The transcriptional regulatory repertoire of Corynebacterium(2)

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).