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
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18
Maruyamaetal．
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