Distribution of Archaea in a Black Smoker Chimney Structure

12 Pages·2001·0.31 MB·English

APPLIEDANDENVIRONMENTALMICROBIOLOGY,Aug.2001,p.3618–3629 Vol.67,No.8
0099-2240/01/$04.0010 DOI:10.1128/AEM.67.8.3618–3629.2001
Copyright©2001,AmericanSocietyforMicrobiology.AllRightsReserved.
Distribution of Archaea in a Black Smoker Chimney Structure
KENTAKAI,*TETSUSHIKOMATSU,FUMIOINAGAKI,ANDKOKIHORIKOSHI
SubgroundAnimalculeRetrieval(SUGAR)Project,FrontierResearchProgramforDeep-SeaExtremophiles,
JapanMarineScienceandTechnologyCenter,Yokosuka237-0061,Japan
Received26February2001/Accepted30May2001
Archaeal community structures in microhabitats in a deep-sea hydrothermal vent chimney structure were
evaluated through the combined use of culture-independent molecular analyses and enrichment culture
methods. A black smoker chimney was obtained from the PACMANUS site in the Manus Basin near Papua
NewGuinea,andsubsampleswereobtainedfromverticalandhorizontalsections.Theelementalcomposition
of the chimney was analyzed in different subsamples by scanning electron microscopy and energy-dispersive D
X-ray spectroscopy, indicating that zinc and sulfur were major components while an increased amount of o
w
elementaloxygeninexteriormaterialsrepresentedthepresenceofoxidizedmaterialsontheoutersurfaceof
n
the chimney. Terminal restriction fragment length polymorphism analysis revealed that a shift in archaeal lo
ribotypestructureoccurredinthechimneystructure.ThroughsequencingofribosomalDNA(rDNA)clones a
d
from archaeal rDNA clone libraries, it was demonstrated that the archaeal communities in the chimney
e
structure consisted for the most part of hyperthermophilic members and extreme halophiles and that the d

distribution of such extremophiles in different microhabitats of the chimney varied. The results of the f
r
culture-dependentanalysissupportedinparttheviewthatchangesinarchaealcommunitystructuresinthese o
m
microhabitatsareassociatedwiththegeochemicalandphysicaldynamicsintheblacksmokerchimney.

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p
Sincethediscoveryofdeep-seahydrothermalventsin1979 several microbiological, geochemical, and geophysical obser- :/
/
(12, 15), various microorganisms have been isolated from vations, the possible existence of a subvent biosphere popu- a
e
globaldeep-seahydrothermalventenvironments(26,27,29). lated by hyperthermophilic microorganisms was predicted m
Hyperthermophiles and thermophiles, including members of (14). Also, microbial ribosomal DNA (rDNA) showing sub- .a
both the bacterial and archaeal domains, are the most fre- stantialdiversitywasrecoveredfromblacksmokerventwater s
m
quentlyisolatedmicroorganisms,andtheirphysiologicalprop- intheIhayaBasin,OkinawaTrough(51),atatemperatureof .
ertieslikelyreflecttheextraordinaryenvironmentalsettingsof .300°C, which is far above the upper temperature limit for or
g
thedeep-seahydrothermalvents(9,10,18,23,28,42,43,53, growth of even the most hyperthermophilic archaeon, Pyrolo- /

56,60).Inaddition,recentculture-independentmolecularap- bus fumarii (113°C) (9). The microbial rDNA may serve as a on
proaches have revealed the presence of as-yet-uncultivated genetic signature indicative of the microorganisms thriving in A
thermophilesshowingsubstantialphylogeneticdiversityinthe the subvent habitats, conveyed there by vent water. Hence, a p
r
deep-seahydrothermalventenvironments(27,38,39,44,51). hydrothermalventchimneyisanenvironmentanalogoustothe il
Relativelylittleisknownabouttheecologicalsignificanceand subventbiosphere,andelucidationofitsmicrobialcommunity 11
geomicrobiological function of the extremophilic microbial structure is likely to provide great insight into features of the ,
2
communitiesfound,astheoccurrence,abundance,anddistri- subventmicrobialecosystem. 0
bution of the microbial communities associated with the for- 1
The black smoker chimney structure investigated in the 9
mation of diverse geochemical and physical gradients remain present study was obtained from a hydrothermal vent field b
tobeelucidated. y
located at the PACMANUS site in the Manus Basin near
Adeep-seahydrothermalventchimney,formedbychemical g
PapuaNewGuineaatadepthof1,644m.Thishydrothermal u
interaction between cold seawater and hot vent water, is a e
fieldwasdiscoveredin1991(7),andageologicalsurveyofthe s
distinctivestructureinthedeep-seahydrothermalfieldsandis fieldandgeochemicalcharacterizationoftheventwatershave t
largely composed of sulfide materials. In the chimney struc-
both been performed (6, 7, 16, 17). The results of the micro-
turesandtheunderlyingsulfidemounds,steepenvironmental
biological assessment have not yet been reported. Archaeal
gradients of temperature, pH, oxidation-redox potential, and
communitystructuresinmicrohabitatspresentinthechimney
variouschemicalscanbeformedbyequilibrationbetweenthe
structurewereevaluatedthroughthecombineduseofculture-
ventwaterandtheseawater,andtheseprovidediversemicro-
independent molecular analyses and enrichment culture
habitats for microbial communities. In addition, the presence
methods. The molecular techniques used in this study are
ofsimilarenvironmentalgradientsinthesubventenvironment
PCR-based terminal restriction fragment length polymor-
beneaththeactivehydrothermalseafloorisevident.Basedon
phism (T-RFLP) and rDNA clone analyses, and the data
provided by these methods should be carefully evaluated.
However, the PCR-mediated molecular techniques will allow
*Corresponding author. Mailing address: Deep-Sea Microorgan- the possible detection and evaluation of a probably very low
isms Research Group (DEEP-STAR), Japan Marine Science and
biomassofarchaealcommunitiesinthehardlyaccessibledeep-
Technology Center (JAMSTEC), 2–15 Natsushima-cho, Yokosuka
237-0061,Japan.Phone:[phone].Fax:[phone].E-mail: sea hydrothermal vent chimney. The distribution of archaeal
[email protected]. communitiesassociatedwiththeoccurrenceofdiscretemicro-
3618
VOL.67,2001 DISTRIBUTION OF ARCHAEA IN A BLACK SMOKER CHIMNEY 3619
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FIG. 1. Schematicdrawingsofthemorphologicalandstructuralfeaturesofablacksmokerchimneyandthesubsamplingmethodused.(A) u
e
Appearanceofthesurface.(B)Verticalsectionofthewholechimneystructure.(C)Horizontalsectionoftherootpartofthechimney.Thepart s
whereeachofthesubsampleswastakenandthepreliminarymorphologicalandstructuralfeaturesofthesubsamplesareindicated. t
habitatsinthechimneystructureandimplicationsforthepos- ofthesubsamplesisshowninFig.1.Subsamplingwasperformedinananaerobic
siblesubventbiospherearediscussed. chamberinanatmosphereof10%H2and90%N2.Eachofthesubsampleswas
subjectedtochemicalanalysis,nucleicacidextraction,microscopicobservation,
andcultivationofmicroorganisms.SubsamplesIandIIwereobtainedfromthe
MATERIALSANDMETHODS
toppartofthechimney.Asurfacelayer(thickness,1to2mm)withwhiteorgray
Samplecollection,subsampling,andchemicalanalysis.Achimneystructure weathering(subsampleII)wasmoisturizedandgrazedfromthetoppartofthe
wasobtainedfromablacksmokerfoundinahydrothermalfieldatthePAC- chimney,andtherest(subsampleI)waslessmoisturizedandpulverizedusinga
MANUSsiteintheManusBasin(03°43.8169S,151°40.0169E)atadepthof1,644 mortarandpestlewhichhadbeensterilizedbyheatinginadryoven(200°C).A
mbymeansofthemannedsubmersibleShinkai2000inadivein1999(diveno. surface layer (thickness, 1 to 2 mm) with white, orange, or gray weathering
1151).Theinsitutemperatureoftheeffluentblacksmokerventwaterwasfound (subsampleIII)wasalsoobtainedfromthemiddlepartofthechimney.Thevent
to be over 250°C. The chimney structure was immediately cooled and stored surface(subsampleIV),whichwasblack,solid,andcrystallineandcontained
during2weeksinananaerobicbagfilledwith100%N at4°Cpriortosubsam- littlewater,waspulverizedusingamortarandpestle.SubsamplesVandVIwere
2
plinginthelaboratory.Duringthestorage,noapparentchangewasfoundinthe obtainedfromtheinsidestructureoftherootpartofthechimney.Theinner
appearanceofthechimneystructure. structure(subsampleV)wasgray,soft,andporousandcontainedaconsiderable
Adescriptionofthestructureofthechimneyandoftheidentificationfeatures amountofwater.Theouterstructure(subsampleVI)wasblackandsolidand
3620 TAKAI ET AL. APPL.ENVIRON.MICROBIOL.
muchlessmoisturized.Theouterstructurewaspulverizedusingamortarand thereaction(20and25cyclesfor30cyclesand35and40cyclesfor45cycles),
pestle. noapparentproductwasobtained.
Theelementalcompositionofthesubsampleswasanalyzedbyscanningelec- AmplifiedrDNAfromtwoseparatereactionswaspooledandsubjectedto
tron microcopy and energy-dispersive X-ray spectroscopy (SEM-EDS). The agarosegelelectrophoresis.TheproductswerepurifiedbymeansofaGelSpin
grazedorpulverizedsamplesweredirectlyembeddedonspecimenmountsusing DNApurificationkit(MOBIO).TheDNAwasprecipitatedwithethanoland
adhesive tape or electroconductives. After natural drying, the specimen was centrifuged,andthepelletwasresuspendedinthedistilleddeionizedwater.The
coatedwithosmiumplasmatoathicknessof5nmbyusinganosmiumplasma purified rDNA was digested with a restriction enzyme (HhaI). The terminal
coater.ThecoatedspecimenwasobservedusingaJEOLJSV-5800LVscanning restrictionfragments(T-RFs)wereanalyzedusingamodel377automatedse-
electronmicroscopeat15kVandwasanalyzedusinganenergy-dispersiveX-ray quencerequippedwithGeneScansoftware,version3.0(PEAppliedBiosystems,
spectrometer with an ultrathin window at 15 kV. NaCl, BaSO, BaO, BaS, FosterCity,Calif.).ThepreciselengthsofT-RFsweredeterminedbycompar-
4
ZnSOz7HO,ZnO,ZnS,andcolloidalelementalsulfur(S0)(NakaraiTesque, isonwithaninternalsizestandardaddedtoeachdigestedsample.Theelectro-
4 2
Kyoto,Japan)wereusedasreferencesubstances. phoresisconditionsandtheproceduresfollowedwerethosesuggestedinthe
Microscopicobservation.A2-gportionofthesubsamplewassuspendedin6 manufacturer’s protocol. The archaeal T-RFLP profiles from the subsamples
mlofsterilizedsyntheticseawater(MJ)(46,54)containing3.7%(wt/vol)form- wererepeatedlycheckedinadifferentseriesofexperimentsforPCRamplifica-
aldehyde,andthemixturewasvigorouslyagitatedfor2minusingavortexmixer. tion,restrictionenzymereaction,andelectrophoresis.
Thesuspensionsolutionwasverybrieflycentrifuged(3,0003g),andthesuper- CloningandsequencingofarchaealrDNA.ArchaealrDNAwasamplifiedby
natant was filtered with a 0.22-mm-pore-size 13-mm-diameter polycarbonate PCRusingthesameprotocolasforT-RFLPanalysisexceptfortheuseofa D
filter(Advantec,Tokyo,Japan).ThefilterwasstainedbytreatmentwithMJ nonlabeledprimersetofArch21FandArch958R.AmplifiedrDNAfromtwo o
seawatercontaining49,69diamidino-2-phenylindole(DAPI)(10mgml21)oracri- separate reactions was purified as described above. The purified rDNA was w
dineorange(10mgml21)at4°Cfor20min.ThefilterwasbrieflyrinsedinMJ cloned in the vector pCR2.1 using the Original TA cloning kit (Invitrogen, n
seawaterandexaminedunderepifluorescenceusingaNikonOptishotmicro- Carlsbad,Calif.).TheinsertswereamplifiedbydirectPCRfromasinglecolony lo
usingM13primers(36),treatedwithexonucleaseIandshrimpalkalinephos- a
scope.
phatase (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, d
Extractionofnucleicacids.MicrobialDNAwasdirectlyextractedfromeach e
subsample (approximately 5 g) using a Soil DNA Kit Mega Prep (MO BIO UnitedKingdom),anddirectlysequencedbythedideoxynucleotidechain-ter- d
minationmethodusingadRhodaminesequencingkit(PEAppliedBiosystems)
Lpraobtoorcaotlo.rMiesic,rIonbci.a,lSoRlNanAaBweaascehx,tCraacltief.d),ffroolmlowainrgepthliecamteanaulifqaucotutroefr’esascuhggoefsttehde followingthemanufacturer’srecommendations.TheArch21Fprimerwasused fro
inpartialsequencinganalysis. m
samplesusedforDNAextraction.Approximately5gofsamplematerialwas
Sequenceandphylogeneticanalyses.Single-strandsequencesapproximately
suspended in 3.5 ml of extraction buffer (pH 4.2) containing 25 mM sodium h
400nucleotidesinlengthwereanalyzed.Thesequencesimilarityamongallofthe t
acetate,5mMEDTA,and5%(wt/vol)sodiumdodecylsulfate.Then2.5gof t
single-strandsequenceswasanalyzedusingtheFASTAprogramequippedwith p
sterileglassbeads(0.1-mmdiameter;Sigma)and3.5mlofphenolequilibrated :
DNASISsoftware(HitachiSoftware,Tokyo,Japan).TherDNAsequenceshav- /
withextractionbufferwereadded.Thesuspensionwasshakenonabeadbeater ing$98%similarityasdeterminedbytheFASTAprogramwereassignedtothe /a
for2minandincubatedat60°Cfor1h.Theshakingtreatmentwasrepeated,and e
sameclonetype,andarepresentativesequenceofeachclonetypewasapplied m
thesuspensionwascentrifugedatroomtemperature.Thelysatewasextracted
withphenol-chloroform-isoamylalcohol(25:24:1,vol/vol/vol)andwithchloro- t8o).sTeqhueednacteabsiamseilsaruisteydanfoarlygsiaspwpietdh-BdaLtAabSaTseasnbaylytshisewgaeprepethd-eBpLrAokSaTrymoteitchsomda(l1l-, .a
form-isoamylalcohol(24:1,vol/vol).RNAwasprecipitatedbyadding1.5mlof s
subunit(SSU)rRNAdatabaseandthenonredundantnucleotidesequenceda- m
10Mammoniumacetateandthesamevolumeofisopropylalcoholandwas
tabasesfromGenBank,EMBL,andDDBJ. .
recoveredbycentrifugation.Thepelletwaswashedwith70%(vol/vol)ethanol o
Sequences of representative rDNA clones approximately 0.95 kb in length r
anddissolvedinfilter-sterilized,distilled,deionizedwater.ThetotalRNAwas weredeterminedfrombothstrands.ThedatafromtherDNAclonelibrariesand g
purifiedfromthecrudeRNAsolutionusingaRNeasyMidiKitspincolumn thedatafromT-RFLPanalysiswerelinkedbycalculatingtheT-RFlengthfor o/
(Qiagen,Valencia,Calif.).AllplasticwareandallsolutionsusedforRNAex- thesequencedclones.CloneswithaT-RFlengthidentical(within61bp)toone n
tractionandpurificationweretreatedwith0.1%diethylpyrocarbonatetoinac- foundbyT-RFLPanalysiswereassignedtoaT-RFLPribotype.Thesequences A
tivatenucleases.Inordertocheckforlaboratorycontaminants,ablanktube weremanuallyalignedtoprokaryoticSSUrDNAdatafromtheRibosomalData p
(DwNitAhnaondsaRmNpAleeaxdtdraecdt)iowna(s5p7r)o.cReNssAedaansdaDnNegAatcivoencceonnttrraotlioinnsthweerceasmeeoafsubroethd PwreorjeecatlsIoIs(u3b5m)ibttaesdedtooannaplryismisaurysinagndthseeCcohnimdaerryasCtrhuecctkurperocgornasmideorfatthioenRsiabnod- ril 1
usingaspectrophotometer. somalDataProjectIItodetectthepresenceofchimericartifacts.Phylogenetic 1
QuantificationofarchaealrRNAandtherDNApopulation.Quantificationof analyses were restricted to nucleotide positions between the Arch21F and ,
2
archaeal rRNA in the whole microbial RNA assemblages extracted from the Arch958Rprimersthatwereunambiguouslyalignableinallsequences.Neigh- 0
subsampleswasperformedbyRNAdotblothybridization.Adilutionseriesof bor-joining analysis was performed using the PHYLIP package (version 3.5; 1
RNAsamples(1,0.5,and0.25ngml21)with10mMTris-HCl(pH7.5)were obtained from J. Felsenstein, University of Washington, Seattle). Bootstrap 9
prepared,theRNAwasdenaturedat100°Cfor10min,andthesampleswere analysiswasusedtoprovideconfidenceestimatesforphylogenetictreetopolo- b
cooledonice.ThedenaturedRNAsamplesweredottedontoHybond-N1nylon gies. y
g
membranes(AmershamPharmacia)andcross-linkedtothemembranesbyex- EnrichmentandMPNcalculation.Forenrichmentculturestargetingmem- u
posureto120mJofUVlightenergyusingaUVStratalinker1800(Strategene, bersofthegenusThermococcus,MJYPSmedium(56)wasusedandenrichment e
Torrey Pines, Calif.). The oligonucleotide probes (Uni1392R and Arch915R wasperformedat55,75,and90°C.Forenrichmentculturestargetinghalophilic s
t
conjugatedatthe59endstodigoxigenin)(52)andthehybridizationconditions microorganisms,includingmembersofthegenusHaloarcula,themedium(hem-
usedhavebeendescribedpreviously(52).QuantificationofthearchaealrDNA agglutinin[HA]medium)describedbyIharaetal.(24)wasusedandenrichment
populationinthewholemicrobialDNAassemblageswasperformedbyaquan- wasperformedat30and45°C.Thethree-tubemost-probable-number(MPN)
titativefluorescentPCRmethodaspreviouslydescribed(52).AnarchaealrDNA method was employed to assess the populations of viable cells obtained by
mixturecontainingequalamountsofeightkindsofarchaealrDNA(Haloarcula enrichmentfromthesubsamples.Themicroorganismspresentinthemostdi-
japonica,Palaeococcusferrophilus,Pyrobaculumsp.,Sulfurisphaerasp.,pISA42, lutedoftheseriesofculturesofMJYPSandHAmediawereisolatedbythe
pISA48,pISA16,andpMC1A11)wasusedasastandard(52). extinction-dilutionmethod.Thepartialsequencesofthe16SrDNAweredeter-
T-RFLP analysis of archaeal rDNA. In order to rapidly identify archaeal minedandappliedtosequencesimilarityanalysis.
sequencesinthesubsamples,T-RFLPanalysisofrDNAwasperformed(34). Nucleotidesequenceaccessionnumbers.Thesequencesfromthisstudyare
Archaeal rDNA was amplified by PCR using LA Taq polymerase (TaKaRa, availablethroughDDBJunderaccessionnumbersAB052972toAB052993.
Kyoto,Japan).TheoligonucleotideprimersusedwereArch21FandArch958R-
FAM(13).Reactionmixtureswerepreparedinwhichtheconcentrationofeach
RESULTS
oligonucleotideprimerwas0.1mMandthatoftheDNAtemplatewas0.1ng
ml21. Thermal cycling was performed using GeneAmp 9600 (Perkin-Elmer, Descriptionofsubsamplesandchemicalcomposition.Sub-
FosterCity,Calif.),andtheconditionswereasfollows:denaturationat96°Cfor
samplesofthechimneystructurewereobtainedfromvertical
20s,annealingat50°Cfor45s,andextensionat72°Cfor120sforatotalof30
andhorizontalsectionsdistinguishedonthebasisofmorpho-
cycles.Incasesinwhichnoapparentproductwasrecoveredafter30cyclesof
reaction,thenumberofcycleswasextendedto45cycles.Inthefewercyclesof logicalfeatures.Inthecaseofhydrothermalventswithslowly
VOL.67,2001 DISTRIBUTION OF ARCHAEA IN A BLACK SMOKER CHIMNEY 3621
TABLE 1. PrimarychemicalcompositionofsubsamplesfromablacksmokerchimneyobtainedfromthePACMANUSsitea
Chemicalcomposition(atom%)
Subsampleused
O S Ca Ba Fe Zn Other
I 1.2 41.0 ND ND 1.8 58.0 ND
II(white) 5.0 37.5 0.6 3.8 0.6 53.1 ND
II(gray) 6.5 30.9 0.5 1.8 1.1 54.0 5.3
III(gray) 0.5 37.2 0.2 0.4 ND 55.9 ND
III(whiteandorange) 19.3 35.8 ND 19.6 ND 25.1 0.5
IV 0.8 39.4 ND ND 1.4 61.1 ND
V 0.2 39.8 0.2 0.2 1.5 60.2 0.2
VI 2.4 40.0 0.5 ND 1.9 56.1 ND
Subsurfaceorangelayer 18.0 40.1 1.8 23.6 0.2 19.1 ND
aNumberingofsubsampleswasindicatedinFig.1.ND,notdetected.
D
o
flowingeffluent,ithasoftenbeenobservedthatthetoppartof als on the outer surface of the chimney, indicating the occur- w
thechimneyiscoveredwithweaklypackedmaterials,consist- renceofrelativelyoxidativemicrohabitatsintheexteriorzones n
lo
ing of chalcopyrite, pyrrhotite, and anhydrite on solid sulfide ofthechimney.Intheweatheredsurfacepartsthatwerewhite a
materials(21,22,33).Thechimneyinvestigatedinthepresent and orange and in the orange subsurface layer, barium was d
e
studywasobtainedfromablacksmokerwithvigorouslyflow- accumulated at high levels while no significant change in the d

ingeffluent,andnosuchsofthead-structurewasobserved.The amountofcalciumwasobserved. fr
o
vent surface, the interface with vent fluid, was formed by a Microbial population density and nucleic acid extraction. m
solid,crystallineventwall,whichwasobservedthroughouttop The microbial population density was determined by direct
h
to bottom of the chimney structure. The morphological fea- countingofDAPI-andacridineorange-stainedcells(Table2). t
t
p
tures of the top part were similar to those found on the vent The highest population density was observed in the surface :
/
surface.Inthemiddleandrootparts,fourdistinctinnerstruc- layer at the top part of the chimney (subsample II) and was /a
tures were observed (Fig. 1). Considering the features from 3.23107cellsg(wetweight)21.Thetoppartfromwhichthe e
m
insidetowardstheoutersurface,asolid,crystallineventwall, surface was removed (subsample I) and the porous structure
.
a
a grayish, porous soft structure, a black solid structure, and a insidethechimney(subsampleV)hadsomewhathigherpop- s
thin orange layer approximately 1 to 2 mm from the surface ulation densities (1.0 3 106 and 8.0 3 105, respectively) than m
were recognized. The surface of the chimney was weathered didtheothersubsamples(approximately53105). .o
r
andgrayforthemostpart,withwhiteororangeinsomeparts. Nucleic acids were directly extracted from the subsamples. g
/
These morphological features might be associated with geo- Both DNA and RNA were isolated at concentrations in pro- o
chemicalandphysicalgradientsoftemperature,pH,oxidation- portiontotheobservedpopulationdensities(Table1).Based n

A
redoxpotential,andvariouschemicalsformedinthechimney ontheDNAyieldfromeachsampleandassuminganaverage
p
structure. cellular DNA content of 2 fg (2), the calculated microbial r
The primary chemical composition of the subsamples was populationdensityinthesubsampleswas83105to8.53107 il 1
examined by SEM-EDS (Table 1). The whole chimney struc- cellsgwetweight21andwasthusinrelativelygoodagreement 1
,

turewasfoundtocontainmainlyelementalsulfurandzincas withthepopulationdensitiesdetermined(Table1). 2
0
the primary elements, suggesting that zinc sulfide is the main Quantification of archaeal rRNA and rDNA populations. 1
chemical fabric of the chimney. In the subsamples obtained Archaeal rRNA and rDNA in the whole microbial RNA and 9

b
fromthesurfaceofthechimney,ahigheramountofelemental DNA assemblages extracted from the subsamples were quan-
y
oxygenwasobserved.Althoughthemolecularspeciescontain- tified by RNA dot blot hybridization and quantitative fluoro- g
ingoxygenwasnotidentified,itseemedlikelythatthehigher genicPCR(Table2).Throughoutthesubsamples,thepropor- u
e
proportion of oxygen detected in the elemental composition tion of bacterial rRNA or rDNA was higher than that of s
t
wasconsistentwiththeincreasingamountofoxidizedmateri- archaeal rRNA or rDNA in the whole microbial nucleic acid
TABLE 2. PreliminarymicrobiologicalcharacterizationofsubsamplesfromablacksmokerchimneyatthePACMANUSsitea
rRNA/rDNAcomposition(%)
Celldensity DNAyield RNAyield for:
Subsampleno. Totalamt(g)
(cells/g[wetwt]) (ng/g[wetwt]) (ng/g[wetwt])
Bacteria Archaea
I 30 1.03106 5.1 2.2 78.3/92.6 19.9/7.4
II 15 3.23107 169 100 66.8/94.7 30.5/5.3
III 36 4.53105 3.2 1.3 97.6/100 1.8/ND
IV 18 4.53105 1.6 1.4 98.5/97.2 2.8/2.8
V 19.5 8.03105 3.0 4.5 95.1/100 2.2/ND
VI 15 5.13105 1.6 1.7 76.7/92.8 23.1/7.2
aProkaryoticuniversal,bacterial,andarchaealSSUrRNA/rDNAcompositionwasquantifiedbyusingRNAdothybridizationorquantitativefluorogenicPCR(52).
TheproportionsrepresenttheresultbyRNAdothybridizationdividedbytheresultbyquantitativefluorogenicPCR.ND,notdetected.
3622 TAKAI ET AL. APPL.ENVIRON.MICROBIOL.
assemblages.However,theproportionofarchaealrRNAand TABLE 3. DistributionofrepresentativeT-RFLPribotypesand
rDNA varied among the subsamples. In the top part of the clonetypesofarchaealrDNAobtainedfromablack
smokerchimneya
chimneyandtheouterpartoftheinsidestructureoftheroot
part, the proportion of both archaeal rRNA and rDNA was No.ofarchaealrDNAclones
increased while the proportion of the archaeal rRNA and Clonetype(T-RFLPribotype) characterizedpersubsample:
rDNAintheothersubsampleswasafewpercentorbelowthe I II III IV V VI
detection limit, in the whole microbial nucleic acid assem-
MHVG-1
blages. These results suggested that the sizes of the archaeal
pPACMA-Y(9) 1 3 0 0 0 0
communities varied in different microhabitats in the black
smokerchimney.
Molecular phylogenetic analyses. Profiles of archaeal ri- Crenarchaeota
Igniococcales
botypes dominantly recovered from the DNA assemblages of
pPACMA-I(5) 7 2 0 11 0 0
the subsamples were examined by PCR-mediated T-RFLP pPACMA-X(5) 13 4 0 25 3 0
analysis (Fig. 2). Archaeal rDNA clones obtained from the
D
subsamples were characterized by partial sequencing (ca. 400
Euryarchaeota o
nucleotides) and sequence similarity analysis. The number of w
Thermococcales
archaeal rDNA clones characterized varied from 44 to 65 for n
pPACMA-C(6) 19 25 29 0 0 0 lo
each of the samples (Table 3). In addition, sequences of rep- pPACMA-A(6) 0 0 9 0 0 0 a
resentative rDNA clones that were approximately 0.95 kb in pPACMA-B(6) 0 0 2 0 0 0 d
e
length were determined from both strands and were then ap- d
Unknowngroup
plied to the phylogenetic analysis (Fig. 3). The data from the pPACMA-N(8) 1 3 0 0 2 0 fro
representative DNA clone sequences and the data from T- m
RFLPanalysiswerelinkedbycalculatingtheT-RFlengthfor DHVEG
h
thesequencedclones. pPACMA-M(10) 0 5 0 0 0 0 t
t
pPACMA-Q(10) 2 0 1 0 0 0 p
The molecular techniques used in this study were the pPACMA-W(7) 0 1 1 0 0 0 :/
PCR-based T-RFLP and rDNA clone analyses, and the data pPACMA-P(7) 1 2 2 0 0 0 /a
provided by these methods did not completely represent the pPACMA-V(7) 0 3 1 0 0 0 e
m
archaealcommunitystructuresthatoccurredinthemicrohabi-
.
tats of the chimney. In addition, unstable and strongly biased Halobacteriales as
resultshavebeenpointedoutinthenonoptimizedexperiments pPACMA-H(3) 0 0 0 0 9 21 m
pPACMA-T(3) 0 0 0 0 0 7
.
using PCR-mediated T-RFLP and rDNA clone analyses (37, pPACMA-J(4) 0 0 0 1 0 0 o
r
41).Thedataobtainedshouldbecarefullyestimated. pPACMA-L(3) 0 0 0 1 5 8 g
/
InbothT-RFLPandrDNAcloneanalyses,thestructureof pPACMA-U(4) 0 0 0 0 0 5 o
archaealrDNAphylotypeswasvariedinthesubsamplesfrom pPACMA-K(4) 0 0 0 2 0 0 n
pPACMA-E(4) 0 0 0 3 31 10 A
different microhabitats (Fig. 2 and Table 3). In the combined
pPACMA-S(4) 0 0 0 0 1 0 p
cthhearmacatjeorrizTa-tRioFnsoffoTu-nRdFinLPdiaffnedrernDtNsuAbscalomnpeleasnahlaydsetsh,emcoosrtroe-f ppPPAACCMMAA--GF((44)) 00 00 00 10 103 02 ril 1
sponding archaeal phylotypes identified by rDNA clone anal- 1
,

ysis,whilenoarchaealrDNAphylotypecoincidentwithT-RF 2
Total 44 48 45 44 65 53 0
designated as ribotype 1 or 2 was found throughout all the 1
subsamples(Fig.2andTable3).Inaddition,therewerecases aCompositionofarchaealrDNAclonetypeswasdeterminedbypartialse- 9
quencinganalysisofrDNAclonesofarchaealprimerPCRlibraries. b
in which the rDNA clones that corresponded to the T-RFs
y
foundintheT-RFLPanalysiswerenotobtainedfromthesame g
subsample in the rDNA clone analysis. This nonconformity u
e
between the T-RFLP profiles and rDNA clone compositions surface(subsampleIV)(Fig.2).Thearchaealphylotypespre- s
t
might result from the unavoidable cloning bias in Escherichia dominantly obtained from the top part of the chimney were
coli or from the underestimation in the limited number of affiliated with the clusters of Igniococcales, Thermococcales,
rDNAclonessequenced. and the uncultivated Deep-Sea Hydrothermal Vent Eur-
The top part of the chimney (subsamples I and II) and the yarchaeotic Group (DHVEG) (Fig. 3B and C). The archaeal
surface layer of the middle part (subsample III) contained a rDNA clones affiliated with Igniococcales were especially
variety of rDNA clones phylogenetically belonging to both closelyrelatedtothemosthyperthermophilicarchaealisolates,
Crenarchaeota and Euryarchaeota (Table 3). An archaeal suchasPyrolobus(9),Pyrodictium(42),andHyperthermus(60),
rDNAclone,pPACMA-Y,recoveredfromthearchaealrDNA capable of growth at or above 110°C (Fig. 3B). All of the
clonelibrariesofthetoppartofthechimney,wasphylogeneti- ThermococcalesrDNAclonesobtainedfromthechimneywere
cally associated with one of the deepest branches within the closely related with Thermococcus, and no Pyrococcus-type
domain of Archaea conventionally named the Marine Hydro- clones were detected (Fig. 3C). The archaeal rDNA clones,
thermalVentGroup(MHVG)(Fig.3A).TheT-RFprobably pPACMA-M,-Q,-W,-Pand-V,wereclusteredintoacertain
representing the deeply branched archaeal phylotype was de- phylogenetic group consisting of only uncultivated environ-
tectedinthetoppartofthechimney(subsamplesIandII),the mental clones obtained from geologically and geographically
surfacelayerofthemiddlepart(subsampleIII),andthevent distinct deep-sea hydrothermal vent environments (44, 51)
D
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FIG. 2. TypicalelectropherogramsofarchaealT-RFLPgeneratedfromrDNAwithalabeledreverseprimerandHhaIdigestobtainedfrom
subsamples.Thenumbersonthepeaksindicatemajorribotypescommonlyobservedinvarioussubsamples.Shownareatypicalarchaealpattern
fromthetoppartofthechimney(subsampleI)(A),thesurfacelayergrazedfromsubsampleI(subsampleII)(B),thesurfacelayergrazedfrom
themiddlepartofthechimney(subsampleIII)(C),theventsurface(subsampleIV)(D),theinnersoftandporousstructureintherootpartof
thechimney(subsampleV)(E),andtheouterblackandsolidstructureintherootpartofthechimney(subsampleVI)(F).
3623
3624 TAKAI ET AL. APPL.ENVIRON.MICROBIOL.
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VOL.67,2001 DISTRIBUTION OF ARCHAEA IN A BLACK SMOKER CHIMNEY 3625
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FIG. 3. PhylogenetictreesbasedonSSUrDNAsequencesincludingvariousrDNAclonesobtainedfromtheblacksmokerchimneyatthe
PACMANUSsiteintheManusBasin.Eachofthetreeswasinferredbyneighbor-joininganalysisofapproximately600homologouspositionsof
the rDNA sequence. (A) A tree indicating the phylogenetic relationship among the domains of Bacteria and Eucarya, the deep branches of
uncultivated archaea, and the crenarchaeotic and euryarchaeotic kingdoms. (B) A tree indicating the phylogenetic organization among the
hyperthermophiliccrenarchaeota,Thermoproteales,Igniococcales,andSulfolobales.(C)Atreeindicatingthephylogeneticrelationshipwithinthe
hyperthermophilicandthermophiliceuryarchaeotaandthepossiblethermophiliceuryarchaeoticphylotypes.(D)Atreeindicatingthephyloge-
neticorganizationwithinthegenusHaloarcula.Thenumbersonthebranchesrepresentthebootstrapconfidencevalues.Thescalebarsindicate
the expected changes per sequence position. Abbreviations indicate rDNA clones corresponding to uncultivated organisms derived from the
following environments: pMC1A, pMC2A, pISA, and pIVWA from deep-sea hydrothermal vent environments (51); pOWA and pUWA from
shallowmarinehydrothermalventwaterandterrestrialacidichotspringwater,respectively(55);pJPandpSLfromsedimentsinYellowstone
NationalParkhotsprings(3,4);CRAandAPAfromdeep-seasediments(59);pHGPAfromdeepsubsurfacegeothermalwater(50);VC2.1Arc
fromaninsitugrowthchamber(VentCap)deployedatadeep-seahydrothermalvent(44);andpPACMAfromablacksmokerchimneyatthe
PACMANUSsiteintheManusBasin.
3626 TAKAI ET AL. APPL.ENVIRON.MICROBIOL.
(Fig. 3C). The proportion of Igniococcales phylotypes was re- TABLE 4. Viable-cellcountsofThermococcusmembersand
ducedintherDNAclonelibrariesfromthesurfacelayersam- halophilicmicroorganismscalculatedbythree-tube
MPNcultivationmethoda
ples (subsamples II and III), while the proportion of rDNA
clonesphylogeneticallyassociatedwithThermococcusandDH- Viable-cellcountin:
VEGwasgreaterinthesurfacezone(Table3).Thesepropor-
HAmediumfor
tdiiocnatcehdabnygetshefocuhnadngienstohfeTrD-RNFAsigclnoanlse(arnibaolytsyipsewse5r,e6,al7s,oainnd- Subsampleno. MJYPS(mceeldlsiugm[wfeotrwTth]e2r1m)ococcus Halo[waerctuwlat](2c1e)llsg
10)representingthearchaealphylotypes(Igniococcales,Ther- 55°C 75°C 90°C 30°C 45°C
mococcus,andDHVEG)intheT-RFLPanalysis(Fig.2).
I 4.63102 2.43102 3.93101 ND ND
In the vent surface (subsample IV), most of the archaeal II 2.13104 1.53104 9.03103 ND ND
rDNA phylotypes provided by rDNA clone analysis were Ig- III 7.53102 4.33102 7.53101 ND ND
niococcales members (Table 3 and Fig. 3B). The dominant IV 9.03100 9.03100 ND 9.33101 ND
occurrence of these phylotypes was clearly represented by a V ND ND ND 9.33102 9.33101
VI ND ND ND 7.53102 9.03101
largesinglepeakofribotype5intheT-RFLPanalysis(Fig.2).
D
Inaddition,thepresenceofribotype4wasdetectedinthevent aViable-cellcountsobtainedbytheMPNmethodusingHAmediumrepre-
o
surface (Fig. 2), which matched well with the recovery of sentnotHaloarculabuthalophilicbacteriasuchasHalomonasspp,asdescribed w
inResults.ND,notdetected.
rDNA clones phylogenetically related with Halobacteriales n
lo
(Table3). a
In the inside structures of the chimney (subsamples V and the extinction-dilution method. Based on the results of simi- d
e
VI), the major archaeal phylotypes found in the rDNA clone larityanalysisofpartialrDNAsequences,themicroorganisms d

analysis shifted from hyperthermophilic groups of phylotypes thatgrewinMJYPSmediumat50and75°Cwerefoundtobe fr
o
toanextremelyhalophilicgroupofphylotypeswithinHalobac- Thermococcus species (Thermococcus spp. PACM50 and m
teriales (Table 3). These shifts in the archaeal rDNA clone PACM75,showninFig.3C).However,themicroorganismthat
h
community structure were consistent with the shifts in the grewinHAmediumat30°CwasidentifiedasaHalomonassp. tt
p
ribotype structure revealed by the results of T-RFLP analysis (itspartial16SrDNAsequencehad98.5%similaritywiththat :
/
(depletionofsignaturesofribotypes5and6correspondingto of Halomonas meridiana [25]), a member of the Bacteria, not /a
rDNA clones of Igniococcales and Thermococcales, respec- theArchaea.HAmediumcontainsapproximately16%(wt/vol) em
tively, and predominance of ribotypes 3 and 4 representing NaClor20%totalsalts,whichiswithintherangeforgrowthof .
a
Halobacteriales rDNA clones) (Fig. 2). In the phylogenetic not only Haloarcula spp. but also of Halomonas spp. (58). In s
analysis,allofthearchaealrDNAclonesrelatedwithHalobac- addition,noarchaealrDNAwasdetectedbyPCRanalysisof m
.
teriales were located within the phylogenetic cluster of the DNA extracts from any of the enrichment cultures in HA o
r
genusHaloarcula(Fig.3D).Thephylogeneticplacementofthe medium at any cultivation temperature. These results indi- g
/
HaloarcularDNAclonesobtainedfromtheinsidestructuresof catedthattheviablecellcountsobtainedbytheMPNmethod o
thechimneywasdividedmainlyintotwolineages.Thisdisper- in the case of cultures using HA medium represented the n
A
sion and the relatively high divergence of the Haloarcula halophilicbacterialpopulation,nottheHaloarculapopulation. p
rthDeNrARNclAonegesneeqsueonbcseesrvceoduldinbHeadluoeartcoultahespheectieersog(4e0n)e.itTyhoef eArlethdofuroghmnaonyviaobfltehceesllusbosafmthpelegse,nthues Hfinadloinargcsuolabtwaeinreedrefcroomv- ril 1
archaeal rDNA clone pPACMA-N distantly related to any theenrichmentculturessupportedtheviewthataviableTher- 1,

other archaeal rDNA sequences known so far (Fig. 3C) was mococcuspopulationwaspresentinthesurfacelayersandthat 2
0
detectedatasmallproportioninvariousmicrohabitatsbothin preferential colonization of the inside structures of the chim- 1
therDNAcloneanalysisandtheT-RFLPanalysis(Table3and neybyhalophilicmicroorganismsoccurred. 9
b
Fig.2).Theresultsobtainedfromthemolecularphylogenetic
y
analysesindicatedthatdifferentarchaealphylotypestructures DISCUSSION g
consisting mainly of hyperthermophilic and extremely halo- u
e
philicphylotypesweredistributedindifferentmicrohabitatsin Thedistributionofarchaeainablacksmokerchimneystruc- s
t
theblacksmokerchimney. turewasanalyzedbyculture-independent,molecularphyloge-
EnrichmentandMPNcalculations.Inordertosupportthe netictechniques.ThecombineduseofrRNAdotslothybrid-
apparent distribution of different archaeal community struc- ization, quantitative fluorogenic PCR, T-RFLP, and rDNA
turesindicatedbyculture-independentmolecularphylogenetic cloneanalysisrevealedthatthearchaealphylotypecommunity
analyses,enrichmentculturesofviablehyperthermophilicand was significantly altered in terms of size and structure with
halophilicmicrobialpopulationswerepreparedandMPNcal- microhabitats varying at several centimeters distance in the
culationswereperformed.TechniquesforcultivationofTher- deep-sea hydrothermal vent chimney. The occurrence of dis-
mococcus and Haloarcula members, among the archaeal phy- crete archaeal phylotype communities was likely associated
lotypes identified through molecular analyses, are relatively withtheformationofenvironmentalgradientsoftemperature,
wellestablished(5,11,45).Table4showstheviable-cellcounts pH, oxidation-redox potential, and various chemicals in the
ofThermococcusmembersandhalophilicmicroorganismscul- chimney structure, even though the measurement of the gra-
tivatedatseveraltemperaturesinMJYPSandHAmedia.The dientswasnotcompletelydeterminedinthisstudy.
microorganismsthatgrewinthemostdilutedseriesofcultures Theoccurrenceofdiscretemicrobialcommunitiesindeep-
inMJYPSmediumat50or75°CcontainingsubsampleIIorin sea hydrothermal vent environments was first reported by
HAmediumat30°CcontainingsubsampleVwereisolatedby Harmsen et al. (19) after applying enrichment culture and
VOL.67,2001 DISTRIBUTION OF ARCHAEA IN A BLACK SMOKER CHIMNEY 3627
whole-cell fluorescence in situ hybridization (FISH) tech- (57.6to60.2%)oftheuncultivatedDHVEGmayrepresentthe
niques (20) to analysis of black smoker chimneys obtained thermophily in this group of uncultivated archaea. Likewise,
from the Mid-Atlantic Ridge. It was demonstrated that the the deeply branched phylotype pPACMA-Y, phylogenetically
microbial community density was varied in subsamples ob- relatedtotheMHVG,mightatleastrepresentthermophilyas
tainedfromdifferentpartsofachimneystructureandthatthe its distinctive physiological feature, as described previously
topparthadthehighestdensity(19).Inaddition,theincreased (51).
densityofthetotalarchaealpopulationinthetoppartofthe Thediscoveryofasizablepopulationofextremelyhalophilic
chimneywasobservedbyusingthefluorescenceinsituhybrid- archaea in the inside structures of the chimney was the most
ization technique, and the preferred colonization patterns of striking finding in this work. Kaye and Baross have reported
potentialThermococcalesmembersinthesurfaceareaandof findinghalotolerantbacteriabelongingtothegeneraHalomo-
potentialIgniococcalesmembersinthetoppartsandthevent nasandMarinobacterathighincidenceinavarietyofsamples
areawereindicatedbyenrichmentculturetechniques(19).A obtainedfromdeep-seahydrothermalventenvironments(30).
similar distribution pattern of microbial communities was ob- We also obtained here a culturable population of Halomonas
served in our analysis of archaeal community structures in fromtheinsidestructuresofthechimney.Thehalophilicbac-
D
different microhabitats of a black smoker chimney from the teria are abundant in deep ocean water and may be contami-
o
ManusBasin,whichisgeologicallyandgeographicallydistinct natedfromtheambientseawateraroundthedeep-seahydro- w
from those in the Mid-Atlantic Ridge. These features of the thermal vent (30). Given that the halophilic bacterial n
lo
distributionofmicroorganismscommonlyobservedindifferent population,includingHalomonassp.culturedfromtheinside a
deep-seahydrothermalventsystemsprovideanimportantgen- structureofthechimney,isderivedfromtheambientseawater, d
e
eralaspecttobeconsideredinassessmentoftheglobaldeep- there might be obtained a similar or higher abundance of d

seahydrothermalventmicrobialecosystem. cultures for Halomonas spp. from the surface of the chimney fr
o
Significant changes in microbial abundance, diversity, and exposure to the seawater. The discovery of extremely halo- m
community structure associated with environmental gradients philic archaeal rDNA and successful cultivation of halophilic
h
in hydrothermal environments have also been observed in a microorganisms were achieved specifically from the inside t
t
p
series of investigations focusing on a shallow marine hydro- structuresofthechimney.Itseemslikelier,therefore,thatthe :
/
thermalventsiteasamodelsystem(47–49).Throughtheuse halophilicbacterialculturesandthegeneticsignaturesforex- /a
of molecular techniques, an increased abundance of the ar- tremelyhalophilicarchaeaarederivedfrommicrobialcommu- e
m
chaeawasfoundintheactiveventingareaandcomplex,active, nitiesdwellinginthemicrohabitatsthatoccurredintheinside
.
a
bacterial communities became predominant with increasing structuresratherthanfromcontaminatedmicrobialcommuni- s
distancefromtheventingpoint(47–49).Attheshallowmarine ties. Since no positive cultivation of the extremely halophilic m
.
hydrothermalventsite,temperatureandpHrangingfromap- archaea represented by rDNA phylotypes from the inside o
r
proximately70°CatpH5to20°CatpH7wereformedovera structures was achieved, the viability of the extremely halo- g
/
distance of several meters (47). The potential impact of the philic archaeal population remained uncertain. However, the o
environment on a microbial community might be much more discoveryofextremelyhalophilicarchaealrDNAandthesuc- n

A
intenseindeep-seahydrothermalsystems,sincegradientsare cessful cultivation of halophilic bacteria suggest that partially
p
expectedtoexistintherangeofapproximately300°CatpH3 hypersaline microhabitats allowing the survival or growth of r
to4°CatpH6to7inthecaseofablacksmokerinvestigated halophilicarchaeaandbacteriaoccurinthechimneystructure. il 1
(16,17)andfrom350to400°Cto4°Cinthecaseofthetypical Furthermore,therecoveryofhyperthermophilicandextremely 1
,

midocean-ridge systems (21). In the range of such extraordi- halophilic archaeal rDNA phylotypes from the interface with 2
0
nary thermal gradients of the deep-sea vents, the archaeal theventfluidandtheinteriorstructuresofthechimneyleads 1
components may play a much more important role in the tothehypothesisthatthemicrobialcommunitiesinthemicro- 9

b
formation and activity of a microbial community than in the habitatsofthechimneyareprovidedfromthemicrobialpop-
y
shallowvents. ulation thriving beneath the active hydrothermal floor, the g
The existence of a diversity of archaea in the deep-sea hy- subventbiosphere. u
e
drothermalventenvironmentshasbeendemonstratedthrough Based on several microbiological, geochemical, and geo- s
t
anumberofenrichmentandisolationattemptstodate(9,10, physical observations, Deming and Baross proposed the pos-
18, 23, 28, 42, 43, 53, 56, 60). Additionally, culture-indepen- sible existence of a hyperthermophilic subvent biosphere be-
dent,molecularapproacheshaverevealedthatagreaterdiver- neath the active hydrothermal vent fields (14). The relatively
sityofarchaeaarepredominantlypresentandremainuniden- high density of microbial cells and the detection of microbial
tifiedintermsoftheirphysiologicalpropertiesandmetabolic rDNA in the black smoker vent water at a temperature of
functions (44, 51). This study is the first report in which the .300°C also support the occurrence of a sizable microbial
distribution of discrete archaeal communites in different mi- population in the subvent environment of the hydrothermal
crohabitats of a black smoker chimney was demonstrated by system in Ihaya Basin, Okinawa Trough (51). Since the deep
usingculture-independent,molecularphylogeneticanalysis.In ocean water infiltrating this system consistently replenishes
thisstudy,itwasrevealedthatmembersofThermococcusand foreignmicroorganismsandnutrientsintheenvironments,itis
members of the DHVEG are abundant on the chimney sur- difficult to identify the exclusively indigenous microbial com-
face.ThecoexistencewiththehyperthermophilesThermococ- ponentsandactivitiesinthesubventbiosphere.However,ex-
cus,thewidedistributioninthevariousdeep-seahydrothermal tremehalophilesaswellashyperthermophilesarethepossible
vents, the relatively short branch length in the phylogenetic indigenous microorganisms in the subvent biosphere. The
tree(Fig.3C),andrelativelyhighG1CcontentoftherDNA physiological and metabolic diversity of known hyperthermo-