phosphorylationjavmoo.info

RESEARCHOpenAccessMetabolicregulationofEscherichiacolianditsgdhA,glnL,gltB,DmutantsunderdifferentcarbonandnitrogenlimitationsinthecontinuouscultureRahulKumar1,KazuyukiShimizu1,2*AbstractBackground:Itisquiteimportanttounderstandhowthecentralmetabolismisregulatedundernitrogen(N)-limitationaswellascarbon(C)-limitation.
Inparticular,theeffectofC/Nratioonthemetabolismisofpracticalinterestfortheheterologousproteinproduction,PHBproduction,etc.
Althoughthecarbonandnitrogenmetabolismsareinterconnectedandtheoverallmechanismiscomplicated,itisstronglydesirabletoclarifytheeffectsofcultureenvironmentonthemetabolismfromthepracticalapplicationpointofview.
Results:TheeffectofC/NratioonthemetabolisminEscherichiacoliwasinvestigatedintheaerobiccontinuouscultureatthedilutionrateof0.
2h-1basedonfermentationdata,transcriptionalRNAlevel,andenzymeactivitydata.
Theglucoseconcentrationwaskeptat10g/l,whileammoniumsulfateconcentrationwasvariedfrom5.
94to0.
594g/l.
TheresultantC/Nratioswere1.
68(100%),2.
81(60%),4.
21(40%),8.
42(20%),and16.
84(10%),wherethepercentagevaluesinbracketsindicatetheratioofN-concentrationascomparedtothecaseof5.
94g/lofammoniumsulfate.
ThemRNAlevelsofcrpandmlcdecreased,whichcausedptsGtranscriptexpressiontobeup-regulatedasC/Nratioincreased.
AsC/Nratioincreasedcratranscriptexpressiondecreased,whichcausedptsH,pfkA,andpykFtobeup-regulated.
AthighC/Nratio,transcriptionalmRNAlevelofsoxR/Sincreased,whichmaybeduetotheactivatedrespiratorychainasindicatedbyup-regulationsofsuchgenesascyoA,cydB,ndhaswellastheincreaseinthespecificCO2productionrate.
TherpoNtranscriptexpressionincreasedwiththeincreaseinC/Nratio,whichledglnA,L,GandgltDtranscriptexpressiontochangeinsimilarfashion.
ThenactranscriptexpressionshowedsimilartrendasrpoN,whilegdhAtranscriptexpressionchangedinreversedirection.
ThetranscriptionalmRNAlevelofglnB,whichcodesforPII,glnDandglnKincreasedasC/Nratioincreases.
ItwasshownthatGS-GOGATpathwaywasactivatedforgdhAmutantunderN-richcondition.
InthecaseofglnLmutant,GOGATenzymeactivitywasreducedascomparedtothewildtypeunderN-limitation.
InthecaseofgltB,Dmutants,GDHandGSenzymeswereutilizedunderbothN-richandN-limitedconditions.
Inthiscase,thetranscriptionalmRNAlevelofgdhAandcorrespondingGDHenzymeactivitywashigherunderN-limitationascomparedtoN-richcondition.
Conclusion:ThemetabolicregulationofE.
coliwasclarifiedunderbothcarbon(C)-limitationandnitrogen(N)-limitationbasedonfermentation,transcriptionalmRNAlevelandenzymeactivities.
Theoverallregulationmechanismwasproposed.
TheeffectsofknockingoutN-assimilationpathwaygeneswerealsoclarified.
*Correspondence:shimi@bio.
kyutech.
ac.
jp1DepartmentofBioscienceandBioinformatics,KyushuInstituteofTechnology,Iizuka,Fukuoka820-8502,JapanKumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/82010KumarandShimizu;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
BackgroundItisquiteimportanttounderstandhowthecultureenvironmentaffectsthecellmetabolism.
Amongthecultureenvironments,carbonandnitrogensourcesarebyfarimportantinpractice.
Tounderstandtheregula-tionofcentralmetabolisminE.
coli,thecellmetabolismundercarbon(C)andnitrogen(N)limitationshasbeeninvestigatedinthecontinuousculture[1-3].
Thesestu-dieshighlighttheimportanceofcriticalnodalpointslikePEP-PYR-OAAforthecarboncatabolism[4],andglutamineandglutamatesynthesisfornitrogenassimila-tion[5].
Thesubsequentstudiesmadeitclearthatcar-bonmetabolismisnotonlycontrolledbycarbon-derivedsignals,butalsobytheavailabilityofnitrogenandothernutrients[6].
Therefore,severalstudieshavebeenmadeontheregulatoryinterdependenceofdiffer-entmetabolicroutes.
Fromthesestudies,twoofthemajorsignaltransductionsystemsofnitrogenandcar-bonmetabolismhavebeenidentifiedasPII,asmallnitrogenregulatoryproteinandthephosphotransferasesystem(PTS).
Becauseoftheimportantrolesinregula-toryfunctions,PIIandthePTScanberegardedasthecentralprocessingunitsofnitrogenandcarbonmetabo-lism,respectively.
ThePIIproteinsensesa-KGandATP,thuslinkthestateofcentralcarbonandenergymetabolismforthecontrolofnitrogenassimilation[6].
Theglucosecatabolismismodulatedbytheglobalregu-latorsencodedbysuchgenesascra,crp,cya,mlcetc[7,8],whilenitrogenassimilationisregulatedbyPII-NtrsystemtogetherwithglobalregulatorslikeCrp,provid-inganovelregulatorynetworkbetweencarbonandnitrogenassimilationinE.
coli[9].
InE.
coli,assimilationofN-sourcesuchasammonia/ammonium(NH4+)usinga-KGresultsinthesynthesisofglutamateandglutamine(Fig.
1).
Glutaminesynthe-tase(GS,encodedbyglnA)catalyzestheonlypathwayforglutaminebiosynthesis.
Glutamatecanbesynthe-sizedbytwopathwaysthroughthecombinedactionsofGSandglutamatesynthase(GOGAT,encodedbygltBD)formingGS/GOGATcycle,orbyglutamatedehydrogenase(GDH,encodedbygdhA)[10].
TheGS/GOGATcyclehasahighaffinityforNH4+(Km1mM),andisutilizedwhensuffi-cientnitrogenisavailableinthemedium.
WhenextracellularNH4+concentrationislowaround5μMorless,ammoniumentersthecellviaAmtBandiscon-vertedtoglutaminebyGS,andUTaseuridylylatesbothGlnKandGlnB[11].
WhenextracellularNH4+concen-trationismorethan50μM,themetabolicdemandforglutaminepoolrises,andUTasedeuridylylatesGlnKandGlnB.
GlnKcomplexeswithAmtB,therebyinhibitingthetransporterviaAmtB,whereGlnBinter-actswithNtrBandactivatesitsphosphataseactivityleadingtodephosphorylationofNtrC,andNtrC-depen-dentgeneexpressionceases[11].
ThecentralnitrogenintermediatessuchasglutamineandglutamateprovidenitrogenforthesynthesisofalltheotherN-containingcomponents.
About88%ofcellularnitrogencomesfromglutamate,andtherestfromglutamine[5].
TheATPrequiredforthenitrogenassimilationusingGS/GOGATcycleunderN-limitingconditionaccountsfor15%ofthetotalrequirementinE.
coli.
AsignificantamountofNADPHisalsorequiredfornitrogenassimi-lation[5,10].
Theotherpathwaysinvolvedinmaintain-ingcellularnitrogenbalanceunderspecificconditionsincludeaspartate-oxaloacetateandalanine-pyruvateshunts[12,13].
Thecarbonandnitrogenmetabolismarelinkedbyenergymetabolism.
TheglycolyticfluxinE.
coliiscon-trolledbythedemandforATP[14].
Recently,ithasbeenreportedthatthePIIproteincontrolsnitrogenassimilationbyactingasasensorofadenylateenergycharge,whichisthemeasureofenergyavailableformetabolism.
ThesignaltransductionrequiresATPbind-ingtoPII,whichissynergisticwiththebindingofa-KG.
Furthermore,a-KGservesasacellularsignalofcarbonandnitrogenstatus,andstronglyregulatesPIIfunctions[15].
Thestudiesonthecarbonandnitrogenpathwayinterdependencehavesofarfocusedontheconversionofa-KGtoglutamate[16].
Itisevidentthattheregulatorymechanismofthisconversioniscriticalfortheinterdependenceofcarbonandnitrogenassimi-lation.
However,littlehasbeeninvestigatedonhowgene-levelregulationaffectsthecellmetabolismundernitrogenandcarbonlimitations.
Inthepresentresearch,theeffectsofC/NratioonE.
colimetabolismbasedonfermentation,transcriptionalmRNAlevels,andenzymeactivitieswereinvestigated.
SuchinvestigationisalsoofpracticalinterestfortheefficientproductionofPHB,ergosterol,ornithine,arginine,putrescine,GABA,lyco-pene,ε-caprolactone,etc[17-20].
Moreover,themeta-bolicregulationanalysiscanbeutilizedforthedynamicmodelingofN-regulation[21].
Inordertounderstandthemetabolicregulationmechanisminmoredetails,wealsoinvestigatedtheeffectsofdeletingN-assimilatingpathwaygenessuchasgdhA,glnL,gltB,andgltDonthemetabolismascomparedtothewildtypeE.
coli.
ResultsWildTypeFig.
2showstheeffectofC/Nratioonthefermentationcharacteristics,whereFig.
2aindicatesthattheglucoseconcentrationincreases,whereasthecellconcentrationdecreasesasC/Nratioincreases.
Fig.
2aalsoshowsthatKumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page2of17theglucoseconcentrationwasverylowat100%and60%ofNconcentrations(C-limitation),whereasitscon-centrationwashighat20%and10%ofNconcentrations(N-limitation).
Fig.
2bshowstheeffectofC/Nratioonthespecificrates,whichindicatesthatthespecificglu-coseconsumptionrateaswellasthespecificacetateandCO2productionratestendedtoincreaseasC/Nratioincreases.
TherawfermentationdataaregiveninAdditionalfile1.
Inordertointerpretthefermentationcharacteristics,therelativemRNAlevelsweremeasuredfordifferentC/NratiosbyRT-PCR(Fig.
3).
Fig3ashowsthatcrptran-scriptlevelbecamelower(p<0.
01)asC/Nratioincreases,whichcorrespondstothefactthatcAMP-Crpleveldecreasesasglucoseconcentrationincreases.
Inaccordancewiththechangeincrptranscriptlevel,mlclevelchangedinsimilarfashion[8].
Fig.
3aalsoshowsthatthetranscriptlevelsofsuchgenesassoxR/SandrpoSbecamehigherasC/Nratioincreases,whichmaybeduetooxygenstresscausedbyhigherrespiratoryactivityfortheformer[1],alongwithnutrientstressforthelatter[22].
Inrelationtotheup-regulationofsoxR/S,thesodAtranscriptlevelincreasedasC/NratioincreasesexceptatthehighestC/Nratio(Fig.
3d).
Fig.
3ashowsquitehighexpressionofanaerobicregulatorfnratthehighestC/Nratio,whilethetranscriptlevelofarcAwhichcodesformicroaerobicregulatordidnotchangemuch.
ThetranscriptlevelofrpoN,whichencodess54,increasedasC/Nratioincreases(Fig.
3b).
Fig3balsoshowsthattheexpressionsofglnA,glnL,glnG,andgltDgeneschangedinsimilarfashionasrpoN,indicatingtheactivationofGS-GOGATpathwayunderN-limitation.
TheglnBgenewhichcodesforPIIalsochangedinsimi-larfashion,whileglnDwhichcontrolstheuridylylationanddeuridylylationshowssomewhatdifferentbutthetrendseemstobesimilar(Fig.
3b).
PIIparalogueencod-inggene,glnKshowsveryhighexpressionsat20%and10%ofN-limitation(Fig.
3b).
TheexpressionpatternofnacissimilartothatofrpoN,whereasgdhAshowsreversepattern,implyingthatgdhAisrepressedbyNac(Fig.
3b).
AsC/Nratioincreases,thetranscriptlevelofcrpgeneaswellasmlcgenedecreased,whichthencausedthetranscriptlevelofptsGgenetobeincreasedasshowninFig.
3c.
Inrelationtothedecreaseinthetranscriptlevelofcra,thetranscriptlevelsofsuchgenesasptsH,pfkAandpykFincreasedasC/Nratioincreases(Fig.
3c).
Figure1CentralmetabolicpathwaysofE.
coliconcernedwithC-metabolismandN-assimilation.
KumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page3of17Thesecorrespondtotheincreasedspecificglucosecon-sumptionrateasC/Nratioincreases.
Moreover,therespiratorychaingenessuchascyoA,cydB,andndhtogetherwithTCAcyclegenessuchasgltA,icdA,fumC,sdhC,andmdhshowedincreasedexpressionsasC/Nratioincreases(Fig.
3d),whichcorrespondstotheincreaseinthespecificCO2productionrate(Fig.
2b).
Partofthereasonwhythishappenedmaybeduetotheaccumulationofa-KGcausedbythedecreasedactivityofGDH.
AlthoughTCAcyclegenesareundercontrolofArcA(Additionalfile2),Fig.
3ashowslittlechangeinthetranscriptlevelofarcAgene.
SinceferricuptakeregulatorFuractivatessomeoftheTCAcyclegenessuchassdh,suc,andfum[23],partofthereasonmaybeduetoup-regulationofthetranscriptleveloffurgene,whereitisnotclearatthistimewhythetran-scriptlevelofthisgenetendstodecreaseasC/Nratioincreases(Fig.
3a).
Figure2ComparisonoffermentorcharacteristicsofthewildtypeE.
coliatvariousC/Nratios:(a)biomass,glucoseandacetateconcentration(g/l),cellyield(g/g);(b)specificratesofglucoseconsumption,acetateandCO2production(mmol/gDCW.
h).
KumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page4of17MutantsFigs.
4and5showthecomparisonsofthefermentationdataforgdhA,glnL,gltB,andgltDmutantsascomparedtothewildtypeunderN-richandN-limitingcondi-tions,wheretherawdataaregiveninAdditionalfile3aand3b.
Fig.
4aindicatesthatthecellconcentrationsandthecellyieldsofthemutantswerealllowerascom-paredtothoseofthewildtype,whereglucosewasnearlycompletelyconsumedunderN-richcondition.
Fig.
4aalsoindicatesthatacetateconcentrationsincreasedforallthemutantsascomparedtothewildtype.
Amongthemutants,acetateconcentrationwasthelowestforglnLmutantfollowedbygdhA,gltBandgltDmutants.
Fig.
4bindicatesthatthespecificglucosecon-sumptionrate,specificacetateandCO2productionrateswereallincreasedforthemutantsascomparedtothewildtype.
Fig.
5aindicatesthatthecellandacetateconcentrationsandthecellyieldsofthemutantswerealmostsimilarascomparedtothoseofthewildtypeunderN-limitation.
TheglucoseconcentrationwasFigure3ComparisonofthetranscriptionalmRNAlevelsofthewildtypeE.
coligenescultivatedat100%(C/N=1.
68),40%(C/N=4.
21),20%(C/N=8.
42)and10%(C/N=1.
68)N-concentration:(a)globalregulatory,(b)N-regulatory,(c)metabolicpathway,(d)respiratorychain.
KumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page5of17relativelyhigherforthemutantsascomparedtothewildtype(Fig.
5a),althoughthespecificglucosecon-sumptionratesshowedlittlechanges(Fig.
5b).
Thefor-merphenomenonmaybecausedbythelowercellconcentration.
Fig.
5balsoindicatesthatthespecificCO2productionratesforallthemutantswerelowerascomparedtothatofthewildtype.
NotethatthespecificCO2productionratesofglnL,gltB,gltDmutantsaswellasthewildtypewerehigherunderN-limitationascom-paredtothoseunderN-richcondition,whereasthespe-cificCO2productionrateofgdhAmutantwassimilarunderN-limitationascomparedtoN-richcondition.
Inthefollowingsections,thecomparisonsofthetranscriptlevelsbetweenthewildtypeandgdhA,glnL,gltBandgltDmutantsaremade.
Overall,thechangingpatternsofgltBandgltDmutantsweresimilar,whereasgdhAandglnLmutantsshowsomewhatdifferentpatterns.
gdhAmutantInthecasewheregdhAgenewasknockedout,gluta-mateandglutaminewereformedbyGSandGOGATpathwaysunderN-richconditionascanbeseenfromtheup-regulationsofthetranscriptlevelsofglnA,L,G(p<0.
01,p<0.
01,p<0.
01,respectively),andgltDgene(p<0.
01)asshowninAdditionalfile4b.
ThechangingpatternsweresimilartothoseofthewildtypeunderN-limitation(Fig.
3b).
Additionalfile4eindicatesthatPTSandglycolysisgenessuchasptsH,ptsG,pfkA,pykFgeneswereup-regulated(p<0.
01,p<0.
05,p<0.
01,p<0.
01,respectively)andPPpathwayandEDpathwaygenessuchaszwf,gnd,andedagenesaswellaslpdAFigure4ComparisonoffermentorcharacteristicsofthewildtypeE.
coliandnitrogenassimilationrelatedmutants,gdhA,glnL,gltB,andgltDat100%N-concentration(C/Nratio=1.
68):(a)biomass,glucoseandacetateconcentration(g/l),cellyield(g/g);(b)specificratesofglucoseconsumption,acetateandCO2production(mmol/gDCW.
h).
KumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page6of17genewereup-regulated(p<0.
01,p<0.
01,p<0.
05,p<0.
01respectively)forgdhAmutantascomparedtothewildtypeunderN-richcondition.
Moreover,TCAcyclegenessuchasgltA,icdA,fumC,sdhC,mdhwereup-regulated(p<0.
01,p<0.
01,p<0.
01,p<0.
01,p<0.
01,respectively),andglyoxylatepathwaygeneaceAwereup-regulated(p<0.
01)forgdhAmutantascom-paredtothewildtypeunderN-richcondition.
NotethataspCgeneexpressionwasdown-regulatedforthemutantascomparedtothewildtype(p<0.
05),whichmaybeduetothecouplingwithGDHreaction.
More-over,Additionalfile4findicatesthattherespiratorychaingenessuchascydB,cyoA,ndhwereup-regulated(p<0.
01,p<0.
01,p<0.
1,respectively),andsodAgeneexpressionwasalsoup-regulated(p<0.
01),wherethelatterwasconsistentwiththeup-regulationofsoxRgeneexpression(p<0.
05)(Additionalfile4a)forgdhAmutantascomparedtothewildtypeunderN-richcondition.
UnderN-limitation,thetranscriptlevelsofglnA,L,GgenesaswellasrpoNweredown-regulated(p<0.
01,p<0.
01,p<0.
01,p<0.
01,respectively),whereastran-scriptlevelsforgltB,Dgenesweresimilartothoseofthewildtype(Additionalfile4d),indicatingthatonlyGOGATpathwaywasactiveingdhAmutantunderN-limitation.
Itwasalsoconfirmedbytheenzymeactivitymeasurements(Additionalfile4andFig.
6).
UnderN-limitation,thetranscriptlevelofmlcgenewashigher(p<0.
01)(Additionalfile4c)andcorrespondinglyptsHgenewasdown-regulated(p<0.
01)(Additionalfile4g).
Figure5ComparisonoffermentorcharacteristicsofthewildtypeE.
coliandnitrogenassimilationrelatedmutants,gdhA,glnL,gltB,andgltDat20%N-concentration(C/Nratio=8.
42):(a)biomass,glucoseandacetateconcentration(g/l),cellyield(g/g);(b)specificratesofglucoseconsumption,acetateandCO2production(mmol/gDCW.
h).
KumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page7of17Moreover,thetranscriptlevelsofTCAcyclegenessuchasgltA,icdA,sdhCweredown-regulated(p<0.
05,p<0.
01,p<0.
01,respectively)forgdhAmutantascom-paredtothewildtype(Additionalfile4g).
glnLmutantTheoperonglnALGtranscribesasensor-histidinekinase(NtrB/NRII,glnL)whichphosphorylatesresponseregula-tor(NtrC/NRI,glnG)inresponsetothechangeinC/Nratio.
Thisoperontranscribestheonlyglutaminesynthesizingenzyme,glutaminesynthetase(GS,glnA),wheretheGSisanessentialenzyme(glnAmutantfailedtogrowinM9minimalmedium,datanotshown).
IntheglnLmutantunderN-richcondition,thetranscriptlevelofglnAgenewascomparabletothatofthewildtype(Additionalfile5b),whileitwassignificantlyreduced(p<0.
01)underN-limitation(Additionalfile5d).
Additionalfile5ashowsthatthetranscriptlevelsofgenes,whichencodeglobalregulators,suchasfnr,arcA,cra,mlc,fur,soxR,SandrpoSwereup-regulated(p<0.
05,p<0.
01,p<0.
1,p<0.
01,p<0.
01,p<0.
01,p<0.
01andp<0.
01respectively)forglnLmutantascom-paredtothewildtypeunderN-richcondition.
Addi-tionalfile5eshowsup-regulationsofPTSandglycolysisgenessuchasptsH,ptsG,pfkA,pykF(p<0.
01,p<0.
01,p<0.
01,p<0.
01,respectively),andPPpathwaygenessuchaszwfandgndgenes(p<0.
01,p<0.
01,respec-tively)andEDpathwaygeneeda(p<0.
01)ascomparedtothewildtypeunderN-richcondition.
Moreover,Additionalfile5ealsoindicatesthattheTCAcyclegenessuchasgltA,icdA,sdhC,fumC,mdhwereup-regulated(p<0.
01,p<0.
01,p<0.
01,p<0.
01,p<0.
01,respectively)andglyoxylatepathwaygeneaceAaswellaslpdAwereup-regulated(p<0.
01,p<0.
01,respectively)forglnLmutantunderN-richcondition.
Theup-regulationsofTCAcyclegenesareconsistentwiththeincreasedspecificCO2productionrate.
TheaspCgeneexpressionwashigher(p<0.
01)forglnLmutantascomparedtothewildtypeundersuchcondi-tion(Additionalfile5e).
UnderN-limitation,thetranscriptlevelofptsHgenewasdown-regulated(p<0.
1),whileyfiDandgadAgeneswereup-regulated(p<0.
01,p<0.
1,respec-tively).
NotethatAdditionalfile5gindicatesthattheTCAcyclegenessuchasgltA,icdA,sdhC,mdhweredown-regulated(p<0.
01,p<0.
01,p<0.
01,p<0.
01,respectively)andlpdAwasdown-regulated(p<0.
01)forglnLmutantascomparedtothewildtypeunderN-limitation.
Additionalfile5h,however,showsup-regulationofthetranscriptlevelsofrespiratorypath-waygenessuchascydB,cyoA,andndh(p<0.
01,p<0.
01,p<0.
01,respectively)forglnLmutantascom-paredtothewildtypeunderN-limitation,whichisconsistentwiththeincreasedspecificCO2productionrate.
gltBandgltDmutantsThegltBDoperonisresponsibleforthesynthesisofglu-tamatesynthase(GOGAT)enzyme.
UnderN-richcon-dition,GDHandGSmaybeusedtoformglutamateandglutamine.
Additionalfile6bshowssignificantdown-regulationofthetranscriptlevelofgdhAgene(p<0.
01)forgltB,Dmutantsascomparedtothewildtype,wherep-valuesaregivenforthecriticalvaluesofeithergltBorgltDmutantinthefollowings.
Additionalfile6aindicatesthatthetranscriptlevelsoffur,rpoSandsoxR/Swereup-regulated(p<0.
01,p<0.
01,p<0.
01,p<0.
01,respectively),whiletranscriptexpressionofmlcgenewasdown-regulated(p<0.
01)forgltB,Dmutantsascomparedtothewildtype.
Thedown-regu-lationofmlctranscriptcausedup-regulationofptsHtranscript(p<0.
01)asshowninAdditionalfile6e.
ThetranscriptofglnEgenewhichencodesATasewasdown-regulated(p<0.
01)forgltB,DmutantsascomparedtothewildtypeunderN-richcondition(Additionalfile6b).
Moreover,Additionalfile6bshowsthatrpoN,glnB,glnK,glnL,andglnDgeneswereup-regulated(p<0.
01,p<0.
01,p<0.
01,p<0.
01,p<0.
01,respectively)forgltB,Dmutantsascomparedtothewildtype,implyingtheactivationofGSpathway.
UnderN-limitation,thetranscriptlevelsofsuchgenesasfur,rpoSandsoxRwereup-regulated(p<0.
01,p<0.
01,p<0.
01,respectively),andalsomlcandcrageneswereup-regulated(p<0.
05,p<0.
01respectively)forgltB,Dmutantsascomparedtothewildtype(Additionalfile6c).
Additionalfile6gindicatesthatthetranscriptlevelofptsHgenewasdown-regulated(p<0.
01),whilethetranscriptlevelsofpfkAandedageneswereup-regu-lated(p<0.
01,p<0.
05)forgltB,DmutantsascomparedtothewildtypeunderN-limitation.
Moreover,thetran-scriptlevelsoflpdA,gltA,andsdhCgenesweredown-regulated(p<0.
01,p<0.
01,p<0.
01,respectively),whileyfiDandgadAgeneswereup-regulated(p<0.
01,p<0.
05,respectively)forgltB,DmutantsascomparedtothewildtypeunderN-limitation.
Additionalfile6dindicatesthatthetranscriptlevelsofglnA,glnG,glnK,nac,andglnEgenesweredown-regulated(p<0.
01,p<0.
01,p<0.
01,p<0.
1,andp<0.
05,respectively)forgltB,Dmutantsascomparedtothewildtype,whichimpliesthatthecelltriestorepresstheGSactivity.
NotethatgdhAgeneexpressionwashigherascomparedtoN-richcon-dition,andcomparabletothewildtypeunderN-limita-tion.
ThecorrespondingactivityofGSshowedrepression,whilethatofGDHenzymeshowedde-repres-sionascomparedtothewildtypeunderN-limitation(Additionalfile6andFig.
6).
DiscussionTheglucoseconcentrationinthefermentorincreasedwiththeincreaseinC/Nratio(Fig.
2).
TheglucoseKumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page8of17Figure6EnzymeActivitiesofN-assimilationpathwaysuchasGDH,GS,andGOGATinthewildtypeE.
colianditsmutantsinUnits*/mgprotein.
KumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page9of17uptakeismadeviaphosphotransferasesystem(PTS)inE.
coli,wherephosphateofPEPistransferredbythephosphorelayviaenzymeI(EI)encodedbyptsI,histi-dinephosphorylatableproteinHPrencodedbyptsH,glucosespecificenzymeII,EIIAGlcencodedbycrr,andmembraneboundEIICBGlcencodedbyptsG.
Whenglu-coseispresentinexcess,thephosphorylatedEIIAGlctransfersphosphatetoEIICBGlcfortheglucoseuptakewithphosphorylation,andtheunphosphorylatedEIIAGlcisdominatedinthecytosol[24].
Sinceunphosphory-latedEIIAGlcdoesnotactivateCya,thecAMPleveldecreasesunderN-limitationtogetherwithcrpgeneasshowninFig.
3a.
Sincemlcisundercontrolofcrp,thetranscriptlevelofmlcgenedecreasedaswell(Fig.
3a),whichcausedup-regulationoftranscriptlevelsofptsHandptsGgenes(Additionalfile2).
Moreover,increaseintheglucoseconcentrationathigherC/Nratiomayhavecauseddown-regulationofcra,whichcausedup-regula-tionoftheglycolysisgenessuchasptsH,ptsG,pfkA,pykF,togetherwithzwf(Additionalfile2).
TheGDHpathwayisimportantduringglucose-lim-ited(C-limited)condition.
ThispathwayisfavoredwhentheorganismisstressedforenergybecauseGDHdoesnotuseATPasdoesGSpathway[25].
Fig3bshowsthedecreasedexpressionofgdhAasC/Nratioincreases.
LiangandHoughton[26]investigatedtheeffectofNH4ClconcentrationonGDHandGSactivities,andshowedtheup-regulationsofGDHandtranshydrogen-aseactivitiesatlowerNH4Clconcentration.
TheavailabilityofnitrogenissensedbyPIIproteinatthelevelofintracellularglutamine,whereglutamineissynthesizedbyglutaminesynthetase(GS)encodedbyglnA,andistransportedmainlybyGlnHPQ.
TheglnHPQoperonisunderthecontroloftandempromo-terssuchasglnHp1andglnHp2,wheretheformeriss70-dependent,andthelatteriss54-andNtrC-Pdependent[27,28].
Ithasbeenshownthatasthemajortranscriptionaleffectoroftheglucoseeffect,Crpaffectsnitrogenregulation[9].
Namely,glnAp1isactivatedbyCrpwithglutamineasN-source.
ThroughglnHPQ-dependentsignaling,CrpactstodecreasetheamountofthephosphorylatedNtrCactivator,whichinturncausesthedecreaseinglnAp2expression[9].
However,thisregulationismorecomplexasexplainednext.
Ithasbeensuggestedthats54-dependentNtrgenesofE.
coliformagenecascadeinresponsetoN-limitation[29].
ThecentralparticipantsofNtrresponseareNRIorNtrCandNRIIorNtrB,andRNApolymerasecom-plexedtos54.
NRIisthetranscriptionalactivatorofs54-dependentpromoters,whileNRIIisabifunctionalpro-teinthatcaneithertransferphosphatetoNRIorcontrolthedephosphorylationofNRI-phosphate.
N-limitationresultsinthephosphorylationofNRI,whichinturnsti-mulatestheexpressionofglnALGoperon.
TheexpressionoftheglnALGoperoniscontrolledbytan-dempromoterssuchasglnAp1andglnAp2,whereglnAp1isas70-dependentweakpromoteranditstran-scriptioncanbeactivatedbyCrpandblockedbyNtr-P.
Ontheotherhand,glnAp2istranscribedbyRNApoly-merase(Es54)andisactivatedbyNtr-P.
Therefore,glnAp2isresponsibleforactivatingglnAtranscriptionunderN-limitation[30].
Fig3bshowsthattheexpres-sionsofglnA,L,GgeneschangedinsimilarfashionasrpoNgeneexpression.
IthasbeenreportedthatthereisnoNRI-PbindingsitesinthegdhAregulatoryregion[31],anditisunli-kelyforNRItodirectlyrepressgdhApromoter[32].
AsithasbeenshownthatNacisinvolvedinthetranscrip-tionalrepressionofgdhAgeneunderN-limitation[32],NacseemstorepressgdhAgeneasshowninFig.
2b.
Fig2bshowsthatthetranscriptlevelofgdhAgenewaslower,whilegltBandDgeneswerehigherunderN-lim-itationascomparedtoC-limitation.
NADPHisanimportantcofactorinGDHand(GS)-GOGATactivities,andithasbeenreportedthattranshydrogenaseplayssomeroleintheregulationofthesepathways[26].
UnderN-limitation,theglutamateandglutaminesyn-theticpathwaysareexpectedtoberepressedduetoshortageofNH3forthosereactions,andthusNADPHislessutilized,resultinginoverproductionofNADPH.
PartofthismaybeconvertedtoNADHbytranshydro-genaseandtheconvertedNADHtogetherwithotherNADHformedmaybeutilizedforATPproductionthroughrespiratorychain.
OverproductionofNADPHrepressessuchpathwaysasG6PDH,6PGDHandICDHinE.
coli.
However,zwfwasactivatedinFig.
3c,whichmaybeduetosoxR/Scausedbyhigherrespiratoryactiv-ity.
TheICDHactivityisreportedtobeinsensitivetoNconcentration,whereFig.
3calsoshowslittlechangeinicdAgene.
E.
colipossessestwocloselyrelatedPIIparaloguessuchasGlnBandGlnK,whereGlnBisproducedconstitu-tively,anditregulatestheNtrB(NRII)/NtrC(NRI)twocomponentsystem[33].
Ithasbeenshownthattheintra-cellularconcentrationsofNRIandNRIIincreaseduponN-limitation[34-36].
ThephosphorylatedNtrCisanactivatorofvariousnitrogen-controlledgenessuchasglnAwhichcodesforGS[29]andglnKencodingthesec-ondPIIparalogues[36].
TheincreasedNRI,presumablyinthephosphorylatedformsuchasNRI-PactivatestheexpressionofglnKandnacpromotersunderN-limitation[37,38].
Fig3bshowsthatthetranscriptlevelsofglnKandnacgeneincreasedasC/Nratioincreases,whileslightdecreasecanbeseenatthehighestC/Nratio,whereithasbeenreportedthatglnKandnacpromotersaresharplyactivatedwhenammoniaisusedup[36].
ThegltBDFoperonwhichhasbeenfoundtohavebindingaffinitywithglobalregulatorssuchasFnrandKumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page10of17Crpinthepromoterregion[39],wherethetranscriptleveloffnrgenewashigherunderN-limitationwhereascrpgenebecamelower(Fig.
3a).
Theup-regulationofyfiD(Fig.
3c)maybeduetoup-regulationoffnr.
TheNtrsystemiscomposedoffourenzymes(Fig.
7):auridylytransferase/uridylyl-removingenzyme(UTase/UR)encodedbyglnDgene,asmalltrimericprotein,PIIencodedbyglnB,andthetwo-componentsystemcom-posedofNtrBandNtrC.
GlnDcontrolstheactivityofGSbyadenylylation/deadenylylationthroughabifunc-tionalenzymeadenylyltransferase(ATase),theglnEgeneproduct[40-42].
TheactivityofGlnKbecomeshighunderN-limitation(Fig.
3b)andcontributestotheregulationofNtrC-dependentgenes[43].
IthasbeenshownthatonGSadenylylation,ATaseactivityisregu-latedbyUTase/URandPIIsuchthatuponnitrogenlim-itation,UTasecovalentlymodifiesPIIbyadditionofaUMPgroupataspecificresidueandtheresultanturidy-lylatedformofPIIpromotesdeadenylylationofGSbyATase(Fig.
7).
Conversely,underN-richcondition,theuridylyl-removingactivityofGlnDpredominatesandthedeuridylylatedPIIpromotesadenylationofGSbyATase.
AdenylylationbyATaseispromotedbydeuridy-latedPIIwhichisproducedbyURactiononPII(UMP)3underhigherN-concentration(lowC/Nratio)(Fig.
7).
TheseindicatethatUTase/URandPIIactingtogethersensetheintracellularnitrogenstatus[44].
ThePIIsig-naltransductionproteinssuchasGlnBandGlnKareuridylylated/deuridylylatedinresponsetointracellularglutaminelevel,wherelowintracellularglutaminelevel,signallingN-limitation,leadstouridylylationofGlnB[44].
GlnBwasshowntobeallostericallyregulatedbya-KG,andthusGlnBmayplayaroleinintegratingsig-nalsofC/Nstatus.
TheNtrB/NtrCtwocomponentsys-temandGlnEwhichadenylylates/deadenylylatesGSarethereceptorsofGlnBsignaltransduction[43].
Ithasbeensuggestedthatthecarbon/cAMPeffectwasmediatedthroughGlnBuridylylation[43].
ThephosphorylatedNRI/NtrC(NRI/NtrC-P)activatestranscriptionfromN-regulateds54-dependentpromo-tersbybindingtotheenhancers[11,44-46].
PIIandtherelatedGlnKproteincontrolthephosphorylationstateofNRII/NtrBbystimulatingthephosphataseactivityofNRII(Fig.
7).
TheabilityofGlnKandPIItoregulatetheactivitiesofNRIIisinturnregulatedbytheintracellularsignalsofCandNavailabilityviaallostericcontrol[11].
ItisnotclearatthistimewhyTCAcycleisactivatedunderN-limitation.
OneofthepossiblereasonswhyTCAcycletogetherwithrespirationbecameactiveunderN-limitationmaybeduetotheaccumulationofa-KGcausedbytheblockageoftheGDHpathway.
ThisisalsotrueforgdhAmutantevenunderN-richcondition.
ThereasonwhyglycolysiswasactivatedmaybeduetoATPrequirementandthedown-regulationsofcrpandcracausedbytheincreaseinglucoseconcen-trationasmentionedbefore.
WhengdhAgenewasknockedout,thechangingpatternsofnitrogen-regu-latedgenesunderN-richcondition(Additionalfile4b)weresimilartothoseofthewildtypeunderN-limitingcondition(Fig.
3b),indicatingthatGS-GOGATcyclewasmainlyutilized,where2molesofGluwereformedfroma-KGandGlnviaGOGATpathway,andGlnisformedfromGluviaGSpathway.
UnderN-limitation,GSbecamelessactiveforgdhAmutant,whereonlyGOGATpathwaywasactive(Additionalfile4d).
InthecaseofglnLmutant,thetranscriptsofglycoly-sis,PPpathway,andTCAcyclegeneswereup-regulatedunderN-richcondition,whilethosewererepressedunderN-limitation.
Partofthereasonmaybeduetoup-regulationofcrpandmlcgenes.
UnderN-limitation,GluisproducedfrombothGDHandGOGATbuttheformerislessutilizedbecauseoflessavailabilityofNH3,whichmeansthatessentiallyGOGATpathwaywasactive.
ThisphenomenonissimilartothecaseofgdhAmutantunderN-limitation(Fig.
8).
SinceNRIcouldnotbephosphorylatedinglnLmutant,glnAgeneexpressionwaslowerascomparedtothewildtype(Additionalfile5d).
Moreover,thetranscriptlevelofnacgenedidnotchangeevenunderN-limitation,whichmaybeduetolackofphosphorylatedNRI.
TheremightbeanothermechanisminglnLmutant[47].
Namely,acetylphos-phate(AcP)isasignalingmoleculeforglucoseavailabil-ity[48]aswellascAMP,andNRIitselfiscapableofsensingtheAcPlevel,wherethisbecomessignificantonlyintheabsenceofNRII(glnLmutant)[49,50].
IntheabsenceofNRII,NRIsensesAcPlevelandinduceglnAp2.
TheglnGgeneexpressionmaybereflectedinthecarbonlevel,whereNRIbindingsiteoverlapanotherpromoter,glnAp1,whichisregulatedbycAMP-Crp.
TheeffectofglnGgeneknockoutisalsogivenelsewhere[51].
ThosemechanismsarebrieflysummarizedinFig.
9.
InthecaseofgltB,Dmutants,theeffectsofthesegenesknockoutonthemetabolismmaybeconsideredtobeminorunderN-richcondition,sincethispathwayisnotutilizedunderN-richconditioninthewildtype.
However,itwasshownthatgdhAgeneexpressionwassignificantlyreduced,whileGSpathwaywasactivatedascanbeseeninAdditionalfile6b.
Since,thetranscriptlevelofrpoNgenewashighevenunderN-richcondi-tionforgltB,Dmutants(Additionalfile6b),thismayhavecausedNactobeincreased(althoughnotsignifi-cantinAdditionalfile6b)andrepressedgdhAgene.
ItisnotclearatthistimewhythetranscriptlevelsofrpoNgenebecamehigherunderN-richcondition.
UnderN-limitation,nacgenewasrepressed(p<0.
01),andthusthetranscriptlevelofgdhAgenewascompar-abletothewildtypeunderN-limitation.
Thede-KumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page11of17repressionofGDHactivityappearstobedrivenbythecellularrequirementforglutamate(Additionalfile6andFig.
6).
Additionalfile6dshowsthatthetranscriptlevelsofglnAandglnGgeneswerelow(p<0.
01,p<0.
01)andthusGSenzymewasrepressedunderN-limitationascomparedtothewildtype(Additionalfile6andFig.
6).
Additionalfile6eand6gindicatesthatthetranscriptlevelofgadAgenewashigherunderbothC-limitationandN-limitation.
SincegltB,Dmutantsarereportedtobeosmosensitive[52],glutamateandGABAetc.
mayhavebeenaccumulatedandexcretedintothefermenta-tionbroth.
Overall,thede-repressionofGDHenzymeunderN-limitationindicatesthecomplexmechanismofN-regulationinthesemutantsandthemechanisticdetailsofthisde-repressionarenotyetcompletelyknown.
OneofthereasonwhythecellconcentrationsofthemutantsunderC-limitationislowerthanthatofthewildtype(Fig.
4a)maybeduetolowerformationrateofGlucausedbythedeficiencyinN-assimilatingpath-ways.
TheoverallregulationmechanismissummarizedinFig.
7,andtheeffectsofspecificgeneknockoutonthenitrogenassimilatingpathwaysareillustratedinFig.
8.
ItisimportanttonotethattheexperimentalresultsatC/Nratio16.
48showsomewhatdifferentpatternasFigure7ProposedoverallmechanismofN-assimilationinE.
coliunderC-limited(N-rich)andN-limitedconditions.
KumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page12of17comparedtotheotherratiosusedinthepresentstudy.
Theamountofavailablenitrogenseemstobethecru-cialfactorindecidingthemetabolicresponseespeciallyunderN-limitation.
MostthepathwaysusedforN-assimilationunderN-limitationutilizehighamountofATP.
Therefore,itappearscriticalforthecelltoshutdownactivitiesofsuchpathwayslikeGS-(GOGAT)undercertaincircumstancestosaveATPandtopreventexcessiveglutamineproduction.
Thisscenariohasbeenspeculatedforammoniumshocktothecarbonstarvedcells[20].
NotethatthetranscriptlevelsofsuchgenesasglnA,glnL,glnGandgltB,gltDwhichencodesforGS-GOGATpathwayenzymeswerereduced(Fig.
3b).
ConclusionThemetabolicregulationofE.
coliwasclarifiedtosomeextentunderbothC-limitation(N-richcondition)andN-limitationinviewoffermentationcharacteristics,transcriptlevels,andenzymeactivities.
TheoverallmechanismwasfoundtobeasdepictedinFig.
7.
More-over,theeffectsofknockoutofN-assimilationpathwaygenessuchasgdhA,glnL,andgltB,DwereinvestigatedandfoundtobeasshowninFig.
8.
MaterialsandmethodsStrains,mediacomposition,andcultivationconditionsThestrainsusedwereE.
coliBW25113[F-l-rph-1ΔaraBADAH33lacIqΔlacZWJ16rrnBT14ΔrhaBADLD78hsdR514],anditssinglegeneknockoutmutantslackingsuchgenesasgltB(JW3180),gltD(JW3180),gdhA(JW1750),andglnL(JW3840).
Thesesinglegeneknock-outmutantswereobtainedfromKeiocollection[53].
AllthestrainswerefirstpreculturedintheLuria-Ber-tanimedium.
ThesecondprecultureandthemainFigure8ProposedschematicillustrationofN-assimilationpathwaysforthewildtypeE.
colianditsmutantsunderC-limiting(N-rich)andN-limitingconditions.
KumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page13of17culturewerecarriedoutusingM9minimalmediumcontaining10gofglucosetogetherwiththefollowingcomponents(perliter):6.
81gNa2HPO4,2.
99gKH2PO4,0.
58gNaCland5.
94g(NH4)2SO4.
Thefol-lowingcomponentswerefiltersterilizedandthenadded(perliter)with1mlof1MMgSO4.
7H2O,1mlof0.
1mMCaCl2.
2H2O,1mlof1mg/lthiamineHCland10mloftraceelementsolutioncontaining(perliter):0.
55gCaCl2.
2H2O,1.
67gFeCl3.
6H2O,0.
1gMnCl2.
4H2O,0.
17gZnCl2,0.
043gCuCl2.
2H2O,0.
06gCoCl2.
2H2O,and0.
06gNa2MoO4.
2H2O.
Thenitrogenconcentra-tionsusedinthepresentexperimentswere0.
594g/l,1.
188g/l,2.
376g/l,3.
564g/l,and5.
94g/lof(NH4)2SO4,whereastheconcentrationsofalltheothermediumcomponentswerethesame.
Thecontinuousculturewasconductedina1-lfermenter(MDL100,MarubishiCo.
,Tokyo,Japan)withaworkingvolumeof500ml.
ThepHwascontrolledat7.
0±0.
05using2NHClor2NNaOH,andthetemperaturewassetat37°C.
Theairflowratewas1vvm(airvolume/workingvolume/min),andtheagitationspeedwas350rpmtomaintainthedissolvedoxygenconcentrationtobeat35-40%(v/v)ofairsaturation[22].
TheCO2andO2concentrationsweremonitoredusinganoff-gasanalyzer(BMJ-02PI,ABLECo.
,Japan).
Thedilutionratewas0.
2h-1forallthecontinuouscultures.
Thesampleswerecollectedatthesteadystatewhichwasconfirmedbytheconstantoff-gasandcelldensity.
Itgenerallytook5-6residencetimestoachievethesteadystate.
AnalyticalmethodBacterialgrowthwasmonitoredbymeasuringtheopti-caldensityoftheculturebrothat600nm(OD600nm)usingaspectrophotometer(Ubet-30,Jasco,Tokyo,Japan).
Itwasconvertedtodrycellweight(DCW)basedontheOD600nm-DCWrelationshippreviouslyobtained[54].
Glucoseandacetateconcentrationsinthemediumweremeasuredusingcommerciallyavailablekits(WakoCo.
,Osaka,Japanforglucose;Roche,MolecularBio-chemical,Mannheim,Germanyforacetate).
RNApreparation,designofPCRprimersTotalRNAwasisolatedfromE.
colicellsbyQiagenRNeasyMiniKit(QIAGENK.
K.
,Japan)accordingtothemanufacturer'srecommendation.
ThequantityandpurityofRNAweredeterminedbytheopticaldensitymeasurementsat260and280nmandby1%formalde-hydeagarosegelelectrophoresis.
Thesequencesofpri-mersforrespectivegenesusedinthisstudywerereportedelsewhere[55],exceptsuchgenesasrpoN,glnA,glnB,glnD,glnE,glnG,glnL,gltDandnac.
Thepri-mersequencesoftheseadditionalgenesareasfollows:rpoNForward:5'GCAACTCAGGCTTAGCCAAC3'Reverse:5'TCCAGCGTTTCACTGTCTTG3'glnAForward:5'ATGTCCGCTGAACACGTACT3'Reverse:5'GCTGTAGTACAGCTCAAACTC3glnBForward:5'CGAAGTGAAAGGCTTTGGTC3'Reverse:5'GCCACGTCAAAGACGAAGAT3'glnDForward:5'CACCTGTTGATGTCGGTGAC3'Reverse:5'GCTTCCAGCTATTCCACAGC3'glnEForward:5'CCCGCACCACCTATTTAGAA3'Reverse:5'GCTGGTAAAGGGTGTTTGGA3'glnGForward:5'ATGCAACGAGGGATAGTCTG3'Reverse:5'TCACTCCATCCCCAGCTCTT3'glnLForward:5'GAGATGGCTCCGATGGATAA3'Figure9ProposedschemeofN-assimilationinE.
coliinresponsetothechangesinC-andN-concentrations.
KumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page14of17Reverse:5'ATGGGTCAGGTAACGCTTTG3'gltDForward:5'CAATTTATCGACCTGCAGCG3'Reverse:5'AACTTCCAGCCAGTTCATAAT3'nacForward:5'TTCAGACGCCTGAAATACTTC3'Reverse:5'TTAGCTCACCAATTGCCACT3'Criteriaforthedesignofthegene-specificprimerpairswerefollowedaccordingtoSambrookandRussell[56].
TheprimersusedinthisstudyweresynthesizedatHokkaidoSystemScienceCo.
(Sapporo,Hokkaido,Japan).
Inallcases,theprimer-suppliedcompanycon-firmedthepurityandabsolutespecificityofprimers.
cDNAsynthesisandPCRamplificationRT-PCRreactionswerecarriedoutinaTaKaRaPCRThermalCycler(TaKaRaTP240,Japan)usingQiagenOneStepRT-PCRKit(QIAGENK.
K.
,Japan).
Thereac-tionmixturewasincubatedfor30minat50°Cforreversetranscription(cDNAsynthesis)followedby15minincu-bationat95°CforinitialPCRactivation.
Then,thepro-cesswassubjectedto30cyclesofamplificationwhichconsistedofadenaturingstep(94°Cfor1min),anannealingstep(approximately5°Cbelowmeltingtem-peratureofprimersfor1min)andanextensionstep(72°Cfor1min),andfinallythereactionmixturewasincu-batedfor10minat72°Cforfinalextension.
Tocheckfornucleicacidcontamination,onenegativecontrolwasrunineveryroundofRT-PCR.
Thiscontrollacksthetem-plateRNAinordertodetectpossiblecontaminationofthereactioncomponents.
5mlofamplifiedproductswererunona1%agarosegel.
Gelswerestainedwith1μgml-1ofethidiumbromide,photographedusingaDigi-talImageStocker(DS-30,FASIII,Toyobo,Osaka,Japan)underUVlightandanalyzedusingGel-ProAnalyzer3.
1(Toyobo,Osaka,Japan)software.
AlthoughthePCRpro-ductsobtainedforallthegenesshowedthepredictedsizesonagarosegel,theidentityofamplifiedfragmentsofsomegeneswasdemonstratedbyDNAsequencing.
InordertodeterminetheoptimalamountofinputRNA,thetwo-folddilutedtemplateRNAwasamplifiedinRT-PCRassaysunderidenticalreactionconditionstocon-structastandardcurveforeachgeneproduct.
WhentheoptimalamountofinputRNAwasdeterminedforeachgeneproduct,RT-PCRwascarriedoutunderidenticalreactionconditionstodetectdifferentialtranscriptlevelsofgenes.
ThegenednaA,whichencodesforDnaA,areplicationinitiationfactorinE.
coliandisnotsubjectedtovariableexpression,i.
e.
abundantexpressionatrela-tivelyconstantrateinmostcells,wasusedasaninternalcontrolfortheRT-PCRdeterminations[55].
Tocalculatethestandarddeviation,RT-PCRwasindependentlyper-formedthreetimesforeachgeneunderidenticalreactioncondition.
Toensurethattheobservedchangesweresta-tisticallysignificant,theStudent'st-testwasapplied.
EnzymeAssaysThecellswereharvestedatthesamestageasthosetakenforRT-PCRanalysisbythecentrifugationat10,000*gfor10min,washedtwiceinice-cold100mMTris-HCl(pH7.
0)buffercontaining20mMKCl,5mMMnSO4,2mMdithiothreitoland0.
1mMEDTA,andthenre-sus-pendedinthesamebuffersolution(ca.
15gwetcellsin50mlbuffersolution)andstoredat-80°Cinaliquotsforatleast30min.
Thecelldisruptionwasachievedbysoni-cationonanultrasonicdisrupter(UD-201,TomyCo.
,Tokyo,Japan)andresultingcrudecellextractswereimmediatelyusedforthemeasurementsofenzymeactiv-itiesorstoredat-80°Cinaliquots.
Allabovementionedoperationswerecarriedoutonice[54].
InthepresentstudyenzymeactivitiesinvolvedintheN-assimilationpathwayweremeasured.
Themeasurementswerecarriedoutonathermostatrecordingspectrophotometer(U-2000A,HitachiCo.
,Japan)at37°C.
Theproteinconcen-trationswereestimatedbytheBradfordassaymethod.
Eachenzymewasmeasuredthreetimesforthesamecul-ture.
GDHwasassayedbyfollowingtheoxidationofNADPHinasolutioncontaining50mMHepes/KOH,pH7.
5,50mMNH4Cl,5mMa-KG,and0.
3mMNADPH.
GOGATwasassayedinthesamereactionmix-turesubstituting5mML-glutamineforNH4Cl[57].
GSassayfollowedthemethodsuggestedbySigma-Aldrichbasedonpreviouslypublishedstudy[58].
Additionalfile1:FermentationparametersforthechemostatculturesofthewildtypeE.
coliatthedilutionrateof0.
2h-1undervariousC/Nratios.
Clickhereforfile[http://www.
biomedcentral.
com/content/supplementary/1475-2859-9-8-S1.
DOC]Additionalfile2:Globalregulatorsandtheirregulatedgenes.
Clickhereforfile[http://www.
biomedcentral.
com/content/supplementary/1475-2859-9-8-S2.
DOC]Additionalfile3:a:FermentationparametersforthechemostatculturesofthewildtypeE.
coliincomparisontothenitrogenregulatorymutantsatthedilutionrateof0.
2h-1at100%nitrogen.
b:FermentationparametersforthechemostatculturesofthewildtypeE.
coliincomparisontothenitrogenregulatorymutantsatthedilutionrateof0.
2h-1at20%nitrogenconcentration.
Clickhereforfile[http://www.
biomedcentral.
com/content/supplementary/1475-2859-9-8-S3.
DOC]Additionalfile4:ComparisonofthetranscriptionalmRNAlevelsbetweenthewildtypeE.
coliandgdhAmutantgenesatC/Nratio1.
68and8.
42.
Clickhereforfile[http://www.
biomedcentral.
com/content/supplementary/1475-2859-9-8-S4.
DOC]Additionalfile5:ComparisonofthetranscriptionalmRNAlevelsbetweenthewildtypeE.
coliandglnLmutantgenesatC/Nratio1.
68and8.
42.
Clickhereforfile[http://www.
biomedcentral.
com/content/supplementary/1475-2859-9-8-S5.
DOC]KumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page15of17Additionalfile6:ComparisonofthetranscriptionalmRNAlevelsbetweenthewildtypeE.
coliandgltB,gltDmutantsgenesatC/Nratio1.
68and8.
42.
Clickhereforfile[http://www.
biomedcentral.
com/content/supplementary/1475-2859-9-8-S6.
DOC]AbbreviationsPEP:Phospho-enol-pyruvate;PYR:Pyruvate;OAA:OxaloaceticAcid;a-KG:2-ketoglutarate;PTS:Phosphotransferasesystem;GS:GlutamineSynthetase;GOGAT:Glutamatesynthase;GDH:Glutamatedehydrogenase;PHB:poly(3-hydroxybutyrate);GABA:Gamma-aminobutyricacid;DCW:DryCellWeight.
AcknowledgementsThisresearchwassupportedinpartbyStrategicInternationalCooperativeProgram,JapanScienceandTechnologyAgency(JST).
TheauthorsarepleasedtomentionaboutthefruitfuldiscussionwithProf.
HansWesterhoffofManchesterUniversity.
Authordetails1DepartmentofBioscienceandBioinformatics,KyushuInstituteofTechnology,Iizuka,Fukuoka820-8502,Japan.
2InstituteofAdvancedBioscience,KeioUniversity,Tsuruoka,Yamagada997-001,Japan.
Authors'contributionsRKcarriedoutfermentationexperiments,assayed,madestatisticalanalysis,analyzedtheresult,anddraftedthemanuscript.
KSparticipatedintheexperimentaldesign,analyzedtheresult,andpreparedmanuscripttogetherwithRK.
Allauthorsreadandapprovedthefinalmanuscript.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Received:4September2009Accepted:27January2010Published:27January2010References1.
HuaQ,YangC,BabaT,MoriH,ShimizuK:ResponseofthecentralmetabolisminEscherichiacolitophosphoglucoseisomeraseandglucose-6-phosphatedehydrogenaseknockouts.
JBacteriol2003,185:7053-7067.
2.
HuaQ,YangC,OshimaT,MoriH,ShimizuK:AnalysisofgeneexpressioninEscherichiacoliinresponsetochangesofgrowth-limitingnutrientinchemostatcultures.
ApplEnvironMicrobiol2004,70:2354-2366.
3.
NanchenA,SchickerA,RevellesO,SauerU:CyclicAMP-dependentcataboliterepressionisdominantcontrolmechanismofmetabolicfluxesunderglucoselimitation.
JBacteriol2008,190:2323-2330.
4.
SauerU,EikmannsBJ:ThePEP-pyruvate-oxaloacetatenodeastheswitchpointforcarbonfluxdistributioninbacteria.
FEMSMicrobiolRev2005,29:765-794.
5.
ReitzerL:NitrogenassimilationandglobalregulationinEscherichiacoli.
AnnuRevMicrobiol2003,57:155-176.
6.
CommichauFM,ForchhammerK,StülkeJ:Regulatorylinksbetweencarbonandnitrogenmetabolism.
CurrOpinMicrobiol2006,9:167-172.
7.
PerrenoundA,SauerU:ImpactofglobaltranscriptionalregulationbyArcA,ArcB,Cra,Crp,Cya,Fnr,andMlconglucosecatabolisminEscherichiacoli.
JBacteriol2005,187:3171-3179.
8.
GossetG,ZhangZ,NayyarS,CuevasWA,SaierMHJr:TranscriptomeanalysisofCrp-dependentcatabolitecontrolofgeneexpressioninEscherichiacoli.
JBacteriol2004,186:3516-3524.
9.
MaoXJ,HuoYX,BuckM,KolbA,WangYP:InterplaybetweenCRP-cAMPandPII-NtrsystemsformsnovelregulatorynetworkbetweencarbonmetabolismandnitrogenassimilationinEscherichiacoli.
NucleicAcidsRes2007,35:1432-1440.
10.
YanD:Protectionoftheglutamatepoolconcentrationinentericbacteria.
ProcNatlAcadSciUSA2007,104:9475-9480.
11.
NinfaAJ,JiangP,AtkinsonMR,PeliskaJA:IntegrationofantagonisticsignalsintheregulationofnitrogenassimilationinEscherichiacoli.
CurrTopCellReg2000,36:31-75.
12.
FischerE,SauerU:MetabolicfluxprofilingofEscherichiacolimutantsincentralcarbonmetabolismusingGC-MS.
EurJBiochem2003,270:880-891.
13.
ZhangX,JantamaK,MooreJC,ShanmugamKT,IngramLO:ProductionofL-alaninebymetabolicallyengineeredEscherichiacoli.
ApplMicrobiolBiotechnol2007,77:355-366.
14.
KoebmannBJ,WesterhoffHV,SnoepJL,NilssonD,JensenPR:TheglycolyticfluxinEscherichiacoliiscontrolledbythedemandforATP.
JBacteriol2002,184:3909-3916.
15.
JiangP,NinfaAJ:EscherichiacoliPIIsignaltransductionproteincontrollingnitrogenassimilationactsasasensorofadenylateenergychargeinvitro.
Biochemistry2007,46:12979-12996.
16.
NinfaAJ,JiangP:PIIsignaltransductionproteins:sensorsofa-ketoglutaratethatregulatenitrogenmetabolism.
CurrOpinMicrobiol2005,8:168-173.
17.
KabirMM,ShimizuK:FermentationcharacteristicsandproteinexpressionpatternsinarecombinantEscherichiacolimutantlackingphosphoglucoseisomeraseforpoly(3-hydroxybutyrate)production.
ApplMicrobiolBiotechnol2003,62:244-255.
18.
ShangF,WenS,WangX,TanT:Effectofnitrogenlimitationontheergosterolproductionbyfed-batchcultureofSaccharomycescerevisiae.
JBiotechnol2006,122:285-292.
19.
ReitzerL,SchneiderBL:Metaboliccontextandpossiblephysiologicalthemesofs54-dependentgenesinEscherichiacoli.
MicrobiolMolBiolRev2001,65:422-444.
20.
MartínezI,ZhuJ,LinH,BennettGN,SanKY:ReplacingEscherichiacoliNAD-dependentglyceraldehyde3-phosphatedehydrogenase(GAPDH)withaNADP-dependentenzymefromClostridiumacetobutylicumfacilitatesNADPHdependentpathways.
MetabEng2008,10:352-359.
21.
YuanJ,DoucetteCD,FowlerWU,FengX-J,PiazzaM,RabitzHA,WingreenNS,RabinowitzJD:Metabolomics-drivenquantitativeanalysisofammoniaassimilationinE.
coli.
MolSysBiol2009,5:302.
22.
RahmanM,HassanMR,ShimizuK:Growthphase-dependentchangesintheexpressionofglobalregulatorygenesandassociatedmetabolicpathwaysinEscherichiacoli.
BiotechnolLett2008,30:853-860.
23.
ZhangZ,GossetG,BaraboteR,GonzalezCS,CuevasWA,SaierMHJr:Functionalinteractionsbetweenthecarbonandironutilizationregulators,CrpandFur,inEscherichiacoli.
JBacteriol2005,187:980-990.
24.
DeutscherJ,FranckeC,PostmaPW:HowPhosphotransferaseSystem-RelatedProteinPhosphorylationRegulatesCarbohydrateMetabolisminBacteria.
MicrobiolMolBiolRev2006,70:939-1031.
25.
HellingRB:PathwaychoiceinglutamatesynthesisinEscherichiacoli.
JBacteriol1998,180:4571-4575.
26.
LiangA,HoughtonRL:Coregulationofoxidizednicotinamideadeninedinucleotide(phosphate)transhydrogenaseandglutamatedehydrogenaseactivitiesinentericbacteriaduringnitrogenlimitation.
JBacteriol1981,146:997-1002.
27.
WillisRC,IwataKK,FurlongCE:RegulationofglutaminetransportinEscherichiacoli.
JBacteriol1975,122:1032-1037.
28.
Claverie-MartinF,MagasanikB:RoleofintegrationhostfactorintheregulationoftheglnHp2promoterofEscherichiacoli.
ProcNadlAcadSciUSA1991,88:1631-1635.
29.
BlauwkampTA,NinfaAJ:PhysiologicalroleoftheGlnKsignaltransductionproteinofEscherichiacoli:survivalofnitrogenstarvation.
MolMicrobiol2002,46:203-214.
30.
MagasanikB:Regulationofnitrogenutilization.
EscherichiacoliandSalmonellatyphimurium:CellularandMolecularBiologyWashington,DC:AmericanSocietyforMicrobiologyNeidhardtFC,others1996,1344-1356.
31.
RibaL,BecerrilB,Servèn-GonzaèlezL,ValleF,BolivarF:IdentificationofafunctionalpromoterfortheEscherichiacoligdhAgeneanditsregulation.
Gene1988,71:233-246.
32.
CamarenaL,PoggioS,GarcèaN,OsorioA:TranscriptionalrepressionofgdhAinEscherichiacoliismediatedbytheNacprotein.
FEMSMicrobiolLett1998,167:51-56.
33.
NinfaAJ,AtkinsonMR:PIIsignaltransductionproteins.
TrendsMicrobiol2000,8:172-179.
34.
ReitzerLJ,MagasanikB:ExpressionofglnAinEscherichiacoliisregulatedattandempromoters.
ProcNatlAcadSciUSA1985,82:1979-1983.
KumarandShimizuMicrobialCellFactories2010,9:8http://www.
microbialcellfactories.
com/content/9/1/8Page16of1735.
AtkinsonMR,NinfaAJ:Mutationalanalysisofthebacterialsignaltransducingproteinkinase/phosphatasenitrogenregulatorII(NRIIorNtrB).
JBacteriol1993,175:7016-7023.
36.
AtkinsonMR,BlauwkampTA,NinfaAJ:Context-dependentfunctionsofthePIIandGlnKsignaltransductionproteinsinEscherichiacoli.
JBacteriol2002,184:5364-5375.
37.
vanHeeswijkWC,HovingS,MolenaarD,StegemanB,KahnD,WesterhoffHV:AnalternativePIIproteinintheregulationofglutaminesynthetaseinEscherichiacoli.
MolMicrobiol1996,21:133-146.
38.
PahelG,RothsteinDM,MagasanikB:ComplexglnA-glnL-glnGoperonofEscherichiacoli.
JBacteriol1982,150:202-213.
39.
PaulL,MishraPK,BlumenthalRM,MatthewsRG:Integrationofregulatorysignalsthroughinvolvementofmultipleglobalregulators:controloftheEscherichiacoligltBDFoperonbyLrp,IHF,Crp,andArgR.
BMCMicrobiol2007,7:2.
40.
ShapiroBM,StadtmanER:Glutaminesynthetasedeadenylylatingenzyme.
BiochemBiophysResCommun1968,30:32-37.
41.
StadtmanER:Discoveryofglutaminesynthetasecascade.
MethodsEnzymol1990,182:793-809.
42.
JaggiR,vanHeeswijkWC,WesterhoffHV,OllisDL,VasudevanSG:Thetwoopposingactivitiesofadenylyltransferaseresideindistincthomologousdomains,withintramolecularsignaltransduction.
EMBOJ1997,16:5562-5571.
43.
MaheswaranM,ForchhammerK:Carbon-source-dependentnitrogenregulationinEscherichiacoliismediatedthroughglutamine-dependentGlnBsignalling.
Microbiol2003,149:2163-2172.
44.
MerrickMJ,EdwardsRA:Nitrogencontrolinbacteria.
MicrobiolRev1995,4:604-622.
45.
JiangP,PeliskaJA,NinfaAJ:TheregulationofEscherichiacoliglutaminesynthetaserevisited:roleof2-ketoglutarateintheregulationofglutaminesynthetaseadenylylationstate.
Biochemistry1998,37:12802-12810.
46.
KustuS,SanteroE,KeenerJ,PophamD,WeissD:Expressionofsigma54(ntrA)-dependentgenesisprobablyunitedbyacommonmechanism.
MicrobiolRev1989,53:367-376.
47.
JavelleA,SeveriE,ThorntonJ,MerrickM:AmmoniumSensinginEscherichiacoli.
JBiolChem2004,279:8530-8538.
48.
FarmerWR,LiaoJC:ImprovingLycopeneProductioninEscherichiacolibyEngineeringMetabolicControl.
NatureBiotechnol2000,18:533-537.
49.
NystrmT:Theglucose-starvationstimulonofEscherichiacoli:inducedandrepressedsynthesisofenzymesofcentralmetabolicpathwaysandroleofacetylphosphateingeneexpressionandstarvationsurvival.
MolMicrobiol1994,12:833-843.
50.
FengJ,AtkinsonMR,McclearyW,StockJB,WannerBL,NinfaAJ:RoleofphosphorylatedMetabolicIntermediatesintheRegulationofGlutamineSynthetaseSynthesisinEscherichiacoli.
JBacteriol1992,174:6061-6070.
51.
SukmariniL,ShimizuK:MetabolicregulationofEscherichiacolianditsglnGandzwfmutantsundernitrogenlimitation.
BiochemEngJ2010,48:230-236.
52.
NandineniMR,LaishramRS,GowrishankarJ:OsmosensitivityassociatedwithinsertionsinargP(iciA)orglnEinglutamatesynthase-deficientmutantsofEscherichiacoli.
JBacteriol2004,186:6391-6399.
53.
BabaT,AraT,HasegawaM,TakaiY,OkumuraY,BabaM,DatsenkoKA,TomitaM,WannerBL,MoriH:ConstructionofEscherichiacoliK-12in-frame,singlegeneknockoutmutants:theKeiocollection.
MolSystBiol2006,2.
54.
PengL,ShimizuK:EffectoffadRgeneknockoutonthemetabolismofEscherichiacolibasedonanalysesofproteinexpressions,enzymeactivitiesandintracellularmetaboliteconcentrations.
EnzymeMicrobiolTechnol2006,38:512-520.
55.
KabirMM,ShimizuK:GeneexpressionpatternsformetabolicpathwayinpgiknockoutEscherichiacoliwithandwithoutphbgenesbasedonRT-PCR.
JBiotechnol2003,105:11-31.
56.
SambrookJ,RussellDW:MolecularCloning:ALaboratoryManualColdSpringHarborLaboratoryPress,ColdSpringHarbor,NY,32001.
57.
MauriziRM,FatimaRasulova:DegradationofL-GlutamateDehydrogenasefromEscherichiacoli:allostericregulationofenzymestability.
ArchBiochemBiophys2002,397:206-216.
58.
KingdonHS,HubbardJS,StadtmanER:Regulationofglutaminesynthetase.
Biochemistry1968,7:2136-3142.
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