Genome-Wide Expression Analysis Indicates that FNR

JOURNALOFBACTERIOLOGY,Feb.2005,p.1135–1160Vol.187,No.30021-9193/05/$08.00
0doi:10.1128/JB.187.3.1135–1160.2005Copyright©2005,AmericanSocietyforMicrobiology.AllRightsReserved.Genome-WideExpressionAnalysisIndicatesthatFNRofEscherichiacoliK-12RegulatesaLargeNumberofGenesofUnknownFunction†YishengKang,1K.DerekWeber,2YuQiu,1PatriciaJ.Kiley,2andFrederickR.Blattner1*DepartmentofGenetics1andDepartmentofBiomolecularChemistry,2UniversityofWisconsin,Madison,WisconsinReceived10June2004/Accepted11October2004ThemajorregulatorcontrollingthephysiologicalswitchbetweenaerobicandanaerobicgrowthconditionsinEscherichiacoliistheDNAbindingproteinFNR.ToidentifygenescontrolledbyFNR,weusedAffymetrixAntisenseGeneChipstocompareglobalgeneexpressionprofilesfromisogenicMG1655wild-typeand
fnrstrainsgrowninglucoseminimalmediaunderaerobicoranaerobicconditions.Wefoundthat297genescontainedwithin184operonswereregulatedbyFNRand/orbyO2levels.Theexpressionofmanygenesknowntobeinvolvedinanaerobicrespirationandfermentationwasincreasedunderanaerobicgrowthconditions,whilethatofgenesinvolvedinaerobicrespirationandthetricarboxylicacidcyclewererepressedasexpected.Theexpressionofnineoperonsassociatedwithacidresistancewasalsoincreasedunderanaerobicgrowthconditions,whichmayreflecttheproductionofacidicfermentationproducts.Ninety-onegeneswithnopresentlydefinedfunctionwerealsoalteredinexpression,includingsevenofthemosthighlyanaerobicallyinducedgenes,sixofwhichwefoundtobedirectlyregulatedbyFNR.Classificationofthe297genesintoeightgroupsbyk-meansclusteringanalysisindicatedthatgeneswithcommongeneexpressionpatternsalsohadastrongfunctionalrelationship,providingcluesforstudyingthefunctionofunknowngenesineachgroup.SixoftheeightgroupsshowedregulationbyFNR;whilesomeexpressiongroupsrepresentgenesthataresimplyactivatedorrepressedbyFNR,others,suchasthoseencodingfunctionsforchemotaxisandmotility,showedamorecomplexpatternofregulation.AcomputersearchforFNRDNAbindingsiteswithinpredictedpromoterregionsidentified63newsitesfor54genes.WesuggestthatE.coliMG1655hasalargermetabolicpotentialunderanaerobicconditionsthanhasbeenpreviouslyrecognized.Formanybacteria,O2playsacriticalroleintheregulationofnumerouscellularprocesses.TheutilizationofO2asaterminalelectronacceptorduringaerobicrespirationresultsinthehighestconservationofcellularenergyandthereforepro-videsagreatenergeticadvantagetomicroorganisms(93).O2inexcess,however,canbedetrimentaltocellsbecauseoftheproductionofreactiveoxygenspecies(35).ManybacteriahaveevolvedadaptivestrategiestobalanceconsumptionofO2forenergymetabolismwithdetoxificationofreactiveoxygenspe-cies.Inaddition,itiswellestablishedthatEscherichiacoliandotherfacultativeanaerobesaregeneticallyprogrammedtoadaptbetweenenvironmentsofhighandlowO2levels(87).InthecaseofE.coli,removalofO2causesmajorchangesintheexpressionofpathwaysthatpromoteenergyconservation(67,93).Forexample,theexpressionofgenesencodingrespiratoryenzymesthatutilizeO2asanelectronacceptor(e.g.,cyto-chromeoxidases)isrepressed,whereastheexpressionofgenesencodingenzymesthatutilizealternativeelectronacceptors,suchasnitrate,fumarate,etc.,orthatpromotefermentationisincreasedunderanaerobicconditions.Nevertheless,whilemanycellularactivitiesthatfunctionunderanaerobiccondi-tionshavebeenuncovered,westilllackacomprehensiveviewoftheanaerobiclifestyleofE.coli,eventhoughoneofitsimportantecologicalnichesisthelow-O2environmentofthegut.FNRandthetwo-componentregulatorysystem,ArcAB,arethetwomajorregulatorysystemsthatrespondtodecreasesinO2levelsinE.coli(29).FNRhasanO2-sensitive[4Fe-4S]2
clusterthatdirectlysensesO2andregulatessite-specificDNAbinding(40).Incontrast,ArcABsensessignalsemanatingfromtheaerobicrespiratorychain,indicatingatleasttwodi-vergentyetfundamentalmechanismsforO2sensinginthisorganism(26).Recentgeneexpressionprofilingstudieshaveidentified55operonsoftheArcAregulon(52).InanattempttofurtherourunderstandingofthemetabolicpotentialofE.coliunderanaerobicgrowthconditions,wesoughttoidentifyadditionalgenesthatarecontrolledbytheglobalregulatorFNRthroughgenome-wideexpressionstudies.PreviousstudieshaveshownthatFNRcontrolstheexpres-sionof100geneproductsinE.coli(67,75).KnownFNR-activatedgenesincludethoseencodingenzymesfortheanaer-obicoxidationofcarbonsources,suchasglycerolandformate,genesencodingenzymesfortheanaerobicreductionofalter-nateterminalelectronacceptors,suchasnitrate,fumarate,anddimethylsulfoxide(DMSO),andgenesencodingproteinsfortransportofthesecarbonsourcesorelectronacceptors.FNRalsorepressestheexpressionofgenesthatareneededforaerobicmetabolism,suchastherespiratoryenzymesNADH*Correspondingauthor.Mailingaddress:DepartmentofGenetics,425HenryMall,UniversityofWisconsin,Madison,WI53706.Phone:[phone].Fax:[phone].E-mail:[e-mail].†Supplementalmaterialforthisarticlemaybefoundat