::1:罐
●i● ●唾●
lSl
Peptide‑1:
D画陰
D
。 含 黙 鋼 畿
FDDVK
AVRTWGNTSPFPLSPEVQTLGDLCFGWVIVTGV R
日g,IV‑6ComparisonofthePredictedAminoAcidSequenceoftheS」 四 θoわoqys"s
PCC6803FBPase‑IProteinwiththeSequencesoftheSynechococcusPCC7942 FBPase‑iandtheSequencesofSixofthePreparedPeptidesfromSynechococcus IeopolienslsFBPaseformB.ThedifferencesintheFBPase‑Iproteinsequencescan beidentifiedbythesubstitutionofanotheraminoacidbelowtheS.0803FBPase‑f sequence.AminoacidresiduesfoundatthesamepositionasS.6803FBPase‑lare showninwhitetet#ersonblack.
Chα π κ・'6πzα〃oη ρブthe8・enornicgeneencodingFBPω6‑1‑一 一TheFBPase‑I geneconsistedofa1164byopenreadingframe(ORF),encodingforapeptideof388
aminoacidresidues.Thecalculatedmolecularmassofthecloned.sequenceinthegene ofFBPase‑Iwas42kDa,whichwasclosetothemolecularmassoftheFBPase‑Isubunit determinedbySDS/PAGE.ThededucedaminoacidsequencecoincidedwiththeN‑
terminusofthenativeFBPase‑IpurifiedFromS.6803.Thededucedaminoacid sequencesofthe5冒.6803FBPase‑Ishared79.6%identitywithS.7942FBPase‑Iand 83.6°lv(110aminoacidresidues)identitywithS.leopoliensisFBPaseformB{Fig.IV‑
6).
Discussion
OnthebasisoftheresultsoftheSouthernhybridizationwithapc'obefromtheS.
7942FBPase‑IgeneandoftheWesternblotwれhanantibodyagainsttheFBPase‑I
protein,itseemslikelythattheFBPase‑Igenerallyexistsincyanobacteriaandrepresents themajor‑orcompletepartofthetotalextractableactivity(Fig.IV‑1and2A).Recently, Kanekoetal.(1996)reportedacloningofthewholegenefromS,6803andtheetistence ofthegene(slrO952)whichwas68%and41%identicaltoFBPase‑IIofS.7942andthe
cytosolicformofspinach.∠4η ζめ α3照wasalsofoundtocontaintheFBPase‑II‑likegene (Newmannetal.1995}.Theseresultssuggesttheoccurrenceoftheprotein
correspondingtotheFBPaSe‑II‑likeenzymeincyanobacteria.However,theFBPase
activitycorrespondingtotheFBPase・ ・Ienz)1mewaselutedasonlyasinglepeakbyMono Qcolumnchromatography.Atthattime,theFBPase‑IIactivitywasnotdefectedinany
fractioninthevicinityofx.29MNaCloftheion‑exchangecolumn.Inaddition,an antibodyagainsttheFBPase‑IIproteinofS.7942didnotcrossreactwiththecrude extractofS.683.ThesedatashowedthattheFBPase‑IIdoesnotetistataprotein levelandthattheFBPase‑IIgenemaynotbeexpressedundernormalgrowthcondition, evenifsolnecyanobacteriacontainthegene.
WithrespecttooptimumpH,theKinvalueforFI'u1,6‑P
2,thesigmoicialeffectof Mg'+,thelackofinhibitionbyFru2,6‑P
2,theFBPase‑IofS.6803hadthesame propertiesastheFBPase‑IoftheS.7942cells.InthePCRcycle,thehydrolysisofFru
1,6‑P
2tofructose6‑phosphateandSed1,7‑Pztosedoheptulose7‑phosphateisthePi‑
releasingstep,whichplaysanimportantregulatoryboleincontrollingthecarbonflutes inthecycle(RobinsonandWalker1981).TheFBPase‑IpurifiedfromS.6803was
foundtohydrolyzeFru1,6‑P2andSed1,7‑P
2withalmostequalspecificactivities.The activitywithbothsugarbisphosphataseshasbeenreportedforS.7942(chapterIII),S.
leopoliensis(Gerblingetal.1986),R加40p3θ 厩 α ηoη αg(SpringgateandStochow1982),
Noarrdin(AmachiandBoqien1979)理y〃088η01ηoη α∫(AbdelalandSchlegel1974), X6〃zthob≪c彪r/7仰acs(GenbankX17252),Alcaligeneseutraphus(GenbankU16791and
UI6792),andハritrobrrctervtclg・Cf」IS(GenbankL22884).Inthecellsof∫.6803grown heterotrophically,theactivitiesofNADP‑GAPDHandPRKinthePCRcyclewerenot deteαed,bttttheFBPase‑Iactivi1ywasdetected,decreasingby16,7%oDmparedw貢h thatgrownphotoautotrophically(Table1}.Accordingly,itseems‑likelythatthe FBPase‑Iofthecyanobacter童aparticipatesinthePCRcycleandgluconθogenesisしmder normalgrowthconditions.
Ihaverecentlydemonstratedthatthephotosynthesisofcyanobacteria,S.603and 5「.7942andeukaryoticalgae,EtcglenaandChl̀rrraỳ10'raontrsisinsusceptibletoH
202up tolmM,incomparisonwiththatofthechloroplastsofhigherplan的(Takedaetal.1995)、
AsdescribedinchapterIIIandVI,thenativeNADP+‑GAPDHandFBPaseisozymes (FBPase‑1,FBPase‑II)purifiedfroms7942cellsandtheirrecombinantenzymes purifiedfromE.solicellswereresistanttoH20.AsshowninFig.IV‑5,thespinach chloroplastFBPasewascomple㎏iyinhibi頓datO.1mMH
202whichwasinagreement 轍h亡heresultspreviouslyreported(Kaiser1976).Incolユtrast,H
202uptolmMonly slightlyinhibitedtheactivitiesofFBPaseandSBPasefromthepurifiedFBPase‑‑IofS.
6803,indicatingthattheenzymeisinsensitivetoH
202.TheenzymesofNADP+‑
GAPDHandPRKfromS.6803alsoshowedresistancetoH
202uptolmM(Takedaet al.1995).Kaiser(197b)hasreportedthatH
ZO2atlo≪levels(10jclvl)inhibitsthethiol‑
modulatedenzymesofthePCRcyclelmhechloroplastsofhlgherplants.
Ma!℃useta1.(1988)ha、 ノereportedthatthespinachchloroplastFBPasecontains twoCysresiduesseparatedbyonly4aminoacidresidues(Cys‑Val‑Vat‑Asn‑Val‑Cys)
andimpliedtllatthisaminoacidsequenceisoneof̀theregulatorysitesof̀thelight‑
dependentactivation.J'acquotetal.(」'acquotetal1997)havedescribedthattheC173S (Cyswasmodifiedintoserine)andCl78Sstronglyaffecledtheredoxregulatory propertiesoftheenzyme,themoststrikingeffectwasobservedwiththeCI53Smutant whichbecamepermanentlyactiveandredoindependent.Accordingly,itseemslikely thatadisulfidebridgebe卵eenCys‑153andCys‑1730rCys‑178inthespinachenzyme
isresponsibleforthe .light‑dark.regulation.Thesepotentialredox‑sensitiveCys residuesarenotconservedinFBPase‑IIinS.6803andS.7942.Accordingly,the
resist<lnceofFBPase‑IItoH202resultsfromthelackoftheCysresiduesinvolvedinthe lightactivationofthechloroP[asticthiol‑modulatedenzymesofhigherplants・The nLlcteotidesequenceoftheFBPase‑1genesfromS.6803andS.7942indicatedthatthe FBPase‑IiscompletelydistinctfromanypreviouslyknownFBPaseandSBPasefrom euh:aryoticcells(chapterIII}.Consequently,though10Cysresidueswerepresentin
FBPase‑Ifrom∫ ・6803,theresistanceoftheFBPase‑ItoH202wasnotexplainedbyしhe
positionsofpotentialredox‑sensitiveCyspairsinthetypicalformsofchloroplastic FBPases(Lietal.1994).
TheseresultssuggestedthatthePCRcycleofcyanobacteriaisnotregulatedby Tightactivationviaathioredoxin‑ferredoxinsystelnlikehigherplants(MarcusetaL1988, Pacoldetal.1995).Previouspapersreportedthatthesystemispresentinsome
cyanobacteriumspecies,SynechococcusandNostoc(Wagneretal.1978,Yeeetal.
1981).Hotwever,thethiol‑modulatedenzymesinS.7942analS.6803werenot affectedbytheDTT‑treatmentandnotlight‑activated(chapterII},unlikethetlaiol‑
modulatedenzymesinhigherplantchloroplasts.Accordingly,thePCRcycleof cyanobacteriamayhaveanalternativeregulationsystemwhichisdifferentfromthelight activalionofthiol‑modulatedenzyInesintbechloroplastsofhigherplants。Itis noteworthythatthenewtypeofFBPase‑IcontainsbothFBPaseendSBPaseaαivities andissensitivetochangesinthelevelofMgC12.TheFBPase‑Iwhichcansynchronize twopathwaysinthePCRcyclemayfunctionastheregulatoryfactorofphotosynthesis insteadofthe.light/darkregulationofhigherplants.
WhydoescyanobacteriumacquirethenewtypeofFBPaseinthePCRcycleand gluconeogenesis?InacomparisonoftheFBPase‑Inucleotideandproteinsequences withrhoseoftlleEMBLgenebanklibrary,theFBPase‑1・genefromS.6803showed 54%identityintO35nucleotidesand40.2%in343aminoacidstoanewgene,glpX, whichbelongstotheglpregulonofE.coliandthetranscriptionisinduciblewithglycerol andsn‑glycerol‑3‑phosphate(Trunigeretal.1992),thoughthefunctionoftheprotein derivedfromtheglpXgeneremainsunknown.Ithasbeenrepoi‑ledthatatypical
FBPaselikeFBPase‑IIispresehtinE.coliandRhorlobruters、 ρh̀tei"Ut̀lns(]HamiltonetaL 1988,Chenetal.1991).However,detailedstudiesonthepossibilitythattheFBPase
likeFBPase‑Iiswidelydistributedinotherprokaryoticorganismsaswellas
cyanobacteriaarenotavailableyet.Atthepresentstage,Iconsiderthatduringevolution thecyanobacteriahaveacquiredthenewtypeofFBPase‑ItoregulatethePCRcycleand
gluconeogenesisandtotolerateoxidativestresscausedbyactiveoxygenspecies.
Summary
AspreviouslydescribedinchapterIII,SynechococcecsPCC794?cellscontaintwo fructose‑1,6‑bisphosphataseisozymes,designatedFBPase‑IandFBPase‑II;‑theformer belongstoanewtypeofFBPase,whilethelatterisatypicalenzymesimilartothe
cytosolicandchloroplastic.formsfromeukaryoticcells.ThegenesofFBPase‑Iand
FBPase‑‑llwerefoundinthreespeciesofcyanobacteria,SynechocystisPCC6803,
Anrtbrrena712x,andPlectonemaboryrrnccrraaccordingtotheresultsof.Southern
hybridizationwithaprobefromthe5.7942FBPase‑IandFBPase‑IIgenes.InWestern
blotlil19,antibodyraisedagainsttheS.7942FBPase‑Icross‑reactedwithaproteinband correspondingtotheFBPase‑Iineachcrudeextractfromcyanobacterialcells,whereas theantibodyagainstFBPase‑IIfailedtocross‑reactwithanyproteinbandcorresponding totheFBPase‑rI.Incyanobacterialcells,onlyoneform‑ofFBPase‑Ihasbeenresolved byioh‑exchangechromatographyatsameconcentrationofNaClasshowninthe FBPase‑IofS.7942.TheFBPase‑IfromSynechocystis6803hasbeenpurifiedto electrophorefiichomogeneity.TheenzymehydrolyzedbothFru1,6‑P2analSed1,7‑P2.
TheapparentKmvaluesoftheenzymeforFrul,6‑P2andSed1,7‑Pwere57±2.4and I80±6.3ACM,respectively.Theenzyme.activitywasinhibitedbyAMPwitha.Ki valueofO.57±0.03mMforFruI,6P2andO.35±0,02μMforSed1,7‑P.The enzymeshowedamolecularmassof168kDawhichwasoomposedoffouridentical subしmits.TheactivitiesofFBPaseandSBPasefromtheFBPase‑Iwereresistantto hydrogenperoxideuptolInM.Thenucleotidesequenceofthe5.6803FBPase‑Igene showedanORFofll64bpthatencodedaproteinof388amlnoacidresldues(approx.
molecularmassof41.6kDa).Thededuced.aminoacidsequenceshadhomologous sequenceswiththe∫.7942FBPase‑1.
CHAPTERV
FunctionalAnalysisoftheFructose‑1,6‑bisphosphataseIsozymes {fbp‑1andfbp‑IIgeneproducts)inCyanobacteria
Fructose‑1,6‑bisphosphatase(FBPase;EC3.1.3.11),anenzymewhichcleaves Fru1,6‑P2intoFru6‑Pandinorganicphosphate,occursinbothautotrophicand heterotrophicorganisms.Inphotoautotrophiceukaryoticcellsincludingalgae,FBPase existsintvvoForms;oneformparticipatesinthePCRcycleinchloroplastsandtheother formisinvolvedingluconeogenesisinthecytoplasm.Thepropertiesofthechloroplast FBPaseareclearlydistinctfromthoseofthecytosolicenzymewithrespecttolight‑
dependentactivatlonbywayofaferredoxin‑thioredoxinsystemandinsensitivitytoAMP inhibition(Zimmermannetal.1976,1978,Marcusetal.1987).
IhavepreviouslydescribedthatSynechococctcsPCC7942cellscontaintwo FBPaseisozymes,designatedFBPase‑IandFBPase‑II;theformerbelongstoanewtype ofFBPaseandcanhydrolyzebothFru1,6‑PzandSed1,7‑P2,whilethelatterisatypical enzymesimilartothecytosolicandchloroplastlcforlnsfromeukaryoticcells(chapterIII).
SynechvcystisPCC6843cellsalsocontainedtheFBPaseisozymegenes(f'bp‑1'andfbp‑
II);however,onlyoneformofFBPase‑Iwasresolvedbyion‑exchangechromatography atthesameconcentrationofNaCIassho≪nintheFBPase‑IofS.7942cells,andthe FBPase‑IIproteinwasnotdetectedinS.6803cellsgrownunderseveralconditions (chapterIV}.ExceptforFBPase‑1,S.7942andS.683cellsdidnotcontainthe enzymeproteinwhichcouldhydrolyzeSed1,7‑P2(chapterIIIandIV).Thisresultwas supportedbythetactthatnohomologueofplantSBPasegenediscernedintheS.b803 genomedatabase(KazusaDNAResearchInstitute}.Thesefactsraisethequestionsas tohowS.7942cellsproperlyusethetwoFBPaseisozymesandwhyS.6803cellsare unabletoexpressthefbp‑IIgene.
TheprincipalexperimentaladvantageofS.794?andS.68030verplantsandmost othercyanobacteriaisthepotentialforgeneticanalysis.S.794?andS.6803areeasily transformed,andproceduresforgenereplacementallowtherapidintroductionof mtitagenizedcopiesofclonedgenesintothegenome(Goldenetal.1987,Thiel1994).
ToanalyzeChephysiologicalfunctionsofFBPaseisozymesincyanobacteria,hereI createdandcharacterizedtheinsertional‑inactivatedmutantsofthefbp‑Iandfbp‑IIgenes inS.7942andS.6803cells.
MaterialsandMethods
Organismrrndcultaare‐ ‐ ‐Wild‑typeandmutantsofcyanobacteriawere culturedinoneliterofAllenlsmediumat27℃for5daysunderillumination(240μlnol
m'2s'1)withthebubblingofsterileairatllitermin冒 且(TakedaelaL1993b).Forplate cultures,Allen'smediumwassuPPlementedwithl.5%(w!v)agar,andkanamycinwas
usedat50μgml‑lwhenrequired.
Generatic)nsρ ブ 加 ∫8171賜ρ加8rnutartts‑一 一ForthegenerationoftheFBPase‑1‑
deficientmutants(ScdF‑1),S.7942cellsweretransformedwithplasmidpScfbp‑1/kanr, whichwasderivedfrompScfbp‑Ibyinterruptingthe乃1)‑1geneattheSmaIsitewitha
1.kbpHincIIkanamycinresistancecartridge(ん αのfromplasmidpUC4K(Pharmacia) (Taylor,andRose1988)(Fig.V‑IA).Also,forthegenerationortheFBPase‑1‑
deficientmutants(SsdF‑1)fromS.6803,plasmidpSsfbp‑1/kanrwasconstructedfrom
pSsfbp‑IinterruptingwithkanrattheHindlllsite.ForthegenerationoftheFBPase‑
II‑deficientmutants(ScdF‑II)fromS.7942,plasmidpScfbp‑II/kanrwasconstilicted frompSctbp‑IIinterruptingwithkanratthePstIsite.Eachplasmidwastransfonned intoS.794?orS.6803cellsbythemethodofGoldenetal.(1987).
A
B
He He P P He He
ncnし 1
則ヲ 則9門 ヲ
/1bP‑1,卜/Ilbp‑"卜/!bp‑'lh
S HP
風 ノ 又 ノ
H
曳 ノ
pScfbp・1!kan「 pScfbp‑II/kan「
‑{::=‑I りP l2.85kbp{
pSsibp‑1/kan「
S.7942wildtypechromosome H
C
,Pbk =一=345330296422
0.56一 12345
藤 藩・郵
饗 購
灘、3.122 .85
°E‑0 .97
翻議醸
鱗
ScdF‑IIchromosome HPHPH
一 亡 吐 ヒ 圏 ■ 曲 ■■ ■iヒ 五 正:}一 一 一 一 一一一一一一一一 一一
卜一 一・097kbp・ 響一・十曽し 3.12kbp i
巳 IkCp
1
Fig.V‑1ConstructionofPlasmidspScfbp‑1/kanr,pScfbp‑IVkanr,andpSsfbp‑Ukanr.
Detailsaredescribedinthetext.H,Hindlll;Hc,Hincll;P,PstI;S,SmaI.(B)
Schematicrepresentationofthefbp‑1/regioninchromosomesofwild‑typeandScdF‑II mutantsofS.7942,inwhichthefbp‑llgenehasbeeninterruptedbyakanrasdescribed inthetext.(C)SouthernblotanalysisofpScfbp‑IVkanr(lane2),totalDNAofwild‑type (lane1),ScdF‑II‑1,ScdF‑II‑2,andScdF‑II‑3(lane3,4,and5,respectively)inwhichthe wild‑typefbp‑IIgenewasreplacedbythefbp‑IVkanrgeneshowninpanelB.TotalDNA wasrestrictedwithHindillandhybridizedwithanfbp‑IIgene.ThesizesofDNA molecularmassstandardsareindicated.
Enzymeα ∬ の ノ3‑一 一Inthewild‑typeandScdF‑IIof5'.7942,theenzyme activitiesinvolvedinthePCRcycleandgluconeogenesisweredetermined.Bothcells
wereharvestedbycentrifugation,washedonce,suspendedin50mMpotassium
phosphatebuffer(pH8.0)containing2.5mMDTT,1mMglutathioneand10°lo(w/v) sucrose,andsonicatedat10KHzforatotaloflminwithfiveintervalsof10seceach.
Thelysateswerecentrifugedat12,000xgfor15mintoobtainthecrudeextractsforthe enzymeassayandWesternblotting.Forglucose‑6‑phosphatedehydi‑ogenase (G6PDH)assay,thechideextractwaspreparedwithSOmMpotassiumphosphatebuffer (pH8.0)containing10%(w/v)sucrose.TheactivitiesofFBPase,SBPase,NADP+‑
GAPDH,PRK,andG6PDHwereassayedaspreviouslydescribedinchapterIIand chapterIV.
Otherniethò1∫ 一 一 一TherateofNaHCO3‑dependentO2evolutionanddarkO2 uptakeweremeasuredusinganoxygenelectrodeaccordingtothemanufacturer's
instructions(HansatechInstrumentsLtd,King'sLynn,UK).
ResultsandDiscussion
Astheplasmidsdonotreplicateinrecipientcells,kanamycinresistancecanbe acquiredonlybygenereplacementorplasmidintegrationintothechromosome.To distinguishbetweenthese瓜vopossibilities,kanamycin‑resistanttransfolmantswere screenedbyDNA‑DNAhybridizationfortheabsenceofthevectorDNA.Following segregation,chromosomalDNAwasisolatedfromthreetypesoftransfonnants(ScdF‑II‑
1,ScdF‑II‑?,ScdF‑II‑3)andwild‑typecells.TheseDNAsweredigestedwithHindlII, andafterSoutherntransfer,probedwithfbp‑II.(Fig.V‑1B,C)Theobtainedresults indicatedThatallchromosomalfbp‑IIalleles(2.85kbp)arecompletelyinterruptedby1.2‑
Abpknǹ,thusproducingtwofragments(3.12andO.97kbp).
(A)
r /bp‑1Wild‑typechr°m°s°me
‑・p・…am1 .2。kbp門d°wnst「eam
H>Kan「
一
め ρ一〃(an「interruptionmutantchromosome
I
<日downstream
l
(B)
2.44Kbp‑
1.20Kbp‑
MWT12
コ
upsterarη 2.44kbp
Fig.V‑2SchematicRepresentationofthefbp‑(RegioninChromosomesofWild‑type (A)andPCRAnalysis(B).M,Marker;WT,wildtype;Lanesland2,ScdF‑1‑1and‑2
Ontheotherhand,onthefirstsegregationoftheScdF‑IandSsdF‑Imutants,PCR amplificationoftbp‑IfromchromosomalDNAisolatedfromtransfonnanisyieldedthe Abp‑1geneandfbp‑1!kan『gene(Fig・V‑2)・Theseresultsindicated{hatapartofthe
chromosomallbp‑1 .allelesisreplacedbythedisruptedgene.Followingsegregation, however,thepScfbp‑1/kanrorpSslbp‑1/kanrtransformantscouldnotgrowinAllen's
mediumsupplementedwithkanamycinundervariouslightintensities(10,40,100,and 240μmolm'2s‑1).TheseresultssuggestedthatFBPase‑Iisessentialforthegrowthand thefbp‑IdisruptionisalethalmutationinS.7942andS.6803cells.
IntheS.7942cells,theactivityratioofFBPase‑ItoFBPase‑IIis12:1and FBPase‑IcanhydrolyzebothFru1,6‑PzandSed1,7‑P2(chapterIII).Theaclivilyof FBPaseinScdF‑IImutan捻decreasedbyapproximatelylO%,correspondingtothe activityofFBPase‑IIcomparedtothatinthewild‑type,whiletheSBPaseac1ivity showednochangeineithercell(Table1).Accordingly,itislikelythattheFBPase‑II activityinScdF‑IImutantsiscompletelydeleCedw曲outaffecUngtheactivityol'FBPase‑
1.ThisresuhwasverifiedbyWesternblotanalysisofthecell.cttractsfromthewild‑
typealldScdF‑II111L11:i117LSwithantiseraagainsuheFBPase‑IandFBPase‑IIfrom∫.7942 (Fig.V‑3).ThebandscorrespondingtobothFBPase‑1(40kDa}andFBPase‑II(37
kDa)weredetectedinthewild‑type.IntheScdF‑IImutants,thebendcorrespondingto FBPase‑Iwasdetected,whilethebandcorrespondingtoFBPase‑IIwasnotdetected.
=,LDOの
Φα珍℃一旧≧
(kDa) 96一
67一
43‑一
騨 郷 ‡ 醜:::iu
3Q一
Fig.V‑31mmunoblotAnalysisofFBPase‑IandFBPase‑!IProteins.Crudeextracts fromwild‑typeandScdF‑limutantsofS.7942weresubjectedtoSDS‑PAGEinslabgel.
FBPase‑IandFBPase‑llwererevealedusingrespectiveantibodiesraisedagainst FBPase‑IandFBPase‑11fromS.7942.
AlthoughFBPase‑IIactivityandproteinswereabsentintheScciF‑IImutants,other enzymeactivitiesinvolvedinthePCRcycleandgluconeogenesisremainedunalteredor onlyslightlychanged(Table1}.TherateofNaHCO3‑dependentO2evolutionwas similarinthewild‑typeandSc;dF‑IImuiantsundervariouslightintensitiesorvarious NaHCO3concentrationsat27°C(Table2).TosustainthephotosyntheticOZevolution ratecorrespondingtoabout106.6±2.9μmolCO2h'1(mgChl)°1inthe∫.7942cells,the FBPaseactivityinthePCRcycleseemstorequire33%(apProximately45μmolh‑1(mg Chl)°1)oftherateofCO2fixation(RobinsonetaLl981).IntheScdF‑IImutants,the FBPaseandSBPaseactivitieswere70.2±1.5and69.2±3,4μmolh'1(mgChl)‑1atlOO μMFBPand200μMSBP,respectively.However,inthepresenceofbothsubstrates,
thebisphosphataseactivityofFBPase。IwaslO5±3.2μmol止 ゴ1(mgCh1)‑1.Thedata
suggestedthattheactivitiesasFBPaseandSBPaseoftheFBPase‑Imightchange dependingonthenumberofFBPandSBPboundontheenzymeinvivo.Accordingly, itseelnslikelythatFBPase‑Icansustain吐hephotosyntheticCO2fixationin&7942even iftheFBPase‑IIactivityisabsent。
TableV‑1Enzymeacliviiiesinwild‑typeandScdF‑IImutantsof SynechococcusPCC7942cells
Activity {μmolh'1(mgChl)‑t)
wild‑Type ScdF‑II
FBPase画 SBPase NADP+‑GAPDH PRK G6PDH
80,2士1.2 72.8土4.2 676.2±18 10250士54
65.7土1.0
70.2土1.5 692土3.4 657.8t19 1020.4t75 693士3.9
ThemeanstSDofthreedeterminationsareshown.Asterisk
indicateschatthedifterencebetweenwild‑typeandScdF‑IImutants wassignificantby1‑test(P<0.05).
TableV‑2RatesofNaHCO3‑dependentO2evolu重ioninwild‑typeandScdF‑11 mutantsofSynechococcusPCC7942cells
light intensify (μmolm‑2S'1)
NaHCO3 conceniraiion
(mM)
02evolution ωmolO2h°1(mgChl)'り wild‑typeScdF‑!!
20 230 720 1600 1600 1600 1600
oso a.so O.50 0.50 0.25 1.0 2.0
65.9士4.8a 93.2t7.25 1034±63bc las,6±2.gc 105.9土4.3c cos.7士tgC 106.5土6.4c
51.8t3.7a 98.4士5.6b 101.5t4.2bc 105.5:上a6c 105.1士3,5c 1063土2.gc 106.1士3,8c
ThevaluesarethemeanstSDofthreereplicatesandthoseineachrowand columnwishthesameLeiterarenatsignificantlydifferenti,yt‑teslatPく0.05.
ThegrowthrateandChlcontentoftheScdF‑IImutantswerecomparedwiththose ofthewild‑typeunderphotoautotrophicconditions.Underilluminationat24014molmユ s‑1,theScdF‑IImutantshowedgrowthsimilartothatot̀thewildtype(Fig.V‑4A).The chlorophyllcontentoftheScaF‑IImutantsinaunitculturevolumewassimilartothatin thewild‑type(Fig.V‑4B).Thoughtheseparameterswereunaffectedbytheintensityof light(10‑240μmolIP.←2s‑1).Aftercellsweregrownfor8days,therateofdarkO2 uptakeinthewild‑typeandScdF‑IImutantswere10.9±0.8and10.9±0.7ycmolh"̀
(1ngChl)'1,respectively.Incondしision,incyanobacteria,FBPase‑Iexclusively participatesinreactionsofFBPaseandSBPaseinthePCRcycleandthereactionof' FBPaseinglttconeogenesis.
Fig,V‑4TheTimeCourseofPhotoautotrophicGrowth(A)andChangesofChI Content(B)inWild‑typeandScdF‑IIMutantsofS.7942.S.7942wasculturedinone literofAllen'smediuma隻27℃underillumina#ion(240μmols『1m・2)wlththebubblingof sterileairatllitermin‑1.
QD730andChlcontent(Lichtenthaler1987weremeasuredbythemethoddescribedin thecitedreference.Eachpointrepresentsthemeanofthreeassays(coefficientof
variation<5%).
WhyistheFBPase‑IIproteinabsentinS.6$03?Ihavepreviouslydescribedthat S.6803containsCwoFBPaseisozymegenes(chapterIV).AnanalysisofthegeneinS.
6803byKaneKoetal.(1996)revealedthepresenceofthefbp‑IIgene(slrO952;68%v
identicaltoS.794?fbp一 刀)jn重heunknowngenecluster.Incontl魎ast,theS.7942/by‑11 genewasclusteredwiththegenesencodinggluoose‑6‑phosphatedehydrogenase(z}サ),
OpcA(opoA),cytochromeb6(pe彦D),andthecytochromeb6fcoInplexsubunitIV(pet・B) (Newmanetal.1995).Thesedataindicatedthattheorganizationorpromotersequences ofthefbp‑IIgeneinS.6803isdifferentfromthatinS.7942.Accordingly,differences ot̀genestructureinthevicinityofthefbp‑11'genemaynotallowthegenetobeexpressed
111S.6803cells.IhavedescribedthatinAǹめ̀rena7120andPlectone〃uiboryanuyn, FBPase‑11doesnotexistattheproteinlevel,evenifthegeneispresent(chapterIV).
Therefore,itisconceivablethatduringevolution,someofthecyanobacteriaabandonthe expressionofthefbp‑IIgenebecausetheactivityofFBPase‑Iissufficienttosustainthe photosynthesisandgluconeogenesis.
Inhigherplantsphotosynthesistakesplaceinsourceorganswhicharemainly leavesandessentiallyallgreen,chloroplast‑containingtissues.Theseorgansarenet exportersofcarbohydratesandsupplyphotosyntheticallyinactivesinkorgans,suchas roofsorseeds,whicharedependentontheimport‑ofreducedcarbon.Thebiochemical pathwayleadingtotheassimilationofinorganiccarbonbymostautotrophicorganislnsis thePCRcycle.Keyregulatorymechanismscoordinatingsupplyanddemandof
carbohydratesinCO2assimilatingleaveseitheraffectthePCRcycledirectlythrough allostericorcovalentmodificationofenzymesinvolved,orindirectlybythesupplyofthe chloroplastswithinorganicphosphate(Pi)。TherearefourenzymesinthePCRcyc1e whicharesu切 ㏄ttoregulation:FBPase,SBPase,PRK,andNADP{‑GAPDH.Allthese activities‑areSt1111tllatedbylight‑dependentredox‑potentialthroughthe
ferredoxin/thioredolinsystem.
Manydatahavebeengeneratedinvitroinordertodeterminelimitingstepsof photosynthesisandfactorswhichinfluencecarbonallocation.Althoughthesestudies Amassedagreatdealofinformationontheregulatorypropertiesof̀enzymeof̀plantin vitro,evidencefortheirindividualcontributionstocontrollingphotosyntheticnux〃zviレo islargelycircumstantial{Scheibe199,Buchanan1991,Wolosiuketal.1993}.Thisis
becauseitisoftendifficulttoextrapolateregulatorypropertiesandkineticcharacteristicsof̀
enzymeinvitrotothecellularenvironmentoftheintactplant.RecombinantDNA technologyandplantgenetictransformationhaveprovideduswiihexcellenttoolstoget aroundsolneoftheseobstacles.Usingthetechniquesorgenesuppressionand
overexpression,it15possibletoaltertheamountofasinglè̀limiting"fbrphotosynthetic fltlY.AquanCitativeassessmentofanenzyme'sroleineachclasscanbemadeusing controlanalysis(KacserandSurns1973}̲
Byfacthemostwidelyusedapproachtothegeneticmanipulationofphotosynthesis todatehasbeenuseofantisenseRNAtechnologytoproducetransgenicplantswith reducedlevelsofkeyphotosyntheticenzymes.Thesetransgenicplantshavelarwider usesthaninthestudyofphotosyntheticflue..Theinterpretationofthephenotypiceffects ofanan丘senseconstructonphotosynthesisandgrowthofthetmnsfonnedplantsisnot alwaysstraightforward.Becausethedifferenceisdesirableforbiochemicalanalysisof carbonfluxcontrol,thiscouldlimittheusefulnessoftheantisensetechniqueforsome application.
Forunderstandingregulationofphotosynthesis,high‑levelexpressionofnativeor heterologousproteintoincreasefluxthroughapathway,orchangetheregulatory
propertiesofanenzymeisalsopowerfultool.However,itisalsogenerallyaccepted thatoverexpressionofgenesinplantsismoredifficultthanantisensesuppressiondueto cosupPression(Napolietal・1990,vanderKroletaL1990)andtheposslbleinactivation oftheoverexpressedproteinbyendogenousregulatorysystems(Sonnewaldetal.1994).
Metabolicengineeringcanbeusedtocontrolnotonlythequantityofenzyme presentintransgenicplantbtttalsoits"quality".Asdescribedpreviously,cyanobacterial thiol‑modulatedenzymeshadparticularproperties;theseshowedresistanttoH202and werenotregulatedbylight.FBPase‑IcouldhydrolyzebothFru1,6P2andSed1,7‑P2.
Moreover,cyanobacterialノ 加 一1genehasnohomologywiththoseofhigherplantFBPase.
Thesepropertiesareveryusefultoinducethegeneintotheplantandanalyzethe regulatorypropertiesofcarbonfluxinhigherplants.
ThechloroplasticFBPase(cp‑FBPase)playsacrucialroleinthephotosynthesis, andcallocationduetoitscentralfunction,firstbecausethethisenzymeisone.ofthe regulatoryenzymesinthePCRcycle,secondlybecausetheproductofthereaction catalysedbycp‑FBPase,i.e.Fru6‑PrepresentsabranchpointbetweenthePCRcycle andtransitorystarchbiosynthesis.Moreoverbecausethecp‑FBPasecontributesonlya minorproportionofthetotalamountofchloroplastproteinanditsactivityisextremely lowincon重rastto巳hoseofotherthioLmodulatedenzymes,itisconsiderablethauhisis oneoftherate‑limitingenzyme.BecauseofthesereasonsIdecidedtomodulatethe expressionofthecp‑FBPaseintransgenicplants.
Forreasonsmentionedabove,nowItrytogeneratetransgenictobacco(1Wco∫'α1?α
∫oう̀3α 〃ηcv,Xanthi)andrice(0ノ ッz̀z3{厩yαcv.Kinmaze)expressingFBPase‑Ieitllerin thechloroplastorcytosol.ToclarifytheindividualcontributionsofFBPase鋤d
SBPasetocontrollingcarbonfluxinvivo,Iwanttoanswerthefollowingquestionswith thehelpoftheseplants.
(1)
(2)
(3)
(4)
(5}
HowdoestheincreaseinFBPaseandSBPaseactivitiesofthe PCRcycleinchloroplastaffectphotosynthesis?
HowdoestheincreaseinFBPaseactivityinthecytosolaffect sucrosebiosynthesis‑andtranslocation?
WhatistheinfluenceofincreasedfluxthroughthePCRcycle onplantgrowthanddevelopment?
WhatistheinfluenceofaincreasedactivitiesofFBPase andSBPaseinchloroplastontheallocationofincreased carbontowardsstarchsynthesisorsucrose(solublesugars) biosynthesis?
DoesaillcreaseintheactivitiesofFBPaseandSBPaseeithel'
inthechloroplastorcytosolaffecttheexpressionofother genesinvolvedinthePCRcycleandsucrose/starchsynthesis?
Summary
S.794?containstwofructose‑1,6‑bisphosphataseisozymes(FBPase‑Ianti FBPase‑II),whileS.6803hasonlyone{FBPase‑1)inspiteoftheoccurrenceoftwo FBPaseisozymegenes{chapterIII,IV).Inowdemonstratethatdisruptionofthegene encodin8FBPase‑II(fbp‑〃)withakanamycinresistancegenecartridgedoesnotaffect cellgrowth,Chlcontent,orcotassimilationin∫.7942,anddisruμionofthegene encodingFBPase‑1(fbp‑1)isaIethalmutationinbothcyanobactelia.Accordingly,itis
clearthatFBPase‑Iisnecessarytosustain ,photosynthesisandgluconeogenesisin cyanobacteria.