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nnrnnnrnrrnrnnrn nnnnrn01234nn541567nrnnr6n6rn24151189122nn


nrnrnrnnnn/9nnr/4/9/5rrrrnnnx-68nnn/rnrn-nrnrnrnrnnnnrrrrnnn-rr8nrnrnnrnrnn-nrr-nrnnnZapEIsaNovelCellDivisionProteinInteractingwithFtsZandModulatingtheZ-RingDynamicsBenoitSMarteynGouzelKarimovaAndrewK

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Document on Subject : "nnrnnnrnrrnrnnrn nnnnrn01234nn541567nrnnr6n6rn24151189122nn"— Transcript:

1  \n \n \r\n \n
 \n \n \r\n \n \n \r \n    \r   \r \n\r\n       \n\r\n !"\n#  $%&$'(\n)$*$+ \n$,'\n\r"\n-..../...0/.1/23. 4.(\n \n541.56 7\n)\r")\n \n  \r - + 6"(\n" - 6/-" (\r(%\n$2415...11/8&&912./:2 "\n\n-.13(!\n...11/   ; \n\r     \n\r \n   \r" ! ""  (\n  )\n((\n, ! \n/9\n\n((\r/&,4%=/9/&,5\r\r\r\r\n \n \n(&#x-6.8;ᙁ$ $"!" \n \n\n/\r\n((\r $\n- \n\r"   \n"\r \n\r \n" " \r (#\n\n( \n"  (\n \r\r \r )\r\n((\n\n-\r\r 8 \n\r\n"\r\n   \n\r \n!!\r\n'$ \n  ! (-  ; \n\r\r"- \n \r\n"" \n\n  "?  ZapEIsaNovelCellDivisionProteinInteractingwithFtsZandModulatingtheZ-RingDynamicsBenoitS.Marteyn,GouzelKarimova,AndrewK.Fenton,AnastasiaD.Gazi,NicholasWest,*LhousseineTouqui,Marie-ChristinePrevost,Jean-MichelBetton,OemerPoyraz,DanielLadant,KennGerdes,PhilippeJ.Sansonetti,ChristophM.TangInstitutPasteur,UnitŽdePathogŽniemicrobienneMolŽculaire,Paris,France;InstitutNationaldelaSanteetdelarechercheMedicaleUnitŽ786,Paris,France;InstitutPasteur,UnitŽdeBiologiedesInteractionsMolŽculaires,Paris,France;CNRSUMR3528,UnitŽdeBiologiedesInteractionsMolŽculaires,Paris,France;UniversityofNewcastle,CentreforBacterialCellBiology,Newcastle-upon-Tyne,UnitedKingdom;InstitutPasteur,UnitŽdeDŽfenseInnŽeetInßammation,Paris,France;InstitutPasteur,Imagopole/PFMU,Paris,France;InstitutPasteur,UnitŽdeMicrobiologieStructurale,Paris,France;KarolinskaInstitutet,DepartmentofMedicalBiochemistry&Biophysics,Stockholm,Sweden;CollgedeFrance,Paris,France;UniversityofOxford,SirWilliamDunnSchoolofPathology,Oxford,UnitedKingdom*Presentaddress:CentenaryInstitute,MycobacterialResearchLaboratory,Newtown,NSW,Australia. ABSTRACT Bacterialcelldivisionrequirestheformationofamaturedivisomecomplexpositionedatthemidcell.Thelocalizationofthedivisomecomplexisdeterminedbythecorrectpositioning,assembly,andconstrictionoftheFtsZring(Z-ring).Z-ringconstrictioncontrolremainspoorlyunderstoodand(tosomeextent)controversial,probablyduetothefactthatthisphenome-nonistransientandcontrolledbynumerousfactors.Here,wecharacterizeZapE,anovelATPasefoundinGram-negativebacte-ria,whichisrequiredforgrowthunderconditionsoflowoxygen,whilelossofresultsintemperature-dependentelonga-tionofcellshape.WefoundthatZapEisrecruitedtotheZ-ringduringlatestagesofthecelldivisionprocessandcorrelateswithconstrictionoftheZ-ring.OverexpressionorinactivationofleadstoelongationofEscherichiacoliandaffectsthedynam-icsoftheZ-ringduringdivision.Invitro,ZapEdestabilizesFtsZpolymersinanATP-dependentmanner. IMPORTANCE Bacterialcelldivisionhasmainlybeencharacterizedinvitro.Inthisreport,wecouldidentifyZapEasanovelcelldivisionproteinwhichisnotessentialinvitrobutisrequiredduringaninfectiousprocess.Thebacterialcelldivisionprocessreliesontheassembly,positioning,andconstrictionofFtsZring(theso-calledZ-ring).Amongnonessentialcelldivisionpro-teinsrecentlyidentiÞed,ZapEistheÞrstinwhichdetectionattheZ-ringcorrelateswithitsconstriction.WedemonstratethatZapEabundancehastobetightlyregulatedtoallowcelldivisiontooccur;absenceoroverexpressionofZapEleadstobacterialÞlamentation.Asisnotessential,wespeculatethatadditio

2 nalZ-ringdestabilizingproteinstransientl
nalZ-ringdestabilizingproteinstransientlyrecruitedduringlatecelldivisionprocessmightbeidentiÞedinthefuture. 24January201427January20144March2014MarteynBS,KarimovaG,FentonAK,GaziAD,WestN,TouquiL,PrevostM-C,BettonJ-M,PoyrazO,LadantD,GerdesK,SansonettiPJ,TangCM.2014.ZapEisanovelcelldivisionproteininteractingwithFtsZandmodulatingtheZ-ringdynamics.mBio5(2):e00022-14.doi:10.1128/mBio.00022-14.B.BrettFinlay,TheUniversityofBritishColumbia©2014Marteynetal.Thisisanopen-accessarticledistributedunderthetermsofthe CreativeCommonsAttribution-Noncommercial-ShareAlike3.0Unported ,whichpermitsunrestrictednoncommercialuse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.AddresscorrespondencetoPhilippeJ.Sansonetti,psanson@pasteur.fr. C elldivisionisafundamentalprocessthatiscentraltocellularmorphologyandreplication.Accuratedivisionrequirespre-cisecontrolofcellenlargementandofthetimingandlocationofthesiteofdivision.TheÞrstproteinfoundtolocalizeatthemid-cellduringdivisionofprokaryoticcellswasFtsZ,aGTPasethatisstructurallyhomologoustoeukaryotictubulin(1).FtsZpolymer-izesatthemidcell,formingalargering-likenetworkatthecellmembraneknownastheZ-ring(2).AmaturedivisomecomplexsubsequentlyformsattheZ-ringandiscomposedofatleast10proteinswhichareessentialforcelldivision(3).TheseproteinsincludeFtsZ,FtsA,FtsK,FtsQ,FtsL,FtsB,FtsW,FtsI,andFtsN.Itisbeingincreasinglyappreciatedthatanumberofaccessorypro-teinsarealsorecruitedtothedivisomeandcontributetocelldi-visionandmaybeessentialforgrowth,dependinguponthespe-ciÞcenvironmentalconditions.SeveraloftheseZ-ring-associatedproteins(Zaps,i.e.,ZapA,ZapB,ZapC,andZapD[4Ð6])modu-latethedynamicsofZ-ringassemblyandstability.Atpresent,littleisknownaboutcelldivisionduringgrowthunderlow-oxygenconditions,eventhoughmanybacteriathriveinanaerobicenvi-ronments,particularlywithinmammalianhosts(7,8).HereweidentifyafurtherZ-ring-associatedproteinnamedZapEwhichisrequiredforcelldivisionunderlow-oxygencondi-tions.ZapEisanATPasepresentinGram-negativebacteriathatappearsattheconstrictingZ-ringlateincelldivision.ZapEaffectsFtsZmultimerizationandpolymerizationinvitroInvivorecruitmentcorrelateswithZ-ringconstriction.IdentiÞcationofZapE,requiredforgrowthintheabsenceofWhilescreeningalibraryofShigellaßexnerimutantsfortheirabilitytocolonizethegastrointestinal(GI)tract(9,10),weidentiÞedamutantthatwasdefectiveforcolonizationwithatransposon12bpupstreamofa1,128-bpopenreadingframe(ORF)(accessionnumberb3232;Fig.1A).TheroleofthisORF RESEARCHARTICLE March/April2014Volume5Issue2e00022-14 ¨ mbio.asm.org1 (heredesignatedZapE)incolonizationwasconÞrmedbycon-structionandanalysisofadeletionmutant(M90T::)andbycomplementation(Fig.1;seealsoFig.S1AandTablesS1AandBinthesupplementalmaterial).Ofnote,examinationoftissuesfrominfectedanimalsdemonstratedthatS.ßexneridisplayedanelongatedcellularphenotypeintheGItract(Fig.1B).TofurthercharacterizethecontributionofZapEtocelldivi-sion,weexaminedthephenotypeofanEscherichiacolimutant(K12::)grownunderavarietyofconditionsrele-vanttosurvivalofbacteriainvivo.Strikingly,whileE.coliZapEhadnodemonstrablegrowthdefectinaerobicconditions,themutantwasdefectiveforgrowthinanaerobiosis(Fig.2A;seealsoFig.S1BandCinthesupplementalmaterial);complementa-tionanalysisconÞrmedthatlossofZapEwasresponsibleforthefailuretogrowinalow-oxygenenvironment.Additionally,growthatelevatedtemperatures(i.e.,42¡C)ledtoanexaggeratedelongationofE.colilackingZapE(Fig.2B).Thetemperature-dependentelongatedphenotypewasalsoobservedinwithorwithoutavirulenceplasmid(INV)ÑaswellasinE.colicomparinggrowthat30¡Cand42¡C(StudentÕs(seeFig.S2AandB).TheK12::phenotypewasconÞrmedusingaK12::straininthepresenceofglucosebutnotofarabinose,allowingfunctionalcomplementationofthephenotype(seeFig.S3A).ZapEisanATPasethatinteractswithcomponentsofthedi-ZapEisfoundinGram-negativebacteria(seeTableS1Cinthesupplemen

3 talmaterial)andispredictedtocontainputat
talmaterial)andispredictedtocontainputativeWalkerA(GGVGRGKT)andWalkerBnucleotide-bindingsites(seeFig.S3Binthesupplementalmaterial).Therefore,weexam-inedthecapacityofZapEtohydrolyzeATPandGTP.WefoundthatpuriÞedrecombinantZapEhydrolyzedATPbutnotGTP(Fig.2C).IntroductionofasingleaminoacidsubstitutionintheATP-bindingsite(inZapE)abolishedATPaseactivity(Fig.2C).Inaddition,wemodeledtheorganizationoftheactivesiteofZapEbysmall-angleX-rayscattering(SAXS)analysis(Fig.2D;seenalsoFig.S3CtoE),usingfoldrecognitionmethodsbasedontheextendedAAAATPasedomainoftheATP-dependentmetalloproteaseFtsH(PDBidentiÞcationnumber[ID]2CE7).AsFtsZisaknownFtsHsubstrate(11,12),thestruc-turalsimilaritybetweenZapEandFtsHsuggeststhatZapEmightinteractdirectlywithFtsZ.TofurtherdeÞnetheroleofZapEduringcelldivision,weexaminedwhethertheproteininteractswithcomponentsofthedivisome,includingFtsZ.Usingabacterialtwo-hybrid(BACTH)system(13),weidentiÞedpotentialinteractionsbetweenZapEandFtsQ,FtsL,FtsI,andFtsN(Fig.3A)(0.01andtest).Incontrast,nointeractionwasobservedwithFtsA,whichformsactin-likeprotoÞlaments(14),andFtsK,whichcoordinatesDNAsegregationwithdivision(15).AsimilarresultwasobtainedusingZapEfromS.ßexneri(M90T)asbaitpairedwiththeseE.colidivisionproteins(Fig.3A).InteractionsofZapEwithFtsZcouldnotbeexaminedbytheBACTHsystem,asexpres-sionofbothT18-orT25-andT18-togetherwastoxic(notshownandreference16).ToconÞrminteractionsidentiÞedbyBACTHanalysis,pull-downassayswereperformedwithE.coliHis-taggedZapEboundtobeadsandgreenßuorescentprotein-FtsQ(GFP-FtsQ),GFP-FtsL,GFP-FtsI,andGFP-FtsNexpressedinE.coli(Fig.3B);nointeractionwasobservedwhenGFPwasexpressedonitsown.Inaddition,pulldownassayswithboundZapEorZapEFtsZ-GFPfromcelllysates(Fig.3B),indicatingthattheZapEATPbindingsitewasnotrequiredforFtsZinteractioninvitroZapEwasfoundinthecytoplasm,andfurtherlocalizationbyelectronmicroscopy(EM)analysisrevealedthatmostofthesignalwasdetectedinthevicinityofthebacterialinnermembrane(seeFig.S4Ainthesupplementalmaterial)(0.001,StudentÕstest);thisobservationisconsistentwithaninteractionwithFtsZ.ZapElocalizestotheconstrictingZ-ringtowardtheendofcelldivision.Next,weperformedtime-lapsemicroscopytodeter-minethetemporalandspatialassociationofZapEinrelationtoFtsZatthesingle-celllevel.Toachievethis,ZapE-mCherrywasexpressedunderthecontrolofitsnativepromoter(seeFig.S4Binthesupplementalmaterial)inamutantcontainingFtsZ-GFP(carriedonpDSW230)(17).Ofnote,ZapEwasdetectedonlyattheZ-ringandexclusivelyduringthelaterstagesofcelldivision(Fig.3C;seealsoMovieS1inthesupplementalmaterial).Indeed,theappearanceofZapEatthemidcellcoincidedwithconstrictionoftheZ-ringandwithitssubsequentdisappearance(seeFig.S4C).IneventsleadinguptothemaximalizationofZ-ringconstriction(deÞnedas),FtsZlevelsincreasedinastepwisefashionasthediameteroftheZ-ringdiminished;theamountofZapEpresentattheZ-ringincreasedprogressivelyuntilmaximalconstrictionof FIG1ZapEisrequiredduringtheinfectiousprocessinvivo.(A)Competitiveindex(C.I.)ofaShigellaßexneritransposonmutant(M90Tmut6),amutant(M90T::),andacomplementedstrain(M90T::-GFPM90T)invivo.TheC.I.assessedtheabilityofeachmutanttocolonizetherabbitilealloopincomparisonwiththewild-typestrain.AC.I.of1indicatesnoattenuation.Datarepresentaveragesoftheresultsofthreeindependentexperiments.(B)ImmunodetectionoftheM90T,M90T::,andM90T::-GFPstrainsintherabbitilealloopmodel.DNAwasstainedwithDAPI(blue)andactinwithRRX-phalloidin(red).strainswerelabeledusing-LPSpolyclonalantibody(pAb)(green).Imageacquisitionwasperformedusingaconfocalmicroscope.Right-handpanelsshowenlargedareas.Barsare5Marteynetal. ¨ March/April2014Volume5Issue2e00022-14 theZ-ringoccurred(Fig.3D)(StudentÕs0.001),dem-onstratingaclearcorrelationbetweentheexpressionandlocaliza-tionofZapEandZ-ringconstrictioninvivoZapEabundancemodulatesZ-ringstability.Wenextexam-inedwhethermodulatinglevelsofZapEwithincellsaffectedtheirsh

4 apeandtheappearanceofZ-rings.Initially,t
apeandtheappearanceofZ-rings.Initially,thelocationofFtsZ-GFPduringcelldivisionwasexaminedineitherthepresenceortheabsenceofZapE.FtsZ-GFPdoesnotfullycomplementanmutant,butlocalizestothemidcell,anddoesnotimpairnormalcelldivisionwhenexpressedatbasallevelsinwild-typebacteria(18).LossofZapEinastrainexpressingFtsZ-GFPledtomarkedÞlamentation,withthelossoforderedZ-ringsincells(Fig.4A[richmedia]andB[minimummedia];seealsoFig.S5AandMovieS2inthesupplementalmaterial[livemicroscopy]).ThisÞlamentationwasmorepronouncedduringgrowthathighertemperatures(Fig.4A)andduringthestationaryphase(Fig.4C).WeconÞrmedthatÞlamentationincellslackingZapEwasmoremarkedfollowingoverexpressionofnativeFtsZinadose-dependentmanner(Fig.4D).LossofZapEinastrainexpressingFtsZ-mCherryledtotheformationofmultipledisorganizedFtsZringsfoundsporadicallyalongthelengthoftheextendedbacteriacomparedtothewild-typestrain(Fig.5A).Furthermore,weexaminedtheeffectofover-expressingZapEunderthecontrolofanIPTG(isopropyl-thiogalactopyranoside)-induciblepromoter.Inwild-typecells,ZapEoverexpressionalsoresultedindose-dependentÞlamenta-tion(Fig.5BandC;seealsoFig.S5Binthesupplementalmate-rial).However,noZ-rings(detectedwithFtsZ-mCherry)weredetectedalongthelengthofÞlamentswhenZapEoverexpressionwasinducedwithhighlevelsofIPTG(Fig.5A;seealsoFig.S5C[asacontrol,withoutIPTG]),althoughadditionaladverseeffectsofZapEoverexpressioncouldnotberuledout.Thiswasnotob-servedincellsoverexpressingZapE,whichexhibitednormalbacterialshapeandsingleorderedZ-rings(Fig.5A).Takento-gether,thesedataareconsistentwithZapEandFtsZhavingantag-onizingrolesattheZ-ringinsidecells.ZapEimpairsthestabilityofpolymerizedFtsZinvitro.weexaminedtheeffectofZapEonpolymerizedFtsZinvitro FIG2inactivationleadstoastress-dependent(anaerobiosis,temperature)elongatedphenotypeofE.coli.zapEencodesanATPase.(A)Anaerobiosis-dependentphenotypeofE.colimutant,andcomplementedstrains.GrowthofK-12,K12::,andK12::-GFPstrainsinminimummediainthepresence()orabsence()ofoxygenat37¡Cisshown.Scalebarsare10m.Arrowsindicateelongatedbacteria.(B)Temperature-dependentphenotypeofK-12,K12::,andK12::-GFPstrains.BacteriaweregrowninrichmediaattheindicatedtemperatureuntilanOD0.5wasreached.Scalebarsare10m.Arrowsindicateelongatedbacteria.**,0.01;*,0.05.(C)ZapEorZapEATPaseactivityassessedbysilicalayerchromatography.ThereactionwasperformedinaTris-HClbuffer(50mM;pH7.4)containing10mCiofradiolabeledATP32(or32),10mMATP,and2.5mMMgClinthepresenceofvariousZapE-HorZapEquantities,asindicated.(D)Mostprobableatommodelof15DAMMINreconstructionsÞttingthedataatupto0.241withan1.683andanNSDvalueof0.70.024,indicatingthestabilityofthesolution.TheWalkerAmotifintheAAAATPaseishighlightedinyellow.N-terminalandC-terminalresiduesofZapEarehighlightedingreenandblue.ZapE,aNovelCellDivisionProteinMarch/April2014Volume5Issue2e00022-14 ¨ mbio.asm.org3 puriÞedrecombinantproteins.Asdescribedpreviously(19),inthepresenceofGTPandCapuriÞedFtsZandFtsZ-GFPformsmallpolymersinvitro(Fig.5D;seealsoFig.S5Dinthesupple-mentalmaterial).TheadditionofZapEpromotedthepolymer-izationofFtsZ/FtsZ-GFPthroughlargehelicalstructures(Fig.5D;seealsoMovieS3).ThesestructureswerenolongerstablewhenATPwasaddedwithZapEaftera3-minincubation(Fig.5D),whileZapEhadnoeffect(Fig.5D),indicatingthatactiveZapEreducesthestabilityofFtsZpolymersinthepresenceofATPthroughamolecularmechanismwhichremainstobedeÞned.Here,weidentiÞedandcharacterizedanovelZ-ring-associatedproteinnamedZapE,whichisanATPasefoundamongGram-negativebacteria(seeTableS1Cinthesupplementalmaterial).ZapEisnotessentialinE.coliorinduringgrowthunderstandardlaboratoryconditions.However,intheabsenceofoxy-genorattemperaturesover37¡C,thecontributionofZapEtocelldivisionbecomesevident,withcellslackingthisproteindisplay-ingagrowthdefectandelongatedphenotype(Fig.2AandB).ThelatterwasdetectedintissuesectionsoftheGItractinfectedwithaShigellazapEmutant;as

5 alikelyconsequenceofthecelldivisionisreq
alikelyconsequenceofthecelldivisionisrequiredforefÞcientcolonizationofGItrack(Fig.1AandB).Therefore,theidentiÞcationofZapEfromstudiesofbacteriainvivoindicatesthatexaminingbacteriaintheenvi-ronmentsthattheyencounteroutsidethelaboratorycouldun-coverthefunctionofotheraccessorycelldivisionproteins.Inthefuture,studiesofbacterialcelldivisionunderothergrowthcon-ditionssuchaslowpH,oxidativestress,oraminoacidstarvationwillrevealnovelaspectsofcelldivisionregulationallowingbacte-rialsurvivalduringhostinvasion.SeveralATPasestargetingFtsZduringcelldivisionsuchas FIG3ZapEisacytoplasmicproteinwhichinteractswithFtsZinvitroinvivo.ZapErecruitmentcorrelateswithZ-ringconstriction.(A)BACTHanalysiswasperformedusingtheT25-K-12(E.coliK-12)orT25-M90T(M90T)versusT18-plasmidconstructswithindicatedgenes.ResultsareexpressedinMillerunitsandwereaveragedfromthreeindependentexperiments.Errorbarsshowthestandarddeviations(SD).ComparingaverageactivitytothatoftheT18negativecontrol,**indicates0.01and***indicates0.001(StudentÕstest).(B)PulldownassaywithK-12ZapE-HandGFPorGFP-taggedproteinsinE.colilysates.TheinteractionbetweenE.coliFtsZ-GFP(pDSW230)andE.coliandZapE,respectively,wasanalyzedusingaHispulldownassay.(C)ExpressionandlocalizationofFtsZ-GFPandZapE-mCherryduringacelldivision.Time-lapseobservationwasperformedonanLB-agarpadat30¡C,usinga200MAxiovertepißuorescencemicroscope(Zeiss).Imageacquisitionwasperformedevery3min(seealsoMovieS1andFig.S4CinthesupplementalmaterialforrawßuorescencequantiÞcation).ThisresultisrepresentativeofÞveindividualobservationsfromthreeindependentexperi-ments.Barsare2m.Max,maximum.(D)Relationshipbetween,respectively,ZapE-mCherryandtheFtsZ-GFPmeansignal(AU)(K12::mCherry)andtheZ-ringdiameter(pDSW230).MeanFtsZ-GFPandZapE-mCherryßuorescentsignalsarerepresentedinFig.S4Cinthesupplementalmaterial.5independentobservations;errorbarsshowtheSD.***indicates0.001(StudentÕstest).Max.Const.,maximumconstriction.Marteynetal. ¨ March/April2014Volume5Issue2e00022-14 FtsH,MinD,andKaiCareinvolvedincelldivisionfunctions.FtsHisanATP-dependentzincmetalloproteasetargetingFtsZinvitro(11),althoughthisactivitycouldnotbeconÞrmedinvivoMinDbelongstotheMinsysteminvolvedinZ-ringpositioning(20).KaiCinhibitsZ-ringformationcontrollingthecircadianclockinSynechococcuselongatus(21).NoneoftheseATPaseshaveprovenfunctionsinthedynamicsofZ-ringconstrictioninvivoIndeed,ifstartingeventsofcelldivision(Z-ringassemblyand FIG4EffectofZapEinactivationonK-12shapeuponFtsZ-GFPandFtsZexpressionmodulation.(A)LocalizationofFtsZ-GFP(pDSW230)inK-12andstrainsgrowninrichmedia(LB)at37¡Cor42¡CintheabsenceofIPTGuntilanOD0.5wasreached.Barsare5m.(B)LocalizationofFtsZ-GFP(pDSW230)inK-12andK12::strainsgrowninminimummedia(M9)at37¡CintheabsenceofIPTGuntilanOD0.5wasreached.Barsare2m.(C)FtsZ-GFP(pDSW230)localizationinK-12andK12::strainsduringthestationaryphaseperformedinLBrichmediaat37¡Cor42¡C.Theseobservationsarerepresentativeoftheresultsofatleastthreeindependentexperiments.Barsare1m.(D)EffectofFtsZ-H(WM971)overexpressiononK-12andK12::shape.BacteriaweregrowninLBat37¡CinthepresenceoftheindicatedconcentrationsofIPTGuntilanOD0.5wasreached.Theseobservationsarerepresentativeoftheresultsofthreeindependentexperiments.Barsare10ZapE,aNovelCellDivisionProteinMarch/April2014Volume5Issue2e00022-14 ¨ mbio.asm.org5 positioning)havebeenwellcharacterized(22),eventsleadingtothedisappearanceoftheringandcompletionofdivisionhavenotbeendeÞnedinasmuchdetailandremaintobediscussed(23).ThismightbeexplainedbythetransiencyoftheZ-ringconstric-tionphenomenon.Consistently,ZapEfusion(mCherry)recruit-mentattheZ-ringcouldbeseeninthisstudyonlybylivemicros-copy(Fig.3C;seealsoMovieS1inthesupplementalmaterial).WeobservedthatinthepresenceofATPandCa,ZapEpromotedbundlingofFtsZ/FtsZ-GFPpolymersintounstablethree-dimensionalstructures(Fig.5D;seealsoMovieS3inthesupple-mentalmaterial).Astructuralhomo

6 logybetweenZapEandFtsHwasfoundbySAXSanal
logybetweenZapEandFtsHwasfoundbySAXSanalysis(Fig.2D).ThisobservationshouldsupportfurtherinvestigationsonhowZapEdirectlyorindirectlyaffectstheeffectofactivityonZ-ringconstriction,accountingforthedestabilizationobservedinvitro(Fig.5D)andinvivoZapEoverexpression(Fig.4A).TheprecisecontroloftheZapElevelappearstobecrucialforZ-ringstabilityanddynamicsasweshowedthateitherZapElossoroverexpression(Fig.4A)alteredZ-ringstabilityandledtobac-terialÞlamentation.FurtherstudiesshouldaimatdeÞningZapE FIG5ZapElevelofexpressionperturbatesZ-ringstabilityandbacterialshape.(A)EffectofinactivationandZapEorZapEoverexpressiononZ-ringstability(FtsZ-mCherry,pAKF133).Bacteriaweregrowninminimummediaat37¡Cinthepresenceofarabinose(0.01%)inadditiontoIPTG(1mM)whenindicated.Dataarerepresentativeoftheresultsoffourindependentobservations(seealsoFig.S5Cinthesupplementalmaterial).(B)ZapE-HlevelofexpressioninstrainK12-pgrowninLBat37¡CinthepresenceoftheindicatedconcentrationsofIPTGuntilanOD0.5wasreached.ThelevelofexpressionofZapE-HwasassessedbyWesternblotanalysisonbacterialwholeextractusingapolyclonal-ZapEantibody.-RecAwasusedasacontrol.(C)PhenotypiccharacterizationofstrainK12-pgrownundertheconditionsdescribedforpanelB.Theseobservationsarerepresentativeoftheresultsofthreeindependentexperiments.Barsare2m.(D)PolymerizationofFtsZ(10M)withFtsZ-GFP(5invitrowasperformedover3min,inthepresenceof10mMCaCl,asdescribedinreferences16and19.ZapE-HorZapEM)wassubsequentlyaddedtothereactionmixture(0),withATPaddedwhenindicated.Confocalimagingwasperformedat0and3min.Dataarerepresentativeoftheresultsofthreeindependentexperiments.Barsare5Marteynetal. ¨ March/April2014Volume5Issue2e00022-14 abundanceregulation(expressionanddegradation)duringthecelldivisionprocessanditsconsequencesforZ-ringstabilitytobettercharacterizetheroleofZapEintheZ-ringdynamicinvivoMATERIALSANDMETHODSBacterialstrainsandgrowthconditions.Thebacterialstrainsandplas-midsusedinthisstudyaredescribedinTableS1AinthesupplementalstrainsweregrowninTrypticasesoy(TCS)brothoronTCSagarplatessupplementedwith0.01%Congored(Sigma).E.colistrainsweregrowninLBmedia.AnaerobicgrowthexperimentswereperformedinaMiniMacsMG-250chamber(DonWithley).-arabinosewereusedataconcentrationof0.2%tomodulatetheexpressionofgenesclonedunderthecontrolofthepBAD33promoter.Expressionplasmidconstruction.ThepSUCvectorconstructionwasachievedbyamplifyingthemCherryfusionfromthepmCherry-N1vec-torusingtheSG150andSG151primerpair(seeTableS1Binthesupple-mentalmaterial),introducingtheBamHIandEcoRIrestrictionsites.ThepSU19vectorwasdigestedwithBamHIandEcoRIrestrictionenzymespriorligationofthedigestedmCherryampliÞedfragment,leadingtothegenerationofthepSUCvector(seeFig.S4B).Thisexpressionvectoral-lowstheexpressionofmCherryproteinfusionsintheCterminusunderthecontrolofthepromoterofthegeneofinterestinE.coliandinExpressionoftheFtsZ-GFPfusionunderthecontrolofawasperformedusingeitherthepDSW230andpDSW231construct(kindlyprovidedbyDavidWeiss)(describedinTableS1Ainthesupple-mentalmaterial).FtsZ-mCherryfusionexpressionunderthecontrolofanarabinose-induciblepromoterwasperformedwithpAKF133(de-scribedinTableS1A).DNAmanipulations.TheinitialtransposoninsertioninS.ßexneriM90Tinwasperformedasdescribedpreviously(9).Theconstruc-tionofinactivatedmutantswasthenperformedinE.coliS.ßex-mutantconstruction.InMG1655,E.coliK-12P1virpagely-satewaspreparedontheJW3201donorstrainfromtheKeiocollection(1,24).IntheJW3201strain,theORFissubstitutedbythekanamycin(Km)resistancemarker(::Km)(2,24).The::KmcassettewasintroducedintoMG1655byP1transduction(3,25),andselectionforkanamycin-resistant(Km)colonieswasmadeonLBplatescontainingkanamycin(50g/ml).Afterreisolation,severalcloneswereveriÞedbyPCRtoconÞrmtherightchromosomalstructureofthe::Kmdele-tion.OneclonewaschosenandnamedK12wasthenobtainedfromK12::Kmbyremovingthekanamycinresistancemarkerfromthe::Kmcassette.Inthis::Kmca

7 ssette,theantibioticresistancemarkerisßa
ssette,theantibioticresistancemarkerisßankedbytworepeats,whicharetherecognitiontargetsforthesite-speciÞcrecombinaseFLP(4,24).Therefore,togetridoftheresistancemarkerfromtheK12::::Kmchromosome,pCP20,atemperature-sensitiveplasmidthatencodestheFLPrecombinase,wasused(5,26).Brießy,::KmcellsweretransformedwithpCP20,andchloramphenicol-resistant(Cm)colonieswereselectedat30¡ConLBplatescontainingthecorrespondingantibiotic(30g/ml).Severaloftheseclonesweregrownovernightonantibiotic-freeLBplatesat42¡C.Tenindependentcolonieswereselected,andaftersingle-colonypassageat30¡C,all10colonieswerenolongerCmandKm,indicatingsimultaneouslossofpCP20andthekanamycinresistancemarkerfromthebacterialchromosome.ThisFLP-catalyzedexcisioncreatedanin-framedeletionoftheORF,leavingbehinda102-bpscarsequence()(6,24).ToconÞrmthecorrectchromosomalstructureofthedeletion,severalCmandKmclonesweretestedbyPCRusingtheNWpr40andNWpr41primerpair(seeTableS1Binthesupplementalmaterial).AfterconÞrmation,oneclonewaschosenandnamedK12::Alternatively,the::KmcassettewasintroducedintoMG1655expressingp)byP1transduction(7,25),andselec-tionforkanamycin-resistant(Km)colonieswasperformedonLBplatescontainingkanamycin(50g/ml).TheK12::::KmpstrainwasInordertoinactivateShigellaßexneri(M90T),aone-stepchromosomalinactivationmethodwasusedtotargethomologousregionforintegration.Therefore,wegeneratedPCRproductswithmuchlongerßankingsequenceusingtheK12::nullmutantasthetemplate.TheM90TwastransformedwithPCRproductsampliÞedfromK12::::KmmutantgenomicDNAusingprimersNWpr23andNWpr24(seeTableS1Binthesupplementalmaterial).TheNWpr23andNWpr24primersweredesignedtoinclude50bpofupstreamanddownstreamsequenceßanking.ThisproductwastransformedintoM90T::pKD46,whichresultedinallkanamycin-resistantcoloniescontainingthe1.50-kbkanamycinresistancegeneasanalyzedbyPCR.Thus,anS.ßex-nullmutant(M90T::)wassuccessfullygenerated.Expressing,respectively,aandafusionun-derthecontrolofthepromoterresultedinthecomplementationoftheM90T::andK12::mutants.InordertoexpressaZapE-GFPfusion,thegenesofE.coliandtheirpromoters(~500bp)wereampliÞedwiththeSG127andSG128primerpair(seeTableS1Binthesupplementalmaterial)andclonedintopFpV25vectordigestedwiththeBamHIandNdeIrestrictionenzymes.ThepM90Tandp-GFPK-12constructswereobtainedandsequenced(Ta-bleS1A).Inordertoexpress,thegeneanditspromoter(~500bp)wereampliÞedwiththeSG219andSG155primerpair(seeTableS1Binthesupplementalmaterial)andclonedinpSUCvectordi-gestedwiththeHindIIIandXbaIrestrictionenzymes(seeFig.S4B).TheK84ApointmutationofwasperformedusingtheSG114andSG115primerpair(seeTableS1B).p-mCherryandpconstructswereobtainedandsequenced(seeTableS1A).InordertooverproducetheZapEandZapEproteinfusionsinanIPTG-dependentmanner,thecorrespondingDNAfrag-mentswereampliÞedbyPCRpriorcloninginthepKJ1plasmiddigestedusingtheNcoIandBamHIrestrictionenzymes(seeTableS1Ainthesupplementalmaterial).wasampliÞedusingtheSG90andSG91primerpair(seeTableS1B),andwasobtainedusingtheSG114and1G115primerpairtointroduceaK84Asinglepointmutation(seeTableS1B).TheresultingpandpconstructswereanalyzedbyPCRandsequenced.Rabbitligatedilealloopmodel.NewZealandWhiterabbitsweight-ing2.5to3kg(CharlesRiverBreedingLaboratories,Wilmington,MA)wereusedforexperimentalinfections.Foreachanimal,upto12intestinalligatedloops,each5cminlength,werepreparedasdescribedpreviously(8,9,27).Fortheevaluationofthecompetitiveindex(C.I.),equalquan-titiesofthewild-typestrainandofthemutantwereinjectedineachloop(correspondingtoatotaldoseof10CFUperloop).After16h,animalsweresacriÞcedandtheluminalßuidwasaspiratedandS.ßexneriered.TheC.I.wascalculatedastheproportionofmutanttowild-typebacteriarecoveredfromanimalsdividedbytheproportionofmutanttowild-typebacteriaintheinoculums,andresultsareexpressedasthemeansoftheresultsdeterminedwithatleast4loopsfromtwoindepen-dentanimals.TheexperimentalprotocolwasapprovedbytheFrenchEthicCommitteeParis1(numb

8 er20070004,9December2007).Forimmunohisto
er20070004,9December2007).Forimmunohistochemicalstaining,infectedrabbitileumsampleswerewashedinphosphate-bufferedsaline(PBS),incubatedat4¡CinPBScontaining12%sucrosefor90minandtheninPBSÐ18%sucroseover-night,andfrozeninoptimumcuttingtemperature(O.C.T.)compound(Sakura)ondryice.Sections(7minthickness)wereobtainedusingaCM-3050cryostat(Leica).Fluorescentstainingwasperformedusingarabbitanti-lipopolysaccharide(LPS)primaryantibody(P.San-sonetti,InstitutPasteur)(1:200dilution)andananti-rabbitßuoresceinisothiocyanate(FITC)-conjugatedsecondaryantibody(1:1,000).Epithe-liumcellnucleiwerestainedwithDAPI(4[prime],6-diamidino-2-ZapE,aNovelCellDivisionProteinMarch/April2014Volume5Issue2e00022-14 ¨ mbio.asm.org7 phenylindole)(1:1,000)andactinstainedwithRhodamineRed-X(RRX)Ðphalloidin(1:1,000).Imageacquisitionwasperformedusinglaserscanningconfocalmicroscopy.ImageanalysiswasperformedusingIm-ageJsoftware.Two-hybridscreen.WeusedtheBACTHsystemthatisbasedontheinteraction-mediatedreconstitutionofanadenylatecyclase(AC)enzymeintheotherwisedefectiveDHM1E.colistrain(9,13,28).Thissystemiscomposedoftworeplication-compatibleplasmids,pKT25andpUT18,respectivelyencodingtheintrinsicallyinactiveN-terminalT25domainandC-terminalT18domainoftheACenzyme.E.coliShigellazapEwasampliÞedusingtheNG1281andNG1282primerpairandclonedinpKT25vector.pKT25andpUT18plasmidsweresubsequentlydoublytransformedtoDHM1tosearchfortheACreconstitutionthatturnsonproduction,leadingtotheproductionofbluecolorafter2daysofgrowthat30¡ConindicatorplatescontainingX-Gal(5-bromo-4-chloro-3--galactopyranoside)(Eurobio)(40mgml-1),isopropyl-1--galactopyranoside(Invitrogen)(0.5mM),Ap,Km,andnali-dixicacid.-Galactosidaseactivitywasmeasuredasdescribedbefore,averagedfromthreeindependentexperiments,andexpressedasMillerunits(10,13,29).-ZapEantibodyproduction.-ZapErabbitpoly-clonalantibodywascollectedfromtwoNewZealandWhiterabbitschal-lengedwithpuriÞedZapE(2mg/mlsolution)onfouroccasionsseparatedby2weeksrest.TheÞrstinjection(intradermal;500l)wasperformedwiththepuriÞedprotein(125g)andwithcompleteFreundÕsadjuvant.Thesecondinjectionwasperformedfollowingasimilarproce-dureinthepresenceofincompleteFreundÕsadjuvant.Thethirdandthelastinjectionswereperformedwithnoadjuvant.TheÞnalbloodcollec-tionwasperformedbycardiacpunctureusingaheparin-freetube.Serawereseparatedfrombloodcellsbycentrifugation(14,000rpm,30min).-FtsZrabbitpolyclonalantibodywaskindlyprovidedbyKennGerdes(4,13,17).TLCanalysis.ATPaseandGTPaseassayswereperformedinthepres-enceofbovineserumalbumin(BSA)(Sigma)(1.25mg/ml),ATP32or32(PerkinElmer)(30Ci),ATPorGTP(Sigma)(50mM),and0.1to10gofpuriÞedZapEandZapEasindicated.TheÞnalreactionmixturevolumewas20linTMDbuffer(25mMTris[pH7.4],10mMMgCl,1mMdithiothreitol[DTT]).Thereactionwasrunduring10minat37¡Candstoppedbytheadditionof20lmethanol.Whenindicated,chromatographywasperformedonthin-layerchromatogra-phy(TLC)plates(ThomasScientiÞc),withamobilephasecontainingamixtureoflithiumchloride(LiCl)andformicacid.Aftermigration,aÞlmwasexposedontheplateandfurtherdeveloped.RadiolabeledPipresencethroughATP32hydrolysiswasthenrevealed.ProteinexpressionandpuriÞcation.andZapEproteinfusionswereexpressedusingthepK-12andK-12constructs(seeTableS1Ainthesupplementalmaterial)expressedinanE.coliBL21(DE3)strain.OvernightculturesweresubculturedinfreshLBmedia(1:100)andgrownat37¡Cuntilanopticaldensityat600nm(OD)of0.5wasreached.Overexpressionwasinducedbytheadditionof0.5mMIPTGandwasperformedovernightatroomtemperature(RT).His-taggedproteinswerepuriÞedonTalonbeads(Clontech)andfurtherpuriÞedbygelÞltrationusingaHiload16/60Superdex200column(GE)andaTrisbuffer(50mM;pH7.5)containing5mMMgCl,1mMEDTA,and0.1MNaCl.FtsZandFtsZ-GFPwerepuriÞedbyionexchangeonaHiload16/10DEAEcolumnusingaTrisbuffer(50mM;pH7.5)containing5mMMgCl,1mMEDTA,and0.1MNaCl(buffer1)andaTrisbuffer(50mM;pH7.5)containing5mM,1mMEDTA,and1MKCl(buffer2)asdescribed

9 previously(14,30),followedbyagelÞltratio
previously(14,30),followedbyagelÞltration,asdescribedabove.Hispulldownassay.orZapEfusions(30g)wereincubatedonTalonbeadsfor1hat4¡Cinresuspensionbuffer(20mMTris-HCl[pH8],100mMNaCl),supplementedwithaproteaseinhibitorcocktail(Roche).Unboundproteinswereremovedbywashingtwiceus-ingtheresuspensionbuffer.ExtractscontainingGFPfusions(FtsZ,FtsQ,FtsL,FtsI,andFtsN)werepreparedfrombacterialculturesgrowninLBat37¡Cinthepresenceof1mMIPTGfor3h.Bacterialpelletswereresus-pendedinaTris-HClbuffer(50mM)supplementedwithaproteasein-hibitorcocktail(Roche)andsonicated.Theresultingsuspensionswerecentrifugedat12,000for30min,andsupernatantswerecollected.Ineachpulldownexperiment,10gofE.colisolubleextractcontainingGFPfusions(FtsZ,FtsQ,FtsL,FtsI,andFtsN)wasincubatedduring1hatAfter3washeswithresuspensionbuffer,2samplebufferwasaddedandthebeadswereboiledandsubjectedtoSDS-PAGEgelanalysis.Ascon-trols,extractswereincubatedwithTalonbeadsonly.FtsZ/FtsZ-GFPpolymerizationinvitro.FluorescentFtsZ/FtsZ-GFPpolymersweregeneratedinabuffercontaining50mMHEPES,50mMKCl,5mMMgCl,and10mMCaClinadditionto1mMGTP.PolymerizationofFtsZ(10M)andFtsZ-GFP(5M)occurredduring0to3minat30¡Conglassslides.Then,ZapE-HorZapEandATP(1mM)wereaddedwhenindicatedtoreachaÞnalvolumeofl.PolymerimagingwasperformedusingaTCSSP5confocalmicro-scope(Leica).Westernblotanalysis.Bacterialextractswerepreparedasfollows.ForFtsZ,ZapE,andRecAdetectioninK-12andK12::strains,overnightbacterialculturesweresubculturedin100mlLBliquidmediaat37¡CuntiltheODatreached0.3.Bacteriawereharvestedbycentrifugationfor15minat3,000,washed,andthenresuspendedin10mlPBS.Cellswerespunagainfor5minat3,000andresuspendedin10mlofPBS.Membrane-associated(Pellet)andsoluble(Sol.)proteinsweresep-aratedbycentrifugationfor20minat12,000TotalproteinconcentrationsweremeasuredbythemethodofBrad-ford(Bio-Rad).Proteinswereseparatedby16%SDS-PAGE,transferredtonitrocellulosemembranes,andincubatedwiththeprimaryantibodiesdilutedinPBSÐ5%milkÐ0.01%Tween20(Sigma)overnight.Mem-braneswerewashedinPBSthreetimesandthenincubatedwithsecondaryantibodiesfor1hbeforewashing.Antibodybindingwasdetectedwithchemiluminescence(ECLkit;GEHealthcare).FtsZpolymersedimentationassay.FtsZpolymers(P)weregeneratedinareactionmixturecontaining50mMHEPES,50mMKCl,and5mMinadditionto1mMGTPduring3minat30¡Candcollectedbyultracentrifugation(11min,80,000rpm)at4¡C(TL-100Ultracentrifuge;Beckman).Thereactivebufferwasthendiscardedandreplacedbyareac-tionmixturecontaining50mMHEPES,50mMKCl,and5mMMgCladditionto1mMATPand0.5g/mlofpuriÞedZapE-HwhenindicatedinaÞnalvolumeof100l.Thereactionwasstoppedafter0,1,or3min,asindicated.FtsZpolymerscontainingthepelletfraction(P)weresepa-ratedfromthesolublefraction(S)byultracentrifugation(11min,80,000rpm)at4¡C(TL-100Ultracentrifuge;Beckman).SampleswereresuspendedinaLaemlibuffer(1Þnalconcentration)andsubsequentlysubjectedtoSDS-PAGEgelanalysisandtransferredontoanitrocellulosemembrane.FtsZandZapE-HweredetectedinbothfractionsbyWesternblottingusingrabbitpolyclonalantibodies.IdentiÞcationofZapEhomologousproteinsamongotherorganismswasperformedusingBlastP.TheZapEsequencecom-parisonswereperformedusingClustalWsoftware.LengthmeasurementofbacteriawasperformedwithMicrobeTrackersuitesoftware(version0.937)(15,17,31),andthedataminingwasperformedusingaMatLabcomputingsystem(R2012versionwithImageProcessingToolboxandStatisticsToolbox).StatisticalanalyseswereperformedusingGraphPadPrism5software.Fluorescentproteinfusionimaging.InordertolocalizeFtsZ-GFPandZapE-mCherryandmutatedversionsofproteinfusionsinbacteria,thecorrespondingexpressionplasmidsweretransformedinE.coliMG1655wild-typeorstrains,asindicated.ThelocalizationwasperformedoneitherÞxedorlivingbacteria.TheÞxationofbacteriawasperformedbyadding4%paraformaldehyde(PFA)followedbyawashinginPBS.TheobservationwasperformedusingaNikonEclipse80ioranMarteynetal. ¨ March/April2014Volume5Issue2e00022-14 epißuor

10 escencemicroscope.TheliveobservationofFt
escencemicroscope.TheliveobservationofFtsZ-GFPandZapE-mCherryduringthecelldivisionprocesswasperformedonanLBagarose(1%)padusinga200MAxiovertepißuorescencemicroscope(Zeiss)equippedwithaLambdaLS300WXenonlampandaCoolSnapHQcharge-coupled-device(CCD)camera.InordertolocalizeFtsZ-mCherry(pAKF133)inK-12andK12::strains,bacteriaweregrowninanM9minimummediaat37¡Cuntilanof0.5wasreachedandwerefurtherincubatedonaminimalmediumagarpadwith0.01%arabinose.Thecelldivisionprocesswasob-servedusinganinvertedmicroscope(NikonTi)equippedwitha100numericalaperture(NA)PL-APOobjectivelens.Imagestackswereac-quiredusingMetamorphsoftware(MDS)andaCCDcamera(Photomet-ricsCoolsnapHQ).AsimilarprocedurewasappliedtoobservetheeffectofZapE-HoverexpressiononFtsZ-mCherrylocalization,throughtheadditionofIPTG(1mM)withintheminimalmediumagarpadwith0.01%arabinose(noIPTGwasaddedduringtheliquidculturegrowth).Imagereconstruction(seeMovieS3inthesupplementalmaterial)wasperformedusingImarissoftware(Bitplane).SUPPLEMENTALMATERIALSupplementalmaterialforthisarticlemaybefoundat http://mbio.asm.org/lookup/suppl/doi:10.1128/mBio.00022-14/-/DCSupplemental MovieS1,MOVÞle,0.4MB.MovieS2,MOVÞle,1.9MB.MovieS3,MOVÞle,5.7MB.FigureS1,JPGÞle,0.1MB.FigureS2,JPGÞle,0.1MB.FigureS3,JPGÞle,0.1MB.FigureS4,JPGÞle,0.2MB.FigureS5,JPGÞle,0.1MB.TableS1,DOCXÞle,0.1MB.TextS1,DOCXÞle,0.1MB.WeacknowledgeaccesstotheProteinScienceFacility,KarolinskaInsti-tutet.WegratefullythankJeffErringtonforhisinitialinterestinthisproject.WethankWilliamMargolinforbiochemistryadvice.WethankDavidWeissforsupplyingthevariousFtsproteinfusions.WethankAr-naudEchardforscientiÞcdiscussionsandadvice.Wegratefullyacknowl-edgethesynchrotronbeamlineandexcellentusersupportattheESRFBioSAXSBM29beamline.WethankCarmenR.Beuz—nL—pezandSpyri-doulaCharovaforofferingpartoftheirbeamtimefortheZapEmeasure-B.S.M.andP.S.J.weresupportedbyEUERCHomeoepithgrantno.272398andEUFP7Tornadograntno.222720.BiEF,LutkenhausJ.1991.FtsZringstructureassociatedwithdivisioninEscherichiacoli.Nature http://dx.doi.org/10.1038/354161a0 deBoerPA.2010.AdvancesinunderstandingE.colicellÞssion.Curr.Opin.Microbiol.730Ð737. http://dx.doi.org/10.1016/j.mib.2010.09.015 LutkenhausJ,PichoffS,DuS.2012.Bacterialcytokinesis:fromZringtodivisome.Cytoskeleton(Hoboken)778Ð790. http://dx.doi.org/10.1002/cm.21054 GalliE,GerdesK.2010.Spatialresolutionoftwobacterialcelldivisionproteins:ZapArecruitsZapBtotheinnerfaceoftheZ-ring.Mol.Micro-1514Ð1526. http://dx.doi.org/10.1111/j.1365-2958.2010.07183.x HaleCA,ShiomiD,LiuB,BernhardtTG,MargolinW,NikiH,deBoer2011.IdentiÞcationofEscherichiacoliZapC(YcbW)asacomponentofthedivisionapparatusthatbindsandbundlesFtsZpolymers.J.Bacte- http://dx.doi.org/10.1128/JB.01245-10 Durand-HerediaJ,RivkinE,FanG,MoralesJ,JanakiramanA.IdentiÞcationofZapDasacelldivisionfactorthatpromotestheassemblyofFtsZinEscherichiacoli.J.Bacteriol.3189Ð3198. http://dx.doi.org/10.1128/JB.00176-12 MarteynB,ScorzaFB,SansonettiPJ,TangC.2011.Breathinglifeintopathogens:theinßuenceofoxygenonbacterialvirulenceandhostre-sponsesinthegastrointestinaltract.Cell.Microbiol.171Ð176.doi: 10.1111/j.1462-5822.2010.01549.x JonesSA,ChowdhuryFZ,FabichAJ,AndersonA,SchreinerDM,HouseAL,AutieriSM,LeathamMP,LinsJJ,JorgensenM,CohenPS,ConwayT.2007.RespirationofEscherichiacoliinthemouseintestine.Infect.Immun.4891Ð4899. http://dx.doi.org/10.1128/IAI.00484-07 WestNP,SansonettiP,MounierJ,ExleyRM,ParsotC,GuadagniniS,PrévostMC,Prochnicka-ChalufourA,DelepierreM,TanguyM,Tang2005.OptimizationofvirulencefunctionsthroughglucosylationofShigellaLPS.Science http://dx.doi.org/10.1126/science.1108472 MarteynB,WestNP,BrowningDF,ColeJA,ShawJG,PalmF,MounierJ,PrévostMC,SansonettiP,TangCM.2010.ModulationofShigellavirulenceinresponsetoavailableoxygeninvivo.Nature http://dx.doi.org/10.1038/nature08970 AnilkumarG,SrinivasanR,AnandSP,AjitkumarP.2001.BacterialcelldivisionproteinFtsZisaspeciÞcsubs

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