themembraneandperiplasmicspace,whichshiftsthecellintoastatethatstimula - PDF document

themembraneandperiplasmicspace,whichshiftsthecellintoastatethatstimulatesandfuelsoxygenradicalgenerationandultimatelyresultsincelldeath.Importantly,wealsodemonstratethattheenvelopestress-responseandredox-responsivetwo-componentsystemsarebroadlyinvolvedinbactericidalantibi-otic-mediatedoxidativestressandcelldeath,providingaddi-tionalinsightintothecommonmechanismofkillinginducedbybactericidalantibiotics.GeneExpressionAnalysisofPathwaysRelatedtoAminoglycosideLethalityWeutilizedgeneexpressionmicroarraysandstatisticalanalyses(seetheExperimentalProcedures)tocomparegeneexpressionprolesofwild-type(MG1655)E.colitreatedwiththeaminogly-cosidegentamicintoexpressionprolesaftertreatmentwiththebacteriostaticribosomeinhibitorspectinomycin(TableS1ableonline).Statisticallysignicantchangesinexpression(zscore)weredeterminedonagene-by-genebasisbycompar-isonofmeanexpressionlevelstoalarge(525)compendiumofE.colimicroarraydatacollectedunderawidevarietyofcondi-tions(seetheExperimentalProcedures).Wethenexaminedtherelativechangesinzscorebetweenbactericidal-andbacterio-static-treatedsamples.Thisallowedustoseparategeneexpres-sionchangesrelatedtoaminoglycosidelethalityandhydroxylradicalformationfromaminoglycosideeffectsoncellgrowth.Toidentifynetworksrelatedtoaminoglycosidelethality,wel-teredthesetofsignicantlychanginggenesthroughanE.coligeneconnectivitymapgeneratedusingtheelatedness(CLR)algorithm(Faithetal.,2007).AmongtheCLR-predictedconnections,onlythosebetweengenesthatweresignicantlyperturbed(onthebasisofzscorechanges)andconnectionswhereonenonperturbedgeneconnectedtwosignicantlyperturbedgenesweremaintained.Thisallowedus Figure1.IdentiÞcationofPathwaysRelatedtoAmi-noglycosideLethalitySignicantlychanginggenesfromacomparisonoftreatmentwithalethalaminoglycosideversusthebacteriostaticribo-someinhibitorspectinomycin,werelteredthroughanE.coligeneconnectivitymapgeneratedwiththehoodofelatednessalgorithm(Faithetal.,2007);theresultantgenenetworkswereanalyzedforfunctionalenrichment.Thestrainsexhibitingdecreasedgrowthorincreasedgrowthfoundfromahigh-throughputscreenofanE.coligenedeletionlibrarytreatedwithgentamicinwereoverlaidonthesenetworks.toextractgroupsoffunctionallyrelatedgenes(ure1)andgaveusgreaterabilitytofocusonspe-cicpathwaysrelatedtoaminoglycosidemodeofaction.Wealsoexaminedpreviouslycollectedex-pressionproles(Kohanskietal.,2007)aftertreat-mentwiththeaminoglycosidekanamycinandcomparedthesetoexpressionprolesaftertreat-mentwithspectinomycin.Amongthegenenet-worksidentied,thereweresixincommonbe-tweengentamicinandkanamycintreatment(Figure2FigureS1),andthesenetworkswereanalyzedforpathwayandtranscriptionfactorenrichment(seetheExperimentalProce-TableS2).Inadditiontoourgeneexpressionanalysis,wealsoutilizedahigh-throughputscreenofanE.coligenedeletionlibrary(Babaetal.,2006)treatedwithgentamicinTableS3;seetheExperimentalProcedures)toidentifypotentialgenetargetswithincreasedsensitivitytogentamicin(TableS4Thegenenetworks(Figure2FigureS1)werefurtheren-richedwiththegrowthdataderivedfromthehigh-throughputscreenoftheE.colisingle-genedeletionlibrary.Amongthenetworksrelatedtoaminoglycosidelethality,oneFigure2A)showedenrichment(p10)forArcA-regulatedel-ementsoftheelectrontransportchain,thetricarboxylicacid(TCA)cycle,andrespiration.ArcAispartoftheArctwo-compo-nentsystem.Ingeneral,two-componentsystems,whichtakepartinimportantbacterialprocessessuchasthecellcycleandvirulence(Hoch,2000),consistofasensorproteinandacog-natetranscriptionfactor;theactivityofthetranscriptionfactorismodulatedbythesensorprotein.TheArctwo-componentsys-temconsistsofaquinone-sensitivesensorkinase,ArcB,whichrespondstotheredoxstateofthecellularquinonepoolbyreg-ulatingthephosphorylationstateofthetranscriptionfactor,ArcAGeorgellisetal.,2001;Malpicaetal.,2004).Inaddition,ArcAac-tivationleadstochangesinexpressionofmanygenesinvolvedinrespirationandmetabolism(LiuandDeWulf,2004WehavepreviouslyshownthatdeletionofsomeoftheTCAcyclegenesinthismetabolism-relatednetwork(Figure2A)at-tenuatebactericidalantibiotic-mediatedcelldeath(etal.,2007).Manyofthesesingle-deletionknockoutsexhibitin-creasedgrowthinthepresenceofgentamicin(Figure2A).Inad-dition,wefound,viaqPCR,thatthevastmajorityofArcA-regulatedTCAcyclegenestestedexhibitedasignicantspikeingeneexpressionwithintherst30minafterexposuretogen-tamicin(FigureS2A).Importantly,thechangesinexpression 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membraneproteins,whichwouldhaveacumulativeeffectontheelectrochemicalpotentialandintegrityofthemembrane.Aminoglycoside-InducedMistranslationofMembraneProteinsInducesHydroxylRadicalFormationandCellOnthebasisofourexpressionanalysis,wefocusedourattentionongenesandpathwaysassociatedwithdelityofmembraneproteinstodeterminewhetherthesesystemsarefunctionallylinkedtoaminoglycoside-inducedoxidativestressandlethality.Ananalysisofourhigh-throughputgrowthscreen(TableS3)re-vealed11single-geneknockoutsexhibitingsignicantde-creasesingrowthaftergentamicintreatment(TableS4).Amongthese11genes,threeofthegeneproducts,HK,HC,andSecG,areinvolvedintheregulationofmembraneproteins,andtwoofthegenes,,showasignicantincreaseinexpres-sionafteraminoglycosidetreatment(TablesS5andS6E.coli,proteintrafckingthroughtheinnermembraneoc-cursviamultiplepathways,includingtheSecandsignalrecog-nitionparticle(SRP)translocationsystems.TheSecandSRPpathwaysutilizeachaperone-basedsystemtoshuttleimmatureproteinstotheSecYEGtranslocaseproteincomplexfortrans-portacrosstheinnermembrane(DaneseandSilhavy,1998b;Economou,1999;LuirinkandSinning,2004;MoriandIto,2001;WicknerandSchekman,2005).Immatureproteinsaretar-getedtotheSectranslocase(SecY,SecE,andSecG)bychap-erones(SecBortheSRPsystem)andprocessedthroughtheSecYEGtranslocasebythetranslocationATPaseSecA(mou,1999;Hartletal.,1990;LuirinkandSinning,2004;MoriandIto,2001).SecGworksinconjunctionwithSecAtopromoteef-cientproteintranslocation(Matsumotoetal.,1998).HKandHCtogethernegativelyregulateFtsH(Kiharaetal.,1996,1997;Saikawaetal.,2004),andlossofHKorHCshoulddi-minishtheabilityofFtsHtodegrademembrane-associatedpro-teins,includingSecYandAkiyamaetal.,1996;Gottesman,1996;Kiharaetal.,1999Todeterminewhethertranslocationofmistranslatedproteinsacrossorintotheinnermembranespecicallyenhanceskillingbyaminoglycosides,weinitiallyfocusedonthreesingle-gene,and.Wetreatedthemwiththemistranslation-inducingaminoglycosidegentamicin,theDNAgyraseinhibitornoroxacin,thecellwallsynthesisinhibitorampi-cillin,andthebacteriostatic,mistranslation-freeaminocyclitol,spectinomycin(seetheSupplementalDataforresultswithspec-tinomycin),respectively.Additionally,weexaminedsurvivalatbothaclinicallyrelevantlevelofgentamicin(5g/ml)andatasub-stantiallyhigherconcentration(15g/ml)(Zaskeetal.,1982,andtreatedwith5g/mlgentamicineachexhibitedasignicantincreaseinboththerateofkillingandover-allcelldeathrelativetowild-typeE.colitreatedwithgentamicinFigure3A).Incontrast,whenthesesingle-deletionstrainsweretreatedwith100ng/mlnoroxacin(Figure3B)or3g/mlampicil-lin(Figure3C),cellkillingwasquitesimilartothatseenwiththewild-type.Thisdemonstratesthattheincreaseincelldeathob-servedin,andisspecictothelethalmech-anismofactionofaminoglycosides.Treatmentofwithahigherconcentrationofgentamicin(15g/ml)sig-nicantlyreducedthedifferenceinviabilitybetweenthesestrainsandthewild-type(FigureS4).Itisworthnotingthattheremainderofthesingle-genedeletionsidentiedinTableS4didnotdisplayincreasedefcacyofkillingbygentamicin(FiguresS5andS6;seeSupplementalDataforfurtherdiscussion).SecGhasaknownroleinpromotingefcientproteintransloca-tion(Matsumotoetal.,1998),whereastheHKCcomplexregu-latesFtsH,therebyaffectingthedegradationofmembrane-asso-ciatedproteins(Akiyamaetal.,1996;Gottesman,1996;Kiharaetal.,1999).ChangestotheseSecG-orHKC-regulatedfunc-ofmistranslatedproteinsacrossthemem-brane,whichmayaccountfortheobservedincreaseincelldeathFigure3A)afteraminoglycosidetreatmentof,or;seetheSupplementalDataforfurtherdiscussion.Weexaminedhowthesesingle-genedeletionsaffecthydroxylradicalformationandmembranedepolarizationtodeterminewhetherthereisarelationshipbetweenthesephenomenaandanincreaseintranslocationofmistranslatedproteinsacrosstheinnermembrane.Bothradicalformation(Kohanskietal.,)andchangesinmembranepotential(BryanandKwan, Figure3.DisruptingMembraneRegulatorySystemsEnhancesAminoglycosideLethalityPercentsurvivalofwild-typeE.coli(blacksquares),(redcircles),(greentriangles),and(bluetriangles)aftertreatmentwith5g/mlgenta-micin(A),100ng/mlnoroxacin(B),or3g/mlampicillin(C).MeanSEMareshownforallgures. ,679–690,November14,20082008ElsevierInc. 1983;Taberetal.,1987)havebeenassociatedwithaminoglyco-side-mediatedlethality.Consistentwiththeincreaseinkillingef-cacyandtrafckingofmistranslatedmembraneproteinsafteraminoglycosidetreatment,wefoundthattherewassignicantlymorehydroxylradicalformation[measuredwiththedye3hydroxyphenyl)uorescein(HPF)(Setsukinaietal.,2003)]in,orthaninthewild-type(Figure4A).Inaddi-tion,hydroxylradicalformationoccurredby1hrafteradditionofgentamicininthesethreemutants,comparedto2hrinthewild-type(Figure4Ifaminoglycoside-inducedhydroxylradicalformationisinfactrelatedtodisruptionoftheelectrochemicalgradientbe-causeofimpropertrafckingofcorruptmembraneproteins,thechangesinmembranepotentialassociatedwithaminogly-cosidetreatmentshouldreectchangesinhydroxylradicalfor-mation.Weobservedagradualincreaseinmembranedepolar-ization[measuredwiththedyeDIBAC(3),whichdiffersfromuorescentintercalatingdyesinthatitcandiffuseacrossdepo-larizedyetintactcellmembranes(Jeprasetal.,1997)]inwild-E.coliby2hr(Figure4B),whichwasconsistentwiththe Figure4.DisruptionofMembraneRegula-torySystemsandtheEnvelopeStressRe-sponseEnhancesAminoglycoside-InducedHydroxylRadicalFormationandMembraneDepolarization(A–D)Fluorescenceforeachstrainrelativetothemaximumuorescenceachievedinthewild-typebackgroundwiththehydroxylradical-detectingdyeHPF(AandC)orthemembranedepolarizationdyeDIBAC(3)(BandD).Hydroxylradicalforma-tion(A)andmembranedepolarization(B)ofwild-typeE.coli(blacksquares),(redcircles),(greentriangles)and(bluetriangles)aftertreatmentwith5g/mlgentamicinareshown.Hydroxylradicalformation(C)andmem-branedepolarization(D)ofwild-typeE.colisquares),(reddiamonds),(greendi-amonds),(reddiamonds),andcircles)aftertreatmentwith5g/mlgentamicinareshown.(E)Fold-changegeneexpressionrelativetobase-line(t=0min)oftheenvelopestress-response(sqaures),and(triangles),forwild-typeE.coli(brown),andDcpxA(green)aftertreatmentwith5gentamicin.onsetofcelldeath(Figure3A)andhy-droxylradicalformation(Figure4,andallexhibitedasignicantincreaseinmembranede-polarizationby1hr(Figure4B),whichcorrelateswiththesignicantincreaseinhydroxylradicalformationbeyondwild-typelevels(Figure4A).Thespikesindepolarizationobservedat1hrfor,andFigure4suggestamorecomplexroleformem-branepotentialasrelatedtomembraneproteintrafckingandtheincreasesincelldeathandradicalInvolvementoftheEnvelopeStress-ResponseandRedox-ResponsiveTwo-ComponentSystemsinDrug-MediatedCellDeathOurresultswith,andpointtowardarelation-shipamongaminoglycoside-inducedchangesinmembraneproteintranslocation,membranedepolarization,andhydroxylradicalformationresultinginrapidcelldeath.Earlychangesinmembranedepolarizationmayreectchangesintheredoxstateofthecellthatareregulated,inpart,bychangesinexpressionofArcA-regulatedmetabolicgenes(FigureS2).Inaddition,mem-branedepolarizationmayalsoreectchangesinmembranepropertiesbecauseofaminoglycoside-inducedtrafckingofmistranslatedproteinsacrosstheinnermembrane.Onceproteinsaretranslocatedacrossthemembrane,severaloverlappingcellenvelopemaintenanceandstress-responsesystems(includingtheCpxenvelopestress-responsetwo-com-ponentsystem)areresponsibleforpeptidequalitycontrol 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triggerforaminoglycoside-inducedhydroxylradicalformationFigures7C–7E).Two-componentsystemsareparticularlyusefulinfasttransitionsbetweendifferentenvironments(Hoch,2000anditisinterestingthatafast,protectiveresponsetomisfoldedenvelopeproteinsstimulatedbyCpxAactivationalsoleadstohydroxylradicalformation(Figure7).Intuitively,wewouldexpecttheobservedupregulationinexpressionoftheperiplasmicpro-Figure4E),tobeaprotectiveresponseagainstcelldeathrelatedtomistranslationandaccumulationofcorruptperi-plasmicproteins.However,removalofandtheenvelopestress-responsetwo-componenttranscriptionfactor,,ledtoadecreaseinradicalformationandasignicantdelayintherateofkillingafteraminoglycosidetreatment(Figures5Aand5B).TheCpxsystemiscontrolledbyatightfeedbackloopthatallowsforfastswitchingbetweenanonandoffstate(etal.,1999).ThisfeedbackmechanisminvolvestheCpxR-regu-latedperiplasmicproteinCpxP(DaneseandSilhavy,1998aCpxP,whichisdegradedbyDegP,negativelyregulatestheac-tivityofCpxA(BuelowandRaivio,2005).Itispossiblethatin-creaseddegradationofCpxPintheperiplasmicspacebyDegPrelievesCpxP-mediatedrepressionofCpxAandenhancestherateofsignalingviaCpxA,ultimatelyleadingtohydroxylrad-icalformation(Figures7C–7E)throughamechanismthatlikelyinvolvesArcA.Ourdata(Figure5)alsosuggestthatoxidativestresstriggeredbyincorporationofmistranslatedproteinsinthecellenvelopeandthefastswitchingcapabilitiesofthetwo-componentsystemsarecriticalfortheinitial,rapid,radical-dependentkillingafteraminoglycosidetreatment.Importantly,ourresultspointtowardabroadrolefortheredoxresponseandenvelopestress-responsetwo-componentsys-temsinbactericidalantibiotic-inducedcelldeath(Figures7and7E).ItislikelythattheroleofArcAhereisthroughmodulationofcellularmetabolism,andadditionaleffortsarerequiredforde-terminationofhowArcA,togetherwithotherregulatoryelementsincludingtranscriptionfactorsandmetabolitecofactors,affectsmetabolismafterantibioticexposure.ArcA-mediatedchangesinmetabolismandrespirationarealsolikelytobeimportantinthecommonoxidativedamagecelldeathpathwayafterexposuretobactericidalantibiotics(Kohanskietal.,2007).TheinvolvementoftheCpxsysteminantibiotic-mediatedcelldeathshowsthatmembraneintegrityiscrucialforbacterialsurvival.Inthecase-lactams,membraneintegrityisaffecteddirectlybythedrug-targetinteraction,whereaswithaminoglycosides,mem-branefunctionandintegrityappearstobemostaffectedbydrug-inducedproteinmistranslation.Therelationshipbetweenquinoloneantibioticsandthemembraneislessclear.TreatmentwiththeDNAgyraseinhibitornalidixicacidhasbeenassociatedwithchangesintheproteintolipidratiointhecellenvelopeDoughertyandSaukkonen,1985).Quinolonesdoinducela-mentation,anditispossiblethatchangesinmembranecompo-sition,function,andintegritybroughtaboutbyanincreaseincellsizeaffectCpxsignalingandcelldeath.Aminoglycoside-inducedmistranslationinE.coliisspeci-callyduetotheinteractionofthedrugwiththeribosomeFigure7A).Variabilityinthestructureoftheribosomemayaffecttheinductionofmistranslationbyaminoglycosidesandmayalsoinducemistranslationbyotherclassesofribosomeinhibitors.In-terestingly,ribosomalRNAsequencesvaryamongbacterialspecies,andsomeribosomeinhibitorsarebacteriostaticincer-tainspeciesandbactericidalinothers.Forexample,chloram-phenicolisbacteriostaticagainstE.colibutbactericidalagainstHaemophilusinßuenzaRahalandSimberkoff,1979).Itispossi-blethatthevariabilityinribosomalRNAsequencescausessomeribosomeinhibitorstoinducemistranslationandsubsequentmisfoldingofmembraneproteins,therebyloweringthethresholdforhydroxylradicalformationandcelldeathincertainbacterialspecies,whereasinotherspeciesthesesameribosomeinhibi-torsdonotinducemistranslationandaremerelybacteriostatic.Adeeperunderstandingofthislethaltriggermayallowustocon-vertbacteriostaticribosomeinhibitorsintobactericidaldrugsbyidentifyingtargetsthatinduceproteinmistranslationandtherebytriggerradical-basedcelldeath.ThemembraneregulatoryproteinsHKCandSecGFigure7B),knockoutsofwhichwehaveshownenhanceamino-glycosidelethality,interactwithessentialproteins(FtsHandSecE/SecY,respectively)butarenotessentialthemselves).Additionally,SecGisnotconservedoutsideofbacteria,whereasSecEandSecYarewellconservedinhumans(Economou,1999).Earlierworkonglycolipidderivatesofvancomycin(Eggertetal.,2001)hasshownthattargetingnonessentialgenesisaviableoptionforen-hancingantibioticefcacy.Ascombinationtherapyapproachesareexploredfurther(CottarelandWierzbowski,2007),itispos-siblethatnonessentialregulatorsofessentialproteinswillturnouttobehigh-qualitydrugtargetsforpotentiationofknownantibacterialdrugs.Aminoglycosidesareapowerful,broad-spectrumclassofan-tibioticswithexcellentactivityagainstcommoninfectionsinvolv-ingGram-negativebacteria.However,thisclassofdrugshasalimitedtherapeuticindexduetonephrotoxicity(LeclercqandTulkens,1999)andototoxicity(Wuetal.,2002)athigherdosages(e.g.,peakserumconcentrationgreaterthang/mlforgentamicin[Zaskeetal.,1982]).Becauseoftheincreasingprevalenceofresistantbacteria,theeffectivenessofthisdrugclassmaybecomelimited.TargetingofHKCorSecGaspartofacombinationtherapycouldenhancethepo-tencyofaminoglycosides,broadeningtheirtherapeuticindexatlower,nontoxicdosagesandpossiblysensitizingclinicallyre-sistantstrains.EXPERIMENTALPROCEDURESMediaandAntibioticsAllexperimentswereperformedinLuria-Bertani(LB)medium(FisherScien-tic,Pittsburgh,PA).Forallexperiments,weusedtheaminocyclitolfamilyan-tibioticskanamycin(FisherScientic),gentamicin(Sigma,St.Louis,MO),orspectinomycin(MPBiomedicals).ScreeningforaminoglycosidesensitivitywasperformedintheE.coligeneknockoutlibrary(BW25113background)(Babaetal.,2006TableS7AllotherexperimentswereperformedwithMG1655-(ATCC700926)()derivedstrains.AllsingleanddoubleknockoutswereconstructedthroughtheuseofP1phagetransduction,andP1stockwasderivedfromtheE.coliknockoutlibrary(Babaetal.,2006TableS7).PositiveP1transductantswereconrmedbyacquisitionofkanamycinresistanceandPCR.Removalofthekanamycin-resistancecassettewasaccomplishedwiththepcp20 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per0.3OD,stabilizedimmediatelywith2volumeofRNAprotectBacterialReagent(QIAGEN)asperthemanufacturer’sinstructions,andstoredover-nightatC.TotalRNAwasextractedwithQIAGENRNeasyMinispincol-umnsaccordingtothemanufacturer’sinstructions.TotalRNAwastreatedwithRNase-freeDNase(Ambion,Austin,TX).Foreachsample,reversetran-scriptionof2gtotalRNAwasperformedwiththeSuperscriptIIIReverseTranscriptasekit(Invitrogen)inatotalvolumeof20laccordingtothemanu-facturer’sinstructions.qPCRprimersforeachtranscriptofinterest(TableS8)andthereferencerrsAweredesignedwithsequencesfoundintheEcoCycdatabase(Karpetal.,2007)andPrimerQuestsoftware(IDT,http://www.).Forallprimerpairs,ampliconsizewas100bp,thecalculatedprimerannealingtemperaturewas60C,andprobabilitiesofprimer-dimer/hairpinformationswereminimized.Primerspecicitywasconrmedwithgelelectrophoresis.qPCRreactionsusingDyNAmoHSSYBRGreenqPCRKit(Finnzymes)werepreparedaccordingtomanufacturer’sinstructionswithlofa1:10dilutionofcDNA(20ngoftotalRNA)inatotalvolumeoflcontaining300nMofforwardprimersand300nMofreverseprimers,SYBRGreenMasterMix,and0.1lROXpassivereferencedye.Re-actionswerecarriedoutin384-wellopticalmicroplates(AppliedBiosystems)withanABIPrism7900HT.Crossing-pointthreshold(Ct)andreal-timeuores-cencedatawereobtainedwiththeABIPrismSequenceDetectionSoftwarev2.0withdefaultsoftwareparameters.ExpressionlevelswereobtainedfromCtvaluesaspreviouslydescribed(Gardneretal.,2003SUPPLEMENTALDATASupplementalDataincludeSupplementalResultsandDiscussion,Supple-mentalExperimentalProcedures,11gures,andeighttablesandcanbefoundwiththisarticleonlineathttp://www.cell.com/supplemental/S0092-8674(08)01195-1ACKNOWLEDGMENTSWethankN.GerryforprocessingthemicroarraysandB.Hayeteforhelpfuldis-cussionsongeneexpressionanalysis.WethankM.DePristoforcommentsonthemanuscript.ThisworkwassupportedbytheNationalInstitutesofHealth(NIH)throughtheNIHDirector’sPioneerAwardProgram,grantnumberDP1OD003644,theNationalScienceFoundation(NSF)FrontiersinIntegrativeBi-ologicalResearchprogram,NSFawardEMSW21-RTG,andtheHowardHughesMedicalInstitute.Received:November13,2007Revised:July14,2008Accepted:September15,2008Published:November13,2008Akiyama,Y.,Kihara,A.,Tokuda,H.,andIto,K.(1996).FtsH(HB)isanATP-dependentproteaseselectivelyactingonSecYandsomeothermembraneproteins.J.Biol.Chem.271,31196–31201.Ashburner,M.,Ball,C.A.,Blake,J.A.,Botstein,D.,Butler,H.,Cherry,J.M.,Davis,A.P.,Dolinski,K.,Dwight,S.S.,Eppig,J.T.,etal.(2000).Geneontology:Toolfortheunicationofbiology.TheGeneOntologyConsortium.Nat.Genet.,25–29.Baba,T.,Ara,T.,Hasegawa,M.,Takai,Y.,Okumura,Y.,Baba,M.,Datsenko,K.A.,Tomita,M.,Wanner,B.L.,andMori,H.(2006).ConstructionofEscheri-chiacoliK-12in-frame,single-geneknockoutmutants:TheKeiocollection.Mol.Syst.Biol.,2006.0008.Bolstad,B.M.,Irizarry,R.A.,Astrand,M.,andSpeed,T.P.(2003).Acompari-sonofnormalizationmethodsforhighdensityoligonucleotidearraydatabasedonvarianceandbias.Bioinformatics,185–193.Boyle,E.I.,Weng,S.,Gollub,J.,Jin,H.,Botstein,D.,Cherry,J.M.,andSherlock,G.(2004).GO:TermFinder–opensourcesoftwareforaccessingGeneOntologyinformationandndingsignicantlyenrichedGeneOntologytermsassociatedwithalistofgenes.Bioinformatics,3710–3715.Bryan,L.E.,andKwan,S.(1983).Rolesofribosomalbinding,membranepotential,andelectrontransportinbacterialuptakeofstreptomycinandgen-tamicin.Antimicrob.AgentsChemother.,835–845.Buelow,D.R.,andRaivio,T.L.(2005).CpxsignaltransductionisinuencedbyaconservedN-terminaldomaininthenovelinhibitorCpxPandtheperiplasmicproteaseDegP.J.Bacteriol.,6622–6630.Camon,E.,Magrane,M.,Barrell,D.,Lee,V.,Dimmer,E.,Maslen,J.,Binns,D.,Harte,N.,Lopez,R.,andApweiler,R.(2004).TheGeneOntologyAnnotation(GOA)Database:SharingknowledgeinUniprotwithGeneOntology.NucleicAcidsRes.,D262–D266.Cottarel,G.,andWierzbowski,J.(2007).Combinationdrugs,anemergingop-tionforantibacterialtherapy.TrendsBiotechnol.,547–555.Danese,P.N.,andSilhavy,T.J.(1998a).CpxP,astress-combativememberoftheCpxregulon.J.Bacteriol.,831–839.Danese,P.N.,andSilhavy,T.J.(1998b).Targetingandassemblyofperiplas-micandouter-membraneproteinsinEscherichiacoli.Annu.Rev.Genet.Danese,P.N.,Snyder,W.B.,Cosma,C.L.,Davis,L.J.,andSilhavy,T.J.(1995).TheCpxtwo-componentsignaltransductionpathwayofEscherichiacolireg-ulatestranscriptionofthegenespecifyingthestress-inducibleperiplasmicprotease,DegP.GenesDev.,387–398.Datsenko,K.A.,andWanner,B.L.(2000).One-stepinactivationofchromo-somalgenesinEscherichiacoliK-12usingPCRproducts.Proc.Natl.Acad.Sci.USA,6640–6645.Davis,B.D.(1987).Mechanismofbactericidalactionofaminoglycosides.Microbiol.Rev.,341–350.Davis,B.D.,Chen,L.L.,andTai,P.C.(1986).Misreadproteincreatesmem-branechannels:Anessentialstepinthebactericidalactionofaminoglyco-sides.Proc.Natl.Acad.Sci.USA,6164–6168.Dougherty,T.J.,andSaukkonen,J.J.(1985).MembranepermeabilitychangesassociatedwithDNAgyraseinhibitorsinEscherichiacoli.Antimicrob.AgentsChemother.,200–206.Duguay,A.R.,andSilhavy,T.J.(2004).Qualitycontrolinthebacterialperi-plasm.Biochim.Biophys.Acta121–134.Farewell,A.,Ballesteros,M.,Taddei,F.,Radman,M.,andNystrom,T.(2000).P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