SURE: Shizuoka University REpository http://ir.lib.shizuoka.ac.jp/ Title Dynamics of Microcystin-Degrading Bacteria in Mucilage of Microcystis Author(s) Kato, Kenji; Maruyama, Tomoko; Yokoyama, A.; Tanaka, T.; Hiraishi, A.; Park, H.D. Citation Issue Date URL Microbial Ecology. 46(2), p. 279-288 2003-06-13 http://hdl.handle.net/10297/568 Version author Rights The original publication is available at www.springerlink.com This document is downloaded at: 2016-02-02T09:39:10Z Maruyamaetal. Dynamicsofmicrocystin−degradingbacteriainmucilageofMc7Vqげtis TbmokoMaruyama,1,2Ke亘iKato,1*AtsushiYbkoyama,2TbshbrukiThnaka,2AkiraHird血,3HodongP癒2 ハハ/′川灯り/り//加圧い肌/hHハし…Jしい././川/日日/\廿仇LV〃二/J日工./J川、日、//し\/J/コJ日工./J二一\−さごり.・旬… 、ハハ′′川…/り//川川州仙川.′/\…い./.′川/日日/\人肌LV川ハ/J′′J川、い//Ll山/、川川小一J・仙\−′・ご/..恒′〃 、ハハ′′川…/日仏−車小l′//′岬虹′岬./川り/仙/JH川、い/日日/八・しゾ冊車∵し/川り/抽/J/+〃い㍉・\一日..恒′〃 *CoITeSPOndingaudlOr:Ke亘iKato,DeparblentOfBiologyandGeosciences,FacultyofScience,Shizuoka Uhiversi椀836q′a,Shizuoka422−8529,Japan Tbl:+81−54−238−4950 Fax:+81−54−238−4950 E一mail:Skkato@ipc.shizuoka.acjp Datesubmitted: Runninghead:Microcystin−degradingbacteriainmucilageofMc7VCygtlS Abstract TbrevealdlePrOCeSSOfdegradationofhepatotoxicmicrocystlnPrOducedinMc7VCygtlSCellsduringdle Mc7VCygtlSbloomperiod,WeuSedfluorescenceinsihJhybridization(nSH)toanalyzedlePOPulation⑪n肌1ics OfmicrocystindegradingbacteriainMc7VCygtlSmuCilage.Wedesignedandappliedanoligonucleotideprobe targetedtodle16SrRNAsequenceofstrainY20famicrocystin−degradingbacterium(MCD−bacterium),Which WaSisolated丘omLakeSuwa,Japan.Inbodldle1998and1999tests,FISHcleadyshoweddlatMCD−bacteria existedindlemuCilageanddlat,Whenahighconcentrationofcell−boundmicrocystinwasdetected,MCD−bacteria exceededlO%ofdleSumOfbacteriahybridizedwidlgrOuP一甲eCificprobes.TheconcentrationofMCD−bacteria WaShighestinsummer1998,Whenatoxicspecies,MtmC鮎,WaSdominant.TherewasahighcoITelation betweendlenumberofMCD−bacteriaindlemuCilageanddleCOnCentrationofcell−boundmicrocys也1indlelake. 0urreSultssuggestdlatMCD−bacteriarespondedtochangesindleCOnCentrationofmicrocystlnanddegradeddle microcystinwhenitwasreleased丘omMc7VCygtlSCells.Ⅵ屯alsoanalyzedchangesindlebacterialcomity StruCtureaSSOCiatedwidldleMc7VCygtlSCOloniesbyuslngdomain−andgroup一甲eCificoligonucleotideprbbes. ChangesindleCOnCentrationsofdleqノttPh御伽bactenumgroupand8−Proteobactena,Whichcandegrade macromolecules derived丘omMc7VCygtlS Cells,Were SynChronizedwidlChangesindle COnCentration of Mc7VCygtlS.The results notonb,SuggeStdle Slglificantrole ofMCD−bacteriaindetoxification,b血alSo demonstrateapossiblesequenceofdegradation丘omMc7VCyStLgCellstomicrocys也lmaintainedindleCell,Which isdlenCaniedo血bybacterialconsortiaindlemuCilage. 2 Maruyamaetal. Introduction Microcystin,aCyClichepatotoxin,CauSeSSeriOusdisease,aSrePOrtedbyJochimsenetal.[20].In1996,540血 Of130pahentsatadialysiscenterinCaruaru,Brazil,died鮎IllWaterfordialysiscontaminatedbymiCrocystlnS, WhichareproducedbysomecyanobacteriaofdlegeneraMc7VCygtlS,Anabaena,Nbstoc,and(九cilk7tOna.Of dleSe,Mc7VCygtlS,Whichbloomsineutrophictropicalandtemperatelakesandponds,isdlemOStCOmmOn CyanObacteriuml10]. Microcystlnisproducedandmaintainedwithinheal叫Cyanobacterialcells.Approximateb,10%to20%of microcystinislost丘omhealthycyanobacterialcellsinCulturel40,41].WhenMc7VCygtlSCellsaredecayed,dle COnCentrationofdissoIvedmicrocystinmayincreasel44].However,dissoIvedmicrocystinconcentrati0nsremain Verylowinlakewater,eVenduringdledecayofMc7VCygtlSl36].JonesandOITl21]usedhigh1)erformanCe liquidchromatography(HPLC)andaproteinpho甲hataseinl1ib止ionassaytodetemlinedlelossofmicrocystin releasedtolakewaterfollowingalgicidaltreahlent.FurdlermOre,Christo飽rsenetal.[14]showedinlaboratory andfieldexperimentsdlatdledegradationofmicrocystinbyindigenousbacteriainlakewaterwasacceleratedby dleeXistenceofdissoIvedorgmicc訂bon(DOC),anddlatdleadditionofcellb,SateSOftoxicMc7VCygtlSelevated bacterialabundance,PrOductionanddhlerSity.However,dlemeChmismofins血degradationofmicrocystin remains to be clarified.Jones etal.[22]isolated a single str血10fmicrocystin−degrading bacterium (MCD−bacterium)identifiedas勒hklgOmOnagSP.丘omdrainage.Boumeetal.[8]usedthisstraininlaboratory experimentsfocusedondlePrOCeSSOfe呵maticdegradationofmicrocystin.However,dleydidnotexamiIledle degradationofmicrocystinbyMCD−bacteriainnaturalwater.In1996,Weindependentb,isolatedastrainof MCD−bacterium,Y2,Whichbelongstoanewgenusrelatedto御iP轡フmOnag,丘omeutrophicLakeSuwain CentralHonshu,Japanl37].InthisstudyiWeeXamiIleddlePrOCeSSOfdegradationofmicrocystinbybacteriaindle naturalenvironment,focusingondlePOPulation⑪n肌1icsofMCD−bacteria. Degradation ofmicrocystlnPrOducedinMc7VCygtlS OCCurS duringdle2−fold decompos止ionprocess of Mc7VCygtlS:(a)dleCelldecaysbyauto吋sis,graZingbyprotozoa,ZOOPl肌ktonandfishl13],OrViral[27]or bacterialacthlity[15,46],and(b)dleCellularPrOductsaredlendegraded bybacterialconso血a.DaRetal.[15] Showeddlatam卿bacteriumisolated丘om丘eshwaterwasabletob,SeMc7VCygtlS.1ねmamotoetal.[46] 3 indicateddlatSOmebacteriaofdlegeneraAk:alwnesandPgeudbmonaganddleqノkph抑bactenum groupalsob,SedMc7VCygtlS.BecausedleSefindingsconcemlngCelldecompos止ionofMc7VCygtlSWereObtained byculture−dependenttechniques,OurknOwledgeisl血血edtodlefunctionofculturablebacteria.However, numerousnon−CulturablebacteriaexistindlenaturalerrviroIrment,eXCeeding99%ofdletOtalbacterialcoultin Varioussystemsl11].Tbcomplementthisgap,dlefluorescenceinsihJhybridizationmedlOdげISH,[2,16])was recentb,developedtoidentifyindhlidualcells,independentofculturabilib 加Ievelsranging丘omspeciesto domainbyusingoligonucleotideprobesspecifictodle16Sor23SrRNAsequence.ModifiedFISHmedlOdsl12, 19,25,26,34]havealsobeendevelopedtovisualizedlemetatX)licfunctiOnofaspecificbacterialgroup. TheaimofthisstudyistoelucidatedlePrOCeSSOfdledegradationofmicrocystinbyMCD−bacteriaunderdle POSSiblecoexistencewidlVariousodlerbacteriawidlVarylngfunctionsduringdlebloomofMc7VCyStlSinLake Suwa.BasedondleFISHmedlOd,Wedesignedanoligonucleotideprbbespecifictodle16SrRNAsequenceof StrainY2,andappliedseveralprobesspecifictophylogeneticgroupstoexamiIleaStruCturalchangesindle associatedbacterialconnnunitywidlMc7VCygtlSCOloniesduringdlePeriodsofaMc7VCyStlSbloomandits decline. Materialsandmethods ・\/岬血.∵り//九ソ川州両肌・/一./し…T./い、目しソ血・./いめlhソ日日、〝、しい/川汀 LakeSuwa,locatedinNaganoPre丘ctureinJapan,lSae血pDPhic,temPerate,Shallowlake.Thelakehasa surhceareaof13・3km2,amaXimumdepdlOf6・4m,andanaveragedepdlOf5m・ SamplesofheterotrophicbacteriaassociatedwidlMc7VCygtlSCOlonieswerecollected丘omdleSurhcewaterof dleCenterOfdlelakeduringMc7VCygtlSbloomson26August,9SeptembeI;and7atober1998,andonll August,25August,8September,and6atober1999.AsdlebiomassofMc7VCygtlSWaSSlgnifiC叫lowerin 1999,WeSligldychangeddleCOllectionprocedureslighdy.In1999,WaterSamPleswereprescreenedwidlanylon netwidlameShsizeof40トuTLandwerekeptlnaglassbotdefora丘whours.ThefloatlngMc7VCygtlSCOlonies andassociatedbacteriaweredlenCOllectedusingapipette.TheisolatedMc7VCygtlSCOlonieswerefilteredthrough a3−トun−POre−SizeNucleporefilteI;andwashed3to5timeswidlPho甲hate−bu飽redsaline(pH7.2,IMbecco’S 4 Maruyamaetal. PBS(」,Nissui,Japan)to eliminate any丘ee−livingbacteria.The washedcolonieswere sonicated3times @RANSONICB1200,45kHz,30W,3smI/1)todisruptthemanddispersedleheterotrophicbacterialcellsand A感C7Pq償如Cells. /ハ.///川=//九ソ川州両肌・/一./し…・/.//川・/川.//し川州./′J/ん州・、し川。し〃い///J/Jl/−′山h///州 HeterotrophicbacterialsamplesfortotalbacterialcotdandFISHwerefixedinparaformaldehydesolution(pH 7.4,finalconcentration3%)forupto24hat4C.TbavoiddestructionofdleMc7VCygtlSCOloniesin19980rdle Mc7VCygtlSCellsandbacterialcellsin1999,fixedsamplesforFISHwerefilteredgendyonaO.22−トunNuclepore filter(25mmdiameteり[23].Cellsondlefilterwererinsed3timeswidlPBSanddehydratedin1mLof50%, 80%,and99%edlanOlfor3mineach,anddlefilterwasdlenair−dried.Filterswerestoredatr20 Cuntil hybddz血on. /川.//し川〃〃り/./、、目し丁.爪・.//一./し人ソT./ Fixedsampleswerestainedwid14′,6′−diamidin0−2−Phe町lindole(DAPI,finalconcentrationO.01ト1g血lLl39]) andfikeredgendyonaO.22−トunNucleporefilter(25mmdiamete弓.AtotalofmoredlanlOOObacterialcellswere enumeratedbyepifluorescencemicroscopy(uliversalepifluorescencemicroscopic野StemBX50−FLA,0恒叩uS, Japan). J用りん・1・J//〃\///J/Jl/−/・/.ユ〟仙椚 The16SrRNAtargetedoligcnucleotideprobesusedinthisstuQ,areShowninThblel.Theycompriseda domain−SPeCific probefor Bacteria(EUB338),PhylogenetiC−grOuP−SPeCific probesfor〔エー,β−,γ−,and 8−Proteobacteria(ALFlb,BET42a,GAM42a,DEL)anddleqノttPh抑bactenumgroup(CF319a),andan MCD−PrObedesignedspecifiCalb,formicrocystin−degradingbacteria(”str血lY2”inthisstuQ,).Theseprobes Werelabeledwidl血od肌liIleObtained丘omThkaraBioteclmologyPALIAN),Japan. 町bridizationstrmgenCyWaSa鴎ustedbyvarylngdleCOnCentrationofformamideindlehybridizationbu飽r andNaClindleWaShingsoluticn.町bridizati0nsWerePerformedat46Cfor90minonfiltersplacedonslides 5 COatedwidlgelatin,widlhybridizationbu飽rcontainingO.9MNaCl,20mMTriS・HCl(pH7.4),0.01%SDS, formamide(20%forEUB338,ALFlb,CF319a,andMCD,35%forBET42a,GAM42aandDEL),and5ngトIL −10fdlereSPeCtivelabeledprobe・Eachfilterwaswashedat48Cfor15mininpre−Warmedwashingbu飽r COntainingNaCl(0.225MforEUB338,ALFlb,andMCD,0.080MforBET42a,GAM42aandDEL),20mM TriS・HCl(pH7.4),5mMEDTA,andO.01%SDS,rinsedwidldistilledwateI;andair−dried.Thepreparations, whichwereco皿terStainedwidlO.1LlgmI/1DAPIl45]onglassslidesfor5min,WereObservedunderauniversal epifluorescencemicroscopicsystem,BX50−FLAwidla3ccDcamera(C5810,HamamatsuPhotonics,Japan) widlanimageanalysis野Stem(SP500F:0恒叩uS).Moredlan500DAPI−Stainedbacterialcellswerecoultedto detemlinedlePrOPO血onofdlePrObe−SPeCifiC−labeledcellsamongdletOtalofdleaSSOCiatedbacteria. TheaccuracyofdleMCD−PrObehadpreviousb,beenexanlinedbyuslngSequenCeSOfdle16SrRNAgene Obtained丘omaGenBank:dleMCD−PrObewasfoundtocontainlmismatchfordle20dlerkn0wnSequenCeSOf dle16S rRNAgeneindle database;bodlSequenCeShadbeenisolated丘ommarine oligotrq3hicbacteria (ABO21704,ABO22713).Thesebacteria,Whichwereobtainedcowte野OfDr.I.YbshinagaofKyotoUhiversity, WerenOtSuCCeededinhybridizationofdledesignedMCD−PrObewidlVariouscond血)nOfhybridizationaqusted byconcentrationofformamideandtemperature.Thereexistsomeargumentsabo血dlebindingstrengdlbetween dleSequenCeSOfdledesignedprbbeanddletargetPOS止ionof16SrRNA.AccordingtodlerelX)rtSOfFuchsetal. [17],dle16SrRNApositiOnofdleMCD−PrObewasnoteasib,hybridized.However,WeCOnfirmeddlatY2strain WaSSuCCeSSfulb,visualizedbyuslngMCD−PrObe. /い州仙川/り〃日日川し川/′川/州、り/lhソ日日、〝、し・l肌。仙′車Jl//〟.〃仙//仙//し・J/−/−川仙/〃仙/日和汀常山′・ 川/しハJL・い/川〃日加・/.山・ TbdetemlinedleCOnCentrationsofMc7VCygtlS,Chlorophylla,andcell−boundandextracellularmicrocystln, Surhcewatersampleswerecollected丘omdleCenterOfdlelakeoncetwoweeksbetweenAprilandDecemberin 1998肌d1999.SamplesforcellcountingofMc7VCygtlSWerefixedinformaldehydesolution(血1alconcentration, 1.5%Wん).TheconcentrationofMc7VCygtlSCellswasestimatedbyusingaFuchs−Rosenthalhemocytometer (Kayagakiworks,Japan)underamicroscope(BH−2,0恒叩uS). 6 Maruyamaetal. TbmeasuredleChlorophyllaconcentration,WaterSamPleswerefilteredthroughaglassfiberfiker(GF/C, Whahan,UK),WhichwasdlenSOakedin10mLofmedlanOlfor24hat4℃.ARerdlat,dlereSiduewas Centrifugedat3000IPmfor15min.MeasurementofdleChlorophyllaconcentration丘omdleSuPematantWaS quantifiedspectrophotometriCalb,tydlemedlOdofMakeretal.[31]. Measurements ofmicrocys也l COnCentration 肌d dle Clean−uP Ofmicrocystln in preparation for high−PerformanCeliquidchromatogr痢(HPLC)werecaniedo血accordingtoPadietal.[36].Formeasurement Ofce11−boundmicrocystin,Mc7VCyStlSCellswereconcentratedonaGF/Cfilter.Thefilterwasdlenhomogenized andextractedwid15%aqueousaceticacid,and,a鮎rcentrifugat10nat4000IPmfor15min,dleSuPematantWaS POuredintoanODScartridge(Baketbond甲e仇tadecyllC18]3mL,USA).Microcystinextracted丘omdle CartridgewidlO.1%trifluoroacetic acid(TFA)一medlanOIwas appliedtodleIiPLC system(LC−9A S−I, Shimadzu,Japan),WhichwasequippedwidlanODScolumn(Cosmosi15C18−AR4.6×150mm,Nacalai,Japan). TheconditionsofIiPLCforanalysisofmicrocys也lWereaSfollows:absorbanceat238nm;medlanOl:0.05M phosdlatebu飽r(58:42;PH3.0)indlemObilephase;anda111止一min.1flowrate. Extracellularmicrocys也lWaSmeaSuredby2medlOdsuslngaGF/Cfiltrate.In1998,dleGF/Cfiltratesample WaSPOuredintoanODScartridge(5g,ChromatorexODS,10ひっ00mesh,FttiiSib,SiaChemiCal,Kasugai, Japan).ThecartridgewasdlenrinsedwidlWateranddlenwid120%medlanOl.Extracellularmicrocystinwas el血ed丘omdleCartridgewid190%medlanOlandevaporatedtodryness.ARerasilicagelcartridge(2g,SepPak) hadbeenprecond追cnedwidlmedlanOl,dlereSiduewasdissoIvedinmedlanOlandappliedtodleCartridge.ARer dleCartridgewasrinsedwidlmedlanOl,eXtraCellularmicrocystlnWaSel血edwid170%medlanOl.Theeluatewas evaporatedtodryness,anddlereSiduewasre−dissoIvedinmedlanOl.ThemedlanOIsolutionwasdlenanalyzedby IiPLCunderdleSameCOnditionsasabove. In1999,eXtraCellularmicrocystinwasanalyzedbye呵me−linkedimmunosotbentassay鱒LISA),Which detemlineddletOtalconcentrationofmiCrocystlnSwidlO血discrimindlngmicrocystinderivatives.Nagataetal. [33]showedvery simi1areStimates丘omELISAandaliquidchromatographicmedlOdfordle analysisof microcystin,aldlOughdleSenS止ivityofdleELISAmedlOdwashigher.Thus,WeuSeddleELISAmedlOda鮎r Nagataetal.[32,33]. 7 Results rl仙川/′川/州、り/lhソ日日、〝、し・l肌。仙′車Jl//こし.仙/し・J/−/−川仙/.仙/Hmn〟〃/./′」〃仇ソ日日、….晶….∵ lh7日。い//\/−/川り〃\ ChangesindleCOnCentrationsofMc7VCygtlS,Chlorophylla,andcell−boundmicrocystlninwaterareglVenin Figurel.In1998Mc7VCygtlSaPPearedindlemiddleofJuneandbloomed,dledominantspeciesbeingM ich如bk7beinJulyiandMtmC鮎丘omAugusttoatober.Relativepropo血onofMc7VCygtlSbiomassformed moredlan99%intotalphytopl肌ktonbiomassduringdlebloomingperiodofMc7VCygtlS.Theconcentrationof Mc7VCygtlSCellshad2peaks:2・4×109cellsI:10n29Julyiandover7×108cellsI:10m9and25SeptembeI; aRerwhichdatesitdecreased(Fig.1A−1).Thechlorophyllaconcentrationalsoshowed2largepeaks:662LlgI/1 0n29叫andover600LlgI/10n9and25SeptemberBy7atoberithaddecreasedmadiedb,tO180LlgI/1 げig.1A−2).Thecell−boundmicrocystinconcentraticnincreasedexponentialb,inSeptemberuptolOOLlgI/1, dlendecreasedto50LlgI/10rlessinatober(Fig.1A−2).AlthoughdleChangesinMc7VCygtlSCellconcentration WereParalleledbydlOSeOfchlorophyllaandcell−boundmicrocysbn,nOnParametricSpearmanStatistiCalanalysis Showedsignificanceonb,betweendleCOnCentrationofMc7VCygtlSCells肌dchlorophylla(r=0.95,P<0.01,n= 12).Theconcentrationofcell−boundmicrocystinfluctuatedinparallelwidldlatOfchlorophylla(r=0.81,P< 0.01,n=13).TheextracellularmicrocystinconcentrationindleWaterWaSlessdlanO.5LlgI/1through0血dle 1998studyperiod(Thble2)−SimilartOdlefindingsofPadietal.[36].Theconcentrationofextracellular microcystinwasdlehigheston29Jub,;by3Decemberithaddecreasedtobelowdledetectionlimit.AldlOughits fluctuationwassimilarinp細emtodlatOfdleMc7VCygtlSCellconcentration,nOSlglificantrelati0nshh3between dletWOWaSdetectedbynonparametricSpearmanStatistiCalanalysis. Incontrastto1998,aminorbloomofMc7VCygtlSWaSObservedinSeptember1999,Showlngdlatrelative PrOPOrtionofMc7VCygtlSbiomassformedmoredlan99%intotalphytopl肌ktonbiomass.ItconsistedofM ich如bk7be,Maenqnosa,MnoWCekti,andMwesenbe密i.However,nOneOfdlOSeStrainsdominated Strikingly.TheabundanceofMc7VCygtlSWaSlorderofmagnitudelowerdlandlatin1998,ranging丘om4.3× 106to6.8×107cellsI/1(Fig.1B−1).Theconcentrationsofchloro如Ilaandcell−boundmicrocystin鮎mll Augustto6仇toberin1999ranged丘om16to48LlgI/1肌d丘omO.54to4.49LlgI/1,reSPeCtiveb,げig.1B−2). 8 Maruyamaetal. Cell−boundmicrocystlnWaSnOtanalyzedbetweenAprilandJulyiaSdlebiomassofMc7VCygtlSWaSinslgnificant. ThetotalconcentrationofextracellularmicrocystinindleWaterWaSlessdlanO.2LlgI/1through0血dle Observation period(Thble2).AldlOughdle Changesindle COnCentrations ofcell−boundmicrocystin and extracellularmicrocystln aPPearedto parallelchangesindleMc7VCyStlS Cellconcentration,nOnParametric SpearmanStatistiCalanalysishiledto血Owanyslgnificantrelati0nships. /叶/J/.///り〃./川./〃仇・、日仏/し…T.相、、目しソ血・./l川/Jlhソ日日、〝、 ThedensityofbacteriaassociatedwidldleMc7VCygtlSCOloniesranged丘om7.2×106to8.5×107cellsmI/1 0flakewaterin1998and丘oml.1×104to3.2×105cellsIllLlin1999(Thble3).Thenumberofbacterialcells associatedwidleaChMc7VCygtlSCellincreased丘om250n26Augustto1180n9September1998,b血didnot Changemadiedb,in1999,ranging丘om2.6to4.7cells.Thehighestconcentrationofassociatedbacteriaindlelake waterwasfoundon9September1998andon8September1999,reaChing8・5×107and3・2×105cellsmLl, respectively.Whereasassociatedbacteriaconstituted95%ofdletOtalbacteriaindleWaterOn9September1998, associatedbacteriaindle1999stuQ,Periodconstitutedonb,0.2%to3.3%ofdletOtalbacteria.Thedi飽rencein dleCOnCentrationofassociatedbacteriabetween1998and1999wasascribedtodlediBtrenceindleSPeCies COmPOSitionofMc7VCygtlS. (九日.阜い〃h川〃〃〃〃〃/い/川し拙/しり/い、目しソ血・.//一./し・/げ/./ ThecomitystructureofbacteriaassociatedwidlMc7VCygtLgWaSeXPreSSedondlebasisofdlenumberof eachbacterialspecies,aSdetemlinedbyrRNA−targetedoligcnucleotideprbbes,PerlO2cellsofMc7VCygtlSげig. 2),becauseanaveragecolonyofMc7VCygtlSinLakeSuwaconsistedofatleastlO2cells,aldlOughkfluctuated ranglng丘om127to529cells.In1998,dleCOnCentrationofdomainBacteriavisualizedwidldlePrbbeEUB338 ranged丘oml・4×103to7・9×103cellsperlO2Mc7VCygtlSCells,anddleBacteriawerecomposedofbetween 56%and69%DAPI−Stainedparticles(datanotshown).α−ProteobacteriaweredleSeCOndmostcommona鮎r β−Proteobacteriaon9Septemberand7atober,increasing丘om2.6×102(26August)to2.0×103(7atobeり cellsperlO2Mc7VCygtlSCells(18%to26%ofdleSumOfthebacteriahybridizedbydleOligonucleotideprobes 9 SPeCific to〔エー,β−,γ−,and 8”Proteobacteria and dle qノttPh抑bactenum group)げig.2A−1). β−ProteobacteriaweredominantduringdlebloomingofMc7VCygtLg,ranging丘om5.1×102to2.7×103cells perlO2Mc7VCygtlSCells(28%to36%).γ−Proteobacteriaincreased丘om3.8×102(26August)tol.3×103(7 atobeりcellsperlO2Mc7VCygtlSCells(17%to27%).8−ProteobacteriamadeupdlelowestpercentageofdleSum OfbacteriahybridizedwidlgrOuP−SPeCificprobeson26August,9September,and7atober(5.3%to13%). However,thisgroupincreased丘omonb,75(26August)to9.9×102(9SeptembeりcellsperlO2Mc7VCygtlS Cells.Thisincreasewasabo血2−tO4−foldhigherdlandlatOfodlergrOuPSindlePeriod丘omAugusttoSeptember. TheconcentrationofdleqノttPh(脚bactenumgroupincreased丘oml.9×102(26August)tol.5×103(7 atobeりcellsperlO2Mc7VCygtlSCells(13%to20%). In1999,dleCOnCentrationsofdomainBacteriavisualizedwidlEUB338ranged丘om3・1×102to3・9×102 cellsperlO2Mc7VCygtlSCells,anddleBacteriawerecomposedofbetween78%and94%IhPI−Stainedparticles (datanotshown).ThesenumberswerehigherdlandlOSein1998,althoughdledensityofassociatedbacteriain 1999wasabo血100timeslessdlanin1998.α_Proteobacteriaexistedatconcentrati0nsOfbetween1.2×102cells andl.4×102cellsperlO2Mc7VCygtlSCellsthrough0血dleStudyperiod(Fig.2B−1),andpredominatedinall SamPles accounhngfor atleast30%of dle Sum Of bacteria hybridizedwidl grOuP一甲eCific probes. β−Proteobacteriaalsoremainedneadyconstantatl.0×102cellsperlO2Mc7VCygtlSCellsduringdleStuQ,Period, COnStitutlng丘om26%to31%ofdleSumOfbacteriahybridizedwidlgrOuP−SPeCificprobes−aSimilarPerCentage todlatin1998.γ−Proteobacteriaranged丘om49to57cellsperlO2Mc7VCygtlSCells(13%to18%).Thedensities OfdleSe3如logeneticgro叩SWereSimilartOeaChodlerduringdle19990bservations.8−Proteobacteria肌ddle qttph(脚bactenumgroupshowedsimilarfluctuati0nswidlregardtorelativeabundanceandcelldensity: bodlParameterSforbodlgrOuPShadincreasedby8Septemberanddecreasedby6仇tober.Thenumberof8− Proteobacteriaranged丘omllto50cellsperlO2Mc7VCygtlSCells,reaChingapeakon8SeptemberThisgroup made up onb,3.5%to13% of dle Sum Of bacteria hybridizedwidl grO叩−SPeCific probes.The qttph(脚bactenumgroupranged丘om13to69cellsperlO2Mc7VCygtlSCells(4.0%to18%),reaChinga maximumOn8September. MCD−bacteriahadincreasedremadiabb,intx)dlrelativeabundanceanddensityofassociatedbacteriaperlO2 10 Maruyamaetal. Mc7VCygtlSCellsby9September1998(Fig.2A−2).MCD−bacteriaincreasedremadiabb,丘om93cellstol.3× 103cellsperlO2Mc7VCygtlSCellsbetween26Augustand9SeptembeI;andby7atoberhaddecreasedto7・8× 102cellsperlO2Mc7VCygtlSCells,thusaccountingfor6・6%to17%ofdletOtalbacteriahybridizedwidl group一甲eCificprobes.Theirrelativeabundanceamong〔エーProteobacteriawasalsohigh,ranging丘om36%to76% (datanotshown).In1999,MCD−bacteriaranged丘om20to41cellsperlO2Mc7VCygtlSCellsandshoweda tendencytoincreaseinnumbersinSeptembeI;aCCOuntlngfor6.1%to11%ofdleSumOfbacteriahybridizedwidl group一甲eCificprbbes(Fig.2B−2).TherelativeabundanceofMCD−bacteriaamong〔エーProteobacteriawasnotas highasin1998,ranging丘om17%to34%.ThehighestproIX)rtionsofMCD−bacteria,inrelationtotx)dldleSum OfbacteriahybridizedwidlgrO叩−SPeCificprobesand〔エーProteobacteria,WereObservedon8September.These results丘om1998and1999indicateddlatdlenumberofMCD−bacteriaassociatedwidllO2Mc7VCygtlSCells increasedinSeptember and dlen decreasedinatobeI;Closeb,Paral1eling changesindlemicrocystln COnCentrationintx)dldleWater(r=1,P<0.01,n=6)andMc7VCygtlSCells(r=0.89,P<0.05,n=6). Discussion StrainsofseveralspeciesofdlegenuSMc7VCygtlSPrOduce60variantsofhepatotoxicmicrocystin[38].M tmdsisIsuchtoxicspeciesinLakeSuwal35,36].Thehighestconcentrationofcell−boundmicrocystinwas ObservedwhenMtmdLgwasdominantinSeptember1998. Mc7VCygtlSissurroundedbymucilage,Whichconsistsma叫ofpob,SaCCharidel3,5]composedofglucose, mannose,fucose,Xylose,galactose,and血amnose.ThethicknessandsolubilityofdlemuCilagevaryamong Mc7VCygtlSSPeCies[4,24]:dlemuCilageofMtmC鮎ishardertodissoIveinwaterdlandlOSeOfdleOdlerSPeCies l4].Duringblooms,numerOuSbacteriaarekn0wntOeXistindlemuCilagel9].FurdlermOre,dleabundanceand COmitystructureofdleembeddedbacteriamiglltdi飽raccordingtodleMc7VCygtlSSPeCies. Whenmicrocystlnisreleased丘omacellofMc7VCygtlS,itisb’aPPedindlemuCilagebecauseofdlemuCilages highviscosity.TbrevealdlePrOCeSSOfdegradationofmicrocystin,Wedlereforefocusedondlefunctionofdle bacteriaembeddedindlemuCilageandtriedtodescribedlelX)Pulationdyn肌1icsofdleMCD−bacteriadlere.0ur resultsrevealeddlatdlenumberofMCD−bacteriaindlemuCilageincreasedinSeptemberofbodlyearSand 11 COITelatedwidldleCOnCentrationofcell−boundmicrocydn,anddlehighestconcentrationofMCD−bacteria existedin1998whenMtmdiswasdominant.Itisremadiableinnaturalsystemsdlatlgenespecificcloneof MCD−bacteriadetectedbyFISHmadeupl/100fdleWholebacterialcomityinSeptember1998and1999. TheseresultssuggestdlatMCD−bacteriarespondedtochangesindleCOnCentrationofmicrocysbn,anddlat MCD−bacteriawereactiveindlemuCilageofMc7VCygtlSWhenproducedmicrocystlnWaSPreSentdlere.Joneset al.[22]reporteddlatmicrocystin−degradingisolatesrequirealagtimeindledegradationofmicrocystinwhendley havenotbeenexposedtomicrocys也laPnOnunderexperlmentalcond追ons.However,We aSSumeddlat MCD−bacteriaindlemuCilagewereon’stand−by’untildledegradationofmicrocys也10ccurred:dleyCOuldbe directb,eXPOSed to anymicrocys也l released丘om cellsindle Mc7VCygtlS COlony.This suggests dlat MCD−bacteriacouldthusinitiatedledegradationofmicrocystlnindlemuCilagewidlin2weekssohrwe examiIled.ThesefindingscanexplainwhyabacterialspeciesbecomespredominantinaglVenSyStemifitexertsa VerySPeCifiCfunctiontodegradeaspecificcompound,SuChasmicrocystln. During dle bloom ofMc7VCygtlS,dle COnCentrations of dle qノttPh脚bactenum group and 8−Proteobacteriawereapparentb,SynChmizedwidldlatOftheMc7VCygtlSCells,widlr=0.89(P<0.05,n=6) andr=0.94(P<0.05,n=6),reSPeCtively.Tbourknowledge,membersofdleqノttPhqp47atvbactenumgroup areabletodegradenotonb,maCrOmOlecularCOmPOundsl12,43],b血alsoMc7VCygtlSCellsl46].ⅥnHannenn l43]suggesteddlatCytophagales,dlerelated16SrRNAsequenceofwhichappearedindenatu血ggradientgel electrophoresisa鮎rdleb,Sisofcyanobacteria,COuldcontributetodegradationofdissohledorgmicmabr(DOM) released丘omthisb,Sis.Recently,CottrellandKirchmanl12]suggested丘omfluorescenceins血hybridization (MCRO−FISH)studiesdlatdlemOdeofbacterialutiliza onofDOMdi飽rsamongphylogeneticgroups:dle qttph抑bactenumgrouptendstopre丘rhigh−mOlecular−WeightDOMsuchasproteins andchitin. Yamamotoetal.[46]Showedbyaculture−dq)endentmethoddlatSOmeMc7VCygtlSWereb,Sedspecifica町ty SOmeStrainsofthisgroupISOlated丘omdleSurhcewatersofLake Suwa.Thesefindingssuggestdlatdle qttph抑bactenum group contrib血estodleb,SISOfMc7VCygtlS anddegrades DOMderived丘om intracellularPrOductsofMc7VCygtlSindlemuCilage.GrilliCaiolaetal.[18]reporteddlatBdblk)tqbn0−1ikebacteria, COnStituentsofdle8−Proteobacteria,in丘ctMc7VCygtlSCells肌ddegradepeptidoglycananddleCellwal1,aldlOugh 12 Maruyamaetal. Bdblk)庚bnoiskn0wntObeabacterialpredatorl6].Thissuggestsdlat8−ProteobacteriamightcontribtAetodle 吋sISOfA必C7Pq償触 α−Proteobacteriaandβ−ProteobacteriatendedtodominateindlemuCilageofMc7VCygtlSduringdlebloomof Mc7VCygtlS.Ofdle〔エーProteobacteria,Cbuk)bacter can ahch to cyanobacteria and take up exudates of Photo町ndleticproducts[42].Ak:alwnesandPgeudbmonag,Whichareβ−Proteobacteria,arekn0wntOb,Se Mc7VCygtlSCellsbyahchingtodleml28,46]. WestudieddlePrOCeSSOfdegradationofmicrocystinindlelightofchangesinbacterialconnnunitystructurein anaturalenvironment,focuslngParticuladyonstrainY20fMCD−bactena,Whichbelongstoanundescribed genusl37].Ⅵ屯founddlatMCD−bacteriaexistedinarestrictedspaceofdlemuCilageofMc7VCygtlS,anddlatdle ChangeinconcentrationofdleSebacteriawassynchronizedwidldleincreaseindleCOnCentrationofcell−bound microcystln.ThissuggestsdlatMCD−bacteriaindlemuCilagerespondedtochangesindleCOnCentrationof Cell−boundmicrocystln;dlemicrocystlnWaSeXuded丘omdleCelloftoxicMc7VCygtlSanddegradedbydle bacteria.TheqノttPhqp47atdactenumgroupand8−Proteobacteriaalsochangeddleirpopulationdensitiesindle mucilage,Sugge血唱dlatdleyCOntribtAedtodledegradationofMc7VCyStlSCells.MucilagelSreVealednotonb,aS acompounddlatbindsMc7VCygtlSCellstogedleI;b血alsoasahabitatforbacteriadlateXertdleirspecifiCfunction toutilizeandthusdegradeMc7VCygtlSCellularmaterialS. 13 Acknowledgments Thiswo止wassupportedinparttyagrant丘omdleMinistryofHealdl,LaborandWelfke,Japan(Researchin ErrvironmentalHealdlH11−Seikatsu−015,andNaganoPre丘ctureTbclm0−High1andDevelopmentOrgmization) h1998. 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Tablel.Probesequencesandtargetsites Probename Target0−ganism Sequence Targetsitea Reference rRNA Position EUB338 domain Bacteria ALFlb: a−Proteobacteria BET42a b−Proteobacteria 5㌧GCCTCCCCACTTCGTTr−3− 23S,1027−1043 29 GAM42a g−Proteobacteria 5㌧GCCTCCCCACATCGTTr−3− 23S,1027−1043 29 DEL: d−Proteobacteria 5㌧CGGCGTCGCTGCGTCAGG−3− 16S,385−402 1 CF319a: MCD: Cytophaga/FlavobacteriuI曙rOuP MCD−bacteria 5’−GCTGCCTCCCGTAGGAGT−3㌧ 5’−CGTTCG(C/T)TCTGAGCCAG−3’ 5㌧TGGTCCGTGTCTCAGTAC−3− 5㌧CGCCACCAAAGCC TAAAAGG−3一 a Escherichia colinumbering.[7] rhblE2_CDnCentIatiDnOfe蒐traCE鮎hr皿i⊂正OCYStn血I.akeSllW且コ199g且ndlg押 血emd訂血CmC野血 ⊂皿仁皿血m〔甚gm ̄1〕・ Jlm.17 0.05 九九29 0.47 A叫E_26 0.24 S印1.25 0.17 0山.ユ1 0.09 DEC_ヨ 刑■.]D.【 M町.ユO mD9・ Jl軋ユB 田_田3 A叫駐11 m田4 血喝_25 0.10 5甲上玉 田_12. S印t_22 mD邑 0Ct.6 0.06 1Mi坤血鮎血dwi也m_SumqfmiCr∝亨邑t血−1且姐d−R胤 恥血刑亡甲血血弛調血血慮腑血血EL脹A. 恥D.,血町∝:y血n鵬血k仁止丘. 19 16S,338−355 1 16S,19−35 29 16S,319−336 30 16S,839−858 This study Table3.NuIl血erof Microcystiscells,丘ee−livingbacteriaandbacteriaassociatedwlthcolo111eSOf Microcystis Concerrtrahonof MicIⅦCy虞is cells Assoclatedbactena □□□ Free−hⅥ喝bactena (cellsmL ̄1)a (cellsⅦ瓜・OCy鵬s cell ̄1) RatlOOraSSOClated bactenatototal (cellsmL ̄1) bactena(%) (cellsmL ̄1) 7刀106 士11□107 251士 368 4(□106 士 11□106 61 Sept9 74□105 85]107 士 18□108 116 士 248 43]106 士 11□106 95 0ct7 22□105 2(□107 士 42□107 118 士193 3犯106 士 9刀105 87 1999A喝11 43□103 11□104 士 30□103 26士 07 75]106 士 15]106 02 Aug25 39□104 15]105 士 77□104 40 士 20 7幻106 士 2(刀106 19 Sept8 68□104 3刀105 士10□105 47士15 9屯106 士 15]106 33 0ct6 11□104 3幻104 士 12□104 35 士11 5′刀106 士 1刀106 07 1998A喝26 29□105 a□ MiQ・OCyStiscellspermLoflakewater□□□bactenaassoclatedwlthMicrocystisperMicrocystiscell□ 20 Maruyamaetal. Legends Fig∬el SeasonalchangesintheconcentrationsofMcfVq岱如cells,Chlorophylla,andcell−boundmicrocystln atthecenterofLake Suwain1998(A)and1999(B).A−1andB−1showtheconcenb−adonsof MavqLS如cellsQiandes).A−2andB−2showtheconcentradonsofchlorophylla(q3enCirdes)and Cell−boundmicrocystin(closedcircles). Fig∬e2 ThecompositionofthebacterialassemblagesinMavq岱如colonies,aSdetectedby瓜NA−targeted OligornlCleotideprdbesspedBcforcL−,β−,Y−,andaゼrotedbacteria,theqノ呼柳ろacieriLml group,andmicrocydn−degradingbacteria.Sampleswerecollected丘●OmthesurfacewatersofLake Suwaon26August,9SeptembeI;and70ctdber1998(A),andon25August,8SeptembeI;and6 0ctdber1999(B),WhenMcfVq岱如wasinbloom. Fig∬e3 Insituhybridizationofbacteriaassociated扇thcoloniesofMcfVq岱如,Viewedbyepifluorescence microscopy.Bacteriainthe colony ofMcfVgAgtLs dnedbyDAPI(A),andhybridizeddtha 血odamine−labeledprbbe speciBctomicrocydn−degradingbacteria(B).Panicles about5トImin diameterareCdlsofMcfVq岱tLs. 21 0 0 0 0 1 1 1 1 9 8 7. 6 ちuO焉﹂盲UUuOU ︵L⊥S亘S=崇、昔gき もuOコ空富UUuOU ︵L⊥S亘S=望互02童 0 10 10 10 1 0 9 8 7. 一一▲− 几〝cmcys〟scell nO⊃Ce⊃tratiO⊃Of Ce〒bOu⊃d∃icrOCySti⊃︵箪Q﹁⊥︶ 9 8 7 6 5 4 3 2 1 0 ⊥ 0 0 0 0 0 0 0 7 6 5 4 3 2 1 ︵﹁・﹂㌍詣結露 nO⊃Ce⊃tratiO⊃Of 0 0 0 0 2 1 Ce〒bOu⊃d∃icrOCySti⊃︵箪Q﹁⊥︶ 0 0 0 8 6 4 0 0 0 0 0 0 6 5 4 ︵﹁・﹂㌍詣結露 D N O S h t A MO J J M A 1 8 9 9 0 0 0 0 0 5 3 2 0 0 0 5 0 0 0 0 0 5 SニUO S、芯ゝ020≦NOこSニUO一望gU帽皿 0 0 0 0 0 5 2 S=むOS葛ゝ00JO≦NOこS=む0■召20品 0 0 5 0 0 0 0 0 0 0 5 4 3 2 1 0 0 0 0 0 5 Oct.6 Sep.8 Date A g u 6 2 Aug.25 Fig.3
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