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

Aderem,A.,2005.Systemsbiology:itspracticeandchallenges.Cell121,511–513.Alam,M.S.,Garg,S.K.,Agrawal,P.,2007.MolecularfunctionofWhiB4/Rv3681c

ofMycobacteriumtuberculosisH37Rv:a[4Fe-4S]clusterco-ordinatingproteindisulphidereductase.Mol.Microbiol.63,1414–1431.

Alon,U.,workmotifs:theoryandexperimentalapproaches.Nat.Rev.

Genet.8,450–461.

Babu,M.M.,Luscombe,N.M.,Aravind,L.,Gerstein,M.,Teichmann,S.A.,2004.Struc-tureandevolutionoftranscriptionalregulatorynetworks.Curr.Opin.Struct.Biol.14,283–291.

Babu,M.M.,putationalapproachestostudytranscriptionalregulation.

Biochem.Soc.Trans.36,758–765.

Balaji,S.,Babu,M.M.,Aravind,L.,2007.Interplaybetweennetworkstructures,

regulatorymodesandsensingmechanismsoftranscriptionfactorsinthetran-scriptionalregulatorynetworkofE.coli.J.Mol.Biol.372,1108–1122.

Balleza,E.,Lopez-Bojorquez,L.N.,Martinez-Antonio,A.,Resendis-Antonio,O.,

Lozada-Chavez,I.,Balderas-Martinez,Y.I.,Encarnacion,S.,Collado-Vides,J.,2009.Regulationbytranscriptionfactorsinbacteria:beyonddescription.FEMSMicrobiol.Rev.33,133–151.

Baumbach,J.,Brinkrolf,K.,Czaja,L.F.,Rahmann,S.,Tauch,A.,2006.CoryneRegNet:

anontology-baseddatawarehouseofcorynebacterialtranscriptionfactorsandregulatorynetworks.BMCGenomics7,24.

Baumbach,J.,Rahmann,S.,Tauch,A.,2009a.Reliabletransferoftranscriptionalgene

regulatorynetworksbetweentaxonomicallyrelatedorganisms.BMCSyst.Biol.3,8.

Baumbach,J.,Wittkop,T.,Kleindt,C.K.,Tauch,A.,2009b.Integratedanalysisand

reconstructionofmicrobialtranscriptionalgeneregulatorynetworksusingCoryneRegNet.Nat.Protoc.4,992–1005.