobiomedatasuggestthatdisruptionof the mucus layer and mucosal bacteria may play an important role in this aspect ofdisease.

9. Usingbacteriatofightbacteria:Parasitisationofferredoxin-uptakereceptorsinPectobacterium

CatrionaThompsonAntibioticresistanceisaglobalproblemthataffectsbothhealthandfoodsecurityaffecting800 million people every year. Gram-negative plant pathogens, such as Pectobacterium,haveadetrimentaleffectoncropyields.Bacteriocinsareproteinantibioticsthatareusuallyactiveonly againstbacteria closely related to theproducing strain; thesebacteriocins areoftenspeciesspecificandthereforeofferanalternative to thebroadspectrumantibioticsusedinclinicalpractice.ThreerecentlydescribedbacteriocinsproducedbyP.carotovorumconsistofa[2Fe-2S]plant-typeferredoxindomainfusedtoabacteriocincytotoxicdomainandtheseparasitizeanexistingferredoxinuptakesystemtogainentryintotargetcells.Thenormalphysiologicalroleofthisuptakesystemistoacquireironfromplantferredoxin.Wehave identified the ferredoxin/pectocin receptor as a TonB-dependent receptor that wehavedesignatedFusA.Bioinformaticanalysisindicatestheexistenceofaferredoxinuptakeoperon, which in addition to the receptor FusA, encodes a protease, an ABC-transporterwhich likely act to cleave ferredoxin and transport liberated iron to the cytoplasm and ahomologueof TonB.Weare currently dissecting the roles of theseproteins in ferredoxinuptakeusingacombinationofstructural(SANSandSAXS)andfunctionalstudies.

10. AdenylatecyclaseAactingonPKAmediatesinductionofstalkformationbyc-di-GMPattheDictyosteliumorganizer

ChristinaSchildeUniversityofDundeec-di-GMP is awell-known prokaryote secondmessengerwhich plays a crucial role in thetransitionof prokaryotes fromaunicellular swarming state to amulticellular biofilm. Theeukaryote Dictyostelium discoideum uses c-di-GMP, synthesised by a prokaryote-typediguanylatecyclase(DgcA),toinducestalkformationinitsmulticellularfruitingbodies.Weused transcriptional profilingofdgca-null structures to identify target genes for c-di-GMPand used these genes to investigate the c-di-GMP signal transduction pathway. Weidentified several c-di-GMP induced genes that were specifically expressed in the stalk.Knock-downofPKAactivityinprestalkcellsreducedinductionofthesestalkgenesbyc-di-GMP, while PKA activation with 8Br-cAMP bypassed the c-di-GMP requirement for stalkgene expression. c-di-GMP caused a persistent increase in cAMP, and this increase alsooccurred inmutants lacking theadenylatecyclasesACGorACR,or the intracellular cAMPphosphodiesterase RegA. However, both inhibition of adenylate cyclase A (ACA) withSQ22536, and incubation of a temperature-sensitive ACA mutant at the restrictivetemperaturepreventedc-di-GMPinducedcAMPsynthesisaswellasc-di-GMPinducedstalkgene transcription.ACAproduces thecAMPsignal that coordinatescellmovementduringDictyostelium fruiting body formation and is highly expressed at the organising tip ofmulticellular structure.Ourdatashowthat themorewidelyexpressedDgcAactivates tip-expressedACA,which then acts on PKA to induce stalk genes. These results explainwhystalkformationinDictyosteliaalwaysinitiatesatthesiteofthemorphogeneticorganizer.

11. Developmentofathree-dimensionalairwayepithelialcellmodeltostudypathogeninteractionswithinthebovinerespiratorytract

DanielCozens1,ErinSutherland1,GeraldineTaylor3,CatherineBerry2andRobertDavies1

1InstituteofInfection,InflammationandImmunity,UniversityofGlasgow,Glasgow,UK2InstituteofMolecular,CellandSystemsBiology,UniversityofGlasgow,Glasgow,UK3ThePirbrightInstitute,Pirbright,SurreyBovine respiratory disease (BRD) complex is a multifactorial infection of cattle that isresponsibleforsubstantialeconomiclossestothelivestockindustries.Variousviral(bovinerespiratorysyncytialvirus,bovineherpesvirus-1,bovineparainfluenzavirus-3,bovineviraldiarrhoea virus) or bacterial (Mannheimia haemolytica,Pasteurellamultocida,Histophilussomni)pathogensareassociatedwithBRD.Althougheachofthesepathogensiscapableofcausing disease individually, BRD is more often characterised as a mixed viral-bacterialinfection (the viral infection preceding the bacterial infection). The complex interactionsbetween bacteria, viruses and the bovine respiratory tract are poorly understood.Contributingfactorstothisincludethelackofphysiologically-relevant,reproducibleinvitromodelsandthecurrentrelianceoninvivoexperimentsinvolvingcattle.Thisprojectaimstooptimiseathree-dimensionalairwayepithelialcellmodel(grownatanair-liquidinterface)ofthebovinerespiratorytract.Primarybovinebronchialepithelialcells,isolatedfromfreshabattoirmaterial,werecultivatedatanair-liquidinterfacetostimulatedifferentiationintotypical airway epithelial cells (e.g. ciliated cells, basal cells and mucus-producing gobletcells). Selected growth factor concentrations (epidermal growth factor, retinoic acid,triiodothyronine) and other parameters (oxygen tension, membrane pore density) wereoptimised to yield a polarised,well-differentiatedpseudostratified epithelium that closelymimicsthenaturalepitheliumofthebovinerespiratorytract.Themodelwascharacterisedat3day intervalsover42daysof cultivation to identifya ‘window’ofoptimumstructureand function which wasmost reflective of the bovine airway epithelium. Themodel willsubsequentlybeusedtomimicandstudyviral/bacterial/hostinteractionsthatoccurinBRD.In this way, the model will replace and reduce the number of animals required forpathogenesisstudies.

12. CompetitivebacterialinteractionsofPseudomonasaeruginosaduringchronicinfection

DanielUnterweger,KevinR.Foster*,AshleighS.Griffin*DepartmentofZoology,UniversityofOxford,Oxford,UK

Pseudomonasaeruginosa is aGram-negativebacteriumthat is commonly found intheenvironmentandanopportunisticpathogen.InfectionscausedbyP.aeruginosaaretheprimary cause for morbidity and mortality among patients with cystic fibrosis (CF), aninheritablediseaseaffectingtheabilityofthelungtoclearbacteria.

Intheenvironmentandthepatient,P.aeruginosaisexposedtoalimitedamountofnutrients and space. To thrive in competitive environments, bacteria employ a variety ofmechanisms to gain an advantage over their competitors. Onemechanism is the type VIsecretion system (T6SS) that forms a contractile nanomachine and enables bacteria totranslocateeffectorproteins intoneighbouringcells.Bacteriadieasaconsequenceof theactivity of T6SS effectors. Antibodies against components of the T6SS in the serumof CFpatientsindicatethatP.aeruginosaengagesincell-to-cellinteractionsduringinfection.Wedecipher changes in bacterial interactions during the course of infection usingbioinformaticsanduseageneticapproachtoanalysetheconsequencesofbacteria-bacteriainteractionsfortheindividualcells.Thesefindingswillimpactourunderstandingofbacterialinteractionsandtheiradaptationduringinfection.*contributedequally

13. RegulationoftypeIIIsecretionsystemsbydirectinteractionwiththeubiquitousbacterialsecondmessenger,cyclic-di-GMP

EleftheriaTrampariCyclic di-GMP (cdG) is a ubiquitous second messenger in bacteria. It regulatestranscriptionalandpost-transcriptionalprocessesaswellasallostericallycontrollingproteinfunction. The activity of cdG in the cell leads to biofilm formation, secondarymetaboliteproduction and the switch between motile and sessile states of living. While themechanisms of cdG metabolism are well understood, the downstream targets of thissignallingmoleculearecurrentlypoorlycharacterised.AspartofourongoingresearchtounderstandtheroleofcdGsignallinginplant-associatedPseudomonas species, we carried out a cdG-capture compound pull-down experiment toidentifypotentialbindingproteinsinthemodelorganismP.fluorescens.One of the top targets identified was the flagella export AAA+ ATPase FliI, which wassubsequentlyshowntobindspecificallytothecdGmoleculewithaKDinthelowμMrange.Interactions between FliI and cdG are verywidespread,with specific binding seenwithindiverse bacterial FliI homologs. Excitingly, high-affinity bindingwas also observed for thetype-IIIsecretionsystemhomologHrcNandthetype-VIATPaseClpB2.AdditionofcdGwasshowntoinhibittheATPaseactivityofbothFliIandHrcNinvitro.Finally,acombinationofmassspectrometry,site-specificmutagenesisandinsilicoanalysiswasusedtopredictaFliI cdGbindingsite inapocketofhighlyconservedresiduesat theinterfacebetweentwoFliIsubunits.Ourresultssuggestanovel,fundamentalroleforcdGincontrollingthefunctionofmultipleimportant bacterial export pathways, through direct allosteric control of export ATPaseproteins[1].Reference:1.TrampariE,StevensonCE,LittleRH,WilhelmT,LawsonDM,MaloneJG.JournalofBiologicalChemistry.2015;290:24470-83.

14. Behaviorandtoxicityofnovelhyper-virulentstrainsofPseudomonasaeruginosa

EmelineReboud1,SylvieElsen1,StéphanieBouillot1,GuillaumeGolovkine1,PaulineBasso1,KatyJeannot2,InaAttrée1,PhilippeHuber11INSERM,U1036,BiologyofCancerandInfection,Grenoble,France/CNRS,ERL5261,BacterialPathogenesisandCellularResponses,Grenoble,France/UniversitéGrenoble-Alpes,France/CEA,DRF,BIG,Grenoble,France2UniversityhospitalofBesançon,Besançon,FrancePseudomonas aeruginosa, is an opportunist pathogen, responsible for chronic and acuteinfections.ThevirulenceofthemostaggressivestrainsmainlyreliesonatypeIIIsecretionsystem(T3SS),injectingvarioustoxinsdirectlyintothecytoplasmoftargetcells.Wehaverecently isolatedanovelhyper-toxicclinicalstrainofP.aeruginosa,namedCLJ1,that does not possess this secretion system, but instead releases a membrane-permeabilizingtoxincalledExlA.CLJ1belongstoarecentlydiscoveredandhighlydivergentcladeofP.aeruginosa,whosememberspossessanexlAgeneinsteadoftheT3SS.To further describe the virulence potential of exlA-positive strains, we collected 18 exlA-positivestrainsaroundtheworld,obtainedfromvariousorigins,e.g.humaninfectionsandenvironmental samples. We examined the capacity of these strains to display virulence-associatedphenotypesindifferentmodels.In addition to membrane toxicity, exlA-positive strains demonstrated high proteolyticactivitiestargetingVEandE-cadherins,twointercellular-junctionadhesiveproteinsrequiredforendotheliumandepitheliumintegrity.WeinvestigatedthemolecularmechanismsofE-cadherincleavagebyExlA.OurpreliminarydatashowthatVE/E-cadherincleavageisrapidlyinduced by ExlA and mediated by ADAM10, an eukaryotic protease located at the cellmembrane.

15. StreptomycescoelicolorRieske:anunusualTatsubstrate FionaTookeandTracyPalmerTherearetwomajorpathwaysforproteininsertionintotheinnerbacterialmembrane;thegeneralsecretorysystem(Sec)andthetwin-argininetranslocation(Tat)system.Thesetwosystemsoperateinparallelwithdistinctdifferences.TheSectranslocationmachineryisableto insertmultipletransmembranedomainproteins intothecytoplasmicmembrane.Theseproteinsareinsertedco-translationally,preventingtoxicaggregationofhighlyhydrophobicproteins in the cytoplasmic compartment. In comparison the Tat machinery translocatesfullyfoldedproteinsacrossthemembraneandisonlyknowntointegrateproteinsthatcarryasingletransm-embranehelix.The Rieske protein is a critical membrane-bound iron-sulphur protein found in bacterialrespiratoryandphotosyntheticelectrontransportchains.InbacteriaassemblyoftheRieskeprotein is Tat-dependent, as iron-sulphur co-factor insertionoccurs in the cytoplasm, andthe protein usually contains a single transmembrane helix anchor. However, inActinobacteriathisproteincomprises3transmembranehelicesfollowedbytheiron-sulphurco-factor containing domain. Actinobacteria encompass both pathogenic andnon-pathogenic organisms, includingMycobacterium tuberculosis, the causative agent ofTuberculosisandthenon-pathogenicmodelorganismStreptomycescoelicolor.The S.coelicolor Rieske protein was shown to be dual-targeted to the cytoplasmicmembrane,requiringboththeSecandTatexportpathwaysforcorrectinsertion.Thishighlyunusualmechanismisbeinginvestigatedtounderstandthespecificfeaturesofthisproteinthat facilitate release by the Sec machinery and its subsequent recognition by the Tatmachinery,tocompletelyinsertintothemembrane.Reporterconstructshaveallowedtheassessment of the interaction of the transmembrane domains of the Rieske proteinwitheither the Sec or Tat pathway. Results so far, indicate that topological determinantsalongside the positive inside-out rule are crucial to the Secmachinery releasing the thirdtransmembranedomain, incomparison, theTatmachineryonlyrequirestheTatmotif forproteinrecognition.

16. Membraneinteractionsandself-associationofcomponentsoftheEss/TypeVII

secretionsystemofStaphylococcusaureusFranziskaJägerThe Type VII/ESX-1 protein secretion system was first described in MycobacteriumtuberculosiswhereitwasshowntosecreteESAT6(earlysecretedantigen,6kDa)andCFP10(culture filtrate protein, 10 kDa). Both of these proteins are important T-cell targets andessentialforthevirulenceofM.tuberculosis.A protein secretion system distantly related to the ESX-1 protein secretion system inM.tuberculosis was later identified and characterized in the human and animal pathogenStaphylococcusaureus, sharingESAT6/CFP10-likeandEssC-like components. EssCproteinsaremember of a family of the FtsK/SpoIIIE family of ATPases [1],with three interlockingATPasedomainsattheC-terminus.ItwasrecentlyshownthatmultimerisationofEccC(theEssChomologinactinobacteria)dependsuponthebindingofEsxBtoapocketonthemostC-terminalATPasedomain[2].I havebeen studying the fourmembraneproteinsEssA,EssB, EssCandEsaA,of theTypeVII/Ess protein secretion system in S. aureus.While EssC is related to the actinobacterialEccC, EssA, EssB and EsaA are essential components of the secretionmachinery that areuniquetofirmicutes.CrosslinkingandbluenativePAGEanalysishaveshownthattheEssB,EssC and EsaA proteins individually form homomeric complexes, but do not appear tointeractwithoneanother,orwithEssA.Surprisingly,IwasabletoshowthroughcrosslinkingthattheformationofhighermolecularweightmultimersofEssCinS.aureus isnotrelianton EsxB or EsxA. Therefore it seems that oligomerisation of EssC may be controlleddifferentlytothatofEccC[3].Bioinformatic analysis of EssC has indicated the presence of a conserved serinephosphorylationsiteintheN-terminalregionoftheprotein.MycurrentworkisfocusedonidentifyingwhetherEssCisphosphorylatedandifsowhetherthiscontrolstheactivityofthetypeVIIsecretionsystempost-translationally.Mylatestresultswillbepresented.References:[1]M.J.Pallen,“TheESAT-6/WXG100superfamily--andanewGram-positivesecretionsystem?,”TrendsMicrobiol.,vol.10,no.5,pp.209–12,May2002.[2]O.S.Rosenberg,D.Dovala,X.Li,L.Connolly,A.Bendebury,J.Finer-Moore,J.Holton,Y.Cheng,R.M.Stroud,andJ.S.Cox,“SubstratesControlMultimerizationandActivationoftheMulti-DomainATPaseMotorofTypeVIISecretion,”Cell,pp.1–12,2015.[3]F.Jäger,M.Zoltner,H.Kneuper,W.N.Hunter,andT.Palmer,“Membraneinteractionsandself-associationofcomponentsoftheEss/TypeVIIsecretionsystemofStaphylococcusaureus,”FEBSLett.,vol.590,no.3,pp.349–357,2016

17. Alkyl-quinolone-dependentquorumsensingcontrolsprophageactivation,

autolysisandantibioticresistanceinPseudomonasaeruginosabiofilm GiuliaGiallonardi1,MarcoMessina1,EmanuelaFrangipani1,StephanHeeb2,MiguelCámara2,FabioPolticelli1,LiviaLeoni1,GiordanoRampioni1,andPaulWilliams21Dept.ofScience,UniversityRomaTre,Rome,Italy;2CentreforBiomolecularSciences,UniversityofNottingham,Nottingham,UK.ThepqssystemofPseudomonasaeruginosa isresponsiblefortheproductionofdiverse2-alkyl-4-quinolones (AQs), some of which are quorum sensing signal molecules. A recenttranscriptomicanalysisperformed inour laboratory revealed that theAQs2-heptyl-4(1H)-quinolone(HHQ)and2-heptyl-3-hydroxy-4(1H)-quinolone(PQS)influencetheexpressionofmultiple genes, while 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), a compound withknown cytochrome inhibitory activity, has no effect on P. aeruginosa transcriptome.However,itwasreportedbyothergroupsthataP.aeruginosapqsLmutantstrain,impairedinHQNOsynthesis,undergoesautolysiswhengrownascolonybiofilm.ThroughthegenerationandphenotypiccharacterizationofmultipleP.aeruginosamutantsproducingalteredlevelsofAQs,wedemonstratedthatanaccumulationofHHQoccursasaconsequenceofpqsLmutation.ThisinturnleadstothetransitionofthePf4prophagefromthe lysogenic to the lytic phase, ultimately resulting in cell autolysis. Notably, thisphenomenon increases theantibiotic resistanceofP.aeruginosabiofilms.Phenotypicandgenetic analyses revealed that ~ 40% of P. aeruginosa clinical isolates show a lyticphenotypewhen grown as colony biofilms, and that these strains carry pqsL deletion orpointmutations inthisgene.Complementationassaysand insilicomodellingsuggestthatthelyticphenotypeobservedinmostP.aeruginosaclinicalisolatesisduetothelackofpqsLortotheexpressionofinactivePqsLvariants.ThesedataindicatethatthePqsL-mediatedsynthesisofHQNOmightserveasasinktolimitHHQ-induced autolysis mediated by Pf4. However, the lytic phenotype displayed by P.aeruginosa pqsL mutants could confer a selective advantage during chronic infections,characterizedbybiofilmformationandprolongedantibiotictreatment.Overall,lossofpqsLexpression or functionalitymight represent a pathoadaptativemutation in P. aeruginosaclinicalisolates.Generegulationandintracellularsignalling

18. ValidationofanewTypeVIsecretionsystemcargoeffectorinSerratiamarcescensGiuseppinaMariano,KatharinaTrunkandSarahCoulthurstTheTypeVIsecretionsystem(T6SS)belongs to thecategoryofone-stepbacterialproteinsecretion systems and is widely distributed amongst Gram-negative species. T6SSs areencoded by large and variable gene clusters, however within these loci is possible torecognizefourteengenesthatarestronglyconserved.Thesegenesencodefourteenhighlyconserved “core” components, named TssA-M and PAAR, which form a large, envelopespanningmachinery,enablingdeliveryoftoxinsdirectlyintoeukaryoticorprokaryotictargetcells.To date, it has been shown that many anti-bacterial T6SS-dependent toxins can becategorized into different families: peptidoglycan amidase and glycosidase enzymes,phospholipases, probable membrane pore forming proteins and nucleases; while othercandidatetoxinsstillhaveanunknownfunction.DeliveryofthetoxinscanbeaccomplishedthroughassociationwithoneofthecomponentsoftheT6SS’sexpelledpuncturingdevice.This association may be a non-covalent interaction (cargo effectors) or by direct fusion(specializedeffector).Moreover,theseanti-bacterialtoxinsareencodedadjacenttospecificcognate immunity proteins. The presence of immunity proteins that are localized in thecellular compartment inwhich the toxinsdisplay theiractivityprovidesprotectionagainstself-killingandattackfromsistercells.Serratiamarcescensisanopportunisticpathogen,responsiblefornosocomialinfectionsandpneumonia.PreviousworkhasidentifiedoneT6SSinS.marcescensDb10,whichtargetsandefficiently kills other bacterial cells and plays a role in anti-bacterial competition. Manyputative anti-bacterial toxins secreted by this T6SS have been identified. While some ofthese toxins have been characterized and their function has been determined, little isknown about the others. The aim of this project is to characterize one of these newlyidentifiedtoxins,includingvalidationofitsroleasanantibacterialtoxinanditsassociationwith a cognate immunity protein together with initial investigation into its molecularfunction.WeconfirmedthatthetoxinisindeedacargoT6SSeffectorandthatinhibitionofits activity in sister cells depends on direct interactionwith a cognate immunity protein.Additionally,wehavegeneratednewinsightintoitsmodeofactionagainsttargetcells.

19. Theenzyme2-keto-3-deoxy-gluconate-6Paldolase(Eda),partoftheEntner-Douderoffpathwayinbacterialmetabolism,isessentialforvirulenceofPectobacteriumcarotovorumoncallalily

HuanWang1,2†,Zhongling Yang1†, Pengfei Zhang1, LinMa1, ShuoDu1,Yao Liao1,Yujie,Wang1,IanToth2andJiaqinFan1

1.CollegeofPlantProtection,NanjingAgriculturalUniversity,Nanjing,210095,China2.TheJamesHuttonInstitute,Invergowrie,Dundee,DD25DA,Scotland,UK†ThefirsttwoauthorscontributeequallytothisresearchPectobacteriumcarotovorumsubsp.carotovorum(Pcc)isanopportunisticplantpathogenicbacterium causing soft rot disease, which leads to serious economic and yield losses inagriculturalcropsandornamentalplantsworldwide.WhileminimalmediumsupplementedwithplantextractshasoftenbeenusedtomimichosttissueandinduceproteinexpressioninPectobacterium,someproteinsthatareonlyexpressedinplantamaybemissed.HerewecomparedproteinexpressionduringstrainPccS1interactionwithitshostplantZantedischiaelliotiana(callalily)inthreeconditions:LBmedium,LBsupplementedwithplantextracts(invitro) and inoculated into potted plants (in vivo). Fifty three proteins were identified asbeingsignificantlyalteredintheirexpressioninvitroorinvivoorboth.Onlythoseproteinsthatwereover10foldalteredinexpressioninvivowerestudiedfurther.DeletionmutantsingenesclpP,mreB,flgKgreatlyreducedthevirulenceinthehostplantcallalily.However,as ClpP,MreB and FlgK have previously been described in PccS1,we screened additionalproteinsalteredinexpression(over5fold)bothinvivoandinvitro.Aninsertionmutantofthe zwf gene (encoding Glucose-6-phosphate 1-dehydrogenase) showed a reduction inpathogenicity,however,adeletionmutantofthesamegenehadnoeffectonpathogenicity.Genomeanalyse showed that theadjacentgeneeda (encoding2-keto-3-deoxy-gluconate-6P aldolase; an enzymewhich ispart of the Entner-Douderoffmetabolic pathway) is co-transcribedwithzwf.Whenadeletionmutantoftheedagenewasmadeitwassignificantlyattenuated in virulence. To the best of our knowledge, this is the first time thateda hasbeenreportedascontributingtovirulenceinPectobacterium.AcknowledgementThisworkwassupportedbySpecialFundforAgro-scientificResearchinthePublicInterestofChina(GrantNo.201303015).ThescholarshiptotheJamesHuttonInstitutewassupportedbyChinaScholarshipCouncil(CSC).

20. AnanalysisofPseudomonasgenomicdiversityintake-allinfectedwheatfieldsrevealsthelastingimpactofwheatcultivarsonthesoilmicrobiota

JacobMalone

Manipulationof the soilmicrobiotaassociatedwith cropplantshashugepromise for thecontrol of crop pathogens. However, to fully realise this potential we need a betterunderstanding of the relationship between the soil environment and the genes andphenotypesthatenablemicrobestocoloniseplantsandcontributetobiocontrol.Arecenttwo year investigation into the effect of wheat variety on second-year crop yield in thecontextoftake-allfungalinfectionpresentedtheopportunitytoexaminesoilmicrobiomesundercloselydefinedfieldconditions.

Amplicon sequencing of second-year soil samples showed that Pseudomonas spp. wereparticularly affected by the wheat cultivar grown in year one. Consequently, 318rhizosphere-associated Pseudomonas fluorescens strains were isolated and characterisedacrossavarietyofgeneticandphenotypictraits.Again,thewheatvarietygrowninthefirstyearofthestudywasshowntoexertconsiderableselectivepressureonboththeextentandnature of Pseudomonas genomic diversity. Furthermore, multiple significant correlationswere identified within the phenotypic/genetic structure of the Pseudomonas population,andbetweenindividualgenotypesandtheexternalwheatfieldenvironment.Theapproachoutlined here has considerable future potential for our understanding of plant-microbeinteractions,andforthebroaderanalysisofcomplexmicrobialcommunities.

21. Piratingconservedphagemechanismspromotespromiscuousstaphylococcalpathogenicityislandstransfer

JanineBowringTheStaphylococcusaureuspathogenicityislands(SaPIs)aretheprototypicalmembersofanextremely commonand recently identified family ofmobile genetic elements, thephage-inducible chromosomal islands (PICIs) (Penades & Christie, 2015). SaPIs are clinicallyrelevant because they carry and disseminate superantigen genes, for example those fortoxicshock toxinandenterotoxinB. Inabsenceofahelperphagetheyresidepassively inthehostchromosome,underthecontrolofSaPI-codedmasterrepressor,Stl,aDNA-bindingprotein.Our previouswork indicated that SaPI de-repression is effected by specific, non-essential phage proteins that bind to Stl, disrupting the Stl-DNA complex and therebyinitiating the excision-replication-packaging (ERP) cycle of the islands (Tormo-Más, 2010).Different SaPIs encode different Stl repressors, so each SaPI requires a different non-essentialphageproteinforitsde-repression.TakenwithourdataindicatingthatSaPIbov1isde-repressed by both the trimeric dUTPases and the structurally unrelated dimericdUTPases,wehypothesise that theSaPIshaveevolved from targeting specificproteins totargetingphagemechanisms throughdivergenceof theSaPI repressor.Thiswas thecase,and for the first time we have identified an essential phage protein involved in the de-repressionofSaPI2, thephage80αrecombinasesak.Notonly thisbutwehave identifiedtwo further proteins from different phages (52A and 80) capable of de-repressing SaPI2,whicharefromdifferentrecombinasefamiliesbutperformidenticalrolesintheirrespectivephage cycles. The fact that theStl repressors interactwith structurallyunrelatedproteinsperformingthesamefunctionmakethisstrategyuniqueinnatureandextremelyeffective.The processes targeted by the SaPIs are extremely well conserved in the staphylococcalphages,sothefactthatSaPIstargetdifferentversionsoftheproteinsinvolvedinthesamebiological processes ensures the transferability of theseelements. This highlights SaPIs asoneofthemostfascinatingandeffectivesubcellularparasites.References:Penades,J.R.,andChristie,G.E.(2015)Thephage-induciblechromosomalislands:afamilyofhighlyevolvedmolecularparasites.AnnualReviewofVirology,2(1),pp.181-201.Tormo-Más,M.A.,Mir,I.,Shrestha,A.,Tallent,S.M.,Campoy,S.,Lasa,I.,Barbé,J.,Novick,R.P.,Christie,G.E.,andPenades,J.R.(2010)Moonlightingbacteriophageproteinsderepressstaphylococcalpathogenicityislands.Nature,465(7299),pp.779-782.

22. CharacterizationofsubunitinteractionsduringproteintransportbythebacterialTatpathway

JohannHabersetzer,KristofferMooreandTracyPalmerMolecularMicrobiologydepartment,CollegeofLifeSciences,UniversityofDundeeIn bacteria and archaea, the Tat (twin arginine translocation) system transports foldedcomplexesacrossthecytoplasmicmembrane[1].ProteinsubstratesarefirstlyassembledinthecytoplasmandthentargetedtotheTatsystembyN-terminalsignalpeptidescontainingatwinarginineconsensussequencemotif.Tat transport in Escherichia coli requires three integral membrane subunits called- TatA,TatB, and TatC [2-4]. TatC is the central component of the Tat pathway onto which thesubstrateproteinandotherTatcomponentsassemble.Tat system has to transport folded substratewhich size can vary (from 25 to 70 Å). ThisimpliesinteractionsbetweenTatpartnersarehighlydynamictoadaptthesizeoftheporeinfunctionofthesizeofthesubstratewhileinthesametimemaintainingthecellintegrity.TheassemblyofthedifferentcomponentduringthekeystepsoftheproteintranslocationpathwayisaprerequisitetobetterunderstandtheTattranslocationmechanism.Usingmolecularbiologyandbiochemicalapproaches,weprovidedmoleculardetailsabouttheinteractionsbetweenpartnerproteinsTatA,TatBandTatCandhowtheseinteractionsevolveduringtheTattransportcycle.Ourresultsarethefirstdescriptionofthesequentialassemblystepsduringtothesubstratetranslocationasitoccursincells.References:[1]Palmer,T.andBerks,B.C.(2012)NatRevMicrobiol.10,483-496[2]Weiner,J.H.,etal.(1998)Cell.93,93-101[3]Sargent,F.,etal.(1998)EMBOJ.17,3640-3650[4]Sargent,F.,etal.(1999)J.Biol.Chem.274,36073-36082

23. Molecularweaponry:studyoftwohighlypotenttoxinssecretedbytheTypeVIsecretionsystem

JulianaAlcoforadoDiniz,FrancescaRomanaCianfanelli&SarahJaneCoulthurstDivisionofMolecularMicrobiology,UniversityofDundeeBacteriaareoften found inpolymicrobialenvironmentsand, toguaranteeabetter fitnessadvantage, have adopted diverse strategies to fight for their niche. An importantmechanismutilisedbyGram-negativebacteria isprotein secretion,withprotein secretionsystemsefficientlytranslocatingparticularproteinstotheexternalmediumordirectlyintoacompetitortargetcell.Recently,muchprogresshasbeenmadetowardsunderstandingtheroleandmechanismofthefinalmajorsecretionsystemreportedinGram-negativebacteria,theTypeVIsecretionsystem(T6SS).TheT6SSiswidespreadandcandelivertoxinsintobotheukaryotichostcellsandrivalbacteriacells.Intheopportunistichumanpathogen,Serratiamarcescens,wehavedescribedahighlyeffectiveandoffensiveanti-bacterialT6SS.Severalsecretedtoxins,oreffectors,deliveredbythisT6SShavealreadybeenreportedtorepresentefficientweaponsagainstsusceptibletargetcells,actingondiversecellulartargetsandwithdifferent modes of action. In previous work, two of these, from a class of specialisedeffectors,namelypolymorphictoxinscontainingRhsrepeatdomains,havebeenshowntoplay a central role in intraspecies competition. These Rhs-family proteins possess highlyvariableC-terminaltoxindomainsassociatedwithspecificcognateimmunityproteins.WehaveshownthatoneoftheseRhsproteinspossessesanucleasetoxinatitsC-terminusandfurtherreportedanewclassofconservedaccessoryprotein,EagR(Effectorassociatedgene,withRhs).EagRproteinsareessentialfordeploymentofspecificRhseffectorsbytheT6SS.FurtherworkhasaimedtoinvestigatethemodeofactionofthesecondRhsproteinencodedbySerratiamarcescens,whichhasaC-terminaldomainwithnohomologytotoxinsofknownfunction,andtofurtherelucidatethepotentanti-bacterialactivityofthisfamilyofproteins,usingbiochemical,geneticandmicroscopicapproaches.Inaddition,wedescribeadirect interactionbetweenRhsproteinsandtheircognateEagRprotein,togetherwiththeimportance of this accessory protein in the secretion of Rhs proteins by the T6SS. Takentogether, these findingscanhelp tounderstandhowthese important toxinsaredeliveredand act in the target cell and this may aid the identification of novel targets for thedevelopmentofnewantibacterialtherapies.

24. TheepithelialcelllineresponsetoinvasionbytheprokaryoticperiodontalpathogenFusobacteriumnucleatum

JulianaDelatorreBronzato,SchoolofDentistry.(Supervisors;Dr.DavidHEdwards&DrSamCrouch)Fusobacterium nucleatum (FN) as an important pathogen that is central to themicrobialdiversificationofplaque,andtheetiologyofperiodontaldisease.Inrecentyearstheroleoforalmicrobesinextra-oralcancershasbeguntobeestablished.FNhastheabilitytoinvadeepithelial cells and colonize colorectal tumors. Its role in oral cancer is not clear but twostudies have suggested a link to either instigation or progression of some tumours. Ourstudy is concerned with understanding how different FN strains vary in their ability toinvade eukaryotic cell lines, and how this relates to surface adhesins. The experimentalapproach involves the bioinformatic analysis of FN virulence factors and the phenotypiccharacterisation of three sequenced strains isolated from different environments. Aninfection assay using an epithelial human cell line had been developed to determineintracellularinvasion,andhostcellresponsefourhoursafterchallenge.EukaryoticproteinlevelshavebeendeterminedbystandardWesternblottingandalsoatasystemslevelusingthe DigiWest facilities at the MPI in Tuebingen. Preliminary results suggest a moresignificant response associated with FN25 & FNV, and discrete changes in the levels ofeukaryoticproteinsthatareinlinewiththepublishedliterature.

25. AntagonisticactivityofyeaststowardspotentiallyharmfulbacteriaTkachenkoK.S.,FominaM.A.ZabolotnyInstituteofMicrobiologyandVirologyNASofUkraine,154,ZabolotnySt.,Kyiv,MSP,DO3680,UkraineInteraction between microfungi and bacteria is a little studied area of microbiologicalresearch. The yeast-bacterial interactions may play important role in human healthlongevity.Itisknownthathealthylong-livershavesignificantlyincreasedamountsofyeastsin their GIT and the commercially established probiotic yeast Saccharomyces boulardiimanifestsstrongantibacterialactivity.Theaimofthisworkwastostudyantagonisticactivityofyeastculturesisolatedfromhome-madedairy products andGIT of long-livers towards various pathogenic andopportunisticbacteria (genera Staphylococcus,Bacillus, Escherichia, Salmonella,Pseudomonas,Proteus).Out of 108 isolates we found and identified 43 yeasts with some antagonistic activitytowards Gram-positive (90%) and Gram-negative (74%) bacteria and 6 yeasts withbacteriostatic activity. Only 8 strains showed high antagonism with zones of inhibition≥20mm.Wealsoshowedtheabilityofsomeyeaststo inhibitthepigment-formationbyS.aureus. Medium pH affected the antibacterial activity of yeasts and these effects weredifferent for Gram-positive and -negative bacteria. Yeast isolates from authentic huculyogurtmanifestedantagonisticactivityatmoreacidicpHvalues(5.0-5.5).Theantagonisticactivityofyeaststowardspathogenicandopportunisticbacteriamayhavea great potential for applications in health care and veterinary as an alternative or acomplementtothecurrentwaysofbacterialinfectionpreventionsandtreatments.

26. E.coliO157:H7–Watchoutforthesalad,aswellastheundercookedbeefburger!KathrynWright,JacquelineMarshall,AshleighHolmesandNicolaHoldenCellandMolecularSciences,TheJamesHuttonInstitute,Invergowrie,Dundee,DD25DAInrecentyears,colonisationoffreshproduceplantsbythehumanpathogenEsherichiacoliO157:H7hasbeen increasingly implicatedas the sourceofgastro-enteritis infections. It isbecoming clear that this is due, not only to surface contamination resulting from poorpractice in the ready-to-eat food chain, but also due to the E. coli using plants as analternativehost.E. coli, like plant-associated Enterobacteriaceae have the ability to move to the internaltissuesoftheplant,wheretheyposeafood-safetythreatasnormalsanitationprocessareineffective.Herewepresent imagesto illustrateaspectsof thecolonisationofplantsbyE.coli O157:H7 isolate Sakai labelledwithGFP.We show that the ability of the bacteria toproliferate within the leaf apoplast is dependent on the plant species and that coloniesformed demonstrate characteristics of biofilm formation including release of DNA,formationofcurliandinsomecasesformationofcellulose.Theinternalisationofbacteriaintoyoungseedlingsisalsoillustrated,raisingquestionsabouttheabilityofthepathogentointernaliseinrelationtoplantageand/orphysiologicalstatus.

27. SilencingofnaturaltransformationbyanRNAchaperoneandamulti-targetsmallRNAinLegionellapneumophila

LaetitiaAttaiech1,2,@,AïdaBoughammoura1,CélineBrochier-Armanet3,OmranAllatif4,RossA.Edwards5,AyatR.Omar5,AndrewM.MacMillan5,J.N.MarkGlover5andXavierCharpentier1,2

1MAP(CNRSUMR5240,UniversitéLyon1)10rueDubois,69100Villeurbanne2CIRI(InsermU1111,CNRSUMR5308,ENSdeLyon,UniversitéLyon1),10rueDubois,69100Villeurbanne3LBBE(CNRSUMR5558,UniversitéLyon1),43boulevarddu11novembre1918,F-69622Villeurbanne4BIBS,CIRI(InsermU1111,CNRSUMR5308,ENSdeLyon,UniversitéLyon1),46alléed'Italie,69007Lyon5DepartmentofBiochemistry,UniversityofAlberta,Edmonton,ABT6G2H7,Canada

KeywordsGene regulation; RNA chaperone; trans-acting sRNA; Horizontal gene transfer; NaturaltransformationAbstractMechanismsofHorizontalGeneTransfer(HGT),suchasnaturaltransformation,areamajorcontributortogeneticplasticityandadaptationinbacteriaandfavourstheemergenceanddisseminationofvirulencetraitsandantibioticresistance[1,2,3].Legionellapneumophilaisa naturally transformable bacteria, i.e. it possesses the inherent ability to enter aspecialised,geneticallycontrolled,physiologicalstatecalledcompetence,inwhichitisableto internalise exogenous DNA and integrate it into its chromosome. The absence of anyknown competence regulators in L. pneumophila suggested that unusual mechanisms ofregulationcouldbeinvolvedinthisprocess.Indeed,wediscoveredanRNAchaperone(encodedbythegenelpp0148intheParisstrain)andanewtrans-actingnon-codingsRNAthat, together,controlcompetencedevelopmentinL.pneumophila.AΔlpp0148mutantwaspreviously foundhypercompetent [4] andourRNAseqanalysisofthismutantconfirmedtheupregulationofgenesassociatedwithnaturaltransformationinotherbacterialspecies(comEA,comEC,comM,comF,...).lpp0148belongstoalargeandwidelydistributedfamilyofgenesfoundinthecoregenomeofmanyspeciesand of unknown function. These genes encode proteins containing a ProQ/FinO domain,named after the well-studied plasmid-encoded RNA chaperone FinO. FinO represses theconjugation process by facilitating the recognition between the cis-acting antisense RNA(asRNA)FinPanditsmRNAtarget[5].WeusedRNA-Immunoprecipitationcoupledtodeepsequencing and discovered that Lpp0148 interacts with a single intergenic 66-nt sRNA.Lpp0148tightlybindsthissRNAinvitroandprotectsitfromdegradationinvivo.Moreover,adeletionmutantofthissRNApresentsahypercompetencephenotypeidenticaltothatofthe Δlpp0148 mutant. We therefore named it RocR for Repressor of competence RNA.Under specific growth conditions, a growth phase-dependent decrease in expression ofLpp0148, and subsequently of RocR, triggers competence development and naturaltransformation. While during standard growth conditions, Lpp0148 and RocR post-transcriptionallyrepressthecompetenceregulongenes.Thus,ourresultsdescribeLpp0148asthefirstnon-HfqRNAchaperoneofamulti-targetsRNAthatactsbybasepairing.

Global phylogenetic analysis of ProQ/FinO domain-containing proteins revealed that theyare widely distributed amongst most beta- and gamma-proteobacteria. Thus, they couldconstituteapreviouslyunrecognisedbutwellrepresentedclassofsRNAregulatoryproteins.References:[1]Gogarten,J.P.&Townsend,J.P.Nat.Rev.Microbiol.3,679–687(2005).[2]Barlow,M.MethodsMol.Biol.,532,397–411(2009).[3]Arber,W.Life,4,217–224(2014).[4]Sexton,J.A.&Vogel,J.P.J.Bacteriol.186,3814–25(2004).[5]vanBiesen,T.&Frost,L.S.Mol.Microbiol.14,427–436(1994).

28. ControlofhosthemeacquisitiondictatesthevirulenceofStreptococcusagalactiae

LaetitiaJoubert,Jean-BaptisteDagieu,AurélieBobillot,EliseBorezée-Durant,IsabelleFleurot,AlexandraGrussandDelphineLechardeurMicalisInstitute,NationalInstituteofagronomicresearch(INRA),AgroParisTech,UniversitéParis-Saclay,78350Jouy-en-Josas,France.Heme is a redox-reactivemolecule with vital and complex roles in bacterialmetabolism,survival, and virulence. The opportunistic pathogen Streptococcus agalactiae (group BStreptococcus,GBS) is a heme auxotroph bacterium, which acquires exogenous heme toactivate an aerobic respiratory chain. GBS is a harmless component of the intestinal andvaginal flora, but it is also a leading cause of invasive infection in newborns andimmunocompromisedadults.WecharacterizedanoperonhrtBAhssRS that controlshemetoxicity in Streptococcus agalactiae. ThehrtB andhrtA genes encode a permease and anATPase respectively, while hssS and hssR encode the sensor histidine kinase and theresponse regulator of a two component system. A ∆hrtBA mutant exhibited hemehypersensitivityandaccumulatedintracellularhemefromtheenvironmentsuggestingthatHrtBA functions as a heme efflux transporter to regulate heme concentration inside thebacteriathuscontrollingitstoxicity.IntheabsenceofhssRS,theheme-inducedexpressionofHrtBAwasabolisheddemonstratingtheimplicationofthistwo-componentsystemintheinductionofhrtBA.While the importanceofheme forpathogenshasbeendemonstratedfrommainlyinvitroexperiments,theexactimpactofhosthemeontheirvirulenceinvivoisstillunclear.GBSisparticularlysuitedforsuchastudyashemelevelsarefullycontrolledbyexogenoushemepools.WetookadvantageoftheroleofhrtBAhssRSinthemanagementofexogenous heme toxicity to study the real contribution of host heme to Streptococcusagalactiae virulence.Using luminescentS. agalactiae (luxABCDE),we followed in vivo thevirulence of ∆hrtBA mutants in a mouse systemic infection model. We showed that thecapacityofGBStoovercomehemetoxicityinvivohasamajorimpactonitscapacitytogrowin the host. The virulence of GBSwas also severely attenuatedwithmutant defective inrespiration (∆cydA). Using the HssRS heme sensing pathway combined to the heme-dependentinductionof luxABCDE,wevisualizedhemesensing invivoduringthecourseofinfectionandshowedthatitwasdependentontheorgancolonizedbyGBS.Alltogetherourresults demonstrate the importance of host heme in the virulence of S. agalactiae andhighlighttheimportanceofhosthemeinthehostpathogencrosstalk.

29. VgrSRdetectsbothextracellularandintracellularirontocontrolbacterialadaptationinhostplant

LiWang,Zhi-HuiYuan,YuePan,WeiQian1*

InstituteofMicrobiology,ChineseAcademyofSciences,Beijing100101,ChinaIron is a double-edged metal nutrient for life. It is a cofactor absolutely required foressentialcellularprocessesofalmostallorganisms.However,theexcessofironcanbealsoextremely toxic to cells. To cope with this challenge, organisms have to use effectivemolecular mechanisms to achieve iron homeostasis. Here we found that the twocomponent signal transduction system VgrS-VgrR of plant pathogenic bacterium, X.campestris, is a critical modulator to reciprocally modulate iron homeostasis duringpathogenesis.VgrSdetect Fe3+ concentrationanddecreases its autokinaseactivity.Underiron deplete condition, the phosphorylation level of VgrR-VgrS increased and then thephosphorylatedVgrR (VgrR-P) represses thetranscriptionofadownstreamgeneencodingTonB-dependent receptor (TDR) by binding to its 5ʹcis-regulatory element. Since the TDRrepresentsauniquegroupofouter-membranetransporterswhoseactivityisdetrimentaltoironuptakeandbacterialgrowthinhostplant,theVgrR-Pdependentrepressionresultedinnetuptakeofironandenhancementofvirulence.However,whenFe2+isaccumulatedtoahigh concentration in bacterial cells, the binding of Fe2+ to VgrR caused effectivedisassociationbetweenVgrRandthepromoterofTDRgene,whichreleasestherepressionof transcription and that the TDR begin to impede bacterial iron uptake. Therefore, VgrSdetects extracellular iron depletionwhereas VgrR senses intracellular iron repletion. ThisTCSTSsystemregulates thebacterial ironhomeostasisbyheterogeneouslycontrolling theexpressionsofironuptakegenes.

30. ImmunefactorsfoundinthecolonduringBrachyspirahyodysenteriaeinfectioninduceincreasedmucintransportandproductioninamucussecreting,polarizedinvitrocolonicmucosalsurface

MacarenaP.Quintana-Hayashi1,NazaninNavabi1,MaximeMahu2,NeleDePauw2,FilipBoyen2,HarveyR.Fernandez1,AnMartel2,FreddyHaesebrouk2,FrankPasmans2,&SaraLindén1

1Department of Medical Biochemistry and Cell biology, Institute of Biomedicine, SahlgrenskaAcademy,UniversityofGothenburg,Gothenburg,Sweden2DepartmentofPathology,BacteriologyandPoultryDiseases,FacultyofVeterinaryMedicine,GhentUniversity,BelgiumBrachyspira hyodysenteriae colonizes the pig colon causing swine dysentery (SD). The pigcolon is covered by a mucus layer composed by large glycoproteins called mucins. B.hyodysenteriae infection causes changes in the mucin environment characterized by adisorganizedmucus structure and amassivemucus inductionwithde novo expression ofMUC5AC and increased production ofMUC2 in the colon. The aims of the present studyweretodeterminehowfactorsfromtheimmunesystemchangeinthepigcolonduringSDandidentifywhichofthesechangesareimportantformucinproduction.QuantificationofthemRNA levels of several factors from the immune systemby qPCR revealed increasedexpression levels of neutrophil elastase (NE) in the colon tissue of B. hyodysenteriaeinfectedpigs,anine-foldincreaseinthemRNAlevelsofIL-17A,andupregulationofIL-1β,IL-6andIL-8inatleasttwooutoffivepigs.Inordertodeterminetheeffectoftheupregulatedfactors from the immunesystem inmucinproduction,polarizedmucusproducing in vitromucosalsurfacesdevelopedfromHT29-MTX-E12coloniccellsweretreatedwithNE,IL-1β,IL-6, IL-8, and IL-17A, individually and combined, with and without B. hyodysenteriaeinfection.Ourresultsshowedthat thecombinedeffectofB.hyodysenteriae infectionandNEtreatmentincreasedthemucinproductionandtotalnumberofmucincontaininggobletcells in the invitromucosalsurfaces, regardlessof thecytokineenvironment.Overall,ourdata suggests the synergistic effect between bacterial factors and NE inmucin inductionduringB.hyodysenteriaeinfection.

31. TheglobalregulatorShvRofBurkholderiacenocepaciatightlyregulatesvirulenceinzebrafish

MargaridaGomes

Bacteria belonging to the Burkholderia cepacia complex (Bcc) pose serious healthproblemsinvulnerablepatients,particularlyinindividualswithcysticfibrosis(CF)orchronicgranulomatous disease (CGD). Besides, they are increasingly described in health-careassociatedoutbreaks.IntheCFlung,thebacteriacancausechronicinfectionscharacterizedby sudden periods of pulmonary exacerbation, sometimes resulting in rapidly fatalnecrotising pneumonia and septicaemia (Cepacia Syndrome). However, the molecularmechanisms are not known. It has been demonstrated in vitro that Burkholderiacenocepaciaiscapabletosurviveandmultiplyinhostcells.Wehaveshownforthefirsttimein vivo, using a zebrafish infection model, that macrophages provide a critical site forintracellular replication of B. cenocepacia K56-2 and development of acute pro-inflammatoryinfection.

The LysR-type transcriptional regulator ShvR of B. cenocepacia K56-2 has beenshowntoregulategenes important forvirulence inaratmodelof infection.Althoughtheabsence of shvR resulted in strongly reduced lung inflammation, this, paradoxically,correlated with higher bacterial loads. This suggests that the shvR mutant was highlypersistent.Zebrafish larvaewereused to furtherstudya role forShvRduring intracellularstagesand for the inductionof acute fatal inflammation. In theabsenceof the regulator,pro-inflammatory responses and inflammation were strikingly reduced, preventing fatalinfection.Usingrealtimenon-invasiveimagingweobservedthatshvRmutantbacteriawereabletopersistinmacrophagesbut,incontrasttothewildtypeparentstrain,wereunabletodisseminatefrominfectedmacrophages.Iwillpresenthowweusedaninducibleexpressionsystem, to show in vivo that ShvR tightly regulates genesneeded for thedevelopmentofacutepro-inflammatoryinfection.

References:MargaridaC.Gomes1,2,SujathaSubramoni3,PamelaSokol3andAnnetteVergunst1,21INSERM,U1047,UFRMédecineSitedeNîmes,30908Nîmes,France.2UniversitédeMontpellier,U1047,UFRMédecine,30908Nîmes,France.3DepartmentofMicrobiology, Immunology, and InfectiousDiseases,UniversityofCalgary,CalgaryT2N4N1,Canada

32. DecipheringtheroleofEsaBintheregulationoftheTypeVIISecretionSystemof

StaphylococcusaureusMaria-GuillerminaCasabonaandTracyPalmerDivisionofMolecularMicrobiology,SchoolofLifeSciences,UniversityofDundee,ScotlandThe type VII secretion system (T7SS) is a multiprotein machinery used by many Grampositive bacteria to export toxins. It was first described in the pathogenMycobacteriumtuberculosis, where it was shown to secrete substrates of the WXG100 protein familyessential for virulence. T7SSs are encoded by gene clusters that can code for several ofWXG100familyproteinsaswellasanATPasewithapredictedFtsK-SpoIIIEdomain,whoseproductisessentialfortoxinexport.IthasbeenshownrecentlythatStaphylococcusaureuscodes for a functional T7SS called Ess that plays an important role in virulence. Thismachinery is responsible for the export of several toxins of theWXG100 family, namelyEsxA,EsxB,EsxCandEsxD.DifferentreportshavesuggestedaroleofEsaBintheregulationoftheT7SSinS.aureus.EsaBisasmallprotein(<10kDa)predictedtobestructurallyrelatedtoubiquitinhoweveritlackstheC-terminusdoubleglycinemotif.ItisconservedinS.aureusstrainsbut there isnohomolog inM. tuberculosis. In this study,wesought to investigateEsaB as a regulator of the T7SS of S. aureus. Using secretion assays followed bywesternblottingwe have shown that EsaB is an essential component of the T7SS in the RN6390strainsincea∆esaBmutanthasadefectivephenotypeforEsxAandEsxCsecretion.ContrarytowhathasbeenshowninNewmanandUSA300strains,a∆esaBmutantofRN6390doesnotexhibitoverproductionofEsxC.Accordingly,RT-PCRandRNA-seqexperimentsindicatethatEsaBdoesnotaffecttranscription levelsoftheT7SSgenes.ComplementationstudiesindicatethatverylowamountsofEsaBinthecellcanrestoreEsxA,EsxBandEsxCsecretion.Moreover,overproductionofEsaBcanleadtooversecretionofEsxA,arguingthat itmightbeactingasaposttranslational regulatorof themachinery.Site-directedmutagenesishasbeenusedtostudythe importanceof theconservedresiduesofEsaB.Theseexperimentshaveshownthatmanyof theconservedresidues inEsaBaredispensable.FurtherstudieswillaimattheidentificationoftheEsaBinteractome.

PostersessionII·Thursday30thJune2016

33. Cross-talkbetweenaccessoryproteinsduringbiosynthesisof[NiFe]hydrogenase-5

fromSalmonellaentericaMartaAlbaredaandFrankSargentDivisionofMolecularMicrobiology,SchoolofLifeSciences,UniversityofDundee,DowStreet,Dundee,DD15EH,Scotland,UK.

Hydrogenproducednaturallyduringsugarfermentationbythegutmicrobiotaisan

important energy substrate for a number of pathogenic bacteria. In Salmonella entericaserovarTyphimuriumtheenergyconservedbytherespiratoryoxidationofH2iscrucialfortheinfectionprocesswithinthehost(Maieretal.,2004).TheS.entericagenomeencodesthree [NiFe]-hydrogenases that are involved inH2oxidation, twoof themare synthesizedstrictlyunderanaerobicconditions,onebeinganO2-tolerantenzyme(Hyd-1)andtheotherbeing a standard O2-sensitive enzyme (Hyd-2). The third H2-uptake hydrogenase, termedHyd-5, is synthesized aerobically and shares sequence identity and structural similaritieswith Hyd-1 (Parkin et al., 2012). [NiFe] hydrogenases are heterodimeric enzymes thatconsistonalargesubunitthatcontainsthecatalyticsiteoftheenzyme[NiFe(CN-)2CO]andasmall subunit that harbours three FeS clusters throughwhich electrons are conducted totheirprimaryacceptor.BiosynthesisoftheNiFecofactoranditsinsertionintotheprecursorofthe largesubunitrequirestheactionofaccessoryproteinsencodedbythehypoperon.After the insertion of the cofactor, the large subunit is proteolytically processed at its C-terminusbyaspecificprotease.Inorganismsthatcontainseveral[NiFe]-hydrogenaseseachhydrogenaseisoenzymeisprocessedbyitsownspecificendopeptidase(Böcketal.,2006).

Inthisstudy,itisdemonstratedthatthefunctionofHydD,thematurationproteaseof Hyd-5, can be partially replaced by HyaD, the endopeptidase encoded by the Hyd-1operon. Inaddition,geneticandbiochemicalapproachesprovidenewevidenceforstablecomplexes involving both HydD and HyaD with the Hyd-5 large subunit. These stableinteractionsaredependentonthebiosynthesisofafullyfunctional[NiFe]cofactorbuttheydonotrequirethepresenceoftheC-terminalextensiononthelargesubunit.Finally,crosslinkingexperimentsrevealthecontactpointsbetweenthelargesubunitanditsmaturationprotease.References:BöckA,KingPW,BlockeshM,PosewitzMC(2006).Adv.Microbiol.Physiol,51:1-72.MaierRJ,OlczakA,MaierS,SoniS,GunnJ(2004).Infect.Immun.,72(11):6294-6299.ParkinA,BowmanL,RoesslerMM,DaviesRA,PalmerT,ArmstrongFA,SargentF(2012).FEBSLett,586:536-544.

34. ComparativeglobalproteomicanalysisofclinicalStreptococcuspneumoniaeisolatesbyLC-MS/MS.

MustaphaBittayaStreptococcuspneumoniae isresponsibleforavarietyofinvasivediseases,includingsepsisandpneumococcalbacteraemia.Thebacteriarespondtothehostimmunesystemaswellasenvironmentalstressesduringtheirsurvivalinthebloodstreambyalteringtheexpressionofkey proteins that contribute to their persistencewithin the bloodstream. The aimof thisstudy was to identify changes in the protein expression profiles of S. pneumonia thatcorrelatewithinvasiveinfection.TheproteomesofclinicalS.pneumoniaeisolatesrecoveredfrom blood cultures (classified as invasive isolates) were compared to bacteria collectedfrom non-invasive sites such as sputum, skin and ear swabs (classified as non-invasiveisolates). The bacteria were grown in vitro under standard conditions using Brain HeartInfusion (BHI) broth supplemented with 5% (v/v) foetal calf serum. The total cellularproteins were extracted for analysis by LC-MS/MS. Global protein expression profiles ofthree invasiveandthreenon-invasive isolateswereobtained in triplicates foreach isolateusingalabel-freequantitativeLC-MSmethod.TheLC-MSprofilingidentified1200proteinsof which 42 and 96 proteins were significantly up-regulated and down-regulatedrespectively(ANOVA,p<0.05)intheinvasiveS.pneumoniae isolatescomparedtothenon-invasive isolates. Gene Ontology annotation of the differentially synthesised proteinsrevealedthatamajorityoftheproteinswerefunctionally involvedincatalyticactivitiesaswellasinthebindingofsubstratesandmacromolecules.Theup-regulatedproteinsincludedpreviouslydocumentedvirulence factors (suchasPspAandBgaA)andproteinsassociatedwithpathwaysforthesynthesisofsugars,aminoacids,vitamins,andcofactors.Thedown-regulated proteins included ATP-binding cassette (ABC) transporters andphosphotransferase system (PTS) proteins as well as cell envelope proteins involved inmaintaining the integrityof thepneumococcal cell envelope (such asCps4C, EcfA1,MreCandPlsX). These resultswill serveasan important source fromwhichproteinbiomarkerscan be selected for further studies aimed at elucidating the pathogenesis of invasivepneumococcaldiseaseaswellastheidentificationofnewtherapeutictargets.

35. OptimisationofaThree-dimensionalAirwayEpithelialModeloftheOvineRespiratoryTractGrownatanAir-LiquidInterface

NickyO’BoyleThecultureanddifferentiationofrespiratoryepithelialcellsattheair-liquidinterface(ALI)allowsforthedevelopmentofacellstratum,whichishighlyrepresentativeofthetissueinvivo. Lung tissue from domesticated animals such as cattle and sheep can be readilyobtainedfromabattoirmaterialandthusrepresentsacost-effectiveandethicalalternativetoinfectionstudiesusingliveanimals.Theuseoftissuesfrommammalianhostswhichhaverespiratoryepitheliaof similarphysiological structure tohumanepitheliaalsomakes suchmodelsappropriateforthestudyofpathogenswhichinfectthehumanrespiratorytract.Wehavecarriedoutanextensivestudyexaminingavarietyofcriticalfactorswhichregulatetheefficiency of airway epithelial cell differentiation during ALI culture. Growth substrates,medium constituents and gas composition of the culture environmentweremanipulatedand the degree of differentiation assessed by histological analyses, immunofluorescencemicroscopyandscanningelectronmicroscopy.Bycomparingawidevarietyof the factorsthataffectdifferentiation,wehaveselectedtheoptimumgrowthconditionsthatallowforthe development of a well-differentiated epithelial layer, which comprises the keyrespiratoryepitheliumcell types including ciliatedepithelial cells,mucus-producinggobletcellsandbasalstemcells.Thismodelwillproveparticularlyuseful inthestudyofrelevantbacterialandviralpathogensoftheovinerespiratorytract.

36. Aremarkableexampleofconvergentevolutioninvolvingpathogenicityislandsinhelpercosphagesinterference

NuriaCarpena1,3,,AlbertoMarina3,JoséRPenadés1,*.

1InstituteofInfection,ImmunityandInflammation,CollegeofMedical,VeterinaryandLifeSciences,UniversityofGlasgow,GlasgowG128TA,UK;3InstitutodeBiomedicinadeValencia(IBV-CSIC)andCIBERdeEnfermedadesRaras(CIBERER),46010Valencia,Spain.Keywords: capsid morphogenesis, SaPIs, PICIs, small capsids, bacteriophage resistance,bacteriophagepackaging

Staphylococcalpathogenicityislands(SaPIs)arephagesatellitesthatexploitthelifecycleoftheirhelperphagesfortheirownbenefit.Asoccurswiththeirinducinghelperphages,SaPIscanbepackagedbyoneofthetwoclassicalmechanismofphagepackaging.Thus,mostofthecharacterisedSaPIsuse theheadful (pac)packagingmechanism.TheseSaPIs interferewithpacphagereproductionusingcomplementaryandelegantstrategies,includingamongotherstheproductionofsmallSaPI-sizedcapsid,aprocessthatdependsontheexpressionoftheSaPIcodedcpmAandcpmBgenes.TheotherSaPIsubfamilyisinducedandpackagedbycos-typephages,andalthoughthesecosSaPIsalsoblockthelifecycleoftheir inducingphages, thebases for thismechanismof interference remains tobedeciphered.HerewesolvedthemysteryandidentifiedandcharacterisedthefirstmechanismbywhichtheSaPIsinterferewithcosphages reproduction.Remarkably, thismechanismdependson theccmgene, which as previously reported for the cpmA and cpmB genes, encodes a proteininvolved in the production of SaPI sized capsids. Since the Ccm and CpmAB proteins arecompletely unrelated in sequence, this strategy represents a fascinating example ofconvergentevolution.Moreover,thisresultalsoindicatesthattheproductionofSaPI-sizedparticlesisawidespreadstrategyofphageinterferenceconservedduringSaPIevolution.

37. CharacterizationofoutermembranevesiclesofPasteurellamultocidafromavian,bovine,porcineandovineisolates

NurshahiraSulaiman,DrRichardBurchmore,DrRobertL.DaviesInstituteofInfection,ImmunityandInflammation,CollegeofMedical,VeterinaryandLifeSciences,SirGraemeDaviesBuilding,UniversityofGlasgow

Pasteurella multocida is a Gram-negative pathogen that causes economically significantinfections ina rangeofdifferentdomesticated species. It is theaetiologicalagentof fowlcholera inpoultry,enzooticpneumonia incattle,sheepandpigs,athrophicrhinitis inpigs,and haemorrhagic septicaemia in cattle and buffalo.Outermembrane vesicles (OMVs) orblebsarenaturallyproducedfromthebacterialoutermembraneandcompriseproteinsandothermoleculessuchas lipopolysaccharide(LPS)derivedfromthisbacterialcompartment.Thus, they have the capacity to transport active, specific and highly concentrated solubleand insolublematerials into theextracellularmilieu.Outermembraneproteins (OMPs)ofGram-negativebacteriahavebeenwellstudiedandplay importantroles inhost-pathogeninteractions. Since OMVs are derived from the outermembrane, they contain OMPs andalsohave important roles in pathogenesis includingbacterial survival, nutrient acquisitionand biofilm formation.However, the formation and pathophysiological roles of OMVs inelicitingandsustainingP.multocidainfectionhasnotbeenwelldocumented.Inthepresentstudy,OMVformationwasstudied ineightrepresentativeP.multocida isolatesrecoveredfromdiseasedcattle, sheep,pigsandpoultry.Vesicleswereproducedbyalleight isolateswithanaveragediameterof24nm.Comparisonofoutermembraneandvesiclesproteinand LPS profiles by SDS-PAGE and Western-blotting confirmed that the vesicles werederivedfromtheoutermembranes.Gel-freeproteomicanalysesidentified55OMPsintheeightisolateswhichhadfunctionsinoutermembranebiogenesis,iron-uptake,colonizationandadherencetohostcells.Thesefindingswillcontributetotheidentificationofpotentialantigensforthedevelopmentofimprovedvaccines.

38. Localc-di-GMPsignalingbyanetworkofinteractingGGDEF/EALdomainproteinsinE.colibiofilmcontrol.

OlgaSarenkoandRegineHenggeInstitutfürBiologie/Mikrobiologie,Humboldt-UniversitätzuBerlin,10115Berlin,Germanyc-di-GMP is a ubiquitous bacterial second messenger that in general promotes biofilmformation. E. coli K-12 has 29 proteins with GGDEF/EAL domains, which include 12producersofc-di-GMP(diguanylatecyclasesorDGC)and13degraders(phosphodiesterasesorPDE).Themultiplicityandsometimeshightargetspecificityoftheseenzymeshasledtothe idea of 'local' signaling involving direct protein-protein interactions (1). With thePdeR/DgcM/MlrAmodulehavingbeendemonstratedasaprototypicalexampleofalocallyacting c-di-GMP control, inwhich aDGC and a PDE directly interactwith each other andwithatargetprotein(2),thequestionarosewhetherotherDGCsandPDEsareinvolvedinsimilarlocalactions.Using the BacterioMatch-Two-Hybrid system, we systematically investigated interactionsamong all 29 GGDEF/EAL domain proteins of E. coli K-12. Surprisingly, we did not findadditionalspecificDGC/PDEpairs involved insinglecomplexes,butratherobservedthatasubset of the GGDEF/EAL domain proteins has even multiple interaction partners.Interestingly,inalltheseinteractions,atleastoneofthepartnersbelongstothetwocentralswitchmodules(PdeH/DgcEandPdeR/DgcM/MlrA)knowntoplayakeyroleinthecontrolof the biofilm regulator CsgD and therefore the production of the biofilm matrixcomponents curli and cellulose. Among other GGDEF/EAL domain proteins, however, nointeractionswereobserved.We conclude thatwith respect to direct protein-protein interactions, the complement ofGGDEF/EALdomainproteinsfallintothreeclasses:(i)corecomponentsofthecentralc-di-GMP switch module that control biofilmmatrix synthesis (PdeH, DgcE, PdeR, DgcM), (ii)accessorycomponentsthatmayconditionallymodulatetheoutputofthisswitchbydirectinteractionswithinalargerproteinnetwork,and(iii)DGCsandPDEswithenzymaticactivityonlythatactindependentlyofotherGGDEF/EALdomainproteinstoaffectthecellularc-di-GMPlevelwhichissensedbyPdeRasakeycomponentofcentralswitchmodule.References:(1)Hengge(2009)NatureRev.Microbiol.7,263-273.(2)Lindenbergetal.(2013)EMBOJ.32,2001-2014.

39. ThePseudomonasputidaTypeVIsecretionsystemisusedasabiocontrolweaponagainstphytopathogens

PatriciaBernalImperialCollegeLondonBacterialtypeVIsecretionsystems(T6SSs)arenanomachinesusedtoinjecttoxiceffectorsintotargetcells.Thissecretionsystemplaysanimportantroleininter-bacterialcompetitionproviding colonization advantages to the T6SS producer. The aim of this work was toidentify T6SSs of the soil bacterium Pseudomonas putida, an important biocontrol agentthathas the capacity to colonise the rootof cropplants. This bacteriumprovides growthadvantagestotheplantand,importantly,protectionagainstplantpathogens.SinceT6SSismainly used by environmental bacteria for interbacterial competition, it could play arelevant role in the biocontrol properties of P. putida. Here we report that P. putidacontains three T6SSs (K1, K2 and K3) each encoding the core T6SS proteins along withadditional accessory components including regulatory proteins and toxins-immunity pairs.AdditionalT6SS-relatedgenesare found scatteredon thechromosome.TheP.putida K1-T6SS is functional in our in vitro conditions and secretes anRhs-type effector (Tke2) thatshows cytotoxicitywhen expresses in theEscherichia coli cytosol. The toxic effect can becounteracted by a cognate immunity protein, Tki2. Importantly, we found that P. putidaT6SSs are used by the bacterium to kill awide range of resilient phytopathogens in vitroincludingXanthomonas campestris,Pectobacterium carotovorum,Pseudomonas psyringaeand Agrobacterium tumefaciens. This effect has been also observed in an in plantacoinfectionassay inwhichP.putidauses itsT6SStooutcompeteXanthomonascampestrisandthusprotecttheplant fromthedeleteriouseffectofthispathogen.Ourdatastronglysuggest that T6SS plays an important role in the biocontrol properties of P. putida andengineeringofsuper-T6SSstrainsiscurrentlyconsidered.

40. AssessingtheimportanceofthenasalmicrobiotainsusceptibilitytoStreptococcuspneumoniaecolonization

L.P.Schenck,N.Thevaranjan,D.M.E.Bowdish,M.G.SuretteMcMasterUniversity,Hamilton,Ontario,CanadaStreptococcuspneumoniaeistheprimarycauseofpneumoniaintheelderlyandmeningitisin youth worldwide. Colonization of the nasopharynx is an essential step before thebacteria can disseminate to cause invasive pneumococcal disease. We hypothesize thatbacterial interactions within the nasopharynx could play a role in the susceptibility tocolonization.Amurinemodelofpneumococcalcolonizationisutilizedtoassessinteractionswithin the nasalmicrobiota. Regional and adherent bacteria were assessed in the nasalturbinatestodeterminecomponentsofthenasalmicrobiota.Thenasopharynxishosttoadiversemicrobiota,containingHaemophilus,StaphylococcusandotherStreptococcuswhichhavepreviousdocumentationtointeractwithS.pneumoniaepost-colonization.Youngandaged mice had vastly different microbiota profiles after exposure to S. pneumoniae,includingpersistenceofS.pneumoniaeinelderlymice.Furtherassessmentsintopathogeninteractionsbetweenthesebacteriamayleadtopreventativestrategiesforpneumococcalcolonizationresistance.

41. InteractionsbetweentheTatBCcomplexandTatsignalpeptidesduringproteintransportbythebacterialTatpathway

QiHuang1,FelicityAlcock2,HolgerKneuper1,BenC.Berks2andTracyPalmer1†1DivisionofMolecularMicrobiology,CollegeofLifeSciences,UniversityofDundee,DundeeDD15EH,UK2DepartmentofBiochemistry,UniversityofOxford,SouthParksRoad,OxfordOX13QU,UKThe twin-arginine translocation (Tat) system is a protein export systemwhich transportsfully-foldedproteinsacrossthecytoplasmicmembrane.Itispresentinmostbacteriaaswellasmanyarchaeaandplant chloroplastsbutabsent fromanimal cells. TheTat systemhasbeenreportedtobeinvolvedinmanybacterialphysiologicalprocessesandisimportantforvirulence in several humanandplantpathogens. Thus, the Tat systemcanbe apotentialantibacterialdrugtarget.InEscherichiacoli, theTatsystemconsistsofthreeessentialcomponents-TatA,TatBandTatC,whichareallmembrane-integralproteins.TatBandTatCformareceptorcomplextobindsubstratesandTatAformsachanneltoallowsubstratestopassacrossthemembrane.Tat substrates are targeted to the machinery via their N-terminal signal peptide, whichpossesses an invariant twin-arginine (RR) motif close to the N-terminus followed by ahydrophobicregionandaC-terminalregionharbouringasignalpeptidasecleavagesite.TheRR-motifintheTatsignalpeptideiscriticalforsubstratestobetargetedtotheTatsystem.Substitution of the RR-motif in the substrate with KK or other amino acids abolishesrecognition by the Tat system. We constructed several Tat mutagenesis libraries andisolatedsomeTatvariantswhichcouldsuppressinactivatedsubstitutionsattheRR-motif.Byusingcysteinecross-linking,Blue-nativePAGEandfluorescentimaging,wefoundthatthesignal peptide suppressors are functioning by stabilising an assembled state of the twinargininetranslocase.Moredetailsaboutourlatestfindingswillbepresented.

42. ComprehensiveinsightsintotheprimarytranscriptomeofthexanthanproducerstrainXanthomonascampestrispv.campestrisB100

RabeaaS.Alkhateeb1,Frank-JörgVorhölter1,2,†,ChristianRückert3,#,DanielWibberg2,GerdHublik4,KarstenNiehaus1,AlfredPühler21ProteomeandMetabolomeResearch,FacultyofBiology,CenterforBiotechnology(CeBiTec),BielefeldUniversity,Universitätsstraße27,33615,Bielefeld,Germany2InstituteofGenomeResearchandSystemBiology,CenterforBiotechnology(CeBiTec),BielefeldUniversity,Universitätsstraße27,33615,Bielefeld,Germany†Currentaddress:MVZDr.Eberhard&Partner,Brauhausstr.4,44137,Dortmund,Germany3TechnologyPlatformGenomics,CenterforBiotechnology(CeBiTec),BielefeldUniversity,Universitätsstraße27,33615,Bielefeld,Germany#Currentaddress:SinskeyLaboratory,DepartmentofBiology,MassachusettsInstituteofTechnology,31AmesStreet,Bldg.68-364D,Cambridge,MA02142,USA4JungbunzlauerAG,Austria,Pernhofen1,2064,Wulzeshofen,AustriaXanthomonas campestris pv. campestris (Xcc) is the major producer of theexopolysaccharide xanthan, the commercially most important natural polysaccharide ofmicrobialorigin.Thecurrentworkprovidesextensiveinsights intotheyetuncharacterizedtranscriptomic features of the strain Xcc-B100. Towards this goal, RNA sequencing of alibrarygeneratedbyaselectiveenrichmentofthe5’endsofnativetranscriptswasapplied.This approach resulted in the genomewide identificationof 3067 transcription start sites(TSSs) thatwere classified basedon their genomic positions. Among them, 1545mappedupstreamofanactivelytranscribedCDSand1363wereclassifiedasnovelTSSsrepresentingantisense, internal, and TSSs belonging to previously unidentified genomic features.Mapping the exact positions of TSSs aided in the identification of promoter consensusmotifs, ribosomal binding sites, and enhanced the genome annotation of 159 in silicopredicted translational start (TLS) sites. The global view on length distribution of the 5’untranslated regions (5’-UTRs) pointed to the occurrence of leaderless transcripts andtranscripts with unusually long 5’-UTRs. Concerning the biosynthesis of xanthan, RNAsequencing data shows a complex transcriptional pattern of the gum operon, the majorplayer of xanthan biosynthesis on a genomic level. Under the condition tested, thetranscriptionofthegumclusterappearstobeinitiatedatmultiplestartsites.Furthermore,Northernblotanalysis confirmed thepresenceofcis-encodedantisense transcriptswithinthegumcluster;however,theirpotential involvementinregulatinggeneexpressionisstilltobeexamined.

43. ppGppnegativelyimpactsribosomeassemblyaffectinggrowthandantimicrobialtoleranceinGram-positivebacteria

RebeccaM.Corrigana1,LaurenE.Bellowsb,AlisonWooda,andAngelikaGründlingb1

aDepartmentofMolecularBiologyandBiotechnology,UniversityofSheffield,Sheffield,S102TN,UKbSectionofMicrobiologyandMRCCentreforMolecularBacteriologyandInfection,ImperialCollegeLondon,London,SW72AZ,UK1CorrespondingauthorsThestringentresponseisasurvivalmechanismutilizedbybacteriatodealwithstress.Itiscoordinated by the nucleotides guanosine tetra- and pentaphosphate ((p)ppGpp), whichinteractwithtargetproteinsinordertopromotebacterialsurvival.Althoughthisresponsehasbeenwellcharacterizedinproteobacteria,verylittleisknownabouttheeffectorsofthissignaling system in Gram-positive species. Here we report on the identification of seventarget proteins for the stringent response nucleotides in the Gram-positive bacteriumStaphylococcus aureus. We demonstrate that the GTP synthesis enzymes HprT and GmkbindwithahighaffinityleadingtoaninhibitionofGTPproduction.Inaddition,weidentifiedfive putative GTPases - RsgA, RbgA, Era, HflX andObgE as (p)ppGpp target proteins.WeshowthatRsgA,RbgA,Era,HflXarefunctionalGTPasesandthattheiractivityispromotedinthepresenceofribosomes,butstronglyinhibitedbythestringentresponsenucleotides.Bycharacterizing the function of RsgA in vivo, we ascertain that this protein is involved inribosomeassembly,withanrsgAdeletionstrain,orastraininactivatedforGTPaseactivity,displaying decreased growth, a decrease in the amount ofmature 70S ribosomes and anincreased level of tolerance to antimicrobials. We additionally demonstrate that theinteraction of ppGpp with cellular GTPases is not unique to the Staphylococci, ashomologues from Bacillus subtilis and Enterococcus faecalis retain this ability. Takentogether,thisstudyrevealsribosomeinactivationasanewmechanismthroughwhichthestringentresponsefunctionsinGram-positivebacteria.

44. ThreePseudomonasfluorescensplantgrowthpromotingbacteriaandtheirmutualisticmechanisms

Richard Lally1, Samuel Kiwanuka1, Antonio Sergio Moreira2, Andrew Lloyd1, KieranGermaine1,DavidRyan1andDavidDowling1

1Envirocore,TheDarganCentre,DepartmentofScienceandHealth,InstituteofTechnologyCarlow,Ireland2DundalkInstituteofTechnology,Dundalk,Co.Louth,IrelandPlantgrowthpromotingbacteriahavebeenisolatedfromeveryplantspeciesinvestigated,theycanformsymbiotic,mutualisticorcommensalisticrelationshipswiththeirhostplant.Plant growth promoting bacteria have importance in agriculture as they exhibit plantbeneficial mechanisms important in the growth, protection and development of plants.These include plant protection from pathogens and herbivores, soil nutrient cycling andplantgrowthenhancement fromdirect ‘microbe toplant’ interactions.Genomesequenceanalysis provides the opportunity to examine these important mechanisms helpingunderstandtheplant-bacteriarelationships.Understandingthesesignallingmechanismsatamolecular levelwill optimise theeffectiveuseof thesemicroorganisms inplant growthpromotionandsustainableagriculture.Wehavesequenced thegenomesof threePseudomonas fluorescens strains isolated fromtheleavesofsurfacesterilisedMiscanthuscv.giganteus.Thesestrains,P.fluorescensL321,L111 and L228were chosen for genome analysis as they exhibit plant growth promotingtraitsbothinvitroandingreenhouseexperiments.Thesestrainsarealsoeffectiverootandrhizosphere colonisers and express plant beneficial rhizospheric traits, such as 1-aminocyclopropane-1-carboxylate deaminase production and phosphate solubilisation.Other important plant interactionmechanisms have been attributed to these strains andinclude plant hormone indole-3-acetic acid synthesis, biocontrol activity andmotility/chemotaxis capabilities. Here we present the data collected from plant growthtrials,phenotypeexpressiontestsandsequencecharacteristic tests. Analysisof thethreestrains has revealedmechanisms the strains use to colonise, reside on, andwithin planttissue. This work also gives insights into their plant growth promotion properties andmutualisticrelationshipswithplants.Key words: Pseudomonas fluorescens; endophytes; plant growth promotion; sequenceanalysis

45. ChitinaseSecretioninSerratiamarcescens-AChiX-ratedexplorationintoanessentialendopeptidase

RichardOwenTheopportunisticpathogenSerratiamarcescensdependsonafourgeneoperon,chiWXYZ,forsecretionofthechitinolyticmachinery,ChiA,ChiB,ChiCandCbp21.ThisprojectdetailsthecharacterisationoftheessentialcomponentsChiWandChiX.ChiWisapredictedholinprotein,whileChiXcodesforaputativeendopeptidase.Inthiswork,ChiWhasbeenshownessential forChiXpassagetotheperiplasm,allowingaccessto itspredictedpeptidoglycansubstrate.UsingX-raycrystallography,thestructureoffull-lengthChiXhasbeenresolvedto1.34Å,withstructuralsimilaritiestootherknownlysostaphin-type(LAS)familypeptidases.Furthermore purified ChiX hasbeen shown to possessL-ala D-glu endopeptidase activity,confirmingthepeptidoglycancrosslinkasitssubstrate.FinallyanessentialChiXactivesiteasparticacidhasbeenidentified,allowingforamechanismtobeproposed.

46. Decipheringthecrosstalkbetweenhostgenotype,microbiotastructureandplantgrowthinbarley.

RodrigoAlegriaTerrazasMicrobialcommunitiesthrivingintheroot-soil interface,therhizospheremicrobiota,areapotential resource of functions related to plant nutrition and health. However, itsdeployment in agriculture is impaired by the limited knowledge of the mechanismsregulating plant-microbiota interactions. I hypothesize that the host genotypemodulatestherhizospheremicrobiotatosupportplantgrowthunderlimitednutrientsupply.To test thishypothesis Ihavegrown14wildbarley (Hordeumvulgare subp. spontaneum)populations, representing the three major ecotypes in Israel (i.e., ‘North’, ‘Coast’ and‘Desert’), and a modern variety in an agricultural soil under greenhouse conditions. AnIluminaMiSeqprotocolwasusedtogenerate16SrRNAgeneprofilesoftherhizosphereandunplanted soil. Operational Taxonomic Units (OTUs) were assigned at a 97% sequencesimilarity. Barley rhizosphere microbiota was dominated by members of the phylaProteobacteria, Bacteroidetes, Actinobacteria and Firmicutes whose enrichmentdiscriminatesrhizosphereandsoilprofiles.AmongthedepletedphylaintherhizospherearePlanctomycetesandNitrospiraewhosemembersinterveneinNitrogen(N)cyclingprocessesthatcontributetothelossofNfromthesoil.MicrobiotaprofilesretrievedfromtheDesertecotypeaccessionsclusteredseparatelycomparedwiththosefromotherecotypesandthemodern variety supporting a selective enrichment of taxa from the families:Comamonadaceae,OxalobacteraceaeandChitinophagaceae.Interestingly,host-dependentmicrobiotaprofilescorrelatedwithaplantbiomassgradient. Inparallel, I investigatedthestructureofthemicrobiotaofbarleygenotypesgrownunderdifferentconcentrationsofNfertiliser. The number of prokaryotic taxa selectively recruited in the rhizosphere wasmaximisedatzeroN.DesertgenotypeprofilesenrichedahigherproportionMyxoccocalesand clustered differently at zero but not at higher N concentrations suggesting a plantgenotypemodulationofthemicrobiotaunderlimitedNsupply.Taken together, these data suggest a molecular cross-talk that links members of therhizospheremicrobiotaandplantgrowth,and itsmagnitudeamplifiedunder lownutrientconditions.

47. TranscriptionalregulatorsofcarbonmetabolisminPseudomonas

R.Campilongo1,2,R.Fung3,R.Little1,L.Grenga1,2,J.Malone1,2

1JohnInnesCentre,MolecularMicrobiologyDepartment,NorwichResearchPark,Norwich(UK)2InstitutoPasteur-CenciBolognetti,Roma(IT)3UEAUniversityofEastAnglia,SchoolofBiologicalSciences,NorwichResearchPark,Norwich(UK)Identifying and characterizing the genes that control plant colonisation by Pseudomonasspp.hasclearpotentialwithrespecttotheirfutureexploitationforpathogenbiocontrolandcrop growthpromotion. Previously, the rccR genewas identified in an InVivo ExpressionTechnology (IVET)screen forup-regulated lociduringP. fluorescens interactionwithsugarbeet. The rccR gene encodes a transcriptional regulator with helix-turn-helix and sugarisomerase domains, and shares high sequence homology with hexR, a transcriptionalregulatorofcarbonmetabolicgenes.Toexaminetheroleofthesegenes inthecontrolofthe carbonmetabolismduringplant colonisationweproducedΔrccRandΔhexRmutants,and examined their effect on various phenotypes associated with wheat colonisation.Deletionof rccR results in compromisedgrowth inglucoseandglycerol,whiledeletionofhexRaffectsgrowthinthepresenceofpyruvate,acetateandsuccinate.BothrccRandhexRnullmutantsshowastrongreductioninrhizospherecolonisationefficiencyrelativetowildtypeP.fluorescens.WenextdemonstratedbyqRT-PCRthatHexRisakeyregulatoroftheglucose phosphorylative pathway and the Entner-Doudoroff pathway in P. fluorescens,consistentwithearlierdata.TolookforthetranscriptionaltargetsofRccR,weperformedaChIP-seq experiment. Several target genes were identified and confirmed by qRT-PCR.Interestingly we found that RccR regulates the genes involved in the glyoxylate shuntpathway. Finally, we examined both transcription factors in the human pathogenPseudomonasaeruginosa,and identifieda role forRccR in theabilityof thisbacteriumtoswarm inpresenceof glucose.BothHexRandRccRplay central roles in the regulationofprimary carbon metabolism, adapting bacterial growth and behaviour to match theavailablecarbonsource,eitherintherhizosphereorinthehostpathogenenvironment.

48. TranscriptionalregulationofgeneexpressionbyECFsigmafactorsinBurkholderiacenocepaciainresponsetoextra-cytoplasmicstress

SayaliHaldipurkar,MarkSThomasUniversityofSheffieldBurkholderia cenocepacia belongs to the Burkholderia cepacia complex consisting of 18closelyrelatedspeciesthatarenaturallypresentinsoilandwaterwheretheycanbefoundinassociationwithplantsandfungi.Theycanhavebothbeneficialanddamagingeffectstoplants.B.cenocepaciaisparticularlypredominantincausingseriousopportunisticinfectionsin patients with cystic fibrosis and chronic granulomatous disease. These infections aredifficulttotreatsinceB.cenocepaciaisintrinsicallyresistanttomostantibiotics.In order to infect and survive in host tissues bacteria require effective signallingmechanisms. One of these is through the use of extra-cytoplasmic function (ECF) sigmafactors that are alternative sub-units of RNApolymerase.When activatedunder requiredconditionstheyassociatewithcoreRNAPtorecognisespecificpromoterelementsanddrivetranscription of genes to efficiently respond to the respective environmental stress.Conventionally, ECF sigma factors are associated with a membrane anchored anti-sigmafactorthatregulatesitsactivity,howeveralternativeregulatorymechanismsdoexist.Thehabitatofabacterium is roughly correlated to thenumberofECF sigma factors theypossess. For example, environmental organisms like Pseudomonas and Streptomycescontain scoresof ECF sigma factorswhereas theobligatepathogenNeisseriameningitidishas only four. B. cenocepacia harbours 13 ECF sigma factors, of which 11 remainuncharacterisedasofnow.TheECFsigmafactorOrbSofB.cenocepacia, isexpressedunder irondepletedconditions.Iron is an extremely important nutrient for infection and survival that bacteria need toacquire even though it is absent in the free form in host tissues. Many pathogens arecapableofsynthesizingironchelatingcompoundscalled‘siderophores’thatcanacquireironfromanunusableformandmakeitavailableinabiologicallyusableform.OrbSisinvolvedin transcriptionally regulating genes that biosynthesize the siderophore ornibactin andtransport iron-loaded ornibactin back into the bacterial cytoplasm.We have studied theDNA sequence requirements for promoter utilisation by OrbS using systematic ‘basedeletion’reporterassays.Wehaveidentifiedthe-10and-35promoterelementsaswellasotherimportantDNArequirementsforOrbSactivity.Additionally, we are also interested in identifying the role of the putative sigma factorBCAL3748whichunlikeOrbSseemstobe linkedtoatranscriptionallycoupled‘anti-sigma’factor.WehaveusedtwoapproachesforBCAL3748dependentpromoteridentification:onebasedonbioinformatics followedby reporter-gene fusionassays,and theotheronwholegenome scan by chromatin immunoprecipitation and deep sequencing (ChIP-seq). TheseapproacheshaveallowedustoidentifypotentialBCAL3478-dependentpromoters.

49. It’sastickybusiness–theroleofPseudomonasexopolysaccharidesandmotilityduringplantinfection

SebastianPfeilmeier1,2,JacobMalone2,3,CyrilZipfel1

1TheSainsburyLaboratory,NorwichResearchPark,NR47UH,UK2JohnInnesCentre,NorwichResearchPark,NR47UH,UK3UniversityofEastAnglia,NorwichResearchPark,NR47UH,UKPlasma membrane-localized pattern recognition receptors (PRRs) perceive microbe-associated molecular patterns (MAMPs), leading to pattern-triggered immunity (PTI).Pathogenicandcommensalbacteriamustemploystrategiestoevadeand/orsuppressPTIinorder to successfully colonize host plants. Biofilm formation and motility representcontrasting bacterial lifestyles controlled by the secondary messenger cyclic-di-GMP.Weshow that both production of exopolysaccharides (EPS) and flagella-driven motility areimportantfactorsinPseudomonassyringaepv.tomato(Pto)DC3000duringdifferentstagesof the infection process on Arabidopsis thaliana. Besides surface adhesion and stressprotection,a rolehasalsobeensuggested forEPS inevasionofPTI.During infection, theflagellum has an ambiguous role, acting as virulence factor and also as immunogen as aresultoftherecognitionofitsmainbuildingblock,flagellin,byFLAGELLINSENSING2(FLS2).Therefore, strict control of flagella synthesis is important for plant-associated bacteria.Elevatedcyclic-di-GMPlevelsinthepathogenPtoDC3000andthecommensalP. protegensPf-5 inhibit flagellin synthesis and help the bacteria to evade FLS2-mediated defence.However,highcellularcyclic-di-GMPlevelswereshowntodrasticallyreducethevirulenceof Pto DC3000. We propose that this is a result of reduced flagellar motility and/oradditionalpleiotropiceffectsofcyclic-di-GMPsignallingonbacterialbehaviour.

50. SignalPerceptionandSignalTransductionintheDcuB/DcuSSensorComplexofE.

coli

S.Wörner,C.Monzel,M.Zeltner,G.UndenInstituteforMicrobiologyandWineResearch,JohannesGutenberg-UniversityMainz,Mainz,GermanyTheC4-dicarboxylatesensorkinaseDcuSofEscherichiacoliformsasensorcomplexwiththeC4-dicarboxylate transporters DcuB or DctA under anaerobic or aerobic conditions,respectively[1].OnlybindingoftheeffectorstoDcuS,butnottothetransporterstriggerssensing by the complexes [2]. In addition, DcuS has a defined binding site for C4-dicarboxylates and mutation of the site controls substrate specificity of sensing. Hencechemical or concentration sensing is not performed by DctA or DcuB. Sensing is alsoindependent of the transport function of the transporters since transport deficienttransporters retain their regulatory function in the sensor complex [2, 3]. However, thepresenceoftheDctAorDcuBproteinsperseisrequiredfortransferringDcuStothesensorycompetentstate[2]ThedataaltogetherresultinamodelwhereDctAandDcuBtransformDcuStothesensorycompetentstatebutthetranspotersdonothavesensoryfunctionontheirown.Thescanningsubstitutedcysteinaccessibiltymethod(SCAM)wasusedtoshowthatsignaltransductionbyDcuSinvolvesapistontypemovementoftransmembranehelix2(TM2)[4]. It issuggestedthatcytoplasmicPASdomain(PASC)ofDcuSservesasasilencer[5] that requiresDcuBorDctAasco-regulator.UponstimulationbyC4-dicarboxylates thepistontypemovementofTM2stimulatesthekinase.References:[1]Witanetal.(2012)MolMicrobiol85:846-861[2]Steinmetzetal.(2014)MolMicrobiol94:218-29.[3]Kleefeldetal.(2009)JBiolChem284:265-275[4]Monzel&Unden(2015)ProcNatlAcadSciUSA112:11042-11047[5]Monzeletal.(2013)MicrobiologyOpen2:912-927.

51. ThetrialsandtribufilamentationsofSalmonellaasavehicleforgenedeliveryincancertherapy

SíleAJohnson1,LynseyMeikle1,MargaretMullin2,StephenWGTait3andDónalMWall11InstituteofInfection,ImmunityandInflammation,UniversityofGlasgow,Glasgow,UK2IntegratedMicroscopyFacility,UniversityofGlasgow,Glasgow,UK3CR-UKBeatsonInstitute,InstituteofCancerSciences,UniversityofGlasgow,Glasgow,UKBacteriapossessmanytumouricidalcharacteristicswhichlendthemtobeingstudiedasanalternative cancer therapy option.Of growing interest is the ability of bacteria to delivergeneticmaterialtocancercellsfortheexpressionoftumouricidalgenes.Thisstudyfocusedon investigating the use of the Salmonella Typhimurium to mediate the bactofection ofeukaryoticapoptoticgenesoneurkaryoticexpressionplasmidstocancercellsinvitro.Usinga commerciallyavailableeukaryoticexpressionplasmid,we found that transformedbacteria could exhibited a highly filamentous phenotype, which wasn’t seen when thebacteria were transformed with other plasmids. These filamentous bacterial culturesdisplayed decreased propensities to grow, invade eukaryotic epithelial cells and replicateintracellularly.Wesoughttounderstandwhichfeature(s)oftheplasmidwasresponsibleforenablingthefilamentousphenotypeinthesecultures.Manyoftheplasmidfeaturessuchasthe antibiotic resistance cassette, origin of replication, transgene and phage origin ofreplicationarefoundinanumberofplasmidbackbones.ThisresearchisthereforerelevanttoSalmonellaresearchersingeneralandnotjustthoseusingbactofectionasastrategytocombatcancer.

52. Curliproductioninsoil-adaptedE.colianditscontributiontosoilsurvival YinkaSomorin,FionaBrennan,FlorenceAbramandConorO’ByrneMicrobiology,NationalUniversityofIreland,Galway,IRELANDRecent studies have shown that E. coli can persist and grow in various externalenvironments, a findingwith implications for its continued use as an indicator organism.Soil-adaptedE.coli strainsdisplaystronggeneticdiversityandpossessuniquegrowthandmetabolic characteristics that suggest adaptation to soil conditions. However, themechanismsfortheirsurvivalinthesoilenvironmentremainunclear.SincebiofilmshelpE.colisurviveextremeenvironmentalconditionsandstresses,thisstudy investigatedbiofilmandcurli(whichisrequiredforbiofilmformation)productioninfivegeneticallydistinctsoil-adaptedE.colitodeterminewhethercurlicontributestosoilsurvival.Twoofthesoilstrainsproducedhighlevelsofbiofilmat15°Cwhereastheotherthreeproducedverylowlevelsofbiofilm.Curliproductionwasfoundtocorrelatewithhighbiofilmproduction.Bioinformaticanalysisofregulatorsofcurliproductionrevealedamutation(E620K)intheEALdomainofthecyclicdi-GMPphosphodiesterase(PdeR)amongthethreecurli-deficientstrains,whichalso displayed a poor ability to form biofilms. Thismutationwas found in five additionalstrains amongst a collectionof 170 soil-adapted strains. Thismutationmayhave reducedthephosphodiesteraseactivityofPdeRandrepressedtheabilityofthediguanylatecyclaseDgcMtoactivatetheDNAbindingtranscriptionalactivatorMlrA,todirectcurliproduction.AlthoughΔcsgA (curlinmajor subunitnullmutant)was slightly impaired in survival in soilcomparedto thewildtypeE. coliBW25113 (after84dayspost-inoculation intosoil), curli-deficientsoil-adaptedE.colistrainssurvivedsimilarlytothecurli-positivestrainsinsoilthussuggestingthatcurli isnotrequiredforsoilsurvival.Theheterogeneity incurliproductionmay reflect thegeneticdiversityofE. coli lineagespresent in thesoil,perhaps suggestingthattheyhaveevolvedtooccupydifferentmicro-nichesinthesoil.

53. EffectofthequorumquenchingenzymeMomLonbiofilmformationbyPseudomonasaeruginosaandAcinetobacterbaumannii

YunhuiZhang1,GillesBrackman1andTomCoenye1

1LaboratoryofPharmaceuticalMicrobiology,GhentUniversity,Ottergemsesteenweg460,B-9000Gent,Belgium.Keywords:Biofilm,Quorumquenchingenzyme,Acinetobacterbaumanii,PseudomonasaeruginosaAcinetobacter baumannii andPseudomonas aeruginosa are two importantGram-negativepathogens causing serious infections. Both of them show high potential for biofilmformation, which may contribute to their antibiotic resistance and increased virulence.Quorum sensing (QS), the intercellular signaling system, plays an important role inregulating biofilm formation of these two pathogens, and may be a promising target todisrupt biofilm formation and treat infections. QS of P. aeruginosa and A. baumannii isbased on the production and response to N-acyl-homoserine lactones (AHLs), thusenzymatic degradation of AHLs may effectively block their QS system. Recently, a novelmarine-derivedN-acylhomoserinelactonase,MomL,wasreportedtodegradeawiderangeofAHLsandreducethevirulenceofP.aeruginosa.Here,weinvestigatedtheeffectofMomLonbiofilmsformedbyA.baumaniiandP.aeruginosastrains.MomLwasexpressedinE.coliBL21andpurifiedwithNTA-Nicolumns.TheactivityofMomLonmostcommonlyreportedAHLemployedbyA.baumannii,3-OH-dodecanoyl-homoserinelactone(3-OH-C12-HSL)wastested.BiofilmsofA.baumanniiLMG10520,LMG10531andP.aeruginosaPAO1wereformedin96-wellmicrotiterplates.Biofilmformationwasevaluatedbyplating,crystalvioletstainingandalsofluorescencemicroscopy.MomLwasaddedduringbiofilm attachment and formation, the effect of MomL on the biofilm susceptibility toantibioticswasalsotested.Our results showed thatMomL can degrade 3-OH-C12-HSL besides both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position. Over 30%decreaseinbiofilmbiomassofP.aeruginosaPAO1wasobservedwhentreatedwithMomL(150μg/mL).ForA.baumanniiLMG10531,thebiofilmbiomasswasreducedbyaround40%whentreatedwith5μg/mLMomL.ThesameeffectwasalsoobservedinA.baumanniiLMG10520,butnotinaQSdeficientstrainA.calcoaceticusLMG10517.WhenusingLive-Deadstaining in fluorescence microscopy, a reduction in fluorescence was observed in MomLtreatedbiofilmsofbothP.aeruginosaPAO1andA.baumanniiLMG10531.Moreover,therewasclearlymorereductioninbiofilmsofP.aeruginosaPAO1andA.baumanniiLMG10531treatedbydifferentantibioticsincombinationwithMomLthanwhichtreatedbyantibioticsalone. These results indicate the potential use of MomL in combating bacteria biofilminfections.

54. CharacterisationofasubstrateoftheStaphylococcusaureusTypeVIIproteinsecretionpathway

ZhenpingCao,TracyPalmerProtein secretion is an essential process in bacteria, and the Type VII secretion system(T7SS)isamajorpathwayfoundinGrampositivebacteria.InS.aureustheT7SSisessentialforvirulenceand isencodedby the12-geneess locus,with the firstsixgenesesxA,esaA,esaB,essA,essBandessCcodingforthecorecomponentsofthesecretionapparatus.Themainaimofmyproject istocharacterisetheregulationofessexpressioninS.aureusand to understand how substrates are recognised for secretion. Here I show that EsaD,encodedattheess locus, isa largesecretedsubstrateoftheT7SS. BioinformaticanalysissuggeststhatEsaDhasanucleasedomainatitsC-terminusandinsupportofthisEsaDwasshowntocauseDNAdamagewhenproducedinE.coli.EsaG,encodeddirectlydownstreamofesaDattheess locus, isacytoplasmicproteinandpossessestheabilitytointeractwiththenucleasedomainofEsaDandblock itsactivity.EsaE,a thirdess-encodedproteinalsointeractswithEsaD,butwith theN-terminal region rather than thenucleasedomain.Co-purificationexperimentsshowthatthethreeproteinsformaternarycomplex.EsaEisseento localise tobothmembraneandcytoplasm,and tobind to the integralmembraneT7SScore component EssC. It is co-exported with EsaD when EsaD is overproduced. A modeldescribingtheassemblyandsecretionofEsaDsecretionwillbepresented.

A. Thedistributionpatternofmetabolicmodulesandantibioticresistancegenesrevealsdifferencesintheairwaymicrobiomeofcysticfibrosispatients

G.Bacci1,P.Paganin2,N.Segata3,G.Taccetti5,D.Dolce5,A.DeAlessandri5,P.Morelli5,V.Tuccio6,E.V.Fiscarelli6,V.Lucidi6,A.Mengoni1,A.Bevivino21DepartmentofBiology,UniversityofFlorence,Florence,Italy2 ENEA, Italian National Agency for New Technologies, Energy and Sustainable EconomicDevelopment,SustainableTerritorialandProductionSystemsDepartment,Rome,Italy3CentreforIntegrativeBiology,UniversityofTrento,Trento,Italy4DepartmentofPediatrics,CysticFibrosisCenter,MeyerHospital,Florence,Italy5DepartmentofPediatrics,CysticFibrosisCenter,G.GasliniInstitute,Genoa,Italy6CysticFibrosisMicrobiologyandCysticFibrosisCenter,Children'sHospitalandResearch InstituteBambinoGesù,Rome,ItalyCystic fibrosis (CF) is characterized by chronic airway infections usually made up of acomplex mixture of microorganisms. Though several studies have investigated thetaxonomiccompositionofairwaysmicrobiota,littleisknownaboutthegeneticcompositionof such communities. In this study, we applied a full-genome shotgun metagenomicsanalysis to gain new insights into CFmicrobiomemetabolism and composition, trying tounraveltheunderlyingcausesoftheseverelungdiseaseandidentifypotentialdeterminantsofclinicalchangesinlungfunction.TotalDNAwasextractedfromsputumsamplesof12CFpatientsattendingthreeItalianCFcenters (Bambino Gesù Children's Hospital, Rome; Giannina Gaslini Children's Hospital,Genoa; and Meyer Children's Hospital, Florence). The sequencing step was performedthrough the Illumina HiSeq 2000 platform, producing more than 15 million paired-endsequences for each patient. All patients were clinically stable, without any pulmonaryexacerbationandantibiotictherapy(i.v.ororal)inthepreviousfourweeksbeforespecimencollection.PatientsweregroupedaccordingtotheiraverageannualFEV1valueregisteredinthe12monthsbeforetheenrollment.Inparticular,CFpatientsdisplayingvalueshigherthan70%were included in thenormal/mild group,whereas those reporting values lower than40%wereincludedintheseveregroup.To infer functional and metabolic patterns among patients, we used two differentapproaches. The first one was based on raw data analysis through HUMAnN pipeline,whereas the secondone implied theanalysisof all open reading frames (ORFs)predictedfrom assembled contigs. In particular, readswere assembledwith SPAdes assembler andcontigs longer than 500bp (approximately a half of a bacterial gene) were used to inferfunctional capabilities. ORFs were predicted with MetaGeneMark software, assessingfunctionalpatternsbasedoneggNOGdatabase.Proteinsassignedtotransportercategorieswere further classified using the TCDB database to obtain a more reliable and specificclassification, whereas VIRDB database was used to identify putative virulence factors.Finally,theResfamsdatabasewasusedtoinferantibioticresistance(AR)genesdistributioninallsamples.

Weidentifiedacoresetofbacterialmetabolicpathwaysandfunctionsalongwithmetabolicmodulescharacteristicofaparticulargroupofpatients.Inparticular,increasingvalueoftheKeggpathways“Bacterialchemotaxis”(Ko:02030)and“Flagellarassembly”(Ko:02040)weredetectedintheseveregroup.Interestingly,thesetwopatternswerereportedtobepotentmediatorsofacutepulmonary inflammationenhancing thecolonizationof lung tissuesbypathogenbacteria.Interestingly,theseveregroupshowedamassivepresenceofARgenes,with11antibioticresistance-relatedgenesclasses(outof14)beingmorerepresentedthanin the normal/mild group. These results were confirmed by the VIRDB analysis, whichreportedhigherfrequencyvaluesforalmostallvirulencecategoriesinseverepatients.Inconclusion,wefoundthatthedifferentpulmonaryfunctionalityofCFpatientsco-occurswithpotentiallydifferentmicrobiomecapabilities,mainly related toan increasingnumberof genes for multiple antibiotic resistance and to the presence of metabolic pathwaysputatively correlated with bacterial virulence. Understanding how microbial lungcommunities are able to modify their gene repertoire along with CF disease severity isbecomingmandatoryforthedevelopmentofpersonalizedtreatmentsandcares.Acknowledgements:ThisworkwassupportedbyGrantsfromItalianCysticFibrosisFoundation(FFC#8/2012,FFC#10/2014andFFC#14/2015).

B. Peptoclostridiumdifficile:Covalentattachmenttothehostthroughnoveladhesins

MiriamWeckener,UliSchwarz-LinekPeptoclostridium difficile is a Gram-positive pathogen that mostly infects elderly andhospitalised patientswhohavebeen treatedwith antibiotics for a long time. Since 2000,highly virulent strains have emergedwhich causemore severe cases of colitis associatedwith increasingmortality rates.Due to thehighervirulenceand resistances toantibiotics,treatmentofP.difficileinfectionsbecomesincreasinglydifficult.Theadhesionofpathogenstohosttissuesisoneofthemostimportantstepsinestablishinganinfection,andhasbeenidentifiedasnewtargetfornovelantimicrobialagents.Untilrecently,bacterialadhesionhasbeen understood to involve multivalent, non-covalent interactions, which are difficult toprevent by small molecules. Recently, novel adhesins called Thioester-Isopeptide-Esterdomain (TIE) proteins have been found to bind covalently to their targets. TIE proteindomainscontainintramolecularbonds,whicheitherincreasetheproteins'stability,asintheisopeptideandesterdomains,orconveytheadhesionfunction,asinthethioesterdomain.P.difficile expresses at least three different TIE proteins which differ in their size anddomainarchitecture.StructuralstudiesoftheseTIEproteinsareconductedtogetaninsightonthedeterminantsoftargetspecificityandepithelialcellbindingexperimentsareusedfortargetidentification.

C. Unravelingpost-transcriptionalregulationofE.colibiofilmPatrickRosendahlAndreassenProduction of curli, extracellular protein structures important for Escherichia coli biofilmformation, is governed by an extremely complex regulatory mechanism that integratesmultiple environmental signals through the involvement of numerous proteins and smallRNAmolecules(sRNAs).However,theprecisemechanismbehindsRNA-mediatedregulationof thecurli master regulator, CsgD, is largely unknown. In this study, we identified aconservedA/U-rich region in thecsgDmRNA5’-enduntranslated region,which is cleavedupon binding of the small RNA, McaS, to sites located more than 30 nucleotidesdownstream.Mutational analysis showed that theA/U-rich region aswell as an adjacentstem-loopstructurearerequiredforMcaS-stimulateddegradation,mostlikelyservingasabinding platform for the RNA chaperone Hfq. Prevention of McaS-activated cleavagecompletely relieved repression, suggesting that the endoribonucleolytic cleavage is theprimaryregulatoryeffectexertedbyMcaS.

D. GeneticsandstructuralvalidationoftheAspergillusfumigatusRom2SNHdomainasacellwalltarget

WenfanWei1,WenxiaFang1,AndrewT.Ferenbach2andDaanM.F.vanAalten1,2* DivisionofMolecularMicrobiology1,MRCProteinPhosphorylationandUbiquitylationUnit2,SchoolofSciences,UniversityofDundee,UKFungalinfectioninimmuno-compromisedpatientshasbecomeabigclinicalconcern,partlyduetothelimitedtargetsforeffectiveandselectiveantifungaldrugsdevelopment.Rom2asaRhoGTPaseGEF(guaninenucleotideexchangefactor)proteinhasbeenshownasessentialin many fungal organisms, and playing pivotal roles in fungal cell wall integrity (CWI)pathway.SequencealignmentshowsthatRom2proteincontainsaC-terminalCNHdomainthatisabsentinthehumanorthologous.Here,usinggeneticapproachwecharacterisedthefunction of the CNH domain of Rom2 in the pathogenic fungus Aspergillus fumigatus.ComprehensivecellwallanalysisincludingTEMandchemicalanalysissuggestthisdomainisimportant for both β-glucan and chitin synthesis. The crystal structure of this domainrevealsaseven-bladedβ-propeller.The invitrobindingassayshowsthattheCNHdomainbindstoRho1GTPaseinboththeGDPandGTPform,suggestingit isanoncanonicalRhobindingpartner.Ourworkhasprovidedbothgeneticandstructuralevidencesof theCNHdomaininRom2proteinasanovelfungalcellwalltarget.

“HotSpot”PosterTalks·Thursday30thJune2016

55. Innovativeuseofthetetracyclinestoactivatebacterialsuicidegenesandcombatantibioticresistancemechanisms

Chukwudi,C.U.DepartmentofVeterinaryPathologyandMicrobiology,UniversityofNigeria,NsukkaThetetracyclinesareabroadspectrumgroupofantibioticswhoseantibacterialefficacyhasbeen greatly reduced due to the development of resistance. However, their use in non-bacterial diseases continues to increase though their mechanism of action in theseconditionsisstillpoorlyunderstood.Ourrecentstudiesindicateaneffectonthesecondarystructures,processingandconsequently,functionofcellularRNAsundercertainconditions.Thehok/soklocusisawellestablishedtoxin/antitoxinplasmidstabilityelement,whichhasbeenassociatedwithmulti-drug(ampicillin,chloramphenicol,kanamycin,streptomycinandsulphonamides) resistance plasmids. It is one of the most frequently reported plasmidaddiction systems in ESBL-encoding plasmids, and ensures plasmidmaintenance by post-segregational killing of plasmid-free daughter cells via RN:RNA interactions. Thehok/soklocus also occurs in the chromosomes of enterobacteria, and is more abundant inpathogenic strains. Our recent studies also indicate that thehok/soklocus enhances hostcellsurvival,especiallyinstressfulconditionssuchashightemperature,antibioticstressandlow cell concentrations. Interestingly, we observed that although thehok/soklocusincreasesbacterialtolerancetoβ-lactamantibiotics, it increasesthesusceptibilityofthesebacteriatothetetracyclines(doxycycline)by inducingtheself-killingplasmidmaintenancesystemduetotheinterferenceofthetetracyclineswiththeRNA:RNAprocessingpathway.Hence, thehok/soklocusmay represent a drug target thatwill openupopportunities forthe innovative use of tetracyclines in the global war to contain the rise of antimicrobialresistance.

56. c-di-GMPbindingdomaininvolvedinproductionamixed-linkageβ-GlucaninSinorhizobiummeliloti

DanielPérez-Mendoza1*,DanielaBertinetti1,OliverBertinetti1,JuanSanjuán2andFriedrichW.Herberg1

1DepartmentofBiochemistry,UniversityofKassel,34132Kassel,Germany2Dpto.MicrobiologíadelSueloySistemasSimbióticos,EstaciónExperimentaldelZaidín,CSIC.Granada,Spain.An artificial increase of c-di-GMP levels inSinorhizobium meliloti8530 leads tooverproduction of a mixed-linkage (1→3)(1→4)-β-d-glucan (MLG) that resembles MLβ-glucansfoundincerealsandlichens1.Incontrasttolichenanandbarleyglucans,thisuniquebacterialexopolysaccharidehasadistinctiveprimarystructurewithaperfectalternationofβ(1→3)andβ(1→4) bounds, which may give new interesting biotech properties to thisbiopolymer2. Moreover, the MLG participates in bacterial aggregation and biofilmformation,andisrequiredforefficientattachmenttotherootsofahostplant,resemblingthe biological role of cellulose in other plant associated bacteria1. A two-geneoperonbgsBArequired for production of thisMLG, is conserved amongst several generawithin the order Rhizobiales,wherebgsAencodes a glycosyl transferase(GT)with domainresemblance and phylogenetic relationship to bacterial cellulose synthases (CS).Furthermore,MLGsynthesis isalsosubjectedtobothtranscriptionalandposttranslationalregulation,butincontrasttocellulose:(i)bgsBAtranscriptionisdependentontheExpR/SinIquorum sensing regulatory system and (ii) a novel c-di-GMPbinding domain, different toPilZ, is involved in the posttranslational regulation1.Different Biomolecular InteractionAnalysis (BIA), including Fluorescence Polarisation(FP) and Surface Plasmon Resonance(SPR)3,havebeenusedinthisstudytocharacterisethebindingofBgsAtoc-di-GMP.VarioussitedirectedBgsAmutantshasbeenassayed to identify the residues involved in thec-di-GMPbindingandactivationofMLGproduction.Preliminaryresultsobtainedsofarsuggestadifferentactivationmechanismfromotherc-di-GMP-activatedGTslikecellulosesynthase.1Pérez-Mendoza,D.etal.Novelmixed-linkagebeta-glucanactivatedbyc-di-GMPinSinorhizobiummeliloti.ProcNatlAcadSciUSA112,E757-765,doi:10.1073/pnas.1421748112(2015)2Pérez-Mendoza,D.&Sanjuán,J.Exploitingthecommons:cyclicdiguanylateregulationofbacterialexopolysaccharideproduction.CurrOpinMicrobiol30,36-43,doi:10.1016/j.mib.2015.12.004(2016).3Moll,D.etal.Biomolecularinteractionanalysisinfunctionalproteomics.JNeuralTransm113,1015-1032,doi:10.1007/s00702-006-0515-5(2006).

57. Receptorhistidinekinasedirectlybindsplantchemicaltopromotebacterialadaptationinhostplant

Fang-FangWang,Shou-TingCheng,WeiQian*InstituteofMicrobiology,ChineseAcademyofSciences,Beijing100101,ChinaHow phytopathogenic bacteria recognize host plant derived chemical signals and elicitinfection remains to be an opening question. In bacterial cells, two-component signaltransductionsystem(TCS),consistingofamembrane-boundreceptorhistidinekinase(RHK)andacytosolicresponseregulator,isthecanonicalsense-and-responsemachinerytoreactto various environmental stimuli via protein phosphorylation. Based on large-scalescreening of ligand-receptor interactions, we demonstrated that PcrK-PcrR, a TCS of theplantblackrotdiseasepathogenXanthomonascampestrispv.campestris,directlysensesaimportantplantchemicalandmodulatesstressresponsesinhostduringpathogenesis.PcrKspecificallybindstheplantchemicalwithahighaffinity,andmodulatethephosphorylationlevelofPcrR.PcrRregulatesabout60genesinresponsetotheplantchemicalstimulation.Amongthem,anoutermembraneproteinwasconfirmedtoplayanimportantroleinROSstress response to the host environment. Our study revealed a case of inter-kingdomsignalling transduction process, which will give insight into our understanding on themolecularcoevolutionarymechanismduringhost-pathogeninteractions.

58. InterplaybetweendynamicbacterialvirulencephenotypesofE.coliandhostsusceptibilitydeterminesUTIrisk

H.L.SchreiberIV1,2,M.S.Conover1,W.C.Chou2,M.E.Hibbing1,A.L.Manson2,K.W.Dodson1,T.J. Hannan3, P.L. Roberts4, A.E. Stapleton4, T.M. Hooton5, J. Livny2*, A.M. Earl2*,S.J.Hultgren1,6*1MolecularMicrobiologyandMicrobialPathogenesis,WashingtonUniversity,St.Louis,MO63110,USA.2TheBroadInstituteofMITandHarvard,Cambridge,MA02142,USA.3DepartmentofPathologyandImmunology,WashingtonUniversity,St.Louis,MO63110,USA.4DepartmentofMedicine,DivisionofAllergyandInfectiousDiseases,UniversityofWashington,Seattle,Washington,USA.5DepartmentofMedicine,DivisionofInfectiousDiseases,UniversityofMiamiMillerSchoolofMedicine,Miami,FL33136,USA6CenterforWomen’sInfectiousDiseaseResearch,WashingtonUniversity,St.Louis,MO63110,USAUrinarytractinfections(UTIs)areverycommonandmostoftencausedbyEscherichiacoli.However,unlikemanyentericE.colipathogroups,nogeneticsignaturehasbeenidentifiedforE. coliassociatedwith cystitis. Towards this end,weusedhigh-resolution comparativegenomics to study 43 urine-associatedE. coli(UAEC) strains isolated from 14 womensuffering frequent recurrent UTIs. We found that these UAEC strains were geneticallydiverse, clustering into 21 distinct clonal groups that represented 4 majorE.coliphylogeneticclades.Comparativegenomicanalysisofrepresentativestrainsfromeachof theseclonalgroupand46 referenceE. colirevealed that carriageofpreviouslydefinedputativeurovirulence factorscorrelatedwithphylogenybutnotwithastrain’sassociationwith urinary disease.While UAEC strainswere able to colonize severalmousemodels ofacutecystitissignificantlybetterthangut-associatedE.coli,theurovirulenceofUAECstrainsvaried significantly amongdifferentmouse strains and in singleor competitive infections.NosinglesetofgenesinUAECwaspredictiveofbladdercolonizationinanyofthemousemodels.Incontrast,comparativetranscriptomicandphenotypicstudiesrevealedsignificantvariationsintheexpressionofconservedfunctionsandkeybehavioursamongUAECstrainswhengrownunderthesamecultureconditions,severalofwhichwerepredictiveofbladdercolonization in C3H/HeN mice. These include variations in the expression of genesassociatedwithmotilityandnutrientutilizationanddifferences inbothmannose-sensitiveand mannose-resistant hemagglutination activity. Taken together, our findings suggest anewconceptualmodelofuropathogenesisinwhichUTIriskandoutcomeisdeterminedbycomplex interactions of dynamic host susceptibility determinants and diverse bacterialurovirulencepotentialsthataredrivennotonlybygenecontentbutalsobydifferencesintheexpressionandregulationofconservedfunctions.

59. AdaptiveremodelingofthebacterialproteomebyspecificribosomalmodificationregulatesPseudomonasinfectionandnichecolonisation.

LittleRH1,GrengaL1,2,SaalbachG1,HowatAM2,PfeilmeierS1,3,TrampariE1,MaloneJG1,21DepartmentofMolecularMicrobiology,JohnInnesCentre,NorwichResearchPark,Norwich,UK2SchoolofBiologicalSciences,UniversityofEastAnglia,NorwichResearchPark,Norwich,UK3TheSainsburyLaboratory,NorwichResearchPark,Norwich,UKPost-transcriptional control of protein abundance is a highly important, underexploredregulatoryprocessbywhichorganismsrespondtotheirenvironments.Herewedescribeanimportant and previously unidentified regulatory pathway involving the ribosomalmodification protein RimK, its regulator proteins RimA and RimB, and the widespreadbacterial second messenger cyclic-di-GMP (cdG).Disruption ofrimKaffects motility andsurfaceattachmentinpathogenicandcommensalPseudomonasspecies,withrimKdeletionsignificantly compromising rhizosphere colonisation by the commensal soil bacteriumP.fluorescens, and plant infection by the pathogensP. syringaeandP.aeruginosa. RimKfunctionsasanATP-dependentglutamylligase,addingglutamateresiduestotheC-terminusof ribosomal protein RpsF and inducing specific effects on both ribosome proteincomplement and function. Deletion of rimK inP. fluorescensleads to markedly reducedlevelsofmultipleribosomalproteins,andalsoofthekeytranslationalregulatorHfq.Inturn,reduced Hfq levels induce specific downstream proteomic changes, with significantincreasesinmultipleABCtransporters,stressresponseproteinsandnon-ribosomalpeptidesynthetasesseenforbothΔrimKandΔhfqmutants.TheactivityofRimKisitselfcontrolledby interactions with RimA, RimB and cdG. We propose that control of RimK activityrepresentsanovelregulatorymechanismthatdynamicallyinfluencesinteractionsbetweenbacteria and their hosts; translating environmental pressures into dynamic ribosomalchanges,andconsequentlytoanadaptiveremodelingofthebacterialproteome.

60. ThelunghormoneC-typenatriureticpeptide(CNP)modifiesPseudomonasaeruginosavirulenceandbiofilmformationafterbindingtotheAmiCbacterial

protein:Mechanismofaction.

LesouhaitierO1,ClamensT1,RosayT1,CrépinA2,BazireA2,Diaz3,BouffartiguesE1,ChevalierS1,DufourA2,HarmerNJ3,FeuilloleyMGJ11LaboratoryofMicrobiologySignalsandMicroenvironmentEA4312,NormandieUniv.,Univ.Rouen;IRIB,F-27000Evreux,France.2Univ.Bretagne-Sud,EA3884,LBCM,IUEM,F-56100Lorient,France3SchoolofBiosciences,UniversityofExeter,Exeter,EX44QD,UKThere is now ample evidence that bacterial virulence is modulated by eukaryoticmessengers including hormones1. We have shown previously that the C-type NatriureticPeptide (CNP), a peptide produced by lung, enhancesPseudomonasvirulence2. In thepresentwork,westudiedtheeffectofCNPonbacterialbiofilmformationandwelookedforaputativeP.aeruginosaproteinthatcouldexplaintheeffectofCNPonbacteria.Weusedinsilicoandin vitroapproaches to identify a bacterial sensor for CNP. Additionally,pharmacologicaltoolswereusedasaninnovativestrategytocharacterizethebindingsiteinP.aeruginosa.WeobservedthatCNPstronglyinhibitsP.aeruginosabiofilmformation3.Thiseffect is totally prevented by Isatin, an antagonist of natriuretic peptide receptors ineukaryotic cells. The actions of CNP onP. aeruginosavirulence are mimicked by theeukaryotic receptor NPR-C agonist cANF4-23. Screening and comparing 3D structures ofhumannatriureticpeptidesreceptorsandPseudomonasproteinsrevealedthatthebacterialprotein AmiC shows significant homology with the human C-type eukaryotic natriureticpeptide receptor (hNPR-C).Furthermore, theseanalyses showed thatbothCNPand Isatincan interact with AmiC using the same amino acids as in hNPR-C. Finally, recombinantprotein AmiC was purified and the protein interactions assessed using MicroScaleThermophoresis. The results showed that both CNP and an hNPR-C agonist bind AmiCproteinwithaKDof2µMand lessthan100nMrespectively,whereashNPR-Aagonisthaspoor affinity forAmiC.Using anamiC-mutant strain and its complementedderivative,wedefinitively validated the crucial role of AmiC protein on CNP’s effect onP.aeruginosabiofilmformation.AsobservedforhNPR-Cinmammals,AmiCishighlyselectiveand candiscriminatebetween thedifferentnatriureticpeptides, sinceanothernatriureticpeptide(brainnatriureticpeptide,BNP)hasnoaffinitywithAmiC.Inconclusion,ourworkprovidesthefirstdemonstrationthatthebacterialproteinAmiCmaybeanorthologoftheeukaryotic receptor hNPR-C, acting as a CNP sensor inP.aeruginosa. AmiC appears tomodulateaswitchbetweenchronicandacuteinfectionphenotypesdependingonexposureto host factors. The observation that CNP strongly decreases bacterial biofilm formationshouldhavemajorconsequencesforcysticfibrosistreatment.1Lesouhaitieretal.,2009;Sensors9:6967-69902Blieretal.,2011;Microbiology157:1929-19443Rosayetal.,2015;mBio6(4):e01033-15.

61. Pseudomonasaeruginosapore-formingExolysinandTypeIVpilicooperateto

inducehost-celllysis

PaulineBassoCEA,UniversityofGrenobleAlpes,FrancePseudomonas aeruginosa clinical strains lacking Type III secretion systemgenes employ anoveltoxin,Exolysin(ExlA),forhostcelldestruction.Here,wedemonstratedthatExlAusesa predicted outer membrane protein ExlB for its export through two POTRA domainsshowingthatExlA-ExlBrepresentnewmembersofaTwo-PartnerSecretion(TPS)systems.InadditionofTPSsecretionsignals,ExlAharborsseveraldistinctdomains,whichcomprisenotablyhemagglutininFHAdomains, five integrinbindingmotifsArginine-Glycine-Asparticacid (RGD) and a non-conserved C-terminal region. Cytotoxic assays showed that thedeletionoftheC-terminalregionabolisheshost-cellcytolysis.Usingredbloodcellsandlipidvesicles,wedemonstratethatExlAhasapore-formingactivitythatprecedescellmembranedisruption innucleatedcells.Finally,bysettingupaminiaturizedcellular live-deathassay,wescreenedatransposonmutantlibraryofanExlA-producingP.aeruginosaclinicalstrainfor bacterial factors required for ExlA-dependent toxicity. The screen allowed theidentificationofproteinsofTypeIVpiliasbeingabsolutelyrequiredforExlA-dependentcelldestruction.ThisisthefirstexampleofcooperationbetweenaTPSpore-formingtoxinandsurfaceappendagesinhostcellintoxication.

62. CcpAasregulatorofStreptolysinSinStreptococcusanginosus

RichardBauerandBarbaraSpellerbergInstituteforMedicalMicrobiologyandHygiene,UniversityHospitalUlm,GermanyStreptococcus anginosus, as a member of theStreptococcusanginosusgroup, is acommensal of mucosal membranes, but also an emerging human pathogen. SomeS.anginosusstrains, includingthetypestrain,showaprominentβ-hemolyticphenotype.Thegenetic locus (saggene cluster) of the hemolysin expression has recently been describedanditwasshownthatthisvirulencefactorexhibitshighhomologieswiththeStreptolysinS(SLS)ofStreptococcuspyogenes. Itwasobservedthatthehemolyticactivityaswellastheexpression of thesaggene cluster was reduced in the presence of high glucoseconcentrations. The expression of the hemolysinwas thereby investigated in a promoterreporter assay in which the expression of EGFP is driven by thesagpromoter. However,regulationmechanismscontrollingtheexpressionoftheSLSinS.anginosushavenotbeenelucidatedso far.Theuseofdifferent sugars in the reporterassay lead to thehypothesisthat carbon catabolite repression (CCR), a well investigated regulation mechanism inGram+bacteria controls hemolysin expression.A keyplayer inCCR is the regulator calledCatabolitecontrolproteinA(CcpA).InS.pyogenesCcpAregulatestheexpressionoftheSLS,therefore, the role of CcpA in expression of thesaggene cluster ofS. anginosuswasinvestigated. A CcpA insertion mutant was constructed, transformed with the reporterplasmid carrying thesag promoter in front of EGFP and the activity of the promoterwasdetermined in dependency to the glucose concentration of the medium. The previouslydescribedreductionofgeneexpressioninthepresenceofhighglucoseconcentrationinthewild type reporter strain is abolished in the CcpA mutant demonstrating that CcpA isinvolvedintheregulationofthesaggenecluster.Basedonthisobservationthepromoterregion of thesaggene cluster was analysed bioinformatically to identify putative CcpAbinding sites.This resulted in threepotentialCcpAbinding siteswhichwere tested in thepromoter reporter assay by using site directedmutagenesis. The 3 putative binding siteswere separatelymutatedordeletedand cloned in frontof EGFP in the reporterplasmid.Promoters that contain a mutation in the CcpA binding site should not show a glucosedependent reduction of activity.With this assay a potential CcpA binding site,with goodhomologiestotheconsensusbindingsiteofCcpA,couldbeidentified.TheexpressiondatacouldbeverifiedinafunctionalhemolysisassayinwhichtheCcpAmutantwasabletolysehumanerythrocytesinthepresenceofhighglucoseconcentrations.InsummarythisstudycharacterizedforthefirsttimearegulatoroftheemergingpathogenS.anginosus.WecoulddemonstratethatCcpAis involved intheregulationofSLSofS.anginosusand identifiedaputativeCcpAbindingmotifintheSLSpromoterregion.

63. AntimicrobialactivityofhumanskinassociatedStaphylococcusepidermidisSimonBoLassen,RikkeLouiseMeyer,HolgerBrüggemannDepartmentsofBiomedicineandBioscience,AarhusUniversity,Aarhus,DenmarkThe human skin is colonized by a complex mixture of bacteria, fungi and viruses. Somebacterialspeciescanbepredominantdependingonthespecificskinlocation,includingthespeciesStaphylococcusepidermidisandPropionibacteriumacnes.Theinterferencebetweenthesetwospeciesanditsimpactonskinhealthanddiseaseislargelyunknown.Interspeciescompetition can impact on the fine balance of the human skin ecosystem, which mightinitiateorsupportcertainskindisorderssuchasacnevulgaris. Inthisstudyweinvestigatetheantimicrobial activityofhumanskin-associated strainsofS. epidermidis, aimingat theidentification of antimicrobial mechanisms as executors of intercellular competition onhuman skin. Strains ofS. epidermidiswere screened for antimicrobial activity againstP.acnes. Strains capable of inhibitingP. acneswere selected for genome sequencingandanalysis.Genesencodingputativebacteriocinsandotherantimicrobial functionswereidentified.PreliminaryresultsindicatethatagroupofS.epidermidisstrainsencodesasofaruncharacterized group of class II bacteriocins. Another strain, exhibiting an extraordinaryantimicrobial activity against bothP. acnesand otherS. epidermidisstrains, possesses anunusual type VII-like secretion system linked to a polymorphic toxin locus thatmight beinvolvedintheantimicrobialactivity.Theantimicrobialactivitiesofthetestedstrainshaveproven effective in inhibitingP. acnes and could potentially also inhibit othermicroorganisms. These strains or their active compounds could therefore pose a possibleremedy againstP. acnesassociated diseases as well as against other opportunisticinfections.

64. Salmonellaneutralizesahostdefensepathwaywithaone-twopunch

StefaniaSpanòInstituteofMedicalSciences,UniversityofAberdeen,UKHostdefensemechanismsprotectanimalsagainst theattackofmicroorganisms, includinginvasive bacteria. We identified a host trafficking pathway that prevents the human-restrictedpathogenSalmonellaTyphi fromsurviving inmousemacrophagesand thereforeinfectingmice.ThispathwaydependsonthehostGTPaseRab32anditsguaninenucleotideexchange factor BLOC-3. The potential broader role of this pathway in host defense isunknown. We report that the Rab32/BLOC-3 pathway is neutralized by two type-III-secretion effectors delivered by the broad host pathogenSalmonella Typhimurium. Inaddition to GtgE, which is a specific protease cleaving Rab32, SalmonellaTyphimuriumdeliversSopD2,whichisaRabGTPaseactivatingprotein(GAP)thatinactivatesRab32.Eachof these effectors is sufficient to conferSalmonella Typhimurium the ability to infectmice.ASalmonellaTyphimuriumstraindeficientforboththeseeffectorsisunabletoinfectmice, yet it is fully virulent in BLOC-3 deficient mice. These results indicate thattheSalmonellaTyphimurium effectors GtgE and SopD2act redundantly to neutralize apowerfulhostdefensepathwaythatcanprevenSalmonellainfectionsinmice.Interestingly,theRab32/BLOC-3hostdefensepathwaydoesnotrequireknownantimicrobialmechanismsto clearSalmonellainfections suggesting that it functions in conjunctions with novelantimicrobialfactors.