ORIGINAL ARTICLE

Comparative metagenomics of microbialcommunities inhabiting deep-sea hydrothermalvent chimneys with contrasting chemistries

Wei Xie1,2,6, Fengping Wang3,4,6, Lei Guo2,6, Zeling Chen2,6, Stefan M Sievert5, Jun Meng3,Guangrui Huang2, Yuxin Li2, Qingyu Yan2, Shan Wu2, Xin Wang2, Shangwu Chen2,Guangyuan He1, Xiang Xiao3,4 and Anlong Xu2

1China-UK HUST-RRes Genetic Engineering and Genomics Joint Laboratory, Huazhong University of Scienceand Technology, Wuhan, PR China; 2State Key Laboratory of Biocontrol, Guangdong Province Key Laboratoryof Therapeutic Functional Genes, National Engineering Center for Marine Biotechnology of South China Sea,Department of Biochemistry, College of Life Sciences, Sun Yat-Sen (Zhongshan) University, Guangzhou,PR China; 3Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State OceanicAdministration, Xiamen, PR China; 4School of Life Sciences and Biotechnology, State Key Laboratory ofOcean Engineering, Shanghai Jiaotong University, Shanghai, PR China and 5Biology Department, WoodsHole Oceanographic Institution, Woods Hole, Massachusetts, MA, USA

Deep-sea hydrothermal vent chimneys harbor a high diversity of largely unknown microorganisms.Although the phylogenetic diversity of these microorganisms has been described previously, theadaptation and metabolic potential of the microbial communities is only beginning to be revealed.A pyrosequencing approach was used to directly obtain sequences from a fosmid libraryconstructed from a black smoker chimney 4143-1 in the Mothra hydrothermal vent field at theJuan de Fuca Ridge. A total of 308 034 reads with an average sequence length of 227 bp weregenerated. Comparative genomic analyses of metagenomes from a variety of environments bytwo-way clustering of samples and functional gene categories demonstrated that the 4143-1metagenome clustered most closely with that from a carbonate chimney from Lost City. Bothare highly enriched in genes for mismatch repair and homologous recombination, suggestingthat the microbial communities have evolved extensive DNA repair systems to cope with theextreme conditions that have potential deleterious effects on the genomes. As previously reportedfor the Lost City microbiome, the metagenome of chimney 4143-1 exhibited a high proportion oftransposases, implying that horizontal gene transfer may be a common occurrence in the deep-seavent chimney biosphere. In addition, genes for chemotaxis and flagellar assembly were highlyenriched in the chimney metagenomes, reflecting the adaptation of the organisms to the highlydynamic conditions present within the chimney walls. Reconstruction of the metabolic pathwaysrevealed that the microbial community in the wall of chimney 4143-1 was mainly fueled by sulfuroxidation, putatively coupled to nitrate reduction to perform inorganic carbon fixation through theCalvin–Benson–Bassham cycle. On the basis of the genomic organization of the key genes of thecarbon fixation and sulfur oxidation pathways contained in the large genomic fragments, bothobligate and facultative autotrophs appear to be present and contribute to biomass production.The ISME Journal (2011) 5, 414–426; doi:10.1038/ismej.2010.144; published online 7 October 2010Subject Category: microbial population and community ecologyKeywords: metagenomics; deep sea; chimney; fosmid; pyrosequencing

Introduction

Deep-sea hydrothermal vent chimneys that formby interactions between hot fluids and cold sea-water are regarded as biogeochemical hot spots,with reactive gases, dissolved elements andthermal and chemical gradients operating overspatial scales of millimeters and centimeters up tometers (Schrenk et al., 2003; Kristall et al., 2006).These chemical and thermal gradients along andinside the sulfide chimneys provide a wide range of

Received 11 January 2010; revised 5 July 2010; accepted 5 July2010; published online 7 October 2010

Correspondence: A Xu, State Key Laboratory of Biocontrol,Guangdong Province Key Laboratory of Therapeutic FunctionalGenes, National Engineering Center for Marine Biotechnology ofSouth China Sea, College of Life Sciences, Department ofBiochemistry, Sun Yat-Sen University, 135 W.xingang RD,Guangzhou 510275, PR China or X Xiao, School of Life Sciencesand Biotechnology, State Key Laboratory of Ocean Engineering,Shanghai Jiaotong University, 800 Dongchuan RD, Shanghai200240, PR China.E-mails: lssxal@mail.sysu.edu.cn or xoxiang@sjtu.edu.cn6These authors contributed equally to this work.

The ISME Journal (2011) 5, 414–426& 2011 International Society for Microbial Ecology All rights reserved 1751-7362/11

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microhabitats for chemolithoautotrophic microor-ganisms that fix inorganic carbon using chemicalenergy obtained through the oxidation of reducedinorganic compounds contained in the hydrother-mal fluids, converting the geothermally derivedenergy into microbial biomass (for example,Reysenbach and Shock, 2002).

Since the discovery of deep-sea hydrothermal ventsystems, the microbial diversity in these systems hasbeen the subject of studies using both cultivation andcultivation-independent molecular methods (forexample, Nakagawa et al., 2005; Huber et al., 2007).These studies have reported a remarkable phylo-genetic diversity of microbes inhabiting the chimneywalls, yet the metabolic diversity and physiologicalpotential of these microbial communities are onlybeginning to be revealed. Progress in understandingthis diversity has been made largely because of therecent developments in high-throughput genomictechnologies that enable microbial ecologists toaddress complex evolutionary and ecological hypo-theses at a community scale (Tyson et al., 2004; Tringeet al., 2005; DeLong et al., 2006; Grzymski et al.,2008). With advances in sequencing technologies,large-scale genomic surveys of microbial communities(metagenomics) are becoming routine, making deci-phering the genetic and functional ‘differences thatmake a difference’ within and among microbialhabitats increasingly feasible. Using metagenomicanalysis, the metabolic potential and environmentaladaptation strategies of epi- and endosymbionts ofdeep-sea hydrothermal vent invertebrates have beenelucidated (Newton et al., 2007; Grzymski et al.,2008). The metagenome of a Lost City carbonatechimney biofilm, which is so far the only publishedmetagenome from any deep-sea vent chimney, wasfound to contain a remarkable abundance anddiversity of genes potentially involved in lateral genetransfer (Brazelton and Baross, 2009). However, itremains elusive whether lateral gene transfer is acommon occurrence in chimney biofilms, or whetherit is restricted to certain deep-sea hydrothermal ventenvironment, such as Lost City. Comparative meta-genomic analysis of chimney biofilms from eitherdifferent locations and/or with different geochemistrywould provide valuable information on identifyingunique traits of these largely unknown deep-seamicrobial communities, in particular in comparisonwith those from other environments.

Here, we present the first comparative meta-genomic analysis of two different deep-sea hydro-thermal vent chimney microbiomes: one from acarbonate chimney at the ultramafic hosted LostCity vent site that is characterized by relativelylow temperature and high pH (o90 1C, pH 9–11), theother one from sulfide chimney 4143-1 at the basalt-hosted Mothra hydrothermal vent field on the Juande Fuca Ridge that is characterized by venting ofhigh temperature (4300 1C) and low pH (pH 2–3)fluids. This comparative metagenomic approach isproviding very useful information on the adaptation

and metabolic potential of the whole microbialcommunity in the chimney wall.

Materials and methods

Samples collection, DNA isolation and fosmid libraryconstructionThe sulfide chimney (4143-1) was collected in 2005by the submersible Alvin, supported by the R/VAtlantis, from the Mothra Field of the Juan de FucaRidge, located B300 km west of Vancouver Island,Canada. Precautions were taken during sampling andhandling of the chimney samples to preserve theirintegrity for microbiological analyses. The chimneyswere stored at �20 1C immediately following collec-tion, kept on dry ice during transportation, and storedat �80 1C until further analysis. The genomic DNAwas isolated from the outer portion of the chimneyand used for fosmid library construction as previouslydescribed (Meng et al., 2009). A total of 2880 fosmidclones were grown overnight in Luria–Bertani broth,extracted separately using the Axyprep-96 Plasmid kit(Axygen, Union City, CA, USA) and pooled in equalquantities of 20 ng/ml for pyrosequencing.

Sequencing, assembly and annotationSequencing was performed using pyrosequencingon 454 Life Sciences GS FLX system platforms witha practical limit of 250 bp. The total amount ofsequence data obtained for 4143-1 fosmid library areas follows: 578 567 reads, containing 133 MB of rawsequence. SeqClean software (http://compbio.dfci.harvard.edu/tgi/software/) was used to eliminatesequences of host genome (Escherichia coliEPI300) (31 MB) and fosmid sequence (31 MB), thisreduced the data set to: 308 034 reads, containing71 MB of sequence. Those data were assembled withTGICL software (http://compbio.dfci.harvard.edu/tgi/software/) (Zhang et al., 2000). The result is31 405 reads with average length of 484 bp. And22 968 singletons with average length of 196 bpcannot be assembled. Initially, those contigs andsingletons assembly was mapped to a database ofknown 16S rDNA sequences (Ribosomal DatabaseProject release 9.3.3) using Blastn algorithm. These16S rDNA anchors that longer than 50 wereclassified into respective taxonomic groups usingthe Ribosomal Database Project Classifier. Openreading frames (ORFs) were predicted by glimmer3.02 software for those contigs longer than 1000 bp.The predicted ORFs, contigs without any putativeORFs by glimmer 3.02 and singletons were com-pared against the National Center for BiotechnologyInformation non-redundant database using Blastxwith an expectation value cut-off of o10�5. Thosesequences that had reliable hits with the non-redundant database were compared against theKyoto Encyclopedia of Genes and Genomes (KEGG)sequence database and Clusters of OrthologousGroup sequence database by using an expectation

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value cut-off of o10�5. The range, mean, median ofthe expectation values were shown in Supplemen-tary Table S1.

Two-way clustering analysis and bootstrap resamplingmethodologiesFor taxonomic binning, all genomic sequences fromLost City hydrothermal field, acid mine drainage,gutless worm consortium, Peru Margin sub-seafloorsediments, whale falls and North Pacific Gyre waterwere analyzed with the method described in theprevious paragraph. Blast results were tabulated andthe percentage of sequences within each KEGGpathway or Clusters of Orthologous Group categorywas calculated. Dendrograms were generated byusing CLUSTER (http://rana.lbl.gov/EisenSoftware.htm) and visualized with TREEVIEW (http://rana.lbl.gov/EisenSoftware.htm). To test whethersignificant difference exists between any two pro-tein sequence bins for a particular KEGG subsystem,a bootstrap sampling method (DeLong et al., 2006)was used and is summarized in the following. Forany two protein sequence bins to be compared, theprocess includes three steps: (1) 10 000 sequenceswere sampled from each bin and the difference inthe number of KEGG subsystems was calculated. Werepeated this process 1000 times and recorded themedian difference for each subsystem; (2) two setswith 10 000 sequences were sampled from either binwith equal probability, and the difference in thenumber of KEGG subsystems between these two setsof 10 000 was calculated. This process was alsorepeated 1000 times and (3) the P-value for aparticular subsystem was calculated as the numberof differences in step (2) larger than the mediandifference from step (1), divided by the number ofreplicates in step 2 (that is, 1000 here).

Screening for target fosmid clonesEight fosmids carrying genes coding for Calvin–Benson–Bassham cycle (CBB), reductive tricarbo-xylic acid (rTCA), sulfur oxidation or denitrificationfunctions were identified by PCR with specificprimers designed from target contigs or singletons.Information is shown in Supplementary Table S2.Positive clones were grown overnight in Luria–Bertani broth, extracted using the Axyprep plasmidminiprep kit and sequenced by primer walking withABI 3730 XL from Applied Biosystems (Foster City,CA, USA). Those sequences were also assembledwith TGICL software found with the glimmer 3.02software. Then, Blastx was used to compare all thepredicted ORFs the National Center for Biotechno-logy Information non-redundant database, using anexpectation value cut-off of o10�5.

Reconstructions of the microbiome’s metabolismIdentified genes were assigned KEGG Orthologynumbers using the latest release of KEGG (v. 47),

which allowed us to assign identified genes to KEGGmaps. The odds ratio was then used to define if a genewas enriched in the environment. The odds ratio canbe thought of as the likelihood of observing a givengene in the sample relative to the comparison data set.We calculated the odds ratios using (A/B)/(C/D)where A is the number of hits to a given gene in thedeep-sea hydrothermal vent metagenome, B is thenumber of hits to all other genes in the deep-seahydrothermal vent metagenome, C is the number ofhits to a given gene in the comparison data set and Dis the number of hits to all other genes in thecomparison data set (Gill et al., 2006). To minimizethe error caused by the database size, both of thechimneys metagenomes were resampled to 3 000 000to approximate the size of the KEGG database.

Accession numbersThe GenBank Sequence Read Archive accessionnumber for the source sequences of 4143-1 Fosmidlibrary is SRA009990.1. The accession numbers ofeight fosmids are GU191796-803.

Results and discussion

Fosmid library construction and sequencingThe sample 4143-1 represented the outer-layer of anactively venting (316 1C) black-smoker chimneyfrom the Mothra hydrothermal vent field on theEndeavour segment of the Juan de Fuca Ridge. From4143-1, a fosmid library was constructed thatcontained 2880 fosmid clones without amplifica-tion. The sizes of the inserted fragments of theclones in the library were checked by enzymedigestion and gel electrophoresis. It was calculatedthat the average insert size was about 20 kb. Each ofthe fosmid clones was extracted separately andmixed in equal quantity for direct pyrosequencing.A total of 308 034 reads with an average sequencelength of 227 bp were generated after removing thepCC1FOS vector sequence and the chromosomesequence contamination from the host strain.Sequence data were assembled with TGICLsoftware. The clustering is performed by aslightly modified version of National Center forBiotechnology Information’s megablast, and theresulting clusters are then assembled using CAP3assembly program (Details see the Materials andmethods). The sequenced microbiome of the hydro-thermal vent sample consisted of 31 405 contigs and22 968 singletons, a total of 15.3 MB. The averageG-C% of the sequences was determined to be 49%,and a total of 21 836 ORFs were predicted from thedata set, more than 70% of which could be classifiedinto function sets in the Clusters of OrthologousGroup category (Table 1).

The taxonomic diversity of the metagenomicsequence was assessed by SSU rRNA gene sequenceanalysis. All 26 contigs and 11 singletons that

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could be assigned to SSU rRNA gene sequences wereidentified as bacterial 16S rRNA gene sequences. Thesebacterial 16S rRNA gene sequences were found to bemainly affiliated with Gammaproteobacteria (25.5%);Bacteroidetes (15.7%); Alphaproteobacteria (13.7%);Betaproteobacteria (7.8%); and Planctomycetes andDeltaproteobacteria (5.9% each) (SupplementaryFigure S1), supporting our previous 16S rRNAgene library analysis that also had predominantlyGammaproteobacteria (Wang et al., 2009).

Comparative metagenome analysisThe predicted proteins from the 4143-1 chimneymetagenome were classified into functional cate-gories at higher order cellular processes from theKEGG database. Comparative metagenomic analysesusing the two-way clustering method (DeLong et al.,2006) were conducted. The metagenomes beingcompared were obtained from diverse environ-ments, including a biofilm of a carbonate chimneyfrom the Lost City hydrothermal vent field (901C,pH 9–11 fluids); two biofilm samples from acidmine drainage (Tyson et al., 2004), a gutless wormconsortium (Woyke et al., 2006); a whale fallmicrobial community (which also represents adeep-sea reducing environment with similarities tohydrothermal vents (Tringe et al., 2005)); deeplyburied marine sediments (Biddle et al., 2008), andfinally pelagic microbial communities from theNorth Pacific Subtropical Gyre (DeLong et al., 2006).

The two-way clustering of samples and KEGGfunctional pathway, in which categorized sequencespercentage is indicated by yellow shading, is dis-played in Figure 1. The Hawaii Ocean water samplesclustered together and predicted protein sequenceswere differentially distributed in photic zone anddeep water as has been shown before (DeLong et al.,2006). The sub-seafloor sediment samples formed onegroup, and also had a depth-related clusteringtendency. The clustering pattern of the three whalefall samples is also consistent with the one that hasbeen reported before (Tringe et al., 2005). The twosamples from chimneys clustered most closely andthey formed a group with the acid mine drainagebiofilm, whereas the Gutless worm consortiumgrouped with the whale fall samples (Figure 1). Avery similar grouping pattern was obtained byclustering of samples and Clusters of OrthologousGroup functional gene categories (SupplementaryFigure S2). Although the physicochemical conditionsof the two chimney environments are strikinglydifferent (for example, the venting fluid of 4143-1 isacidic, around 310 1C, whereas that of Lost Citysample is o90 1C, pH 9–11), they both representchimney wall samples that are continuously exposedto highly dynamic conditions characterized by vigor-ous mixing of reduced, high-temperature fluids ofextreme pH with oxygenated, cold sea water of pH 8.The two samples from chimneys are enriched ingenes associated with mismatch repair and homo-logous recombination (Figure 1 and Table 2),

Table 1 Summary of sequences from the chimney 4143-1

Item Gene Value

Base pairs 71 064 900GC content (%) 49%Average read length 227Singletons 22 968Average singleton length 196Contigs 31 405Average contig length 484Best hit blastx contigs with non-redundance database 21 836Genes in each Clusters of Orthologous Group category RNA processing and modification 6

Transcription 642Replication, recombination and repair 1631Cell-cycle control, cell division and chromosome partitioning 290Defense mechanisms 427Signal transduction mechanisms 676Cell wall/membrane/envelope biogenesis 1236Cell motility 134Intracellular trafficking, secretion and vesicular transport 273Posttranslational modification, protein turnover and chaperones 958Energy production and conversion 1567Carbohydrate transport and metabolism 849Amino-acid transport and metabolism 1690Nucleotide transport and metabolism 580Coenzyme transport and metabolism 743Lipid transport and metabolism 521Inorganic ion transport and metabolism 962Secondary metabolites biosynthesis, transport and catabolism 237General function prediction only 1643Function unknown 984

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suggesting that the microbial communities thereinhave evolved extensive DNA repair systems to copewith the extreme environment that subjects thegenomes to damaging effects by physical and toxicchemical agents (such as high concentrations ofhydrogen sulfide, trace metals, high temperatureand radionuclides) in the deep-sea hydrothermal ventenvironment (Pruski and Dixon, 2003).

Beside the abundant transposases in the Lost Citycarbonate chimneys metagenome as described byBrazelton and Baross (2009), the percentage trans-posases in the metagenomes of the chimney_4143-1,acid mine drainage biofilm and gutless wormconsortium are 1.10, 2.27 and 2.8%, respectively,which are higher than other samples with little or nobiomass contribution from biofilms. Brazelton andBaross (2009) have shown that the possible carriersof transposases in the Lost City sample are smallextracellular DNA fragments (Brazelton and Baross,2009), similar to our findings for chimney 4143-1(Supplementary Figure S3).

The two samples from chimneys have a highpercentage of genes for chemotaxis and flagellarassembly, commonly used by prokaryotes to senseand respond to environmental cues (Figure 1 andTable 2). The relatively high abundance of methyl-accepting chemotaxis proteins in the two meta-genomes from vent chimneys could be seen asan adapting strategy of the microbial communitiesin response to the steep chemical and thermalgradients observed at hydrothermal vents.

Besides the similarities between the two meta-genomes from chimneys, there are also distinctdifferences. For example, the sulfide chimney4143-1 appeared more enriched in genes in nitrogenmetabolism (Figure 1 and Table 2). Nevertheless,the comparative metagenome analysis clearlydemonstrates that functional gene profiling is auseful and reliable proxy to delineate biologicalcommunities from different environments.

Autotrophic carbon metabolismThe assimilation of carbon dioxide (CO2) intoorganic material is globally the most importantbiosynthetic process. There are six pathways forCO2 fixation, that is, the CBB cycle, rTCA cycle(Arnon cycle), the 3-hydroxypropionate (3-HP)cycle, the reductive acetyl coenzyme A (acetyl-CoA) pathway (Wood–Ljungdahl pathway), the3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB)cycle and the dicarboxylate/4-hydroxybutyrate cycle

(Nakagawa and Takai, 2008), the latter two of whichwere only recently characterized in a thermoacidophi-lic archaeon, Metallosphaera sedula and Ignicoccushospitalis, respectively (Huber et al., 2008).

Odds ratios for cbbLS and/or cbbM encodingRubisCO and cbbP encoding phosphoribulokinasein chimney 4143-1 and Lost City carbonate chimneymetagenome are highly enriched relative to theKEGG database (Figure 2a). To obtain the genomiccontext of the CBB cluster in the chimney 4143-1metagenome, four fosmids were positively identi-fied by PCR with specific primers and subsequentlysequenced as described in Materials and methods.The two unique genes for the CBB cycle, cbbLSand/or cbbM encoding RubisCO and cbbP encodingphosphoribulokinase, were detected at high frequen-cies (Figure 2a). One fosmid clone containingcbbL and three clones containing cbbM genes wereselected, and the adjacent regions were partiallysequenced. The obtained cbbL sequence had 91%identity with that from the gammaproteobacterialendosymbiont of the clam Solemya velum, whichexpresses a form IA RuBisCO. The fosmid cbbLSgenes were followed by genes encoding CbbQ andCbbO, which are important proteins implied in theassembly of RubisCO. Upstream of cbbLS, no genesinvolved in the CBB cycle were identified (Figure 3).A similar cbb gene cluster with conserved cbbLSQOgene organization has been found in the chemoauto-trophic Gammaproteobacteria Thiomirospira cruno-gena XCL-2, Hydrogenovibrio marinus and theendosymbiont of Solemya velum (Badger and Bek,2008). It has been observed that the CBB cycle genesof obligate autotrophs apparently do not form CBBoperons that would facilitate coordinated regu-lation, presumably because these genes are constitu-tively expressed and, therefore, do not requireregulation in these obligate autotrophs (Scott et al.,2006). In the obligate autotroph T. crunogena XCL-2,as well as in H. marinus, the genes encoding otherenzymes of the CBB cycle are scattered throughouttheir respective genomes. Similar to T. crunogenaXCL-2, the fosmid Rubisco genes cbbLS do not form aCBB operon with cbbP, suggesting that the fosmidcbbL gene may come from an obligate chemo-autotrophic organism (Figure 3). The three obtainedcbbM genes exhibit 60–70% sequence identity to oneanother. A DNA fragment of approximately 8.6 kbcontaining the cbbM1 was analyzed in greater detail,and it was found that the cbbM1 gene was likely partof a CBB operon containing other CBB genes, as well

Figure 1 Two-way Clustering analysis of samples and Kyoto Encyclopedia of Genes and Genomes (KEGG) categories on the basis of thepercentage of KEGG-annotated sequences found in each category. The yellow shading is proportional to the percentage of identifiedsequences falling into each sample. KEGG categories with a standard deviation greater than 0.2 of observed values, having 40.2 ando8.0 of the total KEGG-categorized genes are shown. KEGG categories mainly discussed in the text are boxed. Hot_10 to Hot_4000: seawater of Hawaii Ocean Time Series station; Peru_Margin: Peru Margin subseafloor biosphere project (D04684S01, D04684S02,D3EISHW01, D3EISHW02, EC6HCKB01); Whale_Fall_1 to Whale_Fall_3: a rib bone, a microbial mat isolated from gray whale carcass inthe Santa Cruz Basin, and a whale bone in the Southern Ocean, respectively; Lost City: Lost City carbonate chimneys sample; Vent 4143-1:hydrothermal vent sample 4143-1 (this study); Acid mine drainage: Biofilm microbial community from acid mine drainage site at IronMountain, CA; Gutless worm consortium: endosymbionts of gutless worm, Mediterranean Sea.

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as carboxysome structural genes, similar to those ofRhodopseudomonas palustris BisB5, a facultativechemoautotroph (Figure 3). Although only short DNAfragments were sequenced (around 3.4kb each) aroundthe remaining two cbbM genes (Fosmid_aI, Fosmid_contig), they are not likely to constitute CBB operonswith other CBB genes (opposite gene direction of cbbRand cbbM, see Figure 3), and these two cbbM genesmay come from obligate autotrophs. Form I and form IIRuBisCOs have different affinities to CO2, with form IRuBisCOs generally found to be adapted to higher CO2

concentrations. Some autotrophs contain both the formI and form II Rubiscos, indicating the adaptation ofthese organisms to varying CO2 concentrations in theenvironment. Similarly, both form I and form IIRubiscos from putative obligate and facultative auto-trophs were found in the chimney wall, suggesting thatmembers of the microbial community reflect adapta-tions to the highly dynamic physicochemical condi-tions in the chimney wall.

The rTCA cycle has recently been considered asan important CO2 fixation pathway in the deep-seavent environments (Campbell and Cary, 2004;Nakagawa and Takai, 2008). The three key enzymesof rTCA cycle, ATP citrate lyase, 2-oxoglutarate:ferredoxin oxidoreductase and pyruvate:ferredoxinoxidoreductase, were identifiable in the 4143-1metagenome (Figure 2b), suggesting the presenceof the complete rTCA cycle for inorganic carbonfixation by autotrophic organisms in the chimney4143-1 wall. For the Lost City carbonate chimneysequences, fewer putative orthologs involved inrTCA cycle were detected and ATP citrate lyasewas missing. Two gene fragments annotated asputative ATP citrate lyase genes were identified inthe 4143-1 metagenome through KEGG pathwayanalysis, and the fosmid clone named fosmid_aFcontaining the putative aclB-like gene was posi-tively identified and sequenced. Blast analysis ofthis gene showed that it had high sequence identity(70%) with a putative succinyl-CoA synthetaseb-subunit gene (Mmc13639) from the magnetotacticcoccus strain MC-1. Strain MC-1 has been demon-strated to use the rTCA cycle for carbon fixation, butinitially no bona fide ATP citrate lyase could beidentified (Williams et al., 2006). However, recently,the genome sequence of MC-1 has been completed,which has led to the suggestion that the genesMmc13638 and Mmc13639 encode the large (aclA)and small (aclB) subunits of an ATP-dependentcitrate lyase (Schubbe et al., 2009). The aclA gene ofstrain MC-1 contains an inserted portion thatprevented its previous identification as an ATPcitrate lyase. Phylogenetic analysis suggests that theputative aclB contained on the fosmid forms a newaclB cluster together with the putative aclB fromMC-1 (Supplementary Figure S4A). The genomicregion surrounding the putative aclB on the fosmidwas sequenced (approximately 18 kb, Supplemen-tary Figure S4B). A putative malate dehydrogensewas found upstream of the putative aclB, whereasT

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no other genes that may be involved in the rTCAcycle were found throughout the 18 kb DNA se-quence fragment. In particular, no homolog toMmc13638, which represents the large subunit ofthe ATP citrate lyase, could be identified on thefosmid. Up till now, there have been no reportsindicating that the large and the small subunit of theATP citrate lyase may be separately located on thechromosome, making it questionable if the identi-fied aclB-like gene on the fosmid is functional. Ourresults suggest that some microorganisms in thechimney environment may use the rTCA cycle forcarbon fixation, utilizing the novel ATP citrate lyasefor citrate cleavage. The possible presence of an ATPcitrate lyase most closely related to that of the

Gammaproteobacterium Endoriftia persephone andthe magnetotactic Alphaproteobacterium strain MC-1further argues for the importance of Gamma- andAlphaproteobacteria for carbon fixation in the chimneywall. It is possible that organisms use both rTCA andCBB, similar to what has been hypothesized forEndoriftia persephone (Markert et al., 2007).

There are two evolutionarily distinct forms ofcarbon monoxide dehydrogenase (CODH). The aero-bic CODH is used by CO oxidizers to couple theoxidation of CO to oxygen reduction, whereas theanaerobic CODH is used by anaerobic microorga-nisms to couple CO oxidation to sulfate reductionor to reduce CO2 to either acetate (acetogenesis)or methane (methanogenesis) (Wu et al., 2005;

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Figure 2 Enrichment of genes for major carbon fixation pathways. (a) CBB cycle; (b) rTCA cycle; (c) 3-HP cycle; (d) reductive acetyl-CoApathway. Diagrams are based on KEGG pathway maps. When available, enzyme classification numbers for each step were included inboxes. Box color indicated the odds ratio of each enzyme with darker red and green color representing higher odds ratio of respectiveenzyme in the chimney 4143-1 and lost city chimney metagenome, respectively.

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King and Weber, 2007). Comparing with 910sequences coding for CODH in about 61 M of PeruMargin subseafloor biosphere sequences, only sevenand four sequences were found in the 4143-1 andthe Lost City carbonate chimney metagenome,respectively (Figure 2d). There were four genefragments annotated as carbon monoxide dehydro-genase, large subunit or catalytic subunit in the4143-1 metagenome. These genes are most closelyrelated to those of Jannaschia sp. CCS1 (Figure notshown). The fourth gene is closely related to theCODH gene from Methanosaeta thermophila, sug-gesting its origin from a methanogen. At deep-seahydrothermal vents, the majority of microorganismsusing the reductive acetyl-CoA pathway for carbonfixation are likely to be methanogens. The existenceof putative methanogens in the 4143-1 sample wasalso revealed by mcrA gene analysis, indicating thatmethanogens affiliated with Methanomicrobiales,Methanosarcinales and Methanobacteriales exist inthe chimney wall (Wang et al., 2009).

The key enzymes for the 3-HP cycle are acetyl-CoA/propionyl-CoA carboxylase, malonyl-CoAreductase and propionyl-CoA synthase. Not all ofthese enzymes were detected, and the detectionfrequencies of 3-HP genes were not high in the twochimney metagenomes (Figure 2c). Furthermore,4-hydroxybutyryl-CoA dehydratase, the key enzymeof the 3-HP/4-hydroxybutyrate cycle was not found,although it occurs at high frequencies in the GlobalOcean Sampling database (Berg et al., 2007). Ourdata further support the previous observations thatthe 3-HP cycle of the 3-HP/4-hydroxybutyrate may

not be an important carbon fixation pathway atdeep-sea hydrothermal vents.

Sulfur oxidationThe key enzymes involved in both the sulfur-compound oxidation (Sox)-dependent pathwayand the adenosine-50-phosphosulfate-dependent path-way were found to be abundant in the metagenome,whereas sulfite oxidase (EC1.8.3.1) and/or sulfitedehydrogenase (EC 1.8.2.1), which are involved inthe sulfite:cytochrome c oxidoreductase pathway,were not present in the chimney 4143-1 metagenome(Figure 4). A similar sulfur oxidation pathway wasfound in the Lost City carbonate chimney, but inlower abundance compared to chimney 4143-1.

In the metagenome, the discovered sox genesinclude soxA, soxB, soxD, soxX, soxY and soxZ,indicating the presence of chemoautotrophs capableof oxidizing various reduced sulfur compounds tosulfate. To obtain more information about theselithotrophs, two fosmid clones containing soxBgenes were selected and sequenced. The tworetrieved soxB genes are closely related to eachother and clustered with the corresponding genesfrom Halothiobacillus hydrothermalis and H. neapo-litanus, two halophilic obligate sulfur-oxidizingchemolithoautotrophic Gammaproteobacteria (Sievertet al., 2000; Figure 5). The soxB gene from fosmid_soxB1 was separated from other sox genes, as hasbeen observed in T. crunogena (Scott et al., 2006).The soxB from fosmid_soxB2 was linked with othersox genes to form a soxXYZAB cluster on the

500bp

Thiomicrospira crunogena XCL-2 (NC_007520)

Solemya velum gill symbiont (AY531637.1)

Hydrogenovibrio marinus (AB122070) Form IAq

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Rhodopseudomonas palustris BisB5 (NC_007958)

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cbbM3 cbbQtransposase

hphp

fbaC2 cbbP

cbbR

Figure 3 Genomic organization of the CBB cluster. The open reading frame (ORF) Finder program was used to perform ORF analysis.RubisCO genes (cbbLS and cbbM) are green, cbbR genes are blue, other genes encoding CBB cycle enzymes are black, carboxysomestructural genes are gray. hp, hypothetical protein; ribC, friboflavin synthase subunit alpha; tkt, transketolase; fbaC2, fructose-1,6-bisphosphate aldolase, class II; fbp2, fructose-1,6-bisphosphatase, class II; cbbR, transcriptional regulator LysR family; cbbP,phosphoribulokinase genes; cbbQ, P-loop containing nucleoside triphosphate hydrolases; cbbO, Nitric oxide reductase activationprotein; pgcM, phosphatase/phosphohexomutase HAD superfamily; natB, ABC-type transporter permease protein.

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chromosome, but the single sox operon thathas been described in facultative autotrophicsulfur-oxidizers, such as Paracoccus versutus andRhodopseudomonas palustris (Schutz et al., 1999;Friedrich et al., 2000; Figure 5) was not found.Obligate autotrophic sulfur-oxidizers appear to nothave the complete set of the sox genes organized in asingle operon as observed in facultative autotrophs,possibly because the sox genes are constitutivelyexpressed and, therefore, the sox gene organizationinto a single operon may not be strongly evolutio-narily selected (Scott et al., 2006). On the basis ofthese observations, as well as the phylogeny ofsoxB, the two fosmid fragments containing thesox genes are most likely derived from obligatechemoautotrophic sulfur-oxidizing bacteria relatedto the sulfur-oxidizing Halothiobacillus. In themetagenome, the genes encoding the putativesulfide quinone oxidoreductase and the flavocyto-chrome c/sulfide dehydrogenase (FccAB) were bothidentified (Figure 4). Both these enzymes are knownto catalyze the oxidation of sulfide to sulfur,whereas FccAB was hypothesized to be adapted tolow sulfide concentrations (Mussmann et al., 2007).The detection of these enzymes may be a reflectionof different niches corresponding to varying sulfideconcentrations in the chimney wall. The elementalsulfur formed by sulfide quinone oxidoreductase or

fccABsqr

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Figure 4 Sox-dependent and Sox-independent sulfur oxidationpathways identified in this study. SO4

2�, sulfate; SO32�, sulfite; S0,

sulfur; S2O32�, thiosulfate; S2�, sulfide; APS, adenylylsulfate; sqr,

sulfide:quinone oxidoreductase; fccAB, flavocytochrome c/sulfidedehydrogenase; sox, sulfur oxidation multienzyme complex.Box color indicated the odds ratio of each enzyme with darkerred and green color representing higher odds ratio of respectiveenzyme in the chimney 4143-1 and lost city chimney metagenome,respectively. The color scales are the same as Figure 2a.

B

C D

X Y Z A

B

C D

B A Z Y

Y Z A B

X Y Z BAF

X Y Z BA

E Y HC D FF

B

X Y Z BA EC D FR S V W

X Y Z BA

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C

W

B

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X Y Z BA LC DR S V W

B

B

BA

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B

BA X

X

FDCX Z BAV YW F

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ino

sum

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Figure 5 Sox gene cluster organization in proteobacteria. Sox genes are gray, the gene between the sox genes are white. The letters in theboxes are abbreviated from corresponding sulfur oxidizing enzymes. Fosmid_soxB1 and fosmid_soxB2 in this study were marked withblack diamonds. The cladogram was based on an alignment of 820 amino acids of the soxB genes. Sequences were aligned usingClustalW and the dendrogram was constructed using the neighbor-joining method by Mega 4.1.

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FccAB could be further oxidized. The genes encod-ing the dissimilatory sulfate reductase, adenosine-50-phosphosulfate reductase and ATP sulfurylasewere enriched in the metagenome, suggesting thepresence of the so-called ‘reverse dissimilatorysulfate reductase’ pathway for sulfur oxidization(Hipp et al., 1997). From the microbiome, a total of17 gene fragments annotated as partial dissimilatorysulfite reductase subunit A were retrieved. Around16 of them could be potentially placed into the dsrAclusters from sulfide oxidizers (data not shown).The fact that the majority of the dissimilatory sulfitereductases cluster with those from sulfide-oxidizersfurther confirms the presence of a functional reversedissimilatory sulfate reductase pathway for sulfuroxidization in the chimney microbial system.

In general, the oxidation of reduced sulfur com-pounds can be coupled to the reduction of electronacceptors, including oxygen and nitrate. In themetagenome, genes encoding the cbb3-type cyto-chrome c oxidase were identified, whereas genesencoding the widespread aa3-type cytochromec oxidase were not found. The cbb3-type cytochromec oxidase has a higher affinity for oxygen than theregular aa3-type cytochrome c oxidase, suggesting thatmembers of the microbial community in the chimneywall are adapted to microoxic to anoxic conditions,similar to what has been described for the sulfur-oxidizing bacterium T. crunogena (Scott et al., 2006).

Nitrogen metabolismThe nitrogen cycle at deep-sea hydrothermal ventenvironments is less understood than the carbon andsulfur cycles. Intense nitrification and denitrificationhave been detected in some hydrothermal environ-ments (Mehta et al., 2003), and biological nitrogenfixation has been identified as an important processcontributing to the nitrogen cycle at deep-sea vents

(Mehta et al., 2003; Rau, 1981). However, most of themicroorganisms that are involved in the nitrogencycle at vents are still largely unknown. Highammonium and nitrate concentration can be foundin sediment-hosted hydrothermal fields, such as theGuaymas Basin and Okinawa Trough Backarc Basin,as well as the Endeavor segment of the Juan de FucaRidge (Ishibashi et al., 1995; Mehta et al., 2003). Ithas been demonstrated that ammonium is rapidlyconsumed by chemoautotrophs in the hydrothermalplume of vents on the Juan de Fuca Ridge (Lam et al.,2004). However, we did not find genes encoding forammonia monooxygenase (amoA), a key enzyme forammonia oxidization, in the metagenome.

On the other hand, the chimney 4143-1 meta-genome is highly enriched in genes required for thecomplete denitrification pathway (Figures 1 and 6),suggesting that in addition to oxygen, nitrate couldbe an important electron acceptor, possibly coupledto sulfur-oxidation. All the genes, including nar(nitrate reductase), nos (nitrous oxide reductase), nir(nitrite reductase) and nor (nitric oxide reductase),for denitrification could be identified in the meta-genome (Figure 6). The majority of the genesinvolved in denitrification are closely related tothose from Beta and Alphaproteobacteria (data notshown). A fosmid clone (Fosmid_X) containinga narG fragment was selected and sequenced. Theobtained narG has the highest identity with narGfrom Thiobacillus denitrificans (76%), an obligatechemolithoautotrophic bacterium capable of gainingenergy by coupling the oxidation of reduced sulfurcompounds to denitrification, as well as to aerobicrespiration (Figure 7). The DNA fragment furthershowed synteny to Thiobacillus denitrificans, as thegenes narK, narH, narJ and narI were found inidentical order adjacent to narG. These data implythat sulfur oxidation coupled to denitrification is

Urea1.7.3.4HydroxylamineNitrite

1.7.1.31.7.1.4

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1.7.7.11.7.7.2

1.7.2.2

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1.7.99.7 1.7.99.6Nitric oxide Dinitrogen

oxideNitrogen

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1.7.99.1 3.5.1.5

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Figure 6 Components of the nitrogen metabolism pathways identified in this study. Box color indicated the odds ratio of each enzymewith darker red and green color representing higher odds ratio of respective enzyme in the chimney 4143-1 and lost city chimneymetagenome, respectively. The color scales are the same as Figure 2a.

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likely to be an important energy-generating pathwayfueling the microbial community in the outer wall ofthe chimney. However, most of the key enzymes ofdenitrification pathway were not found in theLost City carbonate chimney sequences (Figure 6),indicating that denitrification is not a universalpathway utilized by microbial communities inhabit-ing deep-sea vent chimneys, but is rather specific forparticular vent communities, such as those from theJuan de Fuca Ridge. More analyses of chimneys froma variety of environments and locations should shedmore light on this interesting observation.

In summary, comparative metagenomic analysesdemonstrated that functional gene profiling couldbe a useful and reliable proxy to reflect the specificenvironment in which the biological communitiesreside. The metagenomes from the sulfide chimney4143-1 and the carbonate chimney from Lost Cityclustered most closely. They are especially enrichedin genes for mismatch repair and homologousrecombination, suggesting that the microorganismsin the chimney walls have to cope with a higher rateof DNA damage caused by the extreme conditions oftheir habitat. The high percentage of transposases inthe two samples from chimneys suggests that lateralgene transfer may be a common occurrence in thehigh temperature chimney biospheres. The meta-genome further reveals that autotrophic carbonfixation in the sulfide chimney 4143-1 communitywas mainly a product of the CBB cycle, whichappeared to be primarily driven by the oxidation ofreduced sulfur compounds through both Sox-dependent and adenosine-50-phosphosulfate-depen-dent pathways, using either oxygen or nitrate asterminal electron acceptors. Thus, the availability ofreduced sulfur compounds, as well as oxygen andnitrate, appear to be key parameters structuring themicrobial community. On the basis of the genomicorganization of the key genes of the carbon fixationand sulfur oxidation pathways contained in thelarge genomic fragments, both obligate and faculta-tive autotrophs appear to be present and contri-buting to biomass production. In addition, highabundance of chemotaxis genes in the metagenomereflected the adaptation of the organisms to a highlydynamic environment.

Acknowledgements

This work was supported by the China Ocean MineralResources R&D Association (COMRA DYXM115-05),the National Natural Science Foundation of China(40625016), the State High-Tech Development Project(2006AA09Z433, 2007AA091904 and 2008AA092603),project 2007DFA30840 of International S&T CooperationProgram from the Ministry of Science and Technology ofChina, key project of the Commission of Science andTechnology of Guangdong Province and Guangzhou Cityand project of Sun Yet-sen University Science Foundation.SMS was supported by National Science Foundationgrant OCE-0452333 and a fellowship awarded by theAlfried Krupp Wissenschaftskolleg Greifswald, Germany.We thank Huaiyang Zhou (Tongji University, Shanghai,China), Maurice A. Tivey (Woods Hole OceanographicInstitution, Woods Hole, MA, USA), Marvin D Lilley(University of Washington, Seattle, WA, USA), DeborahKelly (University of Washington, Seattle, WA, USA), KangDing (University of Minnesota, Minneapolis, MN, USA) andall of the crew members from R/V Atlantis/DSV Alvin fortheir efforts and help in sample collection.

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narJnarHnarGnarK2narX

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