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.