Domain-Swapping Analysis of FtsI, FtsL, and FtsQ, Bitopic

基金结构域互换

JOURNALOFBACTERIOLOGY,Aug.1997,p.5094–51030021-9193/97/$04.00?0

Copyright©1997,AmericanSocietyforMicrobiology

Vol.179,No.16

Domain-SwappingAnalysisofFtsI,FtsL,andFtsQ,Bitopic

MembraneProteinsEssentialforCellDivisionin

Escherichiacoli

LUZMARIAGUZMAN,?DAVIDS.WEISS,

AND

JONBECKWITH*

DepartmentofMicrobiologyandMolecularGenetics,HarvardMedicalSchool,Boston,Massachusetts02115

Received24March1997/Accepted11June1997

FtsI,FtsL,andFtsQarethreemembraneproteinsrequiredforassemblyofthedivisionseptuminthebacteriumEscherichiacoli.Cellslackinganyofthesethreeproteinsformlong,aseptate?lamentsthateventuallylyse.FtsI,FtsL,andFtsQarenothomologousbuthavesimilaroverallstructures:asmallcyto-plasmicdomain,asinglemembrane-spanningsegment(MSS),andalargeperiplasmicdomainthatprobablyencodestheprimaryfunctionalactivitiesoftheseproteins.TheperiplasmicdomainofFtsIcatalyzestranspep-tidationandisinvolvedinthesynthesisofseptalpeptidoglycan.TheprecisefunctionsofFtsLandFtsQarenotknown.ToaskwhetherthecytoplasmicdomainandMSSofeachproteinserveonlyasamembraneanchororhaveinsteadamoresophisticatedfunction,wehaveusedmoleculargenetictechniquestoswapthesedomainsamongthethreeFtsproteinsandonemembraneproteinnotinvolvedincelldivision,MalF.InthecasesofFtsIandFtsL,replacementofthecytoplasmicdomainand/orMSSresultedinthelossoftheabilitytosupportcelldivision.ForFtsQ,MSSswapssupportedcelldivisionbutcytoplasmicdomainswapsdidnot.Wediscussseveralpotentialinterpretationsoftheseresults,includingthattheessentialdomainsofFtsI,FtsL,andFtsQhavearoleinregulatingthelocalizationand/oractivityoftheseproteinstoensurethatseptumformationoccursattherightplaceinthecellandattherighttimeduringthedivisioncycle.

TheformationofthecellseptuminthebacteriumEsche-richiacolitakesplaceatapreciseplaceinthecellandataprecisetimeinthedivisioncycle.Anumberofgeneshavebeenidenti?edthatappeartoplayessentialrolesinthisprocess,includingaclusteroffts(temperature-sensitive?lamentation)geneslocatedinthemin-2regionoftheE.colichromosome.TheftsZgeneencodesanabundantcytoplasmicproteinwhichformsaringnearthesiteofcelldivisionpriortotheformationofthecellseptum(2).TheftsAgeneencodesaperipheralmembraneproteinthatmayinteractwithFtsZ(9).Threegenesinthiscluster,ftsI,ftsQ,andftsL,codeforcytoplasmicmembraneproteinswithsimplestructures(4,7,18).Eachofthesethreeproteinshasasimilartopologicalorganization:anamino-terminalhydrophiliccytoplasmicsequence,asinglehy-drophobictransmembranesegment,andahydrophilicperiplasmicdomain(Fig.1).Proteinswiththistopologyhavebeentermedbitopicmembraneproteins.Allthreeproteinsaremadeinverysmallamounts,withestimatesrangingfrom25to150moleculespercell(1,7,11,18).

TheftsIgenecodesforapenicillin-bindingprotein(PBP),PBP3,whichexhibitsatranspeptidaseactivityrelatedtopep-tidoglycansynthesis(22).(WerefertothisproteinasFtsIthroughoutthispaper.)Thisactivityisencodedintheportionofthepolypeptidechainwhichislocalizedtotheperiplasm.MutationsandantibioticsthatinactivateFtsIpreventseptumformationbutdonotinterferewithcellelongation(38).TheenzymaticactivityofFtsIisthuspresumedtohavearoleinpeptidoglycansynthesisonlyduringseptumformation.FtsIislocalizedtothecellseptumatlaterstagesofcellgrowth(42).WhilemutationsintheftsLandftsQgeneshavephenotypesverysimilartothoseintheftsIgene,verylittleinformation

*Correspondingauthor.Phone:(617)432-1920.Fax:(617)738-7664.

?Presentaddress:GenomeTherapeuticsCorp.,Waltham,MA02154.

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aboutthefunctionsofthesetwoFtsmembraneproteinsexists.TheperiplasmicdomainofFtsLiscomposedofanaminoacidsequencewhichhasfeaturesofan?-helicalleucinezipper(18).PreliminarymutagenesisdataonthisdomainofFtsLareconsistentwithitsfunctioningasaleucinezipper,althoughnodirectevidenceforthisproposedrolehasbeenobtained(18a).Noinformationaboutthepossiblefunctionofanyofthedo-mainsofFtsQexists.

Weareinterestedinstudyingtheroleofthissetofmem-braneproteinsinthecelldivisionprocess.Aspartofthesestudies,weareseekingtodeterminethefunctionsofthedo-mainsofeachoftheproteinsthatarelocatedinthethreedifferentsubcellularcompartments—thecytoplasm,themem-brane,andtheperiplasm.Becauseoftheirsimplebitopicstruc-ture,theseproteinscanbereadilydissectedintotheircompo-nentparts.Studiesofmanymembraneproteinsinvolvedinothercellularprocesseshavepointedtoavarietyofdifferentpossiblerolesfortheirmembrane-spanningsegments(MSSs).Amongtheserolesare(i)membraneanchorsforthecytoplas-micand/orexporteddomainofaprotein(reference27andresultsforFtsQdescribedbelow),(ii)domainsforhomodimer-izationoftheprotein(24),(iii)domainsofinteractionwithMSSofothermembraneproteins(3,8),(iv)functionaldo-mainsinproteinsthatactaschannelsorporesinthemem-brane(20),and(v)signalsforsubcellularlocalization,forinstancetotheGolgicompartmentsineukaryoticcells(30,32).Thecytoplasmicdomainsofbitopicmembraneproteinshavealsobeenstudied.Ineukaryoticcells,sequencemotifsinthesedomainssometimesacttolocalizeproteinstospeci?csubcellularcompartmentssuchastheendoplasmicreticulumorthelysosome(17,31,37).Forbitopicmembraneproteinsthatactassignallingmolecules,thecytoplasmicdomainscanactivateregulatorycascadesorevendirectlyregulategeneexpression.Inothercases,thesequencesappeartoactonlyaspartoftheanchoringportionoftheprotein,ensuringitspropertopologyandarrangement.Inthesecases,replacingthe

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VOL.179,1997DOMAINSWAPPINGOFCELLDIVISIONMEMBRANEPROTEINS

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FIG.1.Membranetopologyofproteinsusedinthisstudy(seeintroductionfordetails).FromMalF,onlytheshortamino-terminalcytoplasmicdomainandthe?rstMSSwereused.ThesecanbedeletedwithoutaffectingthetransportactivityofMalF.a.a.,aminoacid.

domainwithanotherunrelatedbutsuitablesequencemayre-sultinaproteinthatisstillfunctional(35).

Inmanycases,theexportedportionofthisclassofmem-braneproteinsencodestheprimaryfunctionalactivity.Exam-plesarePBPsinE.coli(15)andahostofmembranereceptorsineukaryoticcells.

TodeterminetherolesofthevariousdomainsoftheFtsmembraneproteins,wehaveinitiatedanapproachinvolvingtheexchangeofdomainsbetweenthem.Inaddition,wehaveexchangedintotheseproteinsacytoplasmicand/orMSSdo-mainofaproteininvolvedinaprocesstotallyunrelatedtocelldivision.Fortheselatterconstructs,wehavechosenthemem-braneproteinMalF,whichisrequiredformaltosetransportinE.coli(14,21).Thesimpli?edrationalebehindthisapproachisasfollows.Replacinganyonedomainofaproteinwiththatofanotherandassayingtheprotein’sfunctionwouldindicatewhetherthesequenceofthatspeci?cdomainisrequiredforthefunctionoftheprotein.Ifanyofthedomainsarerequiredforsubcellularlocalizationtothecellseptum,thenthosedo-mainsmaybeexchangeableamongtheFtsproteins;however,theMalFdomainwouldnotsuf?ceforproperfunction.Ifanypairorifallthreeoftheproteinsformacomplexvia,e.g.,theirMSSs,thencertainclassesofexchangesmaycomplementeachother,eventhoughasingleexchangemayeliminatethefunc-tion.

Accordingtothissimplerationale,suchstudiescouldrevealtheimportantdomainsofaproteinandthusallowafocusforfurthergeneticandbiochemicalanalyses.Werecognize(seeDiscussion)thattheresultsofsuchananalysiswilllikelybemoredif?culttoevaluatethanthissimpledescriptionwouldsuggest.However,webelieve,asillustratedbytheresultsde-scribedbelow,thatthese?ndingsopennewavenuesofre-searchthatareimportantbothforunderstandinghowtheFtsproteinsfunctionandforunderstandingsomeimportantfea-turesoftheorganizationofmembraneproteinsingeneral.

MATERIALSANDMETHODS

Strains,plasmids,andmedia.ThestrainsandplasmidsusedinthisstudyarelistedinTable1.RichandminimalmediawereNZYandM63,respectively(18,19).Ampicillinwasusedat200?g/mlinrichmediumandat50?g/mlinminimalmedium.Kanamycinwasusedat60or100?g/mlinminimalmediumandat40?0.01%g/mlinleucinerichmedium.and0.005%Minimalisoleucinemediumforcontainedcomplementationthiamine(0.5oftemperature-?g/ml)andsensitive(Ts)mutants,butnotfortransductions.L-Arabinose(0.2,0.02,0.002,and0.0002%)orD-glucose(0.2%)wasaddedtomediumtomodulateexpressionofftsallelesclonedundercontrolofthePBADpromoter(pBADderivatives[19]).Inaddition,minimalmediumalwayscontained0.2%glycerol,whichservedastheprimarycarbonsourceonplatescontaininglowconcentrationsofarabinoseorglucose.

Complementation.(i)ComplementationofTsmutants.SwapconstructsandcontrolplasmidsweretransformedintostrainscarryingTsallelesofeitherftsIorftsQ,selectingforampicillinresistanceonNZYplatesat30°C.IsolatesweretestedforcomplementationbystreakingontoNZY-ampicillinandM63-ampi-cillinplateswithandwithoutarabinoseorglucose(tomodulateexpressionof

the

TABLE1.Bacterialstrainsandplasmidsused

Strainorplasmid

Relevantgeneticmarker(s)or

Sourceorfeature(s)a

reference

StrainsKS272F?38a??phoAlacX74(PvugalEII)

galKthirpsLLMG194KS272?ara714leu::Tn10

19

LMG64KS272ftsI23leu::Tn10recA::catThisstudyMJC129KS272ftsQ(Ts)recA::cat

7aMJC431KS272ftsI::TnphoAI137?IS50R7a(Kanr)/pLMG173(Ampr)LMG145KS272ftsL::TnphoAL81?IS50R18(Kanr)/pLMG180(Ampr)MJC295

KS272ftsQ::TnphoA80(Kanr)/7a

pLMG161(Ampr)

PlasmidspBAD18

Arabinoseregulation,Ampr19pBAD18-KanArabinoseregulation,Kanr19

pLGM130malFThisstudypLMG161ftsQThisstudypLMG173ftsIThisstudypLMG180ftsL

ThisstudypLD30IEag/IMsc/IThisstudypLD45LEag/LMsc/LThisstudypLD47FEag/FMsc/FThisstudypLD39I/IMsc/IThisstudypLD41IEag/I/IThisstudypLD43FEag/FMsc/IThisstudypLD55IEag/IStu/IThisstudypLD57FEag/IMsc/IThisstudypLD59LEag/IMsc/IThisstudypLD61QEag/IMsc/IThisstudypLD63FEag/LMsc/LThisstudypLD65IEag/LMsc/LThisstudypLD67QEag/LMsc/LThisstudypLD75I/IStu/IThisstudypLD88IEag/IMsc/LThisstudypLD90FEag/FMsc/LThisstudypLD92QEag/QMsc/LThisstudypLD94LEag/FMsc/LThisstudypLD96LEag/QMsc/LThisstudypLD98LEag/IMsc/L

ThisstudypLD100LEag/IMsc/L?IEag/LMsc/IThisstudypLD102IEag/IMsc/QThisstudypLD104FEag/FMsc/QThisstudypLD106LEag/LMsc/QThisstudypLD108QEag/FMsc/QThisstudypLD110QEag/IMsc/QThisstudypLD112QEag/LMsc/QThisstudypLD114QEag/QMsc/IThisstudypLD116LEag/LMsc/IThisstudypLD118IEag/FMsc/IThisstudypLD120IEag/QMsc/IThisstudypLD122IEag/LMsc/IThisstudypLD134FEag/QMsc/QThisstudypLD135IEag/QMsc/QThisstudypLD136LEag/QMsc/QThisstudypLD137

QEag/QMsc/QThisstudypLD139(Kanr)ftsI

ThisstudypLD140(Kanr)FEag/FMsc/IThisstudypLD141(Kanr)LEag/IMsc/IThisstudypLD142(Kanr)IEag/QMsc/IThisstudypBS120ss/Iperip4

pLD138ss/Qperip

ThisstudypMAL-c2MBPfusionvectorNewEnglandBioLabspDSW156MBP-FtsI(perip)ThisstudypLMG322MBP-FtsL(perip)

ThisstudypLMG182

PBAD-ftsL-phoAL81fusionThisstudy

a

ss,signalsequence;perip,periplasmicdomain.

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5096GUZMANETAL.plasmid-bornegenebeingtested)(19),andgrowthwasscoredafter18and36hat42°C.Identicalcontrolplateswereincubatedat30°C.ComplementationfortheftsI(Ts)andftsQ(Ts)mutantswasdoneonplatescontaining0.2%glucose,nosugar,and0.002,0.02,and0.2%arabinose.

(ii)Complementationofnullmutantsbytransduction.Swapconstructsweretestedforcomplementationbytransductionessentiallyaspreviouslydescribed(7,19).Brie?y,derivativesofLMG194(ftsL?ftsI?ftsQ?leu::Tn10)carryingplasmidsencodingtheswapallelestobetested(orcontrolplasmids)weretransducedtokanamycinresistanceandleucineprototrophy?onM63plates.ThelysateswerepreparedonLMG145(ftsL::TnphoALeu/pLMG180),MJC431(ftsI::TnphoALeu?/pLMG173),orMJC295(ftsQ::TnphoALeu?/pLMG161).Theplateswereincubatedat37°Candexamineddailyforatleast3days,bywhichtimecomplementingplasmidstypicallyyielded50to100transductantsperplate,whilenoncomplementingplasmidsyieldedfewerthan5transductantsperplate.Someofthetransductantsweremadephagefreeandanalyzed.Isolatesobtainedfromcomplementingplasmidsbredtrueandcontainedtheplasmidoriginallypresentintherecipient,asexpected,whereasisolatesobtainedfromnoncomplementingplasmidsoftengaverisetofaster-growingvariantsuponrestreakingandcontainedthefts?plasmidfromthedonorstrain.

ThedegreesofcomplementationofftsLandftsImutantsbyswapproteinswithP1transductionsweretestedonplatescontaining0.2,0.02,and0.002%arabi-nose,nosugar,and0.2%glucose.ForftsQ,0.2and0.002%arabinoseand0.2%http://wendang.chazidian.complementationwasobservedonlyat0.2%arabinoseforftsLandftsQ,butoverawiderangeofarabinoseconcentrationsforftsI.

Molecularbiologicalproceduresandoligonucleotides.StandardtechniqueswereemployedforcloningandanalysisofDNA,PCR,electroporation,andtransformation(36).EnzymesusedtomanipulateDNAwerefromNewEnglandBioLabs.DNAsequencingwasperformedfordouble-strandedplasmidtem-plateseithermanually(Sequenase;U.S.Biochemicals)orwithanautomatedsequencer(ABI).

Theoligonucleotides(5?33?)usedtomakethedifferentconstructionswereasfollows:1,pBAD18-5?(CTGTTTCTCCATACCCGTT);2,pBAD18-3?alt(GGCTGAAAATCTTCTCT);3,FEag3?(CATCGGCCGGCGTCGCTTTGCCACCAATG);4,FEag5?(GACGCCGGCCGATGGTCAGTGCTAGGTC);5,F3?(TATTCCCCTTGGCCATACATTAAAACA);6,FMsc7,IMsc5?(ATGTATGGCCAAGGGGAATACCTG);8,IEag3?(ACGGCCGCTGATAAAGTTGGCATG);Eag5?(ATCAGCGGCCGTTTTGCGTTGTTATGC);9,IIMsc3?(ACCACGCTACGTGGCCAAGCACAAAAG);10,IMsc5?(TGCTTGGCCACGTAGCGTGGTTACAA);11,IStu3?(ACGCTACGCGGAGGCCTCCCAGAAAAG);12,Stu5?(TGCTTGGCCTCCGCGTAGCGTGGTTACAA);13,L14,L?(CGATTCGGCCGGCEag3?(ACGGCCGCTGATAAAGTTGGCATG);TGCCACTCTGCCTG);15,LEag5(CCACTGGCCACCATACCCGTTTA);Msc3?(CGGTATGGTGGCCAGTGGTTACCAC);16,LMsc5?17,Q18,QEag3?(ACGGCCGCCATTATTGCGGCGAG);QEag5?(AATGGCGGCCGTCTGGCGGGGATCC);19,Msc3?(TGCGCATCTTGGCCAAGGCCCAACACG);20,QMsc5?(GGGCCTTGGCCAAGATGCGCAACGCC);21,FtsI?5?(CGTTTATCCTTATTTGAATTCTACGATATTTTC);22,MBP-LTTTACTGACC);and23,phoAss3?(TGATGGCCATTGGCTTTTGTCAper(CATCATACCCGCAGG).

Constructionofwild-typegenesunderP(malFBADcontrol.PlasmidsthatexpressswapproteinsstartingfrompLMG130),pLMG161(ftsQ),pLMG173(ftsI),andpLMG180(ftsL)wereconstructed.ThesearederivativesofpBAD18andexpressthegenesindicatedunderthecontrolofthearaBADpromoter(19).pLMG130wasconstructedbysubcloninga2.2-kbpEcoRI-HindIIIfragmentcontainingthewild-typemalFgeneandpartofthemalGgenefromplasmidpDHB32(6)intothesamesitesofpBAD18.ToconstructpLMG161,a931-bpEcoRI-KpnI(partialdigest)fragmentfromplasmidpMJC103(7)wasclonedintothesamesitesofpBAD18.TheEcoRIsiteis13bpupstreamoftheribosomebindingsite(RBS)forftsQ,andthesecondKpnIsite,attheendofthefragment,is42bpdownstreamofthestopcodon.ConstructionofpLMG173wasdoneintwosteps.First,aHpaI-NruI(partialdigest)fragmentencodingtheftsIgenefrompZ26::TnphoAL112(7)wassubclonedintotheSmaIsiteofpBAD18.Bothorientationsoftheinsertwereobtained,andaplasmidwithaninsertorientationthatplacedftsIunderPBADcontrolwaschosen.Second,byusingoligonucleotide21andsite-directedmutagenesis,anEcoRIsitewasintroduced9bpupstreamfromtheproposedRBSofftsI.Then,thesequencesupstreamofthisRBSweredeletedbydroppingouttheDNAbetweentheEcoRIsiteofthevectorandthenewEcoRIsite.ThisplacedftsIimmediatelydownstreamofthePpLMG180,whichBADpromoterandeliminatedallnon-ftsIsequences.Plasmidcontainsa412-bpfragmentencodingftsLunderPBADcontrol,hasbeendescribedelse-where(18).AllfourgenesclonedintopBAD18werecontainedbetweentheEcoRIandHindIIIsitesofthevectorandretainedtheirnativeRBSs.

Constructionofswaps.VersionsofthepLMGplasmidsdescribedabove,inwhicheachgenecontainedarestrictionsiteforEagIandMscI?ankingtheMSS,wereconstructedaccordingtothefollowingsteps(heredescribedforftsL)(seeFig.2).

(i)IntroductionofanMscIsiteatthe5?endoftheMSS.ThecytoplasmicdomainandMSSwereampli?edbyPCRwithprimers1(5?pBADprimer)and15withpLMG180asthetemplate.The5?pBADprimeralignstovectorse-quencesbeyondtheRBSofthegeneandtheEcoRIsiteofthevector.Primer15containsanMscIsiteatits5?endandintroducesthissitenearthe3?endofthePCRproduct.Theampli?edDNAfragmentwasmadebluntendwithT4DNA

J.BACTERIOL.

polymerase,digestedwithEcoRI,andthenligatedintopBAD18thathadbeendigestedwithEcoRIandSmaI.TheresultingplasmidwascalledpLD1.

(ii)IntroductionofanEagIsiteatthe3?endoftheMSS.Thecytoplasmicdomainwasampli?edbyPCRwithprimers1(5?pBADprimer)and13withpLMG180asthetemplate.Primer13introducedanEagIsitenearthe3?endofthePCRfragment.Theampli?edfragmentwasmadeblunt-endwithT4DNApolymerase,cutwithEcoRI,andligatedintopBAD18thathadbeendigestedwithEcoRIandSmaI.TheresultingplasmidwascalledpLD11.

(iii)IntroductionofboththeEagIandtheMscIsitesintothesamefragmentofftsL.TheMSSwasPCRampli?edwithprimer14,whichintroducedanEagIsiteatthe5?endoftheMSS,andprimer2(3?pBADprimer)withpLD1asthetemplate.ThefragmentobtainedwasdigestedwithEagIandSphIandligatedintopLD11thathadbeendigestedwiththesameenzymes.TheresultingplasmidwasnamedpLD16.

(iv)ConstructionofacompleteftsLgenecontainingboththeEagIandtheMscIrestrictionsites.TheperiplasmicdomainofftsLwasampli?edbyPCRwithprimers16and2withpLMG180asthetemplate.Primer16introducedanMscIsiteatthe5?endofthefragmentencodingtheperiplasmicdomain.ThefragmentobtainedwasdigestedwithMscIandHindIIIandligatedintopLD16thathadbeencutwiththesameenzymes.TheresultingplasmidwascalledpLD45,anditsgeneisreferredtoasLEagI/LMscI/L(i.e.,Lcyt/Lmss/Lperip)toindicatethepresenceoftherestrictionsitesattheendsofthecorrespondingdomains.LEagI/LMscI/Lwasfunctional,asjudgedbycomplementation,aswereIEagI/IQMscI/I,EagI/QConstructionMscI/Q,andFofEaggenesI/FMscwithI/F.

(v)swappeddomains.ChimericftsLgeneswereconstructedbyexchangingthefragmentsthatcorrespondtothecytoplasmicandMSSdomains.Forinstance,toconstructMSSswapsforFtsL,theEagI-MscIfragmentofpLD45,whichencodestheFtsLMSS,wassubstitutedbyEagI-MscIfragmentsderivedfrommalF,ftsI,andftsQtoproduceLandLEagI/FMscI/L,LIEagI/MscI/L,EagEcoI/QRIMscandI/L.Forcytoplasmicdomainsubstitutions,fragments?ankedbytheEagIsiteswereexchanged,andfordoublesubstitu-tions(cytoplasmicdomainandMSS),EcoRI-MscIfragmentswereexchanged.TheconstructionoftheL/I/LplusI/L/Idouble-swapplasmid(pLD100)wasachievedasfollows.PlasmidpLD98containingtheL/I/LswapwasdigestedwithXbaI,bluntended,anddigestedwithHindIII.BothofthesesitesareattheendsofthehybridL/I/Lgene,withtheXbaI/bluntsitebeingclosest.Then,anEcoRI/blunt-I/L/I-HindIIIfragmentfrompLD122wasclonedintothetreatedpLD98plasmid,placingtheI/L/Igene(withitsownRBS)justdownstreamofL/I/L.

CloningftsIswapsintopBAD18-Kan.Because?-lactamasedestroysthesub-strate?uoresceinhexanoic6-aminopenicillanicacid(FLU-CftsIandthreeswapalleles6-APA)usedtotestpenicillin-bindingactivity,wild-typeweresubclonedintoavectorthatconfersKanrratherthanAmpr.ThiswasaccomplishedbydigestingpLMG173(ftsI),pLD43(FEagI/FMscI/I),pLD59(LEagI/IMscI/I),andpLD120(ImentcarryingEagI/QtheMscftsII/I)withEcoRIandBsaI,gelisolatingthe2-kbpDNAfrag-allele,andligatingthisfragmentintopBAD18-Kan(19)thathadbeencutwiththesameenzymes.TheresultingplasmidswerenamedpLD139to142.

CloningofMBP-fusionproteins.(i)MBP-FtsI(periplasmicdomain).PlasmidpMAL-c2,whichallowsforfusionofproteinstothecarboxylterminusofasignalsequencelessmaltose-bindingprotein(MBP)derivative,wasdigestedwithEcoRI,madebluntendedbytreatmentwithKlenowfragmentinthepresenceofdeoxynucleosidetriphosphates,andcutwithHindIII.Thisvectorfragmentwasthenligatedtothe1.7-kbpMscI-HindIIIfragmentfrompLD30carryingtheperiplasmicdomainofFtsI.TheresultingplasmidwasconvertedfromAmprtoKanrbydigestionwithDraI,whichcutstwicewithintheblagene,andligatingina1.3-kbpfragment(BamHI,thenmadebluntendedwithKlenowfragment)conferringKanrfromplasmidpUC4K(fromPharmaciaBiotech).TheresultingplasmidwasnamedpDSW156.

(ii)MBP-FtsL(periplasmicdomain).DNAencodingtheperiplasmicdomainofftsLwasampli?edfrompLMG180byPCRwithprimers22and2.AftertreatmentwithT4DNApolymeraseandHindIII,theresulting210-bpfragmentwasligatedintopMAL-c2thathadbeendigestedwithXmnIandHindIII.Ampicillin-resistantcloneswerescreenedforexpressionofanMBP-FtsLfusionproteinbyWesternblottingwithantibodyagainstFtsL.Theresultingplasmid,pLMG322,producedafusionwithnopolylinker-encodedaminoacidsbetweenMBPandFtsL.

Antibodies.AntibodiesagainstsyntheticpeptidesderivedfromthesequenceofFtsI(FtsI-1,MKAAAKTQKPKRQEEHC;FtsI-2,CEPDALTTGDKNEFV)orFtsL(FtsL-1,CREQLVLERDALDIE;FtsL-2,CMQHVDPSQENIVVQK)wereraisedinrabbitsbyImmunoDynamics.FtsI-1correspondstothe?rst16residuesofthecytoplasmicdomain,andFtsI-2correspondstothelast14resi-duesofthemature(28)periplasmicdomain.FtsL-1andFtsL-2arederivedfromperiplasmicdomain;FtsL-1’ssequenceisclosetotheMSS,andFtsL-2corre-spondstothecarboxylterminusofFtsL.AstrongreactionoftheantibodiesagainstFtsIwasobservedafter4monthsofboostings,whileforFtsL,8monthswerenecessary.TheantibodyagainstFtsIwasaf?nitypuri?ed(34)againsttheisolatedperiplasmicdomainofFtsI.AntibodyagainstFtsLwassimilarlypuri?edagainstanMBP-FtsL(periplasmicdomain)fusionproteinexpressedbypLMG322.

Westernblotting.Proteinswereseparatedbysodiumdodecylsulfate-polyac-rylamidegelelectrophoresis(SDS-PAGE)andtransferredtonitrocellulose(36).

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ImmunodetectionwasperformedwithantipeptideantibodiesagainstFtsIorFtsLandtheECLWesternblottingsystem(Amersham).Forquantitation,?lmwasscannedwithacomputingdensitometer(MolecularDynamics).

Todeterminethesteady-statelevelsofexpressionofFtsIswapproteins,derivativesofKS272orLMG64carryingappropriateplasmidsweregrownat30°CinNZY-ampicillintoanopticaldensityat600nm(ODinto42°CNZY-ampicillincontaining600)of?0.5.Cultureswerethendiluted?vefoldarabi-nosetoinduceexpressionoftheplasmid-borneswapallele,ifany.After1h,theOD600hadreturnedtoabout0.5,andmicroscopicexaminationofLMG64derivativesrevealedextensive?lamentation(exceptinthecasesofpLMG173andpLD30).Onemilliliterofcellswaspelletedbycentrifugationandresus-pendedin100?lofSDS-samplebufferperODmin,and10?lwasloadedontoa10%600unitof0.5.Sampleswereboiledfor5polyacrylamide-SDSgel.Thesteady-statelevelsofFtsLswapproteinsweredeterminedsimilarly,ex-ceptthatcultureswerealwaysgrownat37°C,andtheswapconstructsweredetectedinftsL?cellsonly(KS272andLMG194).Cellscontainingswapsorcontrolplasmids(pLMG180,pLD45,andpBAD18)andcellscontainingthechromosomalinsertionmutationcomplementedbypLMG180weregrowntomid-logphaseinNZY-ampicillin(plus0.2%arabinoseinthecaseofthenullmutant).Cellswereharvested,resuspendedinfreshmediumwith0.2%arabi-nose,anddiluted10-foldintoNZY-0.2%arabinose;afractionofthecultureofthenullmutantwasresuspendedandgrowninNZY-0.2%glucose.After2.5h,thenullmutanthadformed?lamentsinthepresenceof0.2%glucosebutshowednormalcellsandef?cientcomplementationinthepresenceofarabinose.Atthistime,1mlofcellswaspelletedbycentrifugationandresuspendedin100?10lof?lSDS-samplewasloadedontobufferaper15%ODpolyacrylamide-SDS600of0.5.Sampleswereboiledfor5min,andgel.

DetectionofFtsIwithFLU-Cconcentration6-APA.FLU-Cin506-APAwassynthesizedasde-scribedelsewhere(33),anditsmMsodiumphosphatebuffer(pH7.0)wasdeterminedbyusingE494in?NZY-kanamycin,68mM?1astheextinctioncoef?cient.Thestrainsweregrownovernightinoculated1:500intofreshmedium,andgrownat37°CtoanODplasmid-borneFtsIprotein(ifany)wastheninduced600of?0.5.Expressionofthebyaddingarabinosetoa?nalconcentrationof0.002%,exceptforMBP-FtsIexpressedfrompDSW156,whichwasinducedwith50?Misopropyl-?-D-thiogalactopyranoside.Onehourafterinduction,thecellswereharvestedbycentrifugationandresuspendedinspheroplastingbuffer(50mMTris-HCl[pH8.0],5mMNasucrose).OnemilliliterofcultureatanOD2EDTA,500mMculturesatotherODs,thevolumeofbuffer600of1.0wasresuspendedin50?l;forwasadjustedaccordingly.

SpheroplastingbufferrenderedtheoutermembranepermeanttoFLU-CAPA.Forroutineassays,FLU-Cwasaddedtoa?nalconcentrationof206-?withdrawnM,andassayatvariousmixturestimes,wereaddedincubated6-APAtoanatequal30°Cvolumefor30ofmin.2?SamplesLaemmliweregel-loadingbuffertoquenchthereaction,andboiledfor5min.A10-?lvolumewasloadedontoa10%polyacrylamide-SDSgel,andproteinswereseparatedbyelectrophoresis.ProteinslabelledwithFLU-C6-APAwerevisualizedandquan-titatedwithaMolecularDynamicsFluorImager575.AsecondgelwasruntodeterminethelevelofFtsIbyWesternblotting.SamplesforWesternblottingweregenerallydilutedby10-to100-fold.

FortheexperimentshowninFig.5,proceduresweremodi?edtoallowlysisofthecells.Cellsresuspendedinspheroplastingbufferwereconvertedtosphero-plastsbyincubationoniceinthepresenceof0.2-mg/mllysozymefor20min.FLU-C6-APAwasthenaddedtoa?nalconcentrationof20?M,andsphero-plastswereincubatedat30°Cfor20min,atwhichtimeasamplewastakenandaddedto2?Laemmligel-loadingbuffertoassessbindingofFLU-Cintactspheroplasts.Theremainingspheroplastswere6-APAtoFtsIderivativesinthenpelletedinaMicrofuge,thebuffercontainingFLU-Cwerelysedbyresuspensionin50mM6-APAwasdiscarded,andthespheroplastsTris-HCl(pH8.0)–5mMMgCl2.Afterincubationfor5minat30°Cinthepresenceof1-?g/mlDNaseItoreduceviscosity,anequalvolumeof2?spheroplastingbufferwasadded,followedbyFLU-C6-APAtoa?nalconcentrationof20?M.Samplesweretakenafter10and20minofincubationat30°CtoassessbindingofFLU-Clysedspheroplasts.

6-APAtoFtsIderivativesinRESULTS

Assemblingswapconstructs.InplanningtostudyFtsswapconstructs,weconsideredithighlydesirabletobeabletocontrolthelevelsofexpressionofthehybridproteinsinouranalysisoftheirfunctionality.Ontheonehand,itseemedimportanttobeabletoexaminetheabilitiesofsuchconstructstocomplementftsmutantswhentheyareexpressedatlowerlevelsthatareatleastsomewhatcomparabletowild-typelev-els.Ontheotherhand,someconstructsmightexhibitonlypartialbutsigni?cantfunctionalitythatwouldbeobservedonlyathighexpressionlevels.Furthermore,sincemembranepro-teinsareoftendeleterioustothecellathighlevelsofexpres-sion,tightcontroloftheirexpressioncouldalsobecrucialfor

FIG.2.Constructionofplasmidsencodingproteinswithswappeddomains.Numbers1through5correspondtothe?vestepsdescribedinMaterialsandMethods.cyt,cytoplasmicdomain;mss,MSS;perip,periplasmicdomain.

thisanalysis.Finally,dominant-negativeeffectsoftheswapsmightbeobservedonlyathigherlevelsofexpression.There-fore,allswapconstructsweremadestartingwiththemalFandftsgenesclonedintoaplasmidcontainingthetightlyregulatedpromoterforthearabinoseoperon.TheplasmidthatweusedwaspBAD18asdescribedbyGuzmanetal.(19).

Wewishedtoobtainconstructsinwhichoneortwodomainsofeachmembraneproteinwereswappedwithanother.ForeachoftheftsgenesclonedintopBAD18,weintroducedrestrictionsitesatpositionsinthegene’sDNAsequencecor-respondingtotheamino-terminalandcarboxy-terminalbor-dersofthepresumedtransmembranesegmentusingPCR(Fig.2).(Itshouldberememberedatthispointthat,exceptfortheveryfewmembraneproteinsforwhichthree-dimensionalstructuresexist,thede?nitionoftheexactbordersoftrans-membranesegmentsisbasedonlyonsequenceexaminationandtherulesthathaveevolvedforpredictingtheseborders.)FormalF(6,14),aproteinwitheightMSSs,weintroducedtheserestrictionsitesatthebordersofthe?rstMSSs.ThisMSScanbedeletedwithoutaffectingthetransportactivityofMalF(12).Inplanningtheseconstructs,wealsoattempted

to

基金结构域互换

5098GUZMANETAL.FIG.3.SequenceofcytoplasmicdomainsandMSSofMalF,FtsI,FtsL,andFtsQ.MSSsareboxed.Boldfaceletterstothetopoftheproteinsequencesindicateaminoacid(a.a.)changesresultingfromintroductionofEagIandMscIrestrictionsitesbeforeandaftertheMSS,respectively.Positivelyandnegativelychargedresiduesarealsoindicated.Onlythe?rstMSSofMalFwasusedinthis

study.

ensurethatthechargedistributionateachendoftheMSSsegmentcorrespondedtothatnecessarytomaintaintheorig-inaltopologyoftheseproteins.Thatis,basicaminoacidsattheaminoterminusoftheMSSandacidicresidues(oranetneutralcharge)atitscarboxyterminusaredeterminantsoftheappropriateorientation(5,25,39,41).RelevantaminoacidsequencechangesencodedbytheintroductionofrestrictionsitesintothesegenesareshowninFig.3.

Afterconstructionofthevariousswaps,weveri?edtheirstructuresbyrestrictionmappingandbyDNAsequencingofallgenesegmentsmadebyPCR.Weuseasimplenomencla-turetodescribeeachswapconstruct.Eachswapisdesignatedbythreeletters,suchthatthe?rstletteridenti?estheproteinwhosecodingregioncorrespondstotheaminoterminus(thecytoplasmicdomain)oftheprotein,thesecondcorrespondstotheproteinfromwhichtheMSSwasderived,andthethirdrepresentstheproteinfromwhichtheperiplasmicdomainwasderived.Thus,QLLhasthecytoplasmicdomainofFtsQandtheMSSandperiplasmicdomainsofFtsL.

Expressionandtopologyofswapconstructs.WetestedourFtsIandFtsLconstructstoensurethateachofthemexpresseditsswapprotein.Forproteinscontainingtheperiplasmicdo-mainsofFtsI,wefoundthatallconstructsexpressedsubstan-tiallevelsoftheproteinsasassayedbyWesternblots(Fig.4).Theamountsofproteinvariedaccordingtotheconstructs.Thisvariationwasprobablyduetothedifferingef?cienciesofthetranslationstartsignalsfortheparticularproteinfromwhichtheaminoterminusofaswapderived.WenoteherethatswapconstructsinwhichtheFtsQamino-terminaldomainprecededsequencesfromoneoftheotherFtsproteinscon-sistentlyexpressedlevelsofhybridproteinlowerthanthoseseenwithotherswaps.These?ndingssuggestthattheFtsQtranslationstartsignal,atleastintheseconstructs,isweakerthanthoseofFtsIandFtsL(Fig.4).PreviouslypresentedresultsindicatedthatFtsQhasaweaktranslationstartsignal(26).

ForFtsL,mostoftheconstructsexpressedproteindetect-ableinWesternblotswithanti-FtsLantibody.However,wehaveneverobservedanyproductfromQQL,QLL,orwild-typeLinblots.ForIILandLLL,cross-reactingproteinwasseeninoneofseveralblotsthatweperformed.Theanti-Lantibodydoesnotappeartobeveryeffective,sinceitisalsounabletorevealchromosomallyexpressedFtsLinblots.ThefailuretodetecttheconstructswithaQaminoterminusisperhapsnotsurprising,since,asmentionedabove,suchcon-structsareexpressedatlowerlevelsingeneral.Thefailuretodetectwild-typeFtsLexpressedfromtheplasmidismoresur-prising.However,sincethisplasmideffectivelycomplements

J.BACTERIOL.

FIG.4.Steady-statelevelsofFtsI(A)andFtsL(B)swapproteinsasdeter-minedbyWesternblotting.Molecularmassstandardsareindicatedattheleftofeachblot.Theproteinsexpressedareindicatedaboveeachlane.none,cellscarryingthevectorpBAD18withoutanyinsert.InthecaseofFtsI,vectoralonewasexaminedinboththeTsmutantLMG64(farleft)andwild-typestrainKS272(farright)toallowthesteady-statelevelsofchromosomallyexpressedTsandwild-type(Wt)FtsIproteinstobecomparedwiththelevelsofexpressionofswapproteins.AllFtsIswapswereinducedwith0.002%arabinose,exceptforQQIandQII,whichwereweaklyexpressedandwereinducedwith0.2%arabinose.AllFtsLswapsandtheFtsL81-PhoAfusionproteinwereinducedwith0.2%arabinose.ThelanemarkedLIL?ILIwasincludedbecausethiscombinationofthetwoswapswastestedtoseewhethertheywouldcomplementanFtsLdepletionstrainwhenexpressedinthesamecell.Theydidnot.

anftsLnullmutationandfusionsofphoA(encodingalkalinephosphatase)toftsLinthisplasmidgivehighlevelsofalkalinephosphataseactivity(seebelow),FtsLisobviouslyexpressed.Thealkalinephosphataseactivitylevelsoffusionstowild-typeFtsLandtotheFtsLengineeredwithrestrictionsitesinthegenearesimilartothoseoffusionsderivedfromswapsforwhichwecanreadilydetectprotein(datanotshown).Theinabilitytodetectwild-typeFtsLintheseexperimentsraisesthepossibilitythatwild-typeFtsLisunstablewhiletheswapconstructsarestable.

AlthoughwedonothaveantibodytoFtsQ,sinceallofourFtsQswapconstructscomplementedanftsQtsmutant,theymustbeexpressed(Table2andbelow).Wespeci?callyde-signedtheswapconstructssoastomakeitlikelythatthetopologyofeachproteinwouldbesuchthattheperiplasmicdomainoftheoriginalproteinwasmaintainedintheperiplasm.Nevertheless,wetestedanumberoftheFtsIswapstoverifythisoutcome.ThereasonforfocusingonFtsIis

that