The transcriptional regulatory repertoire of Corynebacterium(8)

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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