Genetic Diversity of Archaea in Deep-Sea Hydrothermal Vent Environments

14 Pages·1999·0.22 MB·English

Copyright(cid:211) 1999bytheGeneticsSocietyofAmerica
Genetic Diversity of Archaea in Deep-Sea Hydrothermal Vent Environments
Ken Takai and Koki Horikoshi
Deep-SeaMicroorganismsResearchGroup,JapanMarineScienceandTechnologyCenter,Yokosuka237-0061,Japan
ManuscriptreceivedJanuary26,1999
AcceptedforpublicationApril12,1999
ABSTRACT
Molecularphylogeneticanalysisofnaturallyoccurringarchaealcommunitiesindeep-seahydrothermal
ventenvironmentswascarriedoutbyPCR-mediatedsmallsubunitrRNAgene(SSUrDNA)sequencing.
As determined through partial sequencing of rDNA clones amplified with archaea-specific primers, the
archaeal populations indeep-sea hydrothermal vent environmentsshowed a great geneticdiversity, and
most members of these populations appeared to be uncultivated and unidentified organisms. In the
phylogenetic analysis, a number of rDNA sequences obtained from deep-sea hydrothermal vents were
placedindeeplineagesofthecrenarchaeoticphylumpriortothedivergenceofcultivatedthermophilic
membersofthecrenarchaeotaorbetweenthermophilicmembersoftheeuryarchaeotaandmembersof
themethanogen-halophileclade.Wholecellinsituhybridizationanalysissuggestedthatsomemicroorgan-
isms of novel phylotypes predicted by molecular phylogenetic analysis were likely present in deep-sea
hydrothermal vent environments. These findings expand our view of the genetic diversity of archaea in
deep-seahydrothermalventenvironmentsandofthephylogeneticorganizationofarchaea.
RECENT molecular phylogenetic analyses based on 1997),Yellowstonehotsprings(Barnsetal.1994,1996),
smallsubunitrRNAgene(SSUrDNA)sequencing shallowmarinehydrothermalventwater,andaJapanese
have indicated that the microbial diversity of naturally acidic hot spring (Takai and Sako 1999). The results
occurringmicrobialcommunitiesismuchgreaterthan of these studies suggested that a number of previously
previously assumed based on standard cultivation and unknownanduncharacterizedmembersofthearchaea
isolationmethods(Stahletal.1984;Giovannonietal. exist in such environments.
1990; Delong 1992; Fuhrman et al. 1992; Ward et al. Todate,anumberofextremethermophilesorhyper-
1992;Barnsetal.1994,1996;Delongetal.1994;Rey- thermophiles have been isolated from terrestrial solfa-
senbachetal.1994;Prestonetal.1996;Bintrimetal. taricfieldsandhotsprings,andshallowordeepmarine
1997;Massanaetal.1997;Munsonetal.1997;Schleper hydrothermal vent environments (Stetter et al. 1990;
etal.1997a;Dojkaetal.1998;Hugenholtzetal.1998). Adams 1993; Adams et al. 1995; Sako et al. 1996a,b).
Although many studies focusing on the identification Thediversityofthermophilicmicroorganismsinterres-
anddiversityofprokaryotesofthedomainBacteriahave trialhotspringsandinashallowmarinehydrothermal
beenconducted,increasingattentionisnowbeingpaid venthasbeeninvestigatedbyboththecultivation-isola-
to the genetic diversity and ecological significance of tion technique and the molecular phylogenetic ap-
prokaryotes of the domain Archaea (Delong 1992; proach (Stetteret al.1990; Adams1993; Adamset al.
Fuhrman et al. 1992; Barns et al. 1994, 1996; Delong 1995;Barnsetal.1994,1996;Sakoetal.1996a,b;Takai
et al. 1994; Preston et al. 1996; Bintrim et al. 1997; andSako1999).Sincethediscoveryofdeep-seahydro-
Kudo et al. 1997; Massana et al. 1997; Mcinerney et thermal vents in 1977, such environments have been
al. 1997; Munson et al. 1997; Schleper et al. 1997a; regarded as one of the major habitats of thermophiles
Vetrianietal.1998;TakaiandSako1999).Molecular and have been also a major source of novel deep-sea
phylogenetic surveys of archaeal diversity have focused thermophilic archaea and bacteria (Gonzalez et al.
onavarietyofenvironments,forinstance,oceanwater 1995, 1998; Prieur et al. 1995; Blo¨chl et al. 1997).
(Fuhrman et al. 1992; Mcinerney et al. 1997), coastal However, the genetic diversity of archaea in deep-sea
water(Delong1992;Massanaetal.1997),apolarsea hydrothermalventenvironmentsispoorlyunderstood,
(Delongetal.1994),sedimentsfromacontinentalshelf andmolecularphylogeneticsurveysofthearchaealpop-
(Vetrianietal.1998),asaltmarsh(Munsonetal.1997), ulationsinsuchenvironmentshavenotyetbeenunder-
a freshwater lake (Schleper et al. 1997a), agricultural taken.
soils(Bintrimetal.1997),paddyfieldsoil(Kudoetal. Inthisstudy,wesoughttodeterminethegeneticdiver-
sity of archaea in deep-sea hydrothermal vent environ-
ments such as effluent vent water and chimney struc-
tures.Thehydrothermalventsystemsinthisstudywere
Correspondingauthor:KenTakai,Deep-SeaMicroorganismsResearch
located at Suiyo Sea Mount and Myojin Knoll in the
Group, Japan Marine Science and Technology Center, 2-15 Natsu-
shima-cho,Yokosuka237-0061,Japan. E-mail:[email protected] OgasawaraareaandatIheyaBasinintheOkinawaarea,
Genetics152:1285–1297(August1999)
1286 K.TakaiandK.Horikoshi
tctzbJhcsheataohayumptemdesidasohmrseipno,neiia,atddcharonaaeeneftdtledolrdtypdmthwhdd-eUseaeipaietfMlrthaatfgehaverAeetrcbhsnahTneheynieonEtdandt1steRsriec9yfoatIfrhs9oArtdtowhoe5efiLmve)emmoSre.rrtaoremsThAnostaieNehhangtrrxyleeeeDeahravamgoaneiMhrgenfndocihEonot1htpg-Tte0tawehhredH0enaaemy.0aptOvrlel–oiphrDr1(gpeioT3eceSorn5annaapml0btveluuyilterelmriaoacnetn1.ntoieso;dmTrcnIgwhogessaesehennyirsotinsiees---- aCharacteristics HightemperaturefluidcontainingrelativelyhighconcentrationsofCa,Sr,Ba,AlComposedofchalcopyrite,sphaler-ite,anhydrite,barite.HighAucontent(cid:122)(cid:56)100clearfluiddischargedfrom(cid:122)(cid:56)180black-coloredfluiddischargedfrom.Composedofbarite,pyrite,andotherminorsulfidesHightemperaturefluidcontainingquitehighconcentrationsofCO,2,andothercarbonhydrates,CH4andHS2Composedofcarbonate-richhydro-thermalprecipitateswithdissemin-atedsulfides
SamplecollectionandDNAextraction:Samplesfromvari-
ousdeep-seahydrothermalventenvironmentswereobtained pth 9m 8m0m
fromthehydrothermalfieldsatSuiyoSeaMountandMyojin e 6 33
d 3 33
KnollintheOgasawaraareaandfromthehydrothermalfields / m 1 11 m
e / // m
amtaInhneeyda sBuabsmineirnsibthleeSOhikninkaaiw2a0a0r0eian,sJeavpearna,lbdyivemse(aDnisveosf9t7h7e, ratur 1369 nside nsidenside 972 1398
1007,1016,and1111).Effluentblacksmokerventwater(11 y e / i ii / /
lsimteorsk;e(cid:122)rc3h0i0m(cid:56);ndeyes(i(cid:122)gn7a0t0edg;adsesSigSnMaVteWd)asaSnSdMthCe)wtiepreocfoallebclatecdk sstud Temp (cid:122)(cid:56)300 (cid:122)(cid:56)300 (cid:122)(cid:56)100(cid:122)(cid:56)180 (cid:122)(cid:56)300 (cid:122)(cid:56)100
fromahydrothermalventsiteatSuiyoSeaMount.Thetipof hi
t
ablacksmokerchimney((cid:122)500g;thetemperatureofthevent n
i E E EE E E
wsmatoekrewracsh(cid:122)im1n8e0y(cid:56);(d(cid:122)e3s0ig0nga;tetdheasteMmCpe2r)aatunrdetohfethtiepvoefntawcaletearr used (cid:57)630 (cid:57)630 (cid:57)004(cid:57)004 (cid:57)900 (cid:57)300
was(cid:122)100(cid:56);designatedasMC1)wereobtainedfromahydro- s 8. 8. 2.2. 3. 8.
e 3 3 55 5 5
twhaetremr(a1l2velintetrssi;t(cid:122)ea3t00M(cid:56);ydojeisnigKnnaotelld.aEsfflIVuWen)tabnldacskedsmimoeknetrfvroenmt ampl ation (cid:56)140 (cid:56)140 (cid:56)139(cid:56)139 (cid:56)126 (cid:56)126
tgIhS;)ethuweperpteeemro1pb–tea1ria0nt-ceumdreflraooyfmetrhatehtestehhdeyimwdareotnethtreswirmamsma1le0vr0ei(cid:56)nn;gtdpseiostieignnatat(t(cid:122)Iehd3e0ya0as ABLE1 alvents Loc (cid:57)4.090N, (cid:57)4.090N, (cid:57)6.208N,(cid:57)6.208N, (cid:57)7.220N, (cid:57)2.800N, 95).
Basin.Thesamplesources,locations,andpropertiesaresum- T m 3 3 00 4 3 9
wmonearriecizeiemadnmidnesdtToiaarbteeldleya1tfr.4oW(cid:56)z,eaanntedrattsha(cid:50)em8tpi5pl(cid:56)esusonfwtceihlreipmriomncemeyssesaedndiad.teselydcimooelnetds ydrother (cid:56)rc28 (cid:56)rc28 (cid:56)32(cid:56)32 (cid:56)27 (cid:56)27 Urabe(1
Nucleic acids were extracted by physical disruption (the h a a
elaborategrindingoffilterwithmortarandpestle)andchemi- ea ra ra arcarc nd
cSvS1(eAaae0nla-kd(cid:109)tlovyMmaws(nioa-1spttu9eeoon9crrf,9teft)T-rohs.oioreAzmkepIcyhNpoeeerl,ailyotcsJaexahxapiBmcoafiacfnalostit)terneh.drlesMyiwna1haignc0ysrddtlosioret6boeq.itt0rahuhs-l(cid:109)eeeopnrmfmmateir-apefatfllitollchyurlvoeeeefisd-nnsliwtttzoebeesrfirletafieTedclsatcketkoahsrtalmrlpieSooacuuapktingeyeehddorr Listofdeep-s Source nt,Izu-Ogasawa nt,Izu-Ogasawa Izu-OgasawaraIzu-Ogasawara MiddleOkinawa MiddleOkinawa omIshibashia
tmw1fhr5eome0rmec-mdatithsmaheemenoNseefwatteCafihrslleh/tceet1erid0lpel0usdwlomoiwtfshmaectNheaEirEcmDeTstnTaabemAteyu)psffiflateewlntrseidc(ore5ssn0e(adA0mni.mdd2mv2esa-Ttn(cid:109)nortmtrsiese,c--d(cid:122)Hp)o.C3arTt0le,0h(cid:50)-speig2Hzefi0(,(cid:56)w8l.t.4ee0I7trn/)s- Suiyoseamou Suiyoseamou MyojinKnoll,MyojinKnoll, IheyaBasin,trough IheyaBasin,trough artreferredfr
ofthesamplematerialwasresuspendedin500mlofMJsyn- p
tthherotiucgsheaaw0a.t2e2r-(cid:109)(Sma-kpooreet-sailz.e19fi9lt6ebr),awndhicahuthoacdlavbeede,nafinldterthede W) arge
satapwannomiddrcepeps-lseeeitdzosetimlmreNae.eitmTtneerthoxiveapfielarlrtwemetsiaruciscalserpncsoredubanasindah6dlee.d0dct-he(cid:109)sileanlmnmst-ofppsrcolofirermenewee-nsatispehzedsieeofictnchlertisrceuoarswuthpeigetadhhdpfeoaacrrh.1mMi10mo0-i(cid:109)crnstrmeeaocyr-- on) water(SSMV ney(SSMC) ney(MC1)ney(MC2) water(IVW) ediments(IS) wereforthel
bteddtbhaniiielaaaesvllmccifrorpfoeorinalbatnlrteoecamtrtdwimicoeiclaniennenbgstsloaalwuenvwlexalteoDsspras(eeNwneTradAciaasmo.hscnlteeelHonentdreacitegrttwedaehisedtatfihamtaol(cid:50)tnlNno.eelr2E1ega.09Tca(cid:56)90Tut.8bi.lhvT2a)uei2ro,sf-fca(cid:109)cewofihmrfienletlitt-etgwcprerhkooirctlrfwweoecDaio-ratssNhnniezotwdxAeaupa,iuntsew4hsrim7eanie-mdsmgidcefernmonaoxors---- Sample(abbreviati Blacksmokervent Blacksmokerchim ClearsmokerchimBlacksmokerchim Blacksmokervent Watersimmerings aCharacteristics
tracted and purified from all filters including the filter em-
ArchaealDiversityinHydrothermalVents 1287
ployed as a negative control as described previously (Takai the same manner as described in this study. A total of 26 of
andSako1999). 33 clones revealed the correct identical rDNA sequence of
Microscopicobservation:Theventwaterandchimneysam- thisorganism.Atotalof6of33cloneshadanerrorsuchas
plesfilteredthrough6.0-(cid:109)m-pore-sizefilterpaperwerefixed asinglenucleotidesubstitution,insertion,ordeletion,and1
for 12 hr in 3.7% formaldehyde, and the fixed microbial of33clones hadbothasinglenucleotide substitutionanda
particles were collected on 0.22-(cid:109)m-pore-size, 13-mm-diame- single nucleotide insertion. The overall error frequency of
ter polycarbonate filters (Advantec). Each filter was rinsed partial sequencing analysis was calculated to be 0.063 error
twiceinMJsyntheticseawater(Sakoetal.1996b),whichhad per100nucleotides.
been filtered through a 0.22-(cid:109)m-pore-size filter and auto- SequencesweremanuallyalignedtoSSUrDNAdatafrom
claved,andthenstainedbytreatmentwithMJseawatercon- the RDP on the basis of primary and secondary structure
taining 4(cid:57),6-diamidino-2-phenylindole (DAPI; 10 (cid:109)g/ml) or considerationsand werealso submittedtoanalysis usingthe
ethidiumbromide(10(cid:109)g/ml)at4(cid:56)for20min.Thefilterwas CHECK CHIMERAprogramtodetectthepresenceofchime-
brieflyrinsedinMJseawaterandexaminedunderepifluores- ric artifacts. Phylogenetic analyses were restricted to nucleo-
cenceusing aLeica DMRBmicroscopewith aLeica MPS30 tide positions that were unambiguously alignable in all se-
camerasystem. quences.Least-squaresdistancematrixanalysis(Olsen1988),
PCRamplificationofrDNA:SmallsubunitribosomalRNA based on evolutionary distances, was performed using the
genes(rDNAs)wereamplifiedbyPCRusingLATaqpolymer- correction of Kimura (1980). Neighbor-joining analysis was
asewithGCbuffer(TaKaRa,Kyoto,Japan),asrecommended accomplishedusingtheODENsoftwarepackage(version1.1;
by the manufacturer. Reaction mixtures were prepared in National Institute of Genetics, Mishima, Japan). Maximum-
whichtheconcentrationofeacholigonucleotideprimerwas likelihoodanalyseswereperformedusingthePHYLIPpackage
0.1(cid:109)mandthatoftheDNAtemplatewas1ng/(cid:109)l.Thermal (version 3.5; obtained from J. Felsenstein, University of
cyclingwasperformedusingtheGeneAmpPCRsystem9600 Washington, Seattle).Bootstrap analysis was usedto provide
(Perkin-Elmer, Foster City, CA) and the conditions were as confidenceestimatesforphylogenetictreetopologies.
follows: denaturation at 96(cid:56) for 20 sec, annealing at 50(cid:56) for Whole-cellhybridizationanalysis:RibosomalRNA-targeted
45sec,andextensionat72(cid:56)for120secforatotalof30cycles. oligonucleotide probes were designed for detection of ar-
Theoligonucleotideprimersequencesandcombinationsused chaeal members that were potentially predominant in the
were as follows: Arch21F (Fuhrman et al. 1992) and 1492R deep-seahydrothermalventenvironments.TheyweretheMGI
(Lane1985)forarchaealrDNA,andUni515FandUni1408R probe (5(cid:57)-AAAYCACTCGGAYTAACCTT-3(cid:57)) and AAG probe
(TakaiandSako1999)forallmicrobialrDNAs. (5(cid:57)-CCTAGCACTCGGGCTCGCGG-3(cid:57)), which corresponded
Cloning and sequencing of amplified rDNA: Amplified topositions391–411and401–421inEscherichiacoli16SrDNA,
rDNAsfromfiveseparatereactionswerepooled,fractionated respectively,andweredesignedfordetectionofmarinegroup
byelectrophoresisona1.2%(w/v)agarosegel,thenextracted I(MGI)crenarchaeoticmembersandafewancientarchaeal
from the gel slicessequentially with phenol, phenol/chloro- group(AAG)membersbasedonthealignmentinthephyloge-
form/isoamyl alcohol and chloroform/isoamyl alcohol, and neticanalysis.Bothprobesequenceswereanalyzedusingthe
precipitated with ethanol. After centrifugation, DNA pellets PROBE CHECKfromtheRDP(Larsenetal.1993)andthe
were resuspended in sterile distilled water. The purified gapped-BLASTsearchalgorithm(Altschuletal.1997;Ben-
rDNAs were cloned in the vector pCR2.1 using the original sonetal.1998)toexaminewhetheranyotherrDNAsequences
TA cloning kit (Invitrogen, Carlsbad, CA), and then the ar- have similarity to these probe sequences. In addition, dot-
chaeal and universal primer PCR libraries were built from hybridizationanalysiswascarriedoutwithrepresentativearch-
DNAobtainedfromeachdeep-seahydrothermalventsample. aeal rDNA clones obtained from the deep-sea hydrothermal
Clonescontaininginsertsoftheappropriatesizewereidenti- vent environments to check the specificity of the probes to-
fied byelectrophoresis of alkaline lysisplasmid preparations wardtheMGIcrenarchaeoticandAAGmembers,respectively.
(Maniatiset al.1982).Denatured, double-strandedplasmid The purified plasmid DNAs (100 ng) containing (cid:122)1.5 kb of
templates were sequenced by the dideoxynucleotide chain- rDNA sequences were blotted onto positively charged nylon
termination method with Taq DNA polymerase FS (Perkin- membranes (Boehringer Mannheim, Indianapolis). The
Elmer)followingthemanufacturer’srecommendations.Uni- membranes were hybridized with the MGI-D probe or the
versal rRNA-specific primers (Lane 1985) and M13 forward AAG-D probe (5 ng/(cid:109)l), which was labeled at the 5(cid:57)-end
and reverse primers (Maniatis et al. 1982) were used in se- with digoxigenin (DIG) and purified by HPLC (Amersham
quencingreactions. PharmaciaBiotech).Afterhybridization,themembraneswere
Sequenceandphylogeneticanalyses: ThepartialrDNAse- washed,and theDIG-labeledoligonucleotide probeshybrid-
quenceswereanalyzedusingSIMILARITY RANKandALIGN izedwiththetargetedrDNAsequencesweredetectedbyusing
SEQUENCE from the Ribosomal Database Project (RDP; the DIG luminescent detection kit for nucleic acids (Boeh-
Larsenetal.1993)andthegapped-BLASTsearchalgorithm ringerMannheim).Allhybridizationandwashingprocedures
(Altschul et al. 1997; Benson et al. 1998) to estimate the werecarriedoutinthesameconditionsasdescribedbelow.
degree of similarity to other rDNA sequences. Sequences of For whole-cell hybridization experiments, the vent water
400bpinlengthdeterminedbymeansofUni515Fprimerin andchimneysamplesfilteredthrough6.0-(cid:109)m-pore-sizefilter
onestrandwereusedinthesimilarityanalysis.Thedatabases paper were fixed for 12 hr in 3.7% formaldehyde, and the
usedforRDPandgapped-BLASTanalysisweretheprokaryotic fixedmicrobialparticleswerecollectedon0.22-(cid:109)m-pore-size,
SSU rRNA database and the nonredundant nucleotide se- 13-mm-diameterpolycarbonatefilters(Advantec).Eachfilter
quence database from GenBank, EMBL, and DDBJ, respec- wasrinsedsequentiallyinMJsyntheticseawaterthathadbeen
tively.Inthisstudy,wetentativelydefinedthat(cid:46)98%similarity filtered through a 0.22-(cid:109)m-pore-size filter and autoclaved in
inUni515F-dependentsequencesrepresentedthesamerDNA 1:1(v/v)MJseawater/deionizeddistilledwater(DDW)and
clone type among rDNA sequences obtained from deep-sea DDW.Thefilterwasdehydratedinanethanolseries(30,60,
hydrothermal vent environments. In addition, error fre- 80, 90, and 95%, v/v) and air-dried (Amann et al. 1990).
quency,whichpresumably occurredthroughPCRamplifica- The hybridization was carried out in hybridization solution
tion,cloning,andpartialsequencing,wasroughlyestimated. containing 20 mm Tris-HCl (pH 7.2), 0.9 m NaCl, 0.1%
The16SrDNAofPyrococcushorikoshiiJCM9974(Gonzalezet (w/v)sodiumdodecylsulfate,and50%(v/v)formamidefor
al. 1998) was amplified, cloned, and partially sequenced in 12 hr at 48(cid:56) in the case of the MGI-F probe (5 ng/(cid:109)l) or at
1288 K.TakaiandK.Horikoshi
55(cid:56) in the case of the AAG-F probe (5 ng/(cid:109)l), which were ducedduringtheDNAextractionandpurificationpro-
each labeled at the 5(cid:57)-end with fluorescein and purified by
cedures,asdescribedbyTanneretal.(1998).However,
HPLC (Amersham Pharmacia Biotech). After hybridization,
norDNAwasamplifiedfromthenegativecontrolsam-
the filter was washed in hybridization solution lacking the
probes,atthesametemperatureasthatemployedforhybrid- ples using either the archaea-specific primers or the
ization,for20mintwiceandstainedwitha10(cid:109)g/mlsolution universal primers under the conditions of this study.
ofDAPIinDDWat4(cid:56)for20min.Thefilterwasbrieflyrinsed The first step in our strategy was to examine the ar-
in DDW and was examined under epifluorescence using a
chaeal populations in naturally occurring microbial
LeicaDMRBmicroscopewithaLeicaMPS30camerasystem.
communitiesinvariousdeep-seahydrothermalventen-
Twotypesofnegativecontrolswereemployedineachexperi-
ment,usingidenticalcellpreparationsandhybridizationcon- vironmentsonthebasisofpartialnucleotidesequence
ditions.Onetypeofnegativecontrolconsistedofsamplesin information obtained through analysis of a number of
whichtheunlabeledMGI-ForAAG-Fprobewasaddedin50- insert-containingplasmids.First,a400-ntsequence(cor-
fold excess (250 ng/(cid:109)l) of the fluorescein-labeled MGI-F or
responding to positions 536–935 of E. coli rDNA num-
AAG-F probe added at the standard concentration (5 ng/
(cid:109)l). Theother type ofnegative control consistedof samples bering) determined in one strand from each plasmid
employed to examine hybridization with cultivated thermo- was obtainedfrom universaland archaealprimed PCR
philic microorganisms, including Aeropyrum pernix JCM9820 libraries of each sample. In this study, 132, 116, 110,
(Sakoetal.1996a),ThermococcuspeptonophilusJCM9653(Gon- 102,and 124clones werepartially sequencedfrom the
zalez et al. 1995), P. horikoshii JCM9974 (Gonzalez et al.
universal primer PCR libraries established from the
1998), Methanococcus jannashii DSM2661 (Jones et al. 1983),
deep-sea hydrothermal vent samples of SSMC, MC1,
RhodothermusobamensisJCM9785(Sakoetal.1996b),andTher-
maerobacter marianensis (Takai et al. 1999). Cells were grown MC2,IVW,andIS,respectively(Table2).Furthermore,
as described in the references given above, and cells in the 104, 164, 139, 145, and 188 clones were partially se-
stationaryphaseofgrowthwereusedforthesehybridization quenced from the archaeal primer PCR libraries of
experiments.
SSMCA(SSMC-Archaeallibrary),MC1A,MC2A,IVWA,
Nucleotide sequence accession numbers: The sequences
reported here have been deposited in the DDBJ database and ISA (Table 3). Gapped-BLAST analysis of these
undertheaccessionnos.AB019714–AB019759. sequences provided a profile of the PCR-dependent
rDNA composition of the various environmental sam-
ples (Tables 2 and 3). As described in materials and
RESULTS methods,wetentativelydefinedthat(cid:46)98%similarityin
Characterization of microbial rDNA recovered from Uni515F-dependentsequencesrepresentedanidentical
deep-seahydrothermalventenvironments:DNAwasiso- rDNAclonetypeamongrDNAsequencesobtainedfrom
lateddirectlyfromsamplescollectedfromvariousdeep- deep-sea hydrothermal vent environments. The error
sea hydrothermal vent environments except for the ef- frequency during the partial sequencing analysis was
fluent water from the black smoker vent (SSMVW) at estimated to be (cid:44)0.1%.
thehydrothermal ventsite atSuiyo SeaMount. Inthat From the black smoker vent chimney of Suiyo Sea
sample, low DNA recovery was expected due to the Mount(SSMC),128ofthe132clones(97%)withinserts
very small number of microorganisms ((cid:44)10 cells/ml) amplifiedbymeansofuniversalprimerswerefoundto
observed in the sample by epifluorescence microscopy be bacterial SSU rDNA sequences (Table 2). Archaeal
after staining with DAPI or ethidium bromide (Table rDNAsequencesweredetectedinalibraryofsequences
2).MicrobialribosomalRNAgeneswereselectivelyam- derivedfromSSMCpreparedbyPCRusingtheuniversal
plified by PCR and cloned. PCR amplification was also primer.All4ofthesecloneswerefoundtohavethesame
carriedoutusingextractspreparedfromnegativecon- rDNAsequence,andthissequencewasthepredominant
trol samples to check for possible contaminants intro- rDNAsequence(pSSMCA1)foundinthearchaealpopu-
TABLE2
RibosomalDNAcompositionofsamplesfromvariousdeep-seahydrothermalventenvironments
Bacterial Archaeal Eucaryal
Sample(no.ofclonessequenced) (%) (%) (%) Microbialcellsa
SSMVW ND ND ND ND
SSMC(132) 97.0 3.0 0 104/gwet
MC1(116) 82.8 17.2 0 107/gwet
MC2(110) 95.5 4.5 0 107/gwet
IVW(102) 73.5 26.5 0 104/ml
IS(124) 96.8 3.4 0 107/gwet
Ribosomal DNA composition was determined by partial sequencing analysis of rDNA clones of universal
primerPCRlibraries.ND,notdetected.
aDirectcountsofmicrobialcellsstainedwithDAPIandethidiumbromide.
ArchaealDiversityinHydrothermalVents 1289
lation of this sample. The archaeal rDNA clones derived foundtoconstitutetwolargephylogeneticassemblages;
fromSSMCconsistedofonlytwoclonetypes,represented one consisted of the very deep lineages of rDNA se-
by pSSMCA1 (99 of 104 clones) and pSSMCA108 (5 of quences within the Crenarchaeota and the other was
104clones),bothofwhichshowedrelativelylowsimilarity placedinanintermediatepositionbetweenthethermo-
((cid:44)80%) to any other rDNA sequences from cultivated philic Euryarchaeota and the methanogens-halophiles
or uncultivated archaea (Table 3). (Figure 1). Most of the rDNA clones obtained from
The proportions of archaeal rDNA sequences were deep-seahydrothermalventenvironmentswereplaced
similar in the universal primer PCR libraries prepared in novel phylogenetic branches distinct from those of
fromtheblacksmokerchimneysamplecollectedfrom cultivated members of the archaea and other environ-
Myojin Knoll (MC2) and the clear water simmering mental clones (Figure 1).
sediment sample from Iheya Basin (IS; Table 2). The Of the many novel rDNA sequences found, several
archaeal primer PCR library prepared from MC2 con- rDNAsequencesrepresentedbypMC2A256,pMC2A249,
sisted of a variety of rDNA sequences, and the same and pMC2A14/pSSMCA1 (AAG) were placed in the
clonetypeasthatrecoveredfromSSMCwasfoundalso deepest branches within the crenarchaeotic phylum.
in this library (Table 3). The archaeal rDNA composi- In this phylogenetic tree, the crenarchaeotic phylum
tion of IS differed significantly from that of the black accommodated korarchaeotic rDNA sequences and
smokerchimneysamplesofSSMCandMC2.Inthecase clonepOWA133,whichwerepreviouslydenotedaspos-
of the archaeal primer PCR library prepared from IS, sible outgroups prior to the divergence of Crenar-
a high proportion of Euryarchaeota was found (Table chaeota and Euryarchaeota, respectively (Barns et al.
3). These euryarchaeotic rDNA clones were unidenti- 1994;TakaiandSako1999).Thepreferenceinplace-
fiedsequencesandconsistedofavarietyofclonetypes ment of these deep lineages of rDNA sequences into
(Table 3). the crenarchaeotic phylum was strongly influenced by
Arelativelyhighproportionofarchaeawasfoundin therDNAsequenceofMethanopyruskandleri.Whenthe
the universal primer PCR libraries prepared from the M.kandlerirDNAsequencewasomittedfromthephylo-
clear smoker chimney sample obtained from Myojin geneticanalysis, thephylogenetictrees obtainedbased
Knoll (MC1)and the effluent blacksmoker vent water on the neighbor-joining and maximum-likelihood
samplefromIheyaBasin(IVW).Intheuniversalprimer methodsshowedfivepossibleseparatearchaealphyletic
PCR library prepared from MC1 and IVW, 20 of 116 groupsatthekingdomlevel;thedeepestoutgroupwas
clones (17.2%) and 30 of 102 clones (26.5%) were ar- represented by the AAG rDNA clones, the second out-
chaeal rDNA sequences, respectively (Table 2). All of groupby thepOWA133clone,and thethirdoutgroup
thesearchaealrDNAcloneswerecloselyrelatedtoma- bythekorarchaeoticclones,priortothedivergenceof
rine planktonic clones, designated as MGI (Delong Crenarchaeota and Euryarchaeota (data not shown).
1992;Fuhrmanetal.1992;Delongetal.1994;Massana TheseverydeepbranchesofrDNAsequencesobtained
et al. 1997; Mcinerney et al. 1997); however, none of fromvarioushotwaterenvironmentshadrelativelylow
these sequences showed (cid:46)98% similarity to the se- bootstrap significance and were sensitive to the se-
quences of MGI clones described previously. In addi- quences used; however, base composition disparities
tion, the archaeal primer PCR library prepared from had little influence on the topology, which was sup-
MC1containedonlytwoclonetypes,bothofwhichwere portedbytransversiondistance-matrixanalysis(Woese
categorized within the MGI (39 clones for pMC1A103 etal.1991).TheseresultsindicatedthattheAAGrDNA
type or 71 clones for pMC1A11 type of 164 clones), clonesrecoveredfromdeep-seahydrothermalventenvi-
and another major unidentified clone type (pMC1A4) ronments undoubtedly represent one of the deepest
categorized within the Euryarchaeota (54 of 164 lineagesofanyarchaealrDNAsequencesderivedfrom
clones),whilethearchaealprimerPCRlibraryprepared cultivated members or unidentified environmental
from IVW mainly consisted of a variety of rDNA clone clones,althoughwhetherthephylogeneticclassification
types categorized within the MGI (Table 3). oftheserDNAclonesintothekingdomCrenarchaeota
Phylogeneticanalyses ofrDNA sequencesrepresent- is appropriate remains uncertain.
ingunidentifiedmicroorganisms:Toinferphylogenetic Inaddition,agreatdiversityofrDNAsequenceswere
affiliationsofarchaealrDNAclonesobtainedfromvari- alliedinpositionsclosetotheThermoplasma-Picrophi-
ous deep-sea hydrothermal vent environments, nearly luscladeandinintermediatepositionsbetweenthermo-
full-length sequences ((cid:122)1400 nt) of representative philic Euryarchaeota branches and methanogen-halo-
rDNAclonetypesweredeterminedandusedforappro- phile branches (DHV euryarchaeotic groups I and II;
priate phylogenetic analysis. Phylogenetic analysis by Figure1).Thesesequencesweremainlyobtainedfrom
the neighbor-joining and maximum-likelihood meth- the black smoker chimney at Myojin Knoll (MC2) and
odsresultedintreeswithsimilartopologies(Figure1). theclearwatersimmeringsedimentsatIS,andmostof
Inanoverviewofthetree,thearchaealrDNAsequences themshowednosignificantsimilaritytoanyotherrDNA
obtainednotonlyfromdeep-seahydrothermalventen- sequences known to date. When the phylogenetic tree
vironmentsbutalsootherhotwaterenvironmentswere was reconstructed by neighbor-joining methods to in-
1290 K.TakaiandK.Horikoshi
TABLE3
DistributionofrepresentativearchaealrDNAclonetypesinvariousdeep-seahydrothermalenvironments
Clonetype
Phylogeneticgroup (positiononblot) SSMC MC1 MC2 IVW IS
Crenarchaeota 39/99a 90/110 46/63 103/141 5/6
Ancientarchaealgroupb 39/99 — 14/22 — —
DHVA1b
pMC2A256(1e) — — 1/2 — —
pMC2A249(1d) — — 1/1 — —
pMC2A14(1c)/pSSMCA1(1a/1b) 39/99 — 12/19 — —
DHVcrenarchaeoticgroupb — — 12/14 — —
DHVC1b
pMC2A15(1f) — — 1/1 — —
pMC2A209(1g) — — 4/5 — —
pMC2A36(1h) — — 4/4 — —
pMC2A308(1i) — — 3/4 — —
MarinegroupI(MGI) — 90/110 20/27 103/141 —
pMC1A103(2a) — 31/39 — — —
pMC1A11(2b) — 59/71 — — —
pMC2A1(2c) — — 20/27 — —
pIVWA1(2d) — — — 21/23 —
pIVWA2(2e) — — — 28/39 —
pIVWA3(2f) — — — 11/16 —
pIVWA5(2g) — — — 29/47 —
pIVWA11(2h) — — — 3/4 —
pIVWA101(2i) — — — 8/9 —
pIVWA108(2j) — — — 3/3 —
THScrenarchaeoticgroupb — — — — 5/6
THSC1b
pISA9(1j) — — — — 4/5
pISA7(3a) — — — — 1/1
Euryarcheaota 3/5 36/54 66/76 3/4 144/182
Methanococcales — — 1/1 — —
pMC2A384(3b) — — 1/1 — —
Archaeglobales — — 3/3 — —
pMC2A228(3c) — — 3/3 — —
DHVeuryarchaeotic
groupIb 3/5 — 39/46 — 52/56
DHVE1b
pMC2A24(3d) — — 17/18 — —
pMC2A203(3e) — — 3/3 — —
pMC2A33(3f) — — 2/2 — —
DHVE2b
pMC2A10/pSSMCA108(3g) 3/5 — 17/23 — —
pISA12(3h) — — — — 23/24
pISA42(3i) — — — — 29/32
DHVeuryarchaeotic
groupIIb — 36/54 23/26 3/4 59/78
DHVE3b
pMC2A25(3j) — — 2/3 — —
pMC2A21(4a) — — 4/4 — —
pMC2A232(4b) — — 8/8 — —
pMC2A211(4c) — — 6/8 — —
DHVE4b
pMC2A17(4d) — — 1/1 — —
pISA35(4e) — — — — 1/1
DHVE5b
pISA18(4f) — — — — 6/7
pISA38(4g) — — — — 4/4
pISA1(4h) — — — — 4/4
(continued)
ArchaealDiversityinHydrothermalVents 1291
TABLE3
(Continued)
Clonetype
Phylogeneticgroup (positiononblot) SSMC MC1 MC2 IVW IS
DHVE6b
pISA3(4i) — — — — 3/5
pMC2A35(4j) — — 2/2 — —
pMC1A4(5a) — 36/54 — — —
pISA48(5b) — — — — 31/41
pISA13/pIVWA104(5c) — — — 3/4 10/16
DHVeuryarchaeotic — 33/48
groupIIIb — — —
DHVE7b
pISA16(5d) — — — — 26/37
pISA14(5e) — — — — 7/11
Total 42/104 126/164 112/139 106/145 149/188
ArchaealrDNAcompositionwasdeterminedbypartialsequencinganalysisofrDNAclonesofarchaealprimerPCRlibraries.
aThis indicates the number of sequence variations per total number of clones designated into each phylogenetic group or
clonetypeonthebasisofpartialsequencinganalysis.
bNovel uncultivated phylogenetic groups shown in Figure 1 and abbreviations are as follows: DHVA, deep-sea hydrothermal
vent ancient archaea; DHVC, deep-sea hydrothermal vent crenarchaeota; THSC, terrestrial hot spring crenarchaeota; DHVE,
deep-seahydrothermalventeuryarchaeota.
cludeotherenvironmental,unidentifiedeuryarchaeotic obtainedfromtheblacksmokerchimney(pMC2A)and
clones based on the partial sequences (corresponding the clear smoker chimney (pMC1A) at Myojin Knoll,
to positions 438–887 inE. coli 16S rDNA), some phylo- andtheblacksmokerventwater(pIVWA)atIheyaBasin
types of sequences (DHVE1) within the DHV euryar- (Figure 3). When the phylogenetic tree was recon-
chaeoticgroupIwerecloselyrelatedwithsomeuniden- structedincludingthesenewMGIclones,twoevolution-
tified euryarchaeotic clones obtained from microbial ary arrays were found in the MGI cluster. The finding
communitiesincoastalsaltmarshandcontinentalshelf of further genetic diversity among the members of the
sediments (salt marsh and marine benthic euryarchae- MGIindeep-seahydrothermalventenvironmentsserves
ota; Munson et al. 1997; Vetriani et al. 1998; Figure asanimportantcluetounderstandingtheubiquityand
2).However,nosignificantphylogeneticrelatednesswas originoftheseuncultivatedmembersofthearchaeain
observed between a variety of euryarchaeotic rDNA se- marine environments.
quencesfromthedeep-seahydrothermalventenviron- Whole-cellhybridizationanalysis:RibosomalRNA-tar-
ments and the marine group II obtained from plank- geted oligonucleotide probes were employed in visual-
tonic microbial communities in ocean water, coastal izationofformalin-fixedmicrobialcellsfromthedeep-
water, and polar sea water (MGII; Delong 1992; seahydrothermalventenvironmentstofacilitateidenti-
Delongetal.1994).Itseemslikely,therefore,thatun- fication of members of MGI or the AAG represented
cultivated and unidentified members of the euryar- bypMC2A249,pMC2A14,andpSSMCA1.Thedesigned
chaeotadisplayinggreatgeneticdiversityarepresentin probes were confirmed to have no specificity to any
themicrobialcommunitiesofvariouscoastalanddeep other rDNA sequences by analyses using the PROBE
sea sedimentary environments, as in the case of mem- CHECK from the RDP (Larsen et al. 1993) and the
bers of MGI in planktonic microbial communities gapped-BLASTsearchalgorithm(Altschuletal.1997;
(Delong1992;Fuhrmanetal.1992;Delongetal.1994; Bensonetal.1998),andwerealsoconfirmedtohybrid-
Massana et al. 1997; Mcinerney et al. 1997). ize specifically with the targeted rDNA sequences of
Figure3showsaphylogenetictreepreparedfocusing MGI and AAG members by dot-hybridization analysis
ontheMGIrDNAclones.TheseMGIrDNAclonesare (Figure4).Usingtheseprobes,whole-cellhybridization
the most frequently occurring rDNA clones in marine analysiswascarriedoutwiththesamplesofSSMC,MC1,
planktonic microbial communities (Delong 1992; MC2,andIVW(Figure5).HybridizationwiththeMGI-
Fuhrman et al. 1992; Delong et al. 1994; Massana et specificfluorescentoligonucleotideprobewasobserved
al. 1997; Mcinerney et al. 1997) and have often been inthesamplesofMC1andIVW,andhybridizationwith
recoveredfrommarinesedimentsamplesandevensedi- the AAG-specific probe was observed in the case of
mentadjacenttoahydrothermalvent(TakaiandSako SSMCandMC2.Thearchaealcellsinthecaseofmem-
1999). A variety of archaeal rDNA sequences closely bers of the AAG were irregular cocci, on average 1.0
related to the sequences of the MGI clones were also (cid:109)m in diameter (Figure 5, C and D, G and H), and
1292 K.TakaiandK.Horikoshi
ArchaealDiversityinHydrothermalVents 1293
Figure 2.—Phylogenetic tree of rDNA clones focused on a variety of environmental, unidentified euryarchaeotic rDNA
sequences. The tree was inferred by neighbor-joining analysis of 436 homologous positions of the rDNA sequence in the case
of each organism or clone. Abbreviations indicate rDNA clones corresponding to uncultivated organisms derived from the
following environments: SBAR16, OARB, and WHARN, from coastal water in North America (Delong 1992); ANTARCTIC5,
fromantarcticmarinepicoplankton(Delongetal.1994);2MT,fromsaltmarshsediment(Munsonetal.1997);andBBA,from
continentalshelfsediments(Vetrianietal.1998).BoldlettersindicaterDNAclonesobtainedfromdeep-seahydrothermalvent
environments.Thescalebarrepresents0.025nucleotidesubstitutionspersequenceposition.Thepercentageof1000bootstrap
resamplingsisindicated.Thefilledcirclesindicatethebranchpointsnotsupportedbymaximum-likelihoodanalysis.
thoseinthecaseoftheMGIwereirregularcocci,(cid:122)1.0 or in hybridization experiments in which MGI-specific
(cid:109)m in diameter and rod shaped, on average 2.0 (cid:109)m orAAG-specificprobeslackingthefluorwereincluded
long and 0.6 (cid:109)m wide (Figure 5, A and B, E and F). simultaneously with fluor-labeled probes, but at a 50-
Preston et al. (1996) reported that marine sponge- fold higher concentration.
associatedMGIcellswererodshaped,0.8(cid:109)mlong,and OfthetotalprokaryoticcellscountedafterDAPIstain-
0.5 (cid:109)m wide, and the fluorescence signals of probes ing, 0.4 and 12.5% were identified as members of the
labeled with Texas Red were detected at both cellular MGI in the case of MC1 (7 of 1753) and IVW (59 of
poles.Inthisstudy,nofluorescentsignalsweredetected 474), respectively, and 2.2 and 0.8% were identified as
at the poles of the short rod-shaped cells. No fluores- membersoftheAAGinthecaseofMMSC(29of1319)
cencewas observedwhenhybridization wasperformed and MC2 (11 of 1375), respectively. The percentages
using the MGI-specific and AAG-specific probes in the of certain rDNA clone types among the total rDNA
case of cultivated bacterial and archaeal thermophiles sequences, based on PCR-mediated partial sequencing
Figure 1.—Phylogenetic tree of rDNA clones derived from various deep-sea hydrothermal vent environments. The tree was
inferredbyneighbor-joininganalysisof1211homologouspositionsoftherDNAsequenceinthecaseofeachorganismorclone.
AbbreviationsindicaterDNAclonescorrespondingtouncultivatedorganismsderivedfromthefollowingenvironments:pOWA
andpUWA,fromshallowmarinehydrothermalventwaterandterrestrialacidichotspringwater,respectively(TakaiandSako
1999);pJP,fromsedimentinaYellowstoneNationalParkhotspring(Barnsetal.1994);andsoilcloneSCA,fromagricultural
soil(Bintrimetal.1997).BoldlettersindicaterDNAclonesobtainedfromdeep-seahydrothermalventenvironments.Thescale
bar represents 0.1 nucleotide substitutions per sequence position. The percentage of 1000 bootstrap resamplings is indicated.
Thefilledcirclesindicatethebranchpointsnotsupportedbymaximum-likelihoodanalysis.
1294 K.TakaiandK.Horikoshi
Figure3.—Phylogenetictree
ofrDNAcloneswithinmarine
group I (MGI) derived from
variousdeep-seahydrothermal
ventenvironments.Thetreewas
inferred by neighbor-joining
analysis of 1211 homologous
positionsoftherDNAsequence
inthecaseofeachorganismor
clone. Abbreviations indicate
rDNAclonescorrespondingto
uncultivated organisms derived
from the following environ-
ments:pJP,fromsedimentina
YellowstoneNationalParkhot
spring(Barnsetal.1994);soil
clone SCA, from agricultural
soil(Bintrimetal.1997);ma-
rineclonesCandPM,fromwa-
ter of the northeast Atlantic
ocean at 500 m depth (Mcin-
erneyetal.1997);marinefos-
mid clone 4B7, from Pacific
oceanwater(Steinetal.1996);
andmarinecloneSB95-57,from
coastal water of the Santa Bar-
bara channel (Massana et al.
1997). Bold letters indicate
rDNA clones obtained from
deep-seahydrothermalventen-
vironments.The scale bar rep-
resents 0.05 nucleotide substi-
tutionspersequenceposition.
The percentage of 1000 boot-
strapresamplingsisindicated.
The topology of this tree was
supported by maximum-likeli-
hoodanalysis.
analysis, were 11.5% MGI cells, 25.8% MGI cells, 2.9% and some were closely related to previously discovered
AAG cells, and 0.7% AAG cells in the whole microbial environmental archaeal clones. Whole-cell hybridiza-
communities of MC1, IVW, SSMC, and MC2, respec- tionanalysistofacilitateidentificationofrepresentative
tively. Although there were differences found between uncultivated environmental clones revealed the exis-
therDNApopulationandthefluorescence-labeledcell tence of organisms of such uncultivated phylotypes in
population, both experiments strongly indicated that the naturally occurring microbial communities of the
uncultivated and unidentified archaeal members are deep-sea hydrothermal vent environments. This is an
presentasacertainproportionofthemicrobialcommu- outstanding example showing that a great variety of
nitiesindeep-seahydrothermalventenvironments,and archaea remain undiscovered and uncharacterized in
the rDNA from theseorganisms was successfully recov- various natural microbial habitats on the earth.
ered in certain proportions from the bulk of DNA di- Samples have been collected from various environ-
rectly extracted from these environments. ments with different geologic, physical, and chemical
properties(Table1;IshibashiandUrabe1995).Envi-
ronmentalcharacteristicshaveanimpactonthemicro-
DISCUSSION
bial communities in a given environment and might
AgreatgeneticdiversityofarchaealrDNAcloneswas be important factors in determining the magnitude,
evident upon analysis of DNA recovered from various content, and diversity of the microbial communities.
deep-seahydrothermalventenvironments.Mostofthe For instance, in the case of the samples of MC2 and
archaealrDNA sequencesobtainedfrom blacksmoker IS, the samples examined contained almost the same
ventwaterandchimneys,aclearsmokerchimney,and number of microbial cells, and the universal primer
clear water simmering sediments represented the se- PCRlibrariesshowedsimilarrDNAcomposition(Table2).
quences of as-yet-uncultivated phylotypes distinct from Thesesamplesalsodisplayedagreaterdiversityofarch-
anyothercultivatedarchaeaandenvironmentalclones, aealrDNAclonesthantheotherdeep-seahydrothermal