The transcriptional regulatory repertoire of Corynebacterium glutamicum

JournalofBiotechnology149 (2010) 173–182

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ThetranscriptionalregulatoryrepertoireofCorynebacteriumglutamicum:Reconstructionofthenetworkcontrollingpathwaysinvolvedinlysineandglutamateproduction

KarinaBrinkrolf,JasminSchröder,AlfredPühler,AndreasTauch

InstitutfürGenomforschungundSystembiologie,CentrumfürBiotechnologie,UniversitätBielefeld,Universitätsstraße27,D-33615Bielefeld,Germany

articleinfoabstract

CorynebacteriumglutamicumisoneofthebeststudiedorganismsofthehighG+CbranchofGram-positivebacteriaandanemergingmodelsystemforthesuborderCorynebacterineae.TogaininsightsintotheregulatorygenecompositionandarchitectureofthetranscriptionalregulatorynetworkofC.glutamicum,componentsofthetranscriptionalregulatoryrepertoirewereintensivelystudiedbymanyscienti cgroupsinrecentyears.Inthismini-review,wesummarizethepresentknowledgeaboutthededucedtranscriptionalregulatoryrepertoireofC.glutamicumandthecurrentstatusoftranscriptionalregulatorynetworkreconstructionwithregardtothegenome-widedetectionoftranscriptionalregula-tions,coregulatoryinteractionsandhierarchicalcross-regulations.Moreover,weprovideanoverviewofthoseregulatorsandtheirtranscriptionalregulationscontrollinggenesinvolvedintheconversionofthecarbonsourcesglucose,fructoseandsucroseintotheindustriallyrelevantproductsl-lysineandl-glutamate.Thisdatawillcontributetoourunderstandingofl-lysineandl-glutamateproductionbyC.glutamicumfromtheperspectiveofsystemsbiologyandmayprovidethebasisforcomputationalmodelingoftherespectivebiotechnologicallyimportantmetabolicpathways.

© 2009 Elsevier B.V. All rights reserved.

Articlehistory:

Received18August2009Receivedinrevisedform19November2009

Accepted1December2009

Keywords:

CorynebacteriumglutamicumTranscriptionregulation

TranscriptionalregulatorynetworkGlutamatebiosynthesisLysinebiosynthesisSystemsbiology

1.Introduction

Thetranscriptionalregulatorynetwork(TRN)ofbacteriaisafundamentalbiologicalsystemcontrollingthe owofinforma-tionfromtheinternalandexternalenvironmenttothegenelevelandthustospeci ccellularfunctions(Seshasayeeetal.,2006).Inconjunctionwithadiversemetabolism,theTRNenablesarapidadaptationofmicroorganismstochangingenvironmentalcon-ditions.ThemaincomponentsofaTRNareregulatoryproteinstermedtranscriptionregulatorsthatsensediversestimuliandrec-ognizeandbindtospeci cDNAsequences(operators)tocontrolandmodulatetheexpressionoftheirtargetgenes(MadanBabuandTeichmann,2003).Thisbasicgeneticprincipleallowstherecon-structionofthis owofinformationinformofadirectedgraph,whichiscommonlyreferredtoastheTRN(Babuetal.,2004).Insuchnetworks,nodesrepresenttranscriptionregulatorsandtheirtargetgenes,anddirectededgesrepresenttheregulatoryinter-actionbetweenthem.Accordingly,transcriptionregulatorsandtargetgenesformregulatoryunitscross-linkedtosimplenetworkmotifsthatarecharacteristictopologicalandfunctionalelements

Abbreviation:TRN,transcriptionalregulatorynetwork.

Correspondingauthor.Tel.:+495211068703;fax:+4952110689041.E-mailaddress:tauch@cebitec.uni-bielefeld.de(A.Tauch).0168-1656/$–seefrontmatter© 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2009.12.004

oftheregulatorynetwork.Thesepatternsofconnectionsincludesingleinputmotifs,bi-fanmotifs,feed-forwardloops,anddenseoverlappingregulons(Dobrinetal.,2004;Shen-Orretal.,2002).Beyondthat,autoregulation,multi-componentloopsandregula-torycascadeswerealsodescribedasnetworkmotifs(Yuetal.,2003).Inthecellularsystem,networkmotifsgenerallyarenotisolatedbutinterconnectedtocomplexstructures,formingthearchitecturalbackboneoftheTRN(Dobrinetal.,2004;MadanBabuetal.,2004).

NovelmethodsofultrafastDNAsequencingandgenome-widetranscriptionalpro lingwithtechniqueslikeDNAmicroarrayhybridizations,aswellascomputationalapproachesgeneratehugeamountsofdataontranscriptionalregulatoryprocessesinabacterialcell(Babu,2008;Ballezaetal.,2009).Overtheyears,considerableinformationhasbeenaccumulatedontranscriptionalregulationintheGram-negativemodelorganismEscherichiacoliandstoredinthereferencedatabaseRegulonDB(Collado-Videsetal.,2009).Currently,theTRNofE.coliisthemostdetailedreconstructionofregulatoryinteractionsinbacteria,providingvaluableinsightsintoitsglobaltopologicalorganization(Balajietal.,2007;Gama-Castroetal.,2008).Thiscomprehensivenetworkreconstructionrevealedamodularandhierarchicalstructureofregulatoryinteractionsatthetranscriptionallevel(Maetal.,2004;Resendis-Antonioetal.,2005).Recentresultsindicatedthatdiffer-entorganizationallevelsoftheTRNofE.coliarebridgedbythe

174K.Brinkrolfetal./JournalofBiotechnology149 (2010) 173–182

presenceofpositiveandnegativefeedbackloops(Freyre-Gonzálezetal.,2008).ThemodularityoftheTRNaccountsfortherobustnessoftheentiresysteminthatwaythatdamageiskeptinacertainpartofthenetwork(Aderem,2005).Cross-genomecomparisonsofthetranscriptionalregulatoryrepertoiresandcorrespondingregulatoryinteractionsamongbacteriafromdiversephylogeneticlineagesindicatedthatTRNsareextremely exibleduringevo-lutionandplayimportantrolesinthephenotypicadaptationofbacteria(Lozada-Chávezetal.,2006).

Anothermicroorganismwithdetailedinformationabouttran-scriptionalregulationistheGram-positivebacteriumCorynebac-teriumglutamicum(Brinkrolfetal.,2007).Itisgenerallycharacterizedbyabroadspectrumofmetaboliccapabilitiesandindustriallyusedforthefermentativeproductionofaminoacids,inparticularl-lysineandl-glutamate(Kimura,2003;Pfefferleetal.,2003).Theavailabilityofthewholegenomesequenceofthewild-typeC.glutamicumATCC13032incombinationwithrecom-binantDNAtechniquesenablednewstrategiesforthedesignofindustrialproductionstrains.The“genomebreeding”strategy,forinstance,wasintroducedtomakeuseofacomparativegenomicsapproachbyscreeningthegenomesequenceofthewild-typestrainandthatofclassicall-lysineproducersforthepresenceofrelevantmutations(Ikedaetal.,2006).Bythismeanssixkeygenes(lysC,hom,pyc,gnd,mqo,andleuC)weredetectedinC.glutamicumtoenabletherationaldesignofanef cientl-lysineproducer(Hayashietal.,2006a;Ikedaetal.,2006;Mitsuhashietal.,2006).Thisstrat-egyalsosuggestedthatalterationsinglobalregulatoryprocessescontributetol-lysineproductioninclassicalstrains(Hayashietal.,2006a,b).Furtherimprovementsofrationallydesignedproductionstrainsmaythereforealsodependongeneticmodi cationsintheTRNofC.glutamicum.

Inthismini-review,wesummarizethecurrentknowledgeaboutthetranscriptionalregulatoryrepertoireandthereconstructedTRNofC.glutamicum.Wealsoprovideanoverviewoftranscriptionreg-ulatorscontrollinggenesinvolvedintheconversionofglucose,fructoseandsucroseintotheindustriallyrelevantproductsl-lysineandl-glutamate.

2.ThetranscriptionalregulatorynetworkofC.glutamicum2.1.DetectionoftherepertoireoftranscriptionregulatorsinC.glutamicum

Thedetectionandsubsequentcharacterizationofthereper-toireofpotentialtranscriptionregulatorsinC.glutamicumwerethekeystepinunderstandingtheTRNinthisbacterium.Thecompletegenomesequenceofthewild-typestrainC.glutamicumATCC13032wasscreenedbydifferentbioinformaticsapproaches,andtheresultingdatawerecombinedwithpublishedknowledgebyperformingliteraturesearches(Bruneetal.,2005).Bythismeansatotalnumberof158geneswasinitiallydetectedasmini-malrepertoirefortranscriptionregulators(Brinkrolfetal.,2007)thatC.glutamicumapparentlyneedstocoordinatetheexpres-sionofaround3000predictedprotein-codinggenesundervaryingenvironmentalconditions(Kalinowskietal.,2003).Basedoncomputationalpredictions,thesegenesareorganizedin2087tran-scriptionunits,including528potentialoperons(Priceetal.,2005).Itshouldbekeptinmindthatthenumberofdetectedtranscrip-tionregulatorsstronglydependsonthecurrentstateofannotationofgeneandproteindatabasesandmaythereforeslightlyvaryinthefuturetobecomemoreandmorepreciseforfollowingreasons.

(i)NovelproteinfamiliesofDNA-bindingtranscriptionregulatorsmaybede nedbasedonexperimentalevidencededucedfromotherbacterialspecies.MembersofthenovelNrtRproteinfamily(Nudix-relatedtranscriptionalregulators),forexam-ple,wereinitiallyannotatedingenomeprojectsasADP-ribosepyrophosphatasesfromtheNudixproteinfamily,butwererecentlyshowntobeinvolvedintheregulationofvariousaspectsofNADbiosynthesisinabroadrangeofbacteria(Huangetal.,2009;Rodionovetal.,2008).InC.glutamicum,thecg1218geneencodesanNrtR-likeregulatoryproteinwithanamino-terminalNudix-likeeffectordomainandacarboxy-terminalhelix-turn-helix-likeDNA-bindingdomainthatmightberesponsibleforthetranscriptionalcontroloftheNADbiosynthesispathway.

(ii)Predictedproteinssupposedtobeinvolvedintranscrip-tionalregulationmayexertothercellularfunctions.TheregulatoryroleofproteinsbelongingtotheWhiBfamily,forinstance,remainsobscureinactinobacteria(denHengstandButtner,2008).ItwasshownthattheWhiB3proteinfromMycobacteriumtuberculosisinteractedwiththecarboxy-terminaldomainoftheprincipleRNApolymerasesigmafactorinayeasttwo-hybridscreening(Steynetal.,2002),suggestingafunctionofWhiB3astranscriptionregulatorbyindirectevi-dence,ter,itwasdemonstratedthatWhiBproteinsfromM.tuberculosis(WhiB1andWhiB4)containredox-sensitiveiron–sulfurclus-tersandcanfunctionasproteindisul dereductases(Alametal.,2007;Gargetal.,2007).However,itcannotbeexcludedatpresentthattheiron–sulfurclustershaveafurtherroleinaffectingtheDNA-bindingpropertiesofWhiBproteins(denHengstandButtner,2008).TheC.glutamicumgenomecon-tainsfourgenesforWhiBfamilyproteins(Bruneetal.,2005).ExperimentalcharacterizationofwhcEsuggestedthatitfunc-tionsastranscriptionregulatorbyactivatingtheexpressionofthetrxBgene(Kimetal.,2005).Likewise,thewhcAgeneofC.glutamicumseemstobeinvolvedinaregulatorypathwaycon-trollinggenesofthecellularheatandstressresponse(Choietal.,2009).FurtherexperimentalworkisthereforenecessarytoelucidatewhethertheWhiBproteinsofC.glutamicumareabletodirectlyinteractwithDNAtargetsites.

(iii)Theregulatorymechanismsofdistinctclassesoftranscrip-tionregulatorsmaydifferbetweenbacterialgenera.Thewell-characterizedpyrimidinebiosynthesisregulatorPyrRofBacillussubtilisisanexampleforactingatthepost-transcriptionallevelbydirectbindingtomRNAtargetsites(HoblandMack,2007).Ontheotherhand,thePyrRproteinofC.glutamicumnegativelycontrolstheexpressionofthepyrHgeneinvolvedinpyrimidinebiosynthesisbydirectbindingtothepyrHpromoter,asdeducedfromelectrophoreticmobilityshiftassayswithpuri edPyrRrepressor(Leeetal.,2006).Likewise,thecold-shockproteinCspAmaypossessDNA-bindingactiv-ityinC.glutamicumtoexertadirectregulatoryeffectongeneexpressionatthetranscriptionallevel(Gualerzietal.,2003;Kimetal.,2007).

(iv)Experimentaldatamaydemonstrateadirectroleinregulatory

processesofproteinswithpredictedfunctionsintranscrip-tionregulation,allowingtheirvalidatedclassi cationintotheregulatoryrepertoire.SuchproteinsinC.glutamicumarerepre-sentedbyCg1340andCg1552thatweregroupedintotheCOGclassesCOG2345andCOG1733,respectively,bothrepresent-ingclustersof“Predictedtranscriptionalregulators”,withoutanybiologicaldatasupportingthisphysiologicalrole(Tatusovetal.,2003).RecentexperimentalworkrevealedthatCg1340(nownamedArnR)speci callybindstothepromoterregionofthenarKGHIJoperonencodingaputativenitrate/nitritetrans-porterandnitratereductase(Nishimuraetal.,2008)andthatCg1552(nownamedQorR)directlyregulatestheexpressionofthequinoneoxidoreductasegeneqor2(Ehiraetal.,2009a).

K.Brinkrolfetal./JournalofBiotechnology149 (2010) 173–182175

Thelatterexampleparticularlyshowsthatthegrowingamountoffunctionalproteindatastoredinpublicdatabaseswillgreatlyfacilitatethedetectionoftheregulatoryrepertoireofbacterialgenomesinthefuture.Inaddition,thesensitivityofcomputa-tionalmodelsgenerallyappliedforthedetectionofDNA-bindingtranscriptionregulators,likehiddenMarkovmodelsthattracktheoccurrenceofconservedDNA-bindingdomainsincandidatepro-teins,willmostlikelyincreaseandtherebycontributetomoreprecisepredictionsofregulatoryrepertoires.

Currently,acollectionof159genesencodingDNA-bindingtran-scriptionregulatorsandsigmafactorscanberegardedastheminimalregulatoryrepertoireusedfortranscriptionalregulationsinC.glutamicumATCC13032(Brinkrolfetal.,2007;Rodionovetal.,2008).Thisnumberofcandidatetranscriptionregulatorsrepre-sents5.3%ofthepredictedprotein-codingregionsofC.glutamicumandisinagreementwithpreviousestimatesthatlessthan10%ofthetotalnumberofproteinsareassociatedwithgeneregulationinbacterialspecies(Pérez-RuedaandCollado-Vides,2000;Rodionov,2007).Thisdatasetprovidedthebasisforthesystematicanalysisofindividualtranscriptionregulatorsandtheirregulonsbyboth,experimentalandcomputationalapproaches.Thedetectedtran-scriptionregulatorsofC.glutamicumATCC13032canbegroupedinto38proteinfamiliesbasedonsequencesimilaritieswithknownbacterialregulatoryproteins,including13two-componentsig-naltransductionsystemsandsevensigmafactors(Brinkrolfetal.,2007;Ehiraetal.,2009a;Koˇcanetal.,2006;Nishimuraetal.,2008;Rodionovetal.,2008).ThelargestfamilyofDNA-bindingtran-scriptionregulatorsinC.glutamicumATCC13032isTetRwith16members,followedbyArsRwith13proteins(Bruneetal.,2005).ThecompletesetofDNA-bindingtranscriptionregulatorsfromC.glutamicumATCC13032wastracedoncompletelysequencedcorynebacterialgenomesconsideringthegenomiccontextinfor-mation,indicatingacoresetofonly24orthologousregulatorsthatareconservedinallcorynebacteria(Brinkrolfetal.,2007;Tauchetal.,2008).Someoftheseregulatorsmayhaveimportanttopo-logicalandregulatoryfunctionsinaputativecoresegmentofacorynebacterialTRN,forinstancetheglobalregulatorynetworknodeGlxRandthetranscriptionregulatorsDtxR,McbR,LexA,andRamA(Bruneetal.,2006;Crameretal.,2006;Jochmannetal.,2009;Kohletal.,2008;Reyetal.,2005).AnextendedcomputationalsearchforsignatureproteinsinthetaxonomicclassActinobacte-riarevealedthatorthologsofWhiB1,WhiB2andCg1631oftheMerRproteinfamilyarepresentinallsequencedactinobacterialspeciesandthattheMerRfamilyproteinCg1633isconservedinallactinobacteria,withtheexceptionofBi dobacteriumlongumandTropherymawhipplei(Gaoetal.,2006).

2.2.Regulatoryinteractions,coregulationsandcross-regulationsinC.glutamicum

Amongthecollectionof159potentialtranscriptionregulatorsdetectedinC.glutamicumATCC13032,knowledgeaboutregula-toryinteractionswasobtainedfor77regulatoryproteins(48%oftheregulatoryrepertoire)fromwet-labexperimentsand/orreli-ablebioinformaticspredictions(Table1).Thesedatainconjunctionwithliteratureinformationwerecompiledandstoredintherefer-encedatabaseCoryneRegNet(Baumbachetal.,2006,2009b).Thesetofcharacterizedtranscriptionregulatorscurrentlycomprise42repressorsand24activators(includingfoursigmafactors),whereastheremainingelevenproteinswereidenti edasdualregulators,exertingpositiveandnegativeregulationsondifferenttargetgenes.Itisnoteworthythatsomeofthedetecteddualregulatorsexerttheirregulatoryfunctioninthatwaythattheydonotdirectlyacti-vateorrepressthetargetgene,butinterferewithDNA-bindingofcoregulators,asdeducedfromthecharacterizationoftheDtxRandLexAregulonsofC.glutamicum(Bruneetal.,2006;Jochmannetal.,2009).Uptonow895regulatoryinteractionsareintegratedinCoryneRegNet5.0(July2009),including621negative(69.4%)and274positiveregulations(30.6%)(Baumbachetal.,2009a).Duetothehighproportionofrepressorproteinsintheregulatoryreper-toireandthehighnumberofnegativeregulatoryinteractionsthatwerehithertodetected,repressionseemstobethemostcommonregulatorymechanisminC.glutamicum.ThistrendisincontrasttoobservationsmadeintheTRNofE.coli(Gama-Castroetal.,2008),butcorrelateswiththe ndingthatmostpromotersareprevalentlyrepressedinbacterialgenomes(Moreno-Campuzanoetal.,2006).

ThecollectionoftranscriptionalregulatoryinteractionsstoredinCoryneRegNetrevealedthattheexpressionofmanyC.glu-tamicumgenesismodulatedbytheactionoffewtranscriptionregulators.Inthecurrentdataset,158genesofC.glutamicumATCC13032areregulatedbytwotranscriptionregulators,46genesbythreetranscriptionregulators,and15genesarecontrolledbyfouror vetranscriptionregulators(Baumbachetal.,2009a).Thishighrateofinterconnectionbetweenregulatorsandtheircommontar-getgenessuggestedthatcoregulationisanimportantprincipleintheTRNarchitectureofC.glutamicumtoenablethecoordinate owofinputinformationfromtheenvironmenttowardsregulonsofdifferentfunctionality.Fig.1representsadiagramshowingthenumberofregulationsexertedbyindividualregulatoryproteinsversusthenumberofcoregulationsoccurringatcorrespondingtar-getgenes.ThisplotleadstotheconclusionthatthetranscriptionregulatorsofC.glutamicumcanberoughlydividedintothreetypes,comprising(i)localregulatorsthatcontroltheexpressionofasmallnumberoffunctionallyrelatedgenesandtendtobeclusteredwiththeirtargetgenes(Rodionov,2007),(ii)masterregulatorsthatcontroltheexpressionofalargenumberofgenesbelongingtoacorrespondingfunctionalmodul,and(iii)theglobalregulatorGlxRthatrepresentsanoutlierduethehighnumberofregulationsandcoregulationsoftargetgenes.Globalregulatorsarede nedaspro-teinsthatcontrolmorethan20genes(includinggenescodingforothertranscriptionregulators)organizedindifferenttranscriptionunitsandbelongingtoaminimumoffourdifferentfunctionalcat-egories(Moreno-Campuzanoetal.,2006;Resendis-Antonioetal.,2005).Inprinciple,itseemsthattheglobalregulatorGlxRand/oronemasterregulatoractinconcertwithamorespeci clocalreg-ulatorto ne-tunetheexpressionofgenesindistinctfunctionalmodulesofC.glutamicum(Kohletal.,2008).

Additionally,wedetected50transcriptionregulatorsofC.glutamicumthatwerereportedtobecross-regulatedbyothertran-scriptionregulators(Baumbachetal.,2009a).Fig.2showsthematrixofregulatoryinteractionsofthesetranscriptionregulators,including54repressions(72%)and21activations(28%).Asimi-lartrendhasbeenobservedforcoregulationsintheregulatorynetworkofE.coli(Pérez-RuedaandCollado-Vides,2000).TheseinterconnectionsmightberelevantforthehierarchicalstructureoftheTRNofC.glutamicumtoestablishdifferentexecutivelevelsinthe owofinformationfromtheenvironmentalsignaltowardstheaffectedgenes.Moreover,32transcriptionregulatorswerefoundtobeautoregulated,mostofthemwerenegative(28)(Fig.2).Negativeautoregulationoccurswhenarepressorcontrolstheexpressionofitsowngene.Inthecaseofpositiveautoregulation,anactivatorenhancesitsownrateofgeneexpression(Alon,2007).Nega-tiveautoregulationgenerallyspeedsuptheresponsetimeoftheregulatorysystem(Rosenfeldetal.,2002),whereastheeffectofpositiveautoregulationisoppositeinslowingdowntheresponsetimeofthecell(MaedaandSano,2006).MostgenesencodingmasterregulatorsinC.glutamicumarenegativelyautoregulated(Fig.2),withtheexceptionofamtRanddtxR(Bruneetal.,2006;Muhletal.,2009).BythismechanismtheymostlikelyenabletheC.glutamicumcelltorapidlyadaptgeneexpressioninthecorrespondingfunctionalcontextto uctuationsintheenviron-ment.

176K.Brinkrolfetal./JournalofBiotechnology149 (2010) 173–182

Table1

FeaturesandphysiologicalrolesoftranscriptionregulatorsinCorynebacteriumglutamicumATCC13032.Codingregioncg0012cg0019cg0051cg0090cg0112cg0156cg0196cg0215cg0313cg0317cg0337cg0350cg0444cg0454cg0500cg0527cg0702cg0800cg0862

Alternativeidenti erNCgl0009NCgl0015NCgl0035NCgl0068NCgl0082NCgl0120NCgl0154NCgl0171NCgl0253NCgl0257NCgl0275NCgl0286NCgl0358NCgl0368NCgl0405NCgl0430NCgl0581NCgl0668NCgl0721

GenenamessuR––citBureRcysRiolRcspAlrparsR2whcAglxRramB–qsuRglyR–prpRmtrA

ProteinfamilyROKLysRAraC

ResponseregulatorMarRROKGntRColdAsnCArsRWhiBCrpHTHTetRLysRArsRLysRHTHResponseregulator

Numberoftargetgenesa

913471810132815055341134

EvidencebEPPEPEEEEEPEEPEEPEE

Regulatoryroleandphysiologicalfunctionoftranscriptionregulatorc

Activatorofsulfonateandsulfonateesterutilizationgenes

Activatorofmembraneproteingenecg0018ActivatorofABC-typetransportergenescg0052–cg0054

ActivatorofcitratetransportergenescitHandtctCBA

RepressorofureasegenesureABCEFGD

Dualregulatorofassimilatorysulfatereductiongenes

RepressorofinositolmetabolismgenesRepressorofmalatesynthasegeneaceB

ActivatorofaminoacidexportergenesbrnFERepressorofarsenitepermeasegenearsB2andarsenatereductasegenearsC2

RepressorofoxidativestressresponsegenesDualregulator(globalregulator)functioningasnetworkhub

Dualregulator(masterregulator)ofcarbonmetabolism

RepressorofMFS-typetransportergenescg0455andcg0456

ActivatorofquinateandshikimateutilizationgenesqsuABCD

ActivatorofserinehydroxymethyltransferasegeneglyA

Activatorofsecondarytransportergenecg0701ActivatorofpropionateutilizationgenesprpD2B2C2

Dualregulatorofgenesinvolvedincellmorphology,antibioticssusceptibilityandosmoprotection

ECFsigmafactor(masterregulator)involvedinglobalstressresponse

ActivatorofthioredoxingeneclustertrxBC-cwlM

ActivatorofpyridoxalphosphatesynthasegenespdxST

RepressorofABC-typetransportergenescg0977andcg0978

Repressor(masterregulator)ofnitrogenmetabolism

Repressorofsecondarytransportergenecg0992Repressorofsecondarytransportergenecg1033RepressorofMmpL-typetransportergenecg1054

Repressorofgenesforironprotein

RepressorofMFS-typetransportergenecg1212RepressorofNADbiosynthesisgenesnadAC-cg1214

ECFsigmafactorinvolvedinresponsestocellsurfacestresses

Repressorofhydroxyquinolpathwaygenescg1310andcg1311

Dualregulator(masterregulator)ofrespirationRepressorofriboseuptakeanduridineutilizationgenes

ActivatorofaminoacidexportergenelysERepressorofleucineandtryptophanbiosynthesisgenes

Repressorofuridineutilizationandriboseuptakegenes

Repressorofquinoneoxidoreductasegeneqor2RepressorofargininebiosynthesisgenesRepressorofABC-typetransportergenescg1652–cg1649andcg1653

RepressorofarsenitepermeasegenearsB1andarsenatereductasegenesarsC1andarsC1’Repressorofaconitasegeneacn

Repressorofiron–sulfurclusterbiogenesisgenes

RepressorofpyrimidinebiosynthesisgenesRepressorofABC-typetransportergenescg1832–cg1834

cg0876cg0878cg0897cg0979cg0986cg0993cg1032cg1053cg1120cg1211cg1218cg1271cg1308cg1340cg1410cg1425cg1486cg1547cg1552cg1585cg1648cg1704cg1738cg1765cg1817cg1831

NCgl0733NCgl0734NCgl0753NCgl0823NCgl0829NCgl0836NCgl0869NCgl0886NCgl0943NCgl1019NCgl1025NCgl1075NCgl1110NCgl1138NCgl1203NCgl1215NCgl1261NCgl1312NCgl1317NCgl1345NCgl1401–

NCgl1483NCgl1504NCg1551lNCgl1563

sigHwhcEpdxR–amtR–––ripA–nrtRsigE–arnRrbsRlysGltbRuriRqorRargRrpiRarsR1acnRsufRpyrR–

SigmafactorWhiBGntRPadRTetRArsRArsRTetRAraCArsRNrtRsigmafactorTetRCOG2345LacILysRIclRLacICOG1733ArgRRpiRArsRTetRArsRPyrRArsR

583233922218141379191029632743

EPEEEPEEEPPEEEEEEEEEPEEEEP

K.Brinkrolfetal./JournalofBiotechnology149 (2010) 173–182

Table1(Continued)Codingregioncg1846cg1861cg1935cg2102

Alternativeidenti erNCgl1578NCgl1590NCgl1650NCgl1844

Genename–relgntR2sigB

ProteinfamilyTetR–

GntR

Sigmafactor

Numberoftargetgenesa

471017

EvidencebEPEE

Regulatoryroleandphysiologicalfunctionoftranscriptionregulatorc

177

cg2103cg2112cg2114cg2115cg2118cg2152cg2268cg2309cg2500cg2502cg2516cg2615cg2624cg2783cg2831cg2888cg2894cg2942cg2965cg3082cg3097cg3202cg3224cg3239cg3253cg3352cg3388cg3420

abc

NCgl1845NCgl1853NCgl1855NCgl1856NCgl1859NCgl1887NCgl1989NCgl2025NCgl2199NCgl2200NCgl2211NCgl2299NCgl2308NCgl2440NCgl2472NCgl2518NCgl2523NCgl2567NCgl2587NCgl2584NCgl2699NCgl2794NCgl2814NCgl2827NCgl2840NCgl2921NCgl2954NCgl2983

dtxRnrdRlexAsugRfruRclgR–bioQznrzurhrcAvanRpcaRgntR1ramAphoR––––hspRfarRlldR–mcbRnagR–sigM

DtxRNrdRLexADeoRDeoRHTH3LysRTetRArsRFurHrcAPadRIclRGntRLuxR

ResponseregulatorTetRAsnCAraCArsRMerRGntRGntRLysRTetRIclRIclR

Sigmafactor

64348405816294312102418211471061465317

EPEEEEPPEEEEEEEEEPPEEEEPEEPE

Repressorofcg1845-cg1844genesandcg1847(p)ppGppsynthetaseinvolvedinsigmafactorcompetition

DualregulatorinvolvedincarbonmetabolismGroup2sigmafactor(masterregulator)

involvedingeneexpressionduringtransitionphaseandunderoxygendeprivationDualregulator(masterregulator)ofironmetabolism

RepressorofribonucleotidereductasegenesnrdHIE

Dualregulator(masterregulator)ofSOSresponse

Repressor(masterregulator)involvedincarbonmetabolism

RepressoroffructosePTSsystemgenes

ActivatorofproteolysisandDNArepairgenesActivatorofmembraneproteingenecg2269Repressorofbiotinuptakeandbiosynthesisgenes

RepressoroftranscriptionregulatorgenezurRepressorofzincuptakesystemgenes

RepressorofheatshockresponsegeneswithCIRCEelements

RepressorofvanillatemetabolismgenesvanABK

RepressorofprotocatechuatemetabolismgenesDualregulatorinvolvedincarbonmetabolismDualregulator(masterregulator)ofcarbonmetabolism

Activator(masterregulator)ofphosphatemetabolism

RepressorofMFS-typetransportergenecg2893ActivatorofLysE-typetranslocatorgenecg2941Activatorofphenoldegradationgenecg2966Repressorofcg3083–cg3085genes

RepressorofheatshockresponsegeneswithHAIRelements

Repressorofarginineandglutamatebiosynthesisgenes

Repressorinvolvedincarbonmetabolism

ActivatorofMFS-typetransportergenecg3240Repressor(masterregulator)ofsulfurmetabolism

Activatorofgentisateuptakeanddegradationgenes

Activatorofhydroxyquinolpathwaygenescg3387–cg3385

ECFsigmafactorinvolvedinexpressionofoxidativestressresponsegenes

CurrentnumberoftargetgenesstoredinCoryneRegNet5.0(July2009).Abbreviations:E,experimentalevidence;P,computationalprediction.

ReferencesforcorrespondingoriginalarticlesareavailablefromtheliteraturesectioninCoryneRegNet.

2.3.CurrentstatusoftranscriptionalregulatorynetworkreconstructioninC.glutamicum

RecentregulatorynetworkreconstructionswithGraphVisandtheentiredatasetofregulatoryinteractionsstoredinCoryneReg-Netincluded544genes,i.e.around20%ofthepredictednumberofC.glutamicumATCC13032genes(KohlandTauch,2009).Thesenetworkreconstructionsrevealedacross-linkageofregulatoryunitstoacomplexnetworkstructure,aso-calledmotifsuper-cluster(Dobrinetal.,2004),including92%ofthehithertoknownregulatoryinteractionsinC.glutamicum.Moreover,amodulararchitectureoftheregulatorynetworkwasobserved,showingsixconnectedunitswithwell-de nedphysiologicalfunctions,includ-ing(i)carbonmetabolism,(ii)sulfurandironhomeostasis,(iii)SOSandstressresponse,(iv)respiration,(v)nitrogenmetabolism,and(vi)phosphatemetabolism.Asexpected,eachofthesefunctionalmoduleswascharacterizedbythepresenceofmasterregulators,suchasMcbR,DtxR,LexA,SigH,ArnR,AmtR,andPhoR,sensingdistinctenvironmentalinputstocontroltheexpressionoffunction-allyrelatedtargetgeneswithintheircorrespondingmodules.ThehierarchicalstructureoftheregulatorynetworkofC.glutamicumwasalsoevidentfromthereconstruction,sincelocalregulatorsweredetectedwithinfunctionalmodules,eitheraspartofthesuperclusterorasseparatemotifswithcorrespondingphysiolog-icalfunctions(Kohletal.,2008).Theonlyregulatoryhub(globalregulatorynetworknode)currentlyknownintheTRNofC.glu-tamicumisGlxR.Recentcomputationalpredictionswithare nedpositionweightmatrixindicatedthatGlxRmightbeinvolvedindirectlycontrollingaround14%oftheentiregenesetofC.glutam-icumATCC13032(KohlandTauch,2009).Thissetincludes23genesfortranscriptionregulators,suggestingthatahighnumberofC.glu-tamicumgenesareunderhierarchicalcontrolbyGlxR,asithasbeenobservedforthehomologousregulatorCRPinE.coli(MadanBabuandTeichmann,2003).

178K.Brinkrolfetal./JournalofBiotechnology

149 (2010) 173–182

Fig.1.CoregulationsinthetranscriptionalregulatorynetworkofC.glutamicumATCC13032.Thediagramvisualizesthenumberofregulatoryinteractionsexertedbydetectedtranscriptionregulatorsandthenumberoftargetgenesthatareunderdirecttranscriptionalcontrolbyotherregulatoryproteins(coregulations).Genenamesandidenti ersareusedforlabeling.Thelowerleftpartofthediagramisshowninmoredetailintheseparatepanel.Symbols:blackdiamond,localregulator;blackcircle,masterregulator;blacksquare,globalregulator.

3.Transcriptionalregulationsinvolvedinl-lysineandl-glutamatebiosyntheses

In2006,Ikedaandco-workerspublishedafeworiginalarti-clesrelatedto“genomebreeding”withC.glutamicum,revealingsixkeygenes(lysC,hom,pyc,gnd,mqo,andleuC)thatareimpor-tanttargetsfortherationaldesignofanindustriall-lysineproducer(Hayashietal.,2006a;Ikedaetal.,2006;Mitsuhashietal.,2006).Takingthisgeneticinformationintoaccount,Fig.3showsthatpartoftheTRNofC.glutamicumATCC13032thatisinvolvedintheconversionofthecarbonsourcessucrose,glucoseandfruc-toseintothebiotechnologicallyrelevantproductsl-lysineandl-glutamate.ThispartialnetworkreconstructionrevealedahighcomplexityofregulatoryinteractionsinthecentralmetabolismofC.glutamicumexertedby19transcriptionregulators,includ-ingtenrepressors,sixdualregulators,oneactivator,andtwosigmafactors(inadditiontothehousekeepingsigmafactorSigA).Highlyregulatednodesinthenetworkstructureare

the

Fig.2.MatrixofregulatoryinteractionsamongtranscriptionregulatorsandregulatorygenesinC.glutamicumATCC13032.Eachcolouredboxshowsthedirectionoftheregulatoryinteractionofthetranscriptionregulatorlistedinthecorrespondingrowontheexpressionoftheregulatorygeneshowninthecorrespondingcolumns.Thediagonalofthematrixrepresentsautoregulatoryinteractions.Colourcode:red,repression;green,activation.

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179

Fig.3.Transcriptionregulatorsandtranscriptionalregulatoryinteractionsinvolvedincontrollingtheconversionofthesugarsubstratessucrose,glucoseandfructoseintotheaminoacidsl-lysineandl-glutamateinC.glutamicumATCC13032.Relevantmetabolicpathwaysareshownschematically.Reactionsinvolvedinglycolysisandthecitratecycleareshownbyblackarrowsandarelabeledbygenenames.Thesixkeygenesdetectedby“genomebreeding”arespeci callymarkedinred.Colourcode:rednode,repressor;greennode,activator;bluenode,dualregulator;yellownode,sigmafactor;dark-graynode,targetgene;light-graynode,genewithoutregulation;redline;repression;greenline,activation;graytrianglenode,antisenseRNA;greendashedline,positivecontrolbyantisenseRNA.

aconitasegeneacnandthesdhABCoperonencodingsuccinatedehydrogenase(Fig.3).Accordingly,acnandsdhABCareappro-priatecandidatesforso-calledintermodulargenes,integratingsignalsattheirpromotersitescomingfromdifferentfunctionalmodulsofthecell(Freyre-Gonzálezetal.,2008).Thereconstruc-tionalsoshowsdifferentexecutivelevelsandthusamulti-layeredhierarchyinthispartoftheC.glutamicumregulatorynetwork(Fig.3).

180K.Brinkrolfetal./JournalofBiotechnology149 (2010) 173–182

Inprinciple,networkmotifscanberegardedasthesimplestorganizationunitsofabacterialTRN(Dobrinetal.,2004).Atleasttworegulatorycascadesareinvolvedincontrollingtheexpressionoftheacngene:(i)SugR–RamA–AcnR–acnand(ii)DtxR–RipA–AcnR–acn.Thisregulatoryhierarchyoftranscriptionregulatorsrepresentsatypicaltopologicalnetworkmotif,knownasregulatorchain(Yuetal.,2003)andallowingacomplexinte-grationofenvironmentalsignalsintotheregulatorynetwork.Thehierarchicalarchitectureofthenetworkisalsocharacterizedbythehighnumberoffeed-forwardloops.Inthisregulatorynetworkmotif,tworegulators,XandY,jointlycontroltheexpressionofthetargetgeneZ,whereasXalsocontrolstheexpressionofY(Alon,2007).TypicalX–Y-pairsoftranscriptionregulatorsconsti-tutingfeed-forwardloopsinthispartoftheC.glutamicumnetworkareDtxR/RipA,GlxR/RamB,RamA/RamB,GlxR/FruR,andSugR/FruR(Fig.3).Anunusualfeatureofthepartialnetworkreconstructionisthepresenceofamulti-componentloop(Yuetal.,2003),consist-ingofthemutualregulatoryinteractionsbetweenSugRandRamA(Engelsetal.,2008;Toyodaetal.,2009).Thecompletenetworkstructurecanbeobtainedbymergingthedifferenttypesofregu-latorynetworkmotifstoasinglemotifsuperclusterofregulatoryinteractions(Dobrinetal.,2004;Shen-Orretal.,2002).

InadditiontotranscriptionalregulationsexertedbyDNA-bindingtranscriptionregulatorsandsigmafactors,wehavetoconsiderfurthermolecularmechanismsthatareinvolvedinreg-ulatoryprocessesinthispartoftheC.glutamicummetabolism.Recently,anantisensemechanismbythesmallnoncodingArnARNAswasreportedtopositivelycontroltheexpressionofthereg-ulatorygenegntR2(Frunzkeetal.,2008;Zemanováetal.,2008).ThisregulatorymechanismisapparentlylinkedtotheglobalstressresponseofC.glutamicum,sincetheextracytoplasmicfunctionsigmafactorSigHisinvolvedinthesynthesisofArnARNAs,espe-ciallyunderheat-shockconditions(Ehiraetal.,2009b;Zemanováetal.,2008).Moreover,thephosphorylationstatusoftheOdhIpro-teinturnedouttoberelevantfortheactivityofthe2-oxoglutaratedehydrogenasecomplexinC.glutamicum(Niebischetal.,2006).PhosphorylationofOdhIiscontrolledbythefourserine/threonineproteinkinasePknA,PknB,PknG,andPknL,withPknGbeingthemostimportantone(Schultzetal.,2009).Initsunphosphory-latedstate,theOdhIproteinbindstotheOdhA(E1)subunitoftheoxoglutaratedehydrogenasecomplex,therebyinhibitingitsactiv-itywhichisessentialforef cientglutamateproduction(Niebischetal.,2006;Schultzetal.,2007).Theseexamplesindicatethatwecanexpectthedetectionofmoreregulatoryelementsatthevariouslevelsofgeneexpressionandprotein–proteininteractionthatcon-tributetothecontrolofmetabolicpathwaysinvolvedinl-lysineandl-glutamateproductioninC.glutamicum.

4.Conclusionsandoutlook

Inrecentyears,tremendousprogresshasbeenmadeintheexperimentalcharacterizationoftranscriptionregulatorsinC.glu-tamicum.Since2007,atleast53originalarticlesrelatedtotheexaminationoftranscriptionregulatorsinC.glutamicumhavebeenpublished(Baumbachetal.,2009a).Currently,almost900regula-toryinteractionshavebeendeducedfromwet-labexperimentsandreliablecomputationalpredictionsandarestoredinthereferencedatabaseCoryneRegNet5.0(Baumbachetal.,2009a).Despitethisrecentprogress,ourknowledgeabouttheTRNofC.glutamicumisfarfrombeingcomplete.Inparticular,thereislackofinformationonglobalregulatorsinC.glutamicum(withtheexceptionofGlxR)thatful llimportantfunctionsinthearchitectureofTRNs.IntheE.colinetworkmodel,mainlynucleoid-associatedproteins,sigmafactorsandtwo-componentsignaltransductionsystemscontributetotherepertoireofglobalregulators(Freyre-Gonzálezetal.,2008;

Maetal.,2004).Threetwo-componentsystemswerecharacterizedinC.glutamicumsofar,includingCitAB,MtrABandPhoRS(Brockeretal.,2009;Mökeretal.,2004;SchaafandBott,2007),butnoneofthesesystemswasattributedaglobalroleintheTRN.PromisingcandidatesforglobaltranscriptionregulatorsinC.glutamicumare,ontheotherhand,thegroup2sigmafactorSigB(Ehiraetal.,2008;Larischetal.,2007)andtheextracytoplasmicfunctionsigmafactorSigH(Ehiraetal.,2009b).However,furtherresearchisnecessarytoestablishaglobalroleoftheseproteinsintheTRNmodelofC.glutamicumbydirectexperimentalevidence.

ThehighnumberofregulatoryinteractionsknowninC.glu-tamicumprovidesthebasisforapplyingnovelcomputationalapproachestothereofdeducethefunctionalarchitectureoftheTRN.Therecentlydescribednaturaldecompositionapproach,forexample,mayshednewlightonthetopologicaldesignprinci-plesoftheTRN,inconjunctionwithde nedsigni cancemeasures,suchasthenovelconnectivityvalueÄ(Freyre-Gonzálezetal.,2008).Bythismeansthehierarchicalandfunctionalcomposi-tionoftheTRNcanbestrengthenedbystatisticalanalysesandmayleadtothedetectionofintermodulargenesthatplayimpor-tantrolesintheintegrationofdifferentphysiologicalresponses(Freyre-Gonzálezetal.,2008).Distinctpartsofthegeneregu-latorynetwork,suchasthemetabolicroutesrelevantfortheindustrialproductionofl-lysineandl-glutamate,mayprovidethebasisforthedesignofintegratedmodelsbycombiningregulatorydatawiththerecentlypublishedcomprehensivemetabolicmod-elsofC.glutamicum(KjeldsenandNielsen,2009;Shinfukuetal.,2009).Thisintegratedmodelcanbeusedforcomputationalsim-ulationstodetectpotentialtargetsformetabolicengineeringandoptimizationofaminoacidproducingstrains.Furthermore,novelhigh-throughputsequencingtechnologiesseemtobeappropriateforlarge-scaleexpressionstudiestovalidateandextendthecur-rentTRNmodelofC.glutamicum(DroegeandHill,2008;Faithetal.,2007;Lemmensetal.,2009).Thistypeofexperimentcanalsohelptoovercomethecurrentstaticaldescription(thatismainlycausedbythelackofknowledgeabouteffectorslinkingthereg-ulatorysystemwiththemetabolicnetwork)andmayresultinadata-driven,dynamicviewonthecontrolofgeneexpressioninselectedpartsoftheC.glutamicumnetwork.Inadditiontomodel-ingapproaches,geneticengineeringoftheregulatorynetworkwillbecomeanimportanttaskintheemerging eldofsyntheticbiologywithindustriallyrelevantmicroorganisms(Choetal.,2007).References

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