Distribution of Archaea in a Black Smoker Chimney Structure Ken Takai, Tetsushi Komatsu, Fumio Inagaki and Koki Horikoshi Appl. Environ. Microbiol. 2001, 67(8):3618. DOI: 10.1128/AEM.67.8.3618-3629.2001. D o w n Updated information and services can be found at: lo a http://aem.asm.org/content/67/8/3618 d e d f r o These include: m h REFERENCES This article cites 47 articles, 30 of which can be accessed free tt p at: http://aem.asm.org/content/67/8/3618#ref-list-1 :/ / a e m CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new . a articles cite this article), more» s m . o r g / o n F e b r u a r y 2 1 , 2 0 1 3 b y P E N N S T A T E U N I V Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ 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 D o methods. A black smoker chimney was obtained from the PACMANUS site in the Manus Basin near Papua w NewGuinea,andsubsampleswereobtainedfromverticalandhorizontalsections.Theelementalcomposition n of the chimney was analyzed in different subsamples by scanning electron microscopy and energy-dispersive lo a X-ray spectroscopy, indicating that zinc and sulfur were major components while an increased amount of d e elementaloxygeninexteriormaterialsrepresentedthepresenceofoxidizedmaterialsontheoutersurfaceof d the chimney. Terminal restriction fragment length polymorphism analysis revealed that a shift in archaeal f r ribotypestructureoccurredinthechimneystructure.ThroughsequencingofribosomalDNA(rDNA)clones o m from archaeal rDNA clone libraries, it was demonstrated that the archaeal communities in the chimney h structure consisted for the most part of hyperthermophilic members and extreme halophiles and that the t t distribution of such extremophiles in different microhabitats of the chimney varied. The results of the p : culture-dependentanalysissupportedinparttheviewthatchangesinarchaealcommunitystructuresinthese // a microhabitatsareassociatedwiththegeochemicalandphysicaldynamicsintheblacksmokerchimney. e m . a s Sincethediscoveryofdeep-seahydrothermalventsin1979 several microbiological, geochemical, and geophysical obser- m (12, 15), various microorganisms have been isolated from vations, the possible existence of a subvent biosphere popu- .o globaldeep-seahydrothermalventenvironments(26,27,29). lated by hyperthermophilic microorganisms was predicted rg Hyperthermophiles and thermophiles, including members of (14). Also, microbial ribosomal DNA (rDNA) showing sub- o/ both the bacterial and archaeal domains, are the most fre- stantialdiversitywasrecoveredfromblacksmokerventwater n F quentlyisolatedmicroorganisms,andtheirphysiologicalprop- intheIhayaBasin,OkinawaTrough(51),atatemperatureof e ertieslikelyreflecttheextraordinaryenvironmentalsettingsof .300°C, which is far above the upper temperature limit for b r thedeep-seahydrothermalvents(9,10,18,23,28,42,43,53, growth of even the most hyperthermophilic archaeon, Pyrolo- ua 56,60).Inaddition,recentculture-independentmolecularap- bus fumarii (113°C) (9). The microbial rDNA may serve as a ry proaches have revealed the presence of as-yet-uncultivated genetic signature indicative of the microorganisms thriving in 2 1 thermophilesshowingsubstantialphylogeneticdiversityinthe the subvent habitats, conveyed there by vent water. Hence, a , 2 deep-seahydrothermalventenvironments(27,38,39,44,51). hydrothermalventchimneyisanenvironmentanalogoustothe 0 Relativelylittleisknownabouttheecologicalsignificanceand subventbiosphere,andelucidationofitsmicrobialcommunity 13 geomicrobiological function of the extremophilic microbial structure is likely to provide great insight into features of the b y communitiesfound,astheoccurrence,abundance,anddistri- subventmicrobialecosystem. P bution of the microbial communities associated with the for- The black smoker chimney structure investigated in the E mation of diverse geochemical and physical gradients remain N present study was obtained from a hydrothermal vent field N tobeelucidated. located at the PACMANUS site in the Manus Basin near S Adeep-seahydrothermalventchimney,formedbychemical PapuaNewGuineaatadepthof1,644m.Thishydrothermal T interaction between cold seawater and hot vent water, is a A fieldwasdiscoveredin1991(7),andageologicalsurveyofthe T distinctivestructureinthedeep-seahydrothermalfieldsandis E fieldandgeochemicalcharacterizationoftheventwatershave largely composed of sulfide materials. In the chimney struc- U both been performed (6, 7, 16, 17). The results of the micro- turesandtheunderlyingsulfidemounds,steepenvironmental N biological assessment have not yet been reported. Archaeal I gradients of temperature, pH, oxidation-redox potential, and V 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:81-468-67-3894.Fax:81-468-66-6364.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 D o w n lo a d e d f r o m h t t p : / / a e m . a s m . o r g / o n F e b r u a r y 2 1 , 2 0 1 3 b y P E N N S T FIG. 1. Schematicdrawingsofthemorphologicalandstructuralfeaturesofablacksmokerchimneyandthesubsamplingmethodused.(A) A Appearanceofthesurface.(B)Verticalsectionofthewholechimneystructure.(C)Horizontalsectionoftherootpartofthechimney.Thepart T whereeachofthesubsampleswastakenandthepreliminarymorphologicalandstructuralfeaturesofthesubsamplesareindicated. E U N I habitatsinthechimneystructureandimplicationsforthepos- ofthesubsamplesisshowninFig.1.Subsamplingwasperformedinananaerobic V 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- D aldehyde,andthemixturewasvigorouslyagitatedfor2minusingavortexmixer. tion,restrictionenzymereaction,andelectrophoresis. ow Thesuspensionsolutionwasverybrieflycentrifuged(3,0003g),andthesuper- CloningandsequencingofarchaealrDNA.ArchaealrDNAwasamplifiedby n natant was filtered with a 0.22-mm-pore-size 13-mm-diameter polycarbonate PCRusingthesameprotocolasforT-RFLPanalysisexceptfortheuseofa lo filter(Advantec,Tokyo,Japan).ThefilterwasstainedbytreatmentwithMJ nonlabeledprimersetofArch21FandArch958R.AmplifiedrDNAfromtwo a seawatercontaining49,69diamidino-2-phenylindole(DAPI)(10mgml21)oracri- separate reactions was purified as described above. The purified rDNA was de dineorange(10mgml21)at4°Cfor20min.ThefilterwasbrieflyrinsedinMJ cloned in the vector pCR2.1 using the Original TA cloning kit (Invitrogen, d seawaterandexaminedunderepifluorescenceusingaNikonOptishotmicro- Carlsbad,Calif.).TheinsertswereamplifiedbydirectPCRfromasinglecolony fr usingM13primers(36),treatedwithexonucleaseIandshrimpalkalinephos- o scope. m phatase (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, Extractionofnucleicacids.MicrobialDNAwasdirectlyextractedfromeach UnitedKingdom),anddirectlysequencedbythedideoxynucleotidechain-ter- h subsample (approximately 5 g) using a Soil DNA Kit Mega Prep (MO BIO t Laboratories,Inc.,SolanaBeach,Calif.),followingthemanufacturer’ssuggested minationmethodusingadRhodaminesequencingkit(PEAppliedBiosystems) tp protocol.MicrobialRNAwasextractedfromareplicatealiquotofeachofthe followingthemanufacturer’srecommendations.TheArch21Fprimerwasused :// inpartialsequencinganalysis. a samplesusedforDNAextraction.Approximately5gofsamplematerialwas e suspended in 3.5 ml of extraction buffer (pH 4.2) containing 25 mM sodium Sequenceandphylogeneticanalyses.Single-strandsequencesapproximately m acetate,5mMEDTA,and5%(wt/vol)sodiumdodecylsulfate.Then2.5gof 400nucleotidesinlengthwereanalyzed.Thesequencesimilarityamongallofthe .a single-strandsequenceswasanalyzedusingtheFASTAprogramequippedwith sterileglassbeads(0.1-mmdiameter;Sigma)and3.5mlofphenolequilibrated s DNASISsoftware(HitachiSoftware,Tokyo,Japan).TherDNAsequenceshav- m wfoirth2emxitnraacntidoninbcuubffaetrewdearte6a0d°Cdefdo.rT1hhe.Tsuhsepsehnaskioinngwtraesasthmaeknetnwoansarebpeeaadtebde,aatnedr ing$98%similarityasdeterminedbytheFASTAprogramwereassignedtothe .o sameclonetype,andarepresentativesequenceofeachclonetypewasapplied r thesuspensionwascentrifugedatroomtemperature.Thelysatewasextracted g tosequencesimilarityanalysiswithdatabasesbythegapped-BLASTmethod(1, / withphenol-chloroform-isoamylalcohol(25:24:1,vol/vol/vol)andwithchloro- 8).Thedatabasesusedforgapped-BLASTanalysisweretheprokaryoticsmall- o form-isoamylalcohol(24:1,vol/vol).RNAwasprecipitatedbyadding1.5mlof n subunit(SSU)rRNAdatabaseandthenonredundantnucleotidesequenceda- 10Mammoniumacetateandthesamevolumeofisopropylalcoholandwas F tabasesfromGenBank,EMBL,andDDBJ. e recoveredbycentrifugation.Thepelletwaswashedwith70%(vol/vol)ethanol Sequences of representative rDNA clones approximately 0.95 kb in length b anddissolvedinfilter-sterilized,distilled,deionizedwater.ThetotalRNAwas weredeterminedfrombothstrands.ThedatafromtherDNAclonelibrariesand ru purifiedfromthecrudeRNAsolutionusingaRNeasyMidiKitspincolumn thedatafromT-RFLPanalysiswerelinkedbycalculatingtheT-RFlengthfor ar (Qiagen,Valencia,Calif.).AllplasticwareandallsolutionsusedforRNAex- thesequencedclones.CloneswithaT-RFlengthidentical(within61bp)toone y tractionandpurificationweretreatedwith0.1%diethylpyrocarbonatetoinac- foundbyT-RFLPanalysiswereassignedtoaT-RFLPribotype.Thesequences 2 1 tivatenucleases.Inordertocheckforlaboratorycontaminants,ablanktube weremanuallyalignedtoprokaryoticSSUrDNAdatafromtheRibosomalData , (withnosampleadded)wasprocessedasanegativecontrolinthecaseofboth Project II (35) based on primary and secondary structure considerations and 2 0 DNAandRNAextraction(57).RNAandDNAconcentrationsweremeasured werealsosubmittedtoanalysisusingtheChimeraCheckprogramoftheRibo- 1 usingaspectrophotometer. somalDataProjectIItodetectthepresenceofchimericartifacts.Phylogenetic 3 QuantificationofarchaealrRNAandtherDNApopulation.Quantificationof analyses were restricted to nucleotide positions between the Arch21F and b y archaeal rRNA in the whole microbial RNA assemblages extracted from the Arch958Rprimersthatwereunambiguouslyalignableinallsequences.Neigh- P subsampleswasperformedbyRNAdotblothybridization.Adilutionseriesof bor-joining analysis was performed using the PHYLIP package (version 3.5; E RNAsamples(1,0.5,and0.25ngml21)with10mMTris-HCl(pH7.5)were obtained from J. Felsenstein, University of Washington, Seattle). Bootstrap N prepared,theRNAwasdenaturedat100°Cfor10min,andthesampleswere analysiswasusedtoprovideconfidenceestimatesforphylogenetictreetopolo- N cooledonice.ThedenaturedRNAsamplesweredottedontoHybond-N1nylon gies. S membranes(AmershamPharmacia)andcross-linkedtothemembranesbyex- EnrichmentandMPNcalculation.Forenrichmentculturestargetingmem- T posureto120mJofUVlightenergyusingaUVStratalinker1800(Strategene, bersofthegenusThermococcus,MJYPSmedium(56)wasusedandenrichment A T Torrey Pines, Calif.). The oligonucleotide probes (Uni1392R and Arch915R wasperformedat55,75,and90°C.Forenrichmentculturestargetinghalophilic E conjugatedatthe59endstodigoxigenin)(52)andthehybridizationconditions microorganisms,includingmembersofthegenusHaloarcula,themedium(hem- U usedhavebeendescribedpreviously(52).QuantificationofthearchaealrDNA agglutinin[HA]medium)describedbyIharaetal.(24)wasusedandenrichment N populationinthewholemicrobialDNAassemblageswasperformedbyaquan- wasperformedat30and45°C.Thethree-tubemost-probable-number(MPN) I V 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 D o aNumberingofsubsampleswasindicatedinFig.1.ND,notdetected. w n lo a d flowingeffluent,ithasoftenbeenobservedthatthetoppartof als on the outer surface of the chimney, indicating the occur- e d thechimneyiscoveredwithweaklypackedmaterials,consist- renceofrelativelyoxidativemicrohabitatsintheexteriorzones f ing of chalcopyrite, pyrrhotite, and anhydrite on solid sulfide ofthechimney.Intheweatheredsurfacepartsthatwerewhite ro m materials(21,22,33).Thechimneyinvestigatedinthepresent and orange and in the orange subsurface layer, barium was studywasobtainedfromablacksmokerwithvigorouslyflow- accumulated at high levels while no significant change in the h t t ingeffluent,andnosuchsofthead-structurewasobserved.The amountofcalciumwasobserved. p : vent surface, the interface with vent fluid, was formed by a Microbial population density and nucleic acid extraction. //a solid,crystallineventwall,whichwasobservedthroughouttop The microbial population density was determined by direct e m to bottom of the chimney structure. The morphological fea- countingofDAPI-andacridineorange-stainedcells(Table2). . a tures of the top part were similar to those found on the vent The highest population density was observed in the surface s m surface.Inthemiddleandrootparts,fourdistinctinnerstruc- layer at the top part of the chimney (subsample II) and was . tures were observed (Fig. 1). Considering the features from 3.23107cellsg(wetweight)21.Thetoppartfromwhichthe or g insidetowardstheoutersurface,asolid,crystallineventwall, surface was removed (subsample I) and the porous structure / o a grayish, porous soft structure, a black solid structure, and a insidethechimney(subsampleV)hadsomewhathigherpop- n thin orange layer approximately 1 to 2 mm from the surface ulation densities (1.0 3 106 and 8.0 3 105, respectively) than F were recognized. The surface of the chimney was weathered didtheothersubsamples(approximately53105). eb andgrayforthemostpart,withwhiteororangeinsomeparts. Nucleic acids were directly extracted from the subsamples. ru a These morphological features might be associated with geo- Both DNA and RNA were isolated at concentrations in pro- r y chemicalandphysicalgradientsoftemperature,pH,oxidation- portiontotheobservedpopulationdensities(Table1).Based 2 redoxpotential,andvariouschemicalsformedinthechimney ontheDNAyieldfromeachsampleandassuminganaverage 1 , structure. cellular DNA content of 2 fg (2), the calculated microbial 2 The primary chemical composition of the subsamples was populationdensityinthesubsampleswas83105to8.53107 01 examined by SEM-EDS (Table 1). The whole chimney struc- cellsgwetweight21andwasthusinrelativelygoodagreement 3 b turewasfoundtocontainmainlyelementalsulfurandzincas withthepopulationdensitiesdetermined(Table1). y the primary elements, suggesting that zinc sulfide is the main Quantification of archaeal rRNA and rDNA populations. P E chemical fabric of the chimney. In the subsamples obtained Archaeal rRNA and rDNA in the whole microbial RNA and N fromthesurfaceofthechimney,ahigheramountofelemental DNA assemblages extracted from the subsamples were quan- N oxygenwasobserved.Althoughthemolecularspeciescontain- tified by RNA dot blot hybridization and quantitative fluoro- S T ingoxygenwasnotidentified,itseemedlikelythatthehigher genicPCR(Table2).Throughoutthesubsamples,thepropor- A proportion of oxygen detected in the elemental composition tion of bacterial rRNA or rDNA was higher than that of T E wasconsistentwiththeincreasingamountofoxidizedmateri- archaeal rRNA or rDNA in the whole microbial nucleic acid U N I V 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 D botypes dominantly recovered from the DNA assemblages of pPACMA-I(5) 7 2 0 11 0 0 o the subsamples were examined by PCR-mediated T-RFLP pPACMA-X(5) 13 4 0 25 3 0 w n analysis (Fig. 2). Archaeal rDNA clones obtained from the lo subsamples were characterized by partial sequencing (ca. 400 a Euryarchaeota d nucleotides) and sequence similarity analysis. The number of e Thermococcales d archaeal rDNA clones characterized varied from 44 to 65 for pPACMA-C(6) 19 25 29 0 0 0 f each of the samples (Table 3). In addition, sequences of rep- pPACMA-A(6) 0 0 9 0 0 0 ro m resentative rDNA clones that were approximately 0.95 kb in pPACMA-B(6) 0 0 2 0 0 0 length were determined from both strands and were then ap- h Unknowngroup tt plied to the phylogenetic analysis (Fig. 3). The data from the p pPACMA-N(8) 1 3 0 0 2 0 : / representative DNA clone sequences and the data from T- /a RFLPanalysiswerelinkedbycalculatingtheT-RFlengthfor DHVEG e m thesequencedclones. pPACMA-M(10) 0 5 0 0 0 0 . pPACMA-Q(10) 2 0 1 0 0 0 a The molecular techniques used in this study were the s pPACMA-W(7) 0 1 1 0 0 0 m PCR-based T-RFLP and rDNA clone analyses, and the data pPACMA-P(7) 1 2 2 0 0 0 . o provided by these methods did not completely represent the pPACMA-V(7) 0 3 1 0 0 0 r g archaealcommunitystructuresthatoccurredinthemicrohabi- / tats of the chimney. In addition, unstable and strongly biased Halobacteriales on resultshavebeenpointedoutinthenonoptimizedexperiments pPACMA-H(3) 0 0 0 0 9 21 F pPACMA-T(3) 0 0 0 0 0 7 e using PCR-mediated T-RFLP and rDNA clone analyses (37, pPACMA-J(4) 0 0 0 1 0 0 b 41).Thedataobtainedshouldbecarefullyestimated. pPACMA-L(3) 0 0 0 1 5 8 ru InbothT-RFLPandrDNAcloneanalyses,thestructureof pPACMA-U(4) 0 0 0 0 0 5 ar pPACMA-K(4) 0 0 0 2 0 0 y archaealrDNAphylotypeswasvariedinthesubsamplesfrom pPACMA-E(4) 0 0 0 3 31 10 2 different microhabitats (Fig. 2 and Table 3). In the combined 1 pPACMA-S(4) 0 0 0 0 1 0 , characterizationofT-RFLPandrDNAcloneanalyses,mostof pPACMA-G(4) 0 0 0 1 0 0 2 0 themajorT-RFsfoundindifferentsubsampleshadthecorre- pPACMA-F(4) 0 0 0 0 13 2 1 3 sponding archaeal phylotypes identified by rDNA clone anal- b ysis,whilenoarchaealrDNAphylotypecoincidentwithT-RF y Total 44 48 45 44 65 53 designated as ribotype 1 or 2 was found throughout all the P subsamples(Fig.2andTable3).Inaddition,therewerecases aCompositionofarchaealrDNAclonetypeswasdeterminedbypartialse- EN quencinganalysisofrDNAclonesofarchaealprimerPCRlibraries. in which the rDNA clones that corresponded to the T-RFs N foundintheT-RFLPanalysiswerenotobtainedfromthesame S T subsample in the rDNA clone analysis. This nonconformity A between the T-RFLP profiles and rDNA clone compositions surface(subsampleIV)(Fig.2).Thearchaealphylotypespre- T E might result from the unavoidable cloning bias in Escherichia dominantly obtained from the top part of the chimney were U coli or from the underestimation in the limited number of affiliated with the clusters of Igniococcales, Thermococcales, N rDNAclonessequenced. and the uncultivated Deep-Sea Hydrothermal Vent Eur- IV 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 o w n lo a d e d f r o m h t t p : / / a e m . a s m . o r g / o n F e b r u a r y 2 1 , 2 0 1 3 b y P E N N S T A T E U N I V 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. D o w n lo a d e d f r o m h t t p : / / a e m . a s m . o r g / o n F e b r u a r y 2 1 , 2 0 1 3 b y P E N N S T A T E U N I V VOL.67,2001 DISTRIBUTION OF ARCHAEA IN A BLACK SMOKER CHIMNEY 3625 D o w n lo a d e d f r o m h t t p : / / a e m . a s m . o r g / o n F e b r u a r y 2 1 , 2 0 1 3 b y P E N N S T A T E U N I V 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 D occurrence of these phylotypes was clearly represented by a V ND ND ND 9.33102 9.33101 ow VI ND ND ND 7.53102 9.03101 n largesinglepeakofribotype5intheT-RFLPanalysis(Fig.2). lo Inaddition,thepresenceofribotype4wasdetectedinthevent aViable-cellcountsobtainedbytheMPNmethodusingHAmediumrepre- a surface (Fig. 2), which matched well with the recovery of sentnotHaloarculabuthalophilicbacteriasuchasHalomonasspp,asdescribed de inResults.ND,notdetected. d rDNA clones phylogenetically related with Halobacteriales f (Table3). ro m In the inside structures of the chimney (subsamples V and the extinction-dilution method. Based on the results of simi- VI), the major archaeal phylotypes found in the rDNA clone larityanalysisofpartialrDNAsequences,themicroorganisms ht t analysis shifted from hyperthermophilic groups of phylotypes thatgrewinMJYPSmediumat50and75°Cwerefoundtobe p: / toanextremelyhalophilicgroupofphylotypeswithinHalobac- Thermococcus species (Thermococcus spp. PACM50 and /a teriales (Table 3). These shifts in the archaeal rDNA clone PACM75,showninFig.3C).However,themicroorganismthat em community structure were consistent with the shifts in the grewinHAmediumat30°CwasidentifiedasaHalomonassp. . a ribotype structure revealed by the results of T-RFLP analysis (itspartial16SrDNAsequencehad98.5%similaritywiththat s m (depletionofsignaturesofribotypes5and6correspondingto of Halomonas meridiana [25]), a member of the Bacteria, not . o rDNA clones of Igniococcales and Thermococcales, respec- theArchaea.HAmediumcontainsapproximately16%(wt/vol) r g tively, and predominance of ribotypes 3 and 4 representing NaClor20%totalsalts,whichiswithintherangeforgrowthof / o Halobacteriales rDNA clones) (Fig. 2). In the phylogenetic not only Haloarcula spp. but also of Halomonas spp. (58). In n analysis,allofthearchaealrDNAclonesrelatedwithHalobac- addition,noarchaealrDNAwasdetectedbyPCRanalysisof F e teriales were located within the phylogenetic cluster of the DNA extracts from any of the enrichment cultures in HA b genusHaloarcula(Fig.3D).Thephylogeneticplacementofthe medium at any cultivation temperature. These results indi- ru a HaloarcularDNAclonesobtainedfromtheinsidestructuresof catedthattheviablecellcountsobtainedbytheMPNmethod r y thechimneywasdividedmainlyintotwolineages.Thisdisper- in the case of cultures using HA medium represented the 2 sion and the relatively high divergence of the Haloarcula halophilicbacterialpopulation,nottheHaloarculapopulation. 1 , rDNA clone sequences could be due to the heterogeneity of Although no viable cells of the genus Haloarcula were recov- 2 0 the rRNA genes observed in Haloarcula species (40). The ered from any of the subsamples, the findings obtained from 1 3 archaeal rDNA clone pPACMA-N distantly related to any theenrichmentculturessupportedtheviewthataviableTher- b other archaeal rDNA sequences known so far (Fig. 3C) was mococcuspopulationwaspresentinthesurfacelayersandthat y detectedatasmallproportioninvariousmicrohabitatsbothin preferential colonization of the inside structures of the chim- P E therDNAcloneanalysisandtheT-RFLPanalysis(Table3and neybyhalophilicmicroorganismsoccurred. N Fig.2).Theresultsobtainedfromthemolecularphylogenetic N analysesindicatedthatdifferentarchaealphylotypestructures S DISCUSSION T consisting mainly of hyperthermophilic and extremely halo- A philicphylotypesweredistributedindifferentmicrohabitatsin Thedistributionofarchaeainablacksmokerchimneystruc- T E theblacksmokerchimney. turewasanalyzedbyculture-independent,molecularphyloge- U EnrichmentandMPNcalculations.Inordertosupportthe netictechniques.ThecombineduseofrRNAdotslothybrid- N apparent distribution of different archaeal community struc- ization, quantitative fluorogenic PCR, T-RFLP, and rDNA IV 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