APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2009, p. 7436–7444 Vol. 75, No. 230099-2240/09/$12.00 doi:10.1128/AEM.01283-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

CO Dehydrogenase Genes Found in Metagenomic Fosmid Clonesfrom the Deep Mediterranean Sea�†

Ana-Belen Martin-Cuadrado, Rohit Ghai, Aitor Gonzaga, and Francisco Rodriguez-Valera*Evolutionary Genomics Group, Departamento Produccion Vegetal y Microbiología, Universidad Miguel Hernandez,

San Juan de Alicante, Spain

Received 3 June 2009/Accepted 28 September 2009

The use of carbon monoxide (CO) as a biological energy source is widespread in microbes. In recent years,the role of CO oxidation in superficial ocean waters has been shown to be an important energy supplement forheterotrophs (carboxydovores). The key enzyme CO dehydrogenase was found in both isolates and metage-nomes from the ocean’s photic zone, where CO is continuously generated by organic matter photolysis. We havealso found genes that code for both forms I (low affinity) and II (high affinity) in fosmids from a metagenomiclibrary generated from a 3,000-m depth in the Mediterranean Sea. Analysis of other metagenomic databasesindicates that similar genes are also found in the mesopelagic and bathypelagic North Pacific and on thesurfaces of this and other oceanic locations (in lower proportions and similarities). The frequency with whichthis gene was found indicates that this energy-generating metabolism would be at least as important in thebathypelagic habitat as it is in the photic zone. Although there are no data about CO concentrations or originsdeep in the ocean, it could have a geothermal origin or be associated with anaerobic metabolism of organicmatter. The identities of the microbes that carry out these processes were not established, but they seem to berepresentatives of either Bacteroidetes or Chloroflexi.

Carbon monoxide (CO) oxidation is a source of energy for awide diversity of prokaryotes and is an important processwithin the global carbon cycle. There is a wide diversity of COoxidation pathways among both archaea and bacteria (27, 28),and their wide distribution attests to both the ecological im-portance and ancient origin of CO oxidation. Most of thesepathways are anaerobic (31, 40) and have been reported inboth archaea and bacteria. However, aerobic CO oxidation isfound only in a few groups of bacteria, specifically in manyActinobacteria and Proteobacteria spp. and in at least one Fir-micutes sp. (for examples, see references 16, 17, 26, 35, 46, and47). Classically, aerobic oxidation of CO has been known to becarried out in soils where, in addition to geological or anthro-pogenic emissions, there are local biological sources connectedto plant roots and animals (15, 18, 19). However, more re-cently, the relevance of CO oxidation processes in the marineenvironment has also become clear, mostly from evidence fromthe fields of genomics and metagenomics (26, 42, 43).

The aerobic oxidation of CO is very amenable to genomicanalysis, since the genes involved are very characteristic, andtheir presence in marine bacterial genomes and in metagenomicdatabases can be considered diagnostic. The genes required foraerobic CO oxidation were first described in detail in chemo-lithoautotrophic Oligotropha carboxidovorans OM5 (10, 35, 36).The enzyme CO dehydrogenase (CODH) catalyzes the oxidationof CO and water to produce carbon dioxide, two electrons, andtwo protons (8, 11). The electrons are transferred to an elec-

tron transfer chain and used to generate a proton gradientacross the membrane. Three genes, coxL, coxM, and coxS (forlarge, medium, and small subunits, respectively), encode thepolypeptides for the CODH enzyme. Two heterotrimers, eachcomposed of one CoxL, CoxM, and CoxS subunit, combine toform a functional aerobic CODH enzyme. The large subunitcontains the molybdenum cofactor, the medium subunit bindsflavin adenine dinucleotide, and the small subunit has twoiron-sulfur clusters (13). In addition to these three genes, anumber of other accessory genes have also been identified(CoxB, CoxC, CoxH, CoxD, CoxE, CoxF, CoxG, CoxI, andCoxK) that are believed to be required in the processes ofregulation, posttranslational modification, and anchorage ofthe CODH complex to the cytoplasmic membrane. A numberof these accessory genes are membrane-bound proteins them-selves (CoxB, CoxC, CoxH, and CoxK), containing severaltransmembrane helices, and indeed, in O. carboxidovoransOM5, the CODH enzyme itself has been observed to associatewith the inner cytoplasmic membrane.

Based on sequence differences, genome organization, andcatalytic properties, there are two types of aerobic molybde-num-based CODH (the anaerobic enzymes are a different classof genes) (20). Both forms can be readily differentiated fromother molybdenum hydroxylases by phylogenetic analysis. Form ICODH (also called OMS, named after Oligotropha, Mycobac-terium, and Pseudomonas) has been conclusively proven bymutagenesis experiments and X-ray crystallography (8, 32, 35)to be the key enzyme in aerobic CO oxidation by carboxydotro-phic bacteria, i.e., those that can grow on CO as the solecarbon and energy source (at �10% CO concentration). Thereaction mechanism has also been clearly defined. Form ICODH large-subunit CoxL can be readily diagnosed by itscharacteristic catalytic site motif AYXCSFR. Moreover, in allthe organisms in which form I CODH genes have been iden-

* Corresponding author. Mailing address: Evolutionary GenomicsGroup, Departamento Produccion Vegetal y Microbiología, Univer-sidad Miguel Hernandez, San Juan de Alicante, Spain. Phone: 34 965919451. Fax: 34 965919576. E-mail: frvalera@umh.es.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 2 October 2009.

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tified so far, the genomic organization of the three subunits isalways M3S3L. The organization of the accessory genes,however, may vary from organism to organism.

There is much less known about the other form, form IICODH (or BMS, after Bradyrhizobium, Mesorhizobium, andSinorhizobium), which was first described in Bradyrhizobiumjaponicum USDA 110 (23), a gram-negative bacterial strainand a nitrogen-fixing symbiont of soybeans. Form II CODHenables these bacteria to grow, albeit slowly, in the presence ofCO as the sole carbon and energy source, but the rate of COoxidation by form II CODH of B. japonicum USDA 110 is 10to 1,000 times lower than that for form I CODH in O. carbox-idovorans OM5 and Pseudomonas carboxydohydrogena OM5.The catalytic site of the form II CoxL large subunit is AYRGAGR. The genome organization of the form II subunits isS3L3M, different from that of form I. The number of acces-sory genes present along with these genes is also variable (20).Form II is often found as a paralogous copy of three subunitsof form I, but without the accompanying set of CODH-relatedgenes. This is not surprising, since in most cases, the genesappear to be already associated with the form I cluster else-where in the genome, like in Rhodothermus marinus DSM4252, Dinoroseobacter shibae DFL 12, and Bradyrhizobium sp.strain BTAi1.

The discovery of the role played by CODH in marine watersis relatively recent. First, it was found in the genome ofSilicibacter pomeroyi DSS-3, a marine alphaproteobacterium ofthe Roseobacter cluster (26), which was concomitantly provento be able to oxidize CO at low concentrations (as should beexpected in marine waters). Later on the process, it was alsofound in metagenomic studies of surface waters for the Sar-gasso Sea metagenome project (26, 45). It has been proposedthat many heterotrophic bacteria in surface waters are litho-heterotrophs and take advantage of the CO released by or-ganic matter photolysis as an alternative energy source to sup-plement the scarce dissolved organic matter in a way akin to

the photoheterotrophy mediated by proteorhodopsin or anoxy-genic photosynthesis.

We recently found evidence of a CODH presence deep inthe Mediterranean Sea by the end sequencing of fosmids froma metagenomic library from a 3,000-m depth in the Ionian Sea(southeast of Sicily, Italy) (24). Here we present the analysis ofnine fully sequenced fosmids that were chosen on the basis ofthe presence of CODH cluster genes at their ends. The resultsconfirm the presence of complete CODH clusters, includingone that has the gene sequence and cluster structure of a formI CODH. Although the source of CO deep in the ocean isunclear, the frequency in which these genes were found andthe retrieval of similar sequences from the deep-ocean met-agenomic database of the Hawaii Ocean Time-Series (HOT)station (7, 21) point toward an important contribution of thislithotrophic metabolism deep in the ocean, similarly relevantto that found in the surface waters.

MATERIALS AND METHODS

Sample collection, metagenomic library construction, sequencing, and assem-bly of genomic clones. Planktonic samples were recovered at the Ionian Km3station (Mediterranean Sea), and a metagenomic library of fosmids was con-structed as described previously (24). Metagenomic DNA fragments (35 to 40kb) were cloned in the pCC1Fos vector and replicated in Escherichia coli EPI300.This collection has a total of 20,757 clones, and the terminal sequences of ca.5,000 inserts were sequenced, generating approximately 7.2 Mb of DNA se-quence (i.e., roughly two prokaryotic genome equivalents). From these fosmidends, those clones with a significant BLASTX hit over 1e�25 (against thenonredundant database) with any of the CODH cluster genes were chosen (atleast 15 sequences gave a good hit with CoxL proteins, and 2 with CoxM). Nineof these clones were chosen, and fosmid DNA was individually isolated using theQIAprep spin miniprep kit (Qiagen). For the sequencing process, the concen-tration of each DNA fosmid was measured using Quant-iT PicoGreen double-stranded DNA reagent (Invitrogen) and pyrosequenced (Roche 454 GS FLXsystem; GATC, Constance, Germany), tagging each one individually using amultiplex identifier adaptor that contains a unique 10-base sequence that isrecognized by the sequencing analysis software and allowing for automatedsorting of multiplex identifier adaptor-containing reads. The average read lengthwas 230 bp, and the average number of reads was �4,500 per fosmid (about 20%

TABLE 1. Main features and Cox subunits present in the analyzed fosmids

Fosmid Length (bp) % GCcontent

Coverage(times) Cox subunit(s) and characteristicsa

KM3-41-E12 44,132 43.05 15.0 CoxL: (f.I) R. marinus DSM 4252 (11,240), 86%; CoxM: T. roseum DSM 5159(A0566), 71%; CoxS: R. marinus DSM 4252 (11,250), 84%; CoxF: R.marinus DSM 4252 (15,230), 60%; CoxG: R. marinus DSM 4252 (15,230),77%; CoxD: T. roseum DSM 5159 (A0563), 56%; CoxE: *T. roseum DSM5159 (1,210), 61%

CoxL: (f.II) R. marinus DSM 4252 (5,520), 69%; CoxM: R. marinus DSM4252 (05510), 67%; CoxS: T. terrenum ATCC BAA-798 (23,480), 84%;CoxG: Pseudovibrio sp. strain JE062 (4,363), 60%

KM3-45-H11 37,813 41.15 34.9 CoxL: *S. thermophilus DSM 20745 (709), 73%; CoxM: R. marinus DSM 4252(5,510), 66%; CoxD: R. marinus DSM 4252 (10), 77%; CoxE: T. roseumDSM 5159 (1,210), 64%

CoxF: Alkalilimnicola ehrlichii MLHE-1 (1,569), 56%KM3-60-B01 37,124 42.72 15.5 CoxL: (f.II) R. castenholzii DSM 13941 (842), 79%; CoxM: R. marinus DSM

4252 (5,510), 65%; CoxS: *Janibacter sp. strain HTCC2649 (8,714), 86%KM3-26-C03 37,247 51.79 35.8 CoxL: (f.II) *R. marinus DSM 4252 (5,520), 70%KM3-28-H12 37,275 58.19 15.2 CoxL: (f.II) T. terrenum ATCC BAA-798 (12,870), 63%KM3-29-C02 36,757 51.48 24.9 CoxL: *S. thermophilus DSM 20745 (709), 66%KM3-54-A05 33,624 53.82 15.1 CoxL: *B. japonicum USDA 110 (3,350), 60%; CoxM: S. thermophilus DSM

20745 (3,193), 60%; CoxG: Nocardioides sp. strain JS614 (232), 55%

a Subunit present, form I (f.I), form II (f.II), truncated protein (�), closest strain hit, number of ORFs (in parentheses), and percent similarity are also shown.

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of them belonged to E. coli EPI300 and the cloning vector). Assembly wasperformed with the program SeqMan (DNASTAR) using the following param-eters: 20 bp of sequence overlapping and 95% similarity. All the fosmids, with theexception of KM3-45-H11, were assembled in one single contig (Table 1).

Annotation and analysis of genome fragments. Protein-coding genes werepredicted using GLIMMER (6) and the SEED server (29) and were furthermanually curated, especially the ends of the fosmids. Spacers were subsequentlysearched against the nonredundant database (http://www.ncbi.nlm.nih.gov/) us-ing BLAST (1, 2) to ensure that no open reading frame (ORF) was missed.Identified ORFs were compared to known proteins in the nonredundant data-base using BLASTX, and all hits of �1e�5 were considered nonsignificant.COGNITOR was used for COG (clusters of orthologous groups) assignmentsand COG functional categories (39). Putative protein transmembrane domainswere predicted using TMHMM 2.0 (22). For comparative analyses, reciprocalBLASTN and TBLASTX searches among the different fosmids were carried out,leading to the identification of regions of similarity. To allow for the interactivevisualization of genomic fragment comparisons, we used Artemis ComparisonTool version 8 (4) and Perl software developed in our laboratory.

Analysis in metagenomic collections. To detect the coxL, coxM, and coxSgenes in environmental sequences, a first screening was done using BLASTXcomparisons in the different metagenomic collections. For the HOT/A Long-Term Oligotrophic Habitat Assessment (ALOHA) (7, 21) and Global OceanSampling (GOS) (34, 45) environmental collections, all the sequences of �1e�5were recovered, and their coding sequences were extracted to confirm the pres-ence of at least one of the two domains of the CoxL (Ald-Xan-dh-C and Ald-Xan-dh-C2), CoxM (flavin adenine dinucleotide-Binding5 and CO-deh-flav-CO), and CoxS (Fer2 and Fer2-2) proteins. This was done using the hmmpfamprogram of the HMMER package (9). The hidden Markov models for theprotein domains were obtained from the Pfam database (http://pfam.sanger.ac.uk). Positive sequences were further examined for the presence of the form Iand form II catalytic site motifs AYXCSFR and AYRGAGR, respectively.Fosmid recruitments were done using TBLASTX comparisons of the meta-genomic libraries against the genomic fragment. A cutoff of 30% similarity in atleast 50% of the environmental sequence was used. In the case of the GOScollections, GS033 (hypersaline lagoon, Punta Cormorant, Floreana Island,Ecuador) and GS020 (freshwater, Gatun Lake, Panama Canal, Panama) sampleswere not used in the analysis. For the metatranscriptome analysis (where se-quence size was only about 107 bp) (37), we considered only the sequences withover 50% similarity in more than 70% of their lengths. Due to the different sizesof the databases, the number of sequences was normalized by dividing by thenumber of megabases sequenced in each collection.

Nucleotide sequence accession numbers. Sequences obtained and annotatedin this study have been deposited in GenBank under the accession numbersGU058051 to GU058057.

RESULTS

We fully sequenced nine fosmids that were selected from adatabase of fosmid ends from the Km3 sample (see Materialsand Methods). The criterium followed was to have a significantBLASTX hit (over 1e�25) to any of the CODH cluster genes.This does not guarantee that a bona fide form I or II CODHis found within the fosmid, since some of the subunits areshared with other protein clusters of different function, and inany case, being at the end of the fosmid, the relevant genesmight be in the other direction and not present in the fosmid.Even with these caveats, seven fosmids that show evidence ofcoding either a form I or form II CODH large subunit werefound. The remaining two fosmid clones had other similar pro-teins not related to CODH but that belonged to the samefamily (data not shown). The genomic fragments cloned in thefosmids had sizes between 44.1 and 36.7 kb, and with theexception of KM3-45-H11, all could be assembled in one singlecontig (Table 1).

CODH genes present in the fosmids. The most interestinggenome fragment found was the one in fosmid KM3-41-E12, inwhich we identified two CODH clusters, one belonging to formI and the other to form II (Fig. 1). The catalytic subunits coxL

contained here were complete, as this fosmid was chosen bythe partial coxE subunit that appeared at one of its ends. Also,in clone KM3-60-B01, it was possible to identify other com-plete coxL genes. In the other fosmids, the coxL genes wereplaced at the ends and were truncated (Fig. 1). However,except in KM3-26-C03, KM3-28-H12, and KM3-29-C02, othersubunit of the CODH cluster were identified, providing morereliable evidence of the presence of a functional CODH path-way. When the diagnostic catalytic site was available, the se-quences of CoxL permitted us to identify putative form I or IICODHs. When the active site was not present in the se-quenced stretch (KM3-41-E12, KM3-60-B01, KM3-26-C03,and KM3-28-H12), we studied the relative positions of theother subunit genes, coxS and coxM, and the similarities withother proteins to classify them in either CODH form I or II.

CoxL form I. Form I of the large subunit of CODH is thebest-studied and the most reliable indicator for the process ofCO oxidation. It has been described in several carboxy-dotrophs (3, 12, 30, 35). The only representative of this type ofCoxL subunit was found in fosmid KM3-41-E12 (Fig. 1). Itshows high overall similarity to other known form I CoxLproteins and has the cysteine-containing catalytic sequencemotif AYXCSFR, which differentiates the CO-oxidizing formI from the larger family of molybdenum hydroxylases (includ-ing form II). In addition, the organization of the coxS and coxMgenes associated with this coxL gene is M3S3L, typical ofform I CODH clusters. Four other accessory genes, coxE,coxD, coxG, and coxF, are also present in the same cluster andprovide good evidence of the presence of a complete andfunctional aerobic CODH complex in the organism to whichthis fosmid belongs. KM3-41-E12 CoxL has the highest simi-larity with form I CoxL of Rhodothermus marinus DSM 4252(86%), a thermophilic Bacteroidetes strain from a shallow ma-rine hot spring in Iceland (unpublished, draft genome), andwith the CoxL plasmidic protein of Thermomicrobium roseumDSM 5159 (82%), an extreme thermophile isolated from aYellowstone National Park hot spring, which has been provenexperimentally to oxidize CO (47). The presence of this formI coxL, along with its accessory genes, could be a strong indi-cator that CO oxidation takes place at a 3,000-m depth in theMediterranean Sea. Interestingly, the same fosmid containsanother cluster of CODH genes with a form II large subunit(see below).

CoxL form II. Among the other fosmids, three CoxL pro-teins could be diagnosed as form II from the identification ofthe conserved catalytic site AYRGAGR. The other CoxL pro-tein encoded by the fosmid KM3-41-E12 is 60% similar to form ICoxL also contained in this fragment, but it was confirmed tobelong to form II. This subunit is also very similar (69%) toform II CoxL found in the R. marinus DSM 4252. The subunitsCoxS and CoxM have their best similarities with “Thermobacu-lum terrenum” ATCC BAA-798 (84%), an unclassified bacte-rium, and again with R. marinus DSM 4252 (67%), respectively.Clone KM3-60-B01 contains the subunits coxS (truncated), coxL,and coxM. These subunits have their best hits within Chloroflexiand Bacteroidetes/Chlorobi bacteria, CoxL has its best hit withRoseiflexus castenholzii DSM 1394167 (79%), and CoxM has itsbest hit again with R. marinus DSM 4252 (65%) (Table 1).Also, the CoxL protein encoded in KM3-28-H12 could beclearly assigned to form II CODH by its catalytic site. This

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subunit is not accompanied by any other subunit related to theCODH cluster and has its best similarity with form II CoxL ofT. terrenum ATCC BAA-798 (63%). It has been suggested thatthe lack of all other subunits, such as those involved in theposttranslational modification, may indicate that this clustercould act over other substrates rather than CO (20), but it

could also happen that the presence in the genome of othercomplete form I or form II clusters could provide the requiredsubunits to assemble a functional CODH.

The remaining fosmids had truncated large subunits that didnot contain the catalytic site. The cluster of KM3-45-H11 con-tained the genes coxL (truncated), coxM, coxD, and coxE (Fig.

FIG. 1. Genes and similarity comparison of environmental fosmids from the Km3 metagenomic library containing CODH genes. CODH clustergenes are highlighted in different colors. Specific additional genes mentioned in the text are also indicated. The two closest genome fragments ofcultivated microbes Rhodothermus marinus DSM 4252 and Bradyrhizobium japonicum USDA 110 are also shown.

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1). Although the CoxL subunit lacks the active site, the genesequence L3M corresponds to the order found in form IIgene clusters. Also, its high similarity (73%) with form II CoxLof Sphaerobacter thermophilus DSM 20745 (Chloroflexi) wouldsupport this hypothesis. The other subunits had also the high-est similarity hits to genes belonging to Chloroflexi genomes(Table 1). Fosmid KM3-54-A05 carried the subunits coxL(truncated), coxM, and coxG. Between the last two genes, thereis a short gene for a transcriptional regulator of the MerRfamily. This CoxL subunit is the most divergent CoxL sequencefound in this work and has its best similarity (60%) with aCoxL-like protein of the alphaproteobacterium B. japonicumUSDA 110 (Fig. 1), the only known microbe lacking form ICoxL that has been proven experimentally to grow with CO asthe sole carbon and energy source (23). The coxM gene in thisfosmid appears upstream of coxL, a gene order that has notbeen found yet in any CODH cluster sequenced. KM3-26-C03and KM3-29-C02 coxL genes (both truncated) are not accom-panied by any other subunit related to the CODH cluster. Thefirst one, KM3-26-C03, had a part of the catalytic site (GAGR)and therefore could be tentatively assigned to form II CoxL.Also, in this case there was a high similarity, 70%, with form IICoxL of R. marinus DSM 4252. In the case of CoxL (truncated)of KM3-29-C02, it is more similar (66%) to form II CoxL of S.thermophilus DSM 20745.

Other genes relevant to CO oxidation. In fosmid KM3-45-H11, we found molybdopterin biosynthesis genes close to theCODH cluster. The CODH cluster is frequently located neargenes related to molybdopterin biosynthesis, i.e., in Alkalilim-nicola ehrlichii MLHE-1 or Jannaschia sp. strain CCS1. Insome cases, the biosynthesis of the molybdopterin cofactor seemsto be coupled to the transcription of the genes of the CODHcluster, so that as soon as molybdopterin is made available, itcan be inserted into the CODH enzyme immediately.

One of the fosmids, KM3-29-C02, was found to contain oneof the key enzymes of the reductive tricarboxylic acid (TCA)cycle, the ATP-dependent citrate lyase (Fig. 1), one of thethree key enzymes essential for fixing carbon through this path-way. At least in Epsilonproteobacteria in deep-sea hydrother-mal vents, it has been shown that functional reductive TCAcycle enzymes are present, and it is believed that they sustainthe predominant primary production in these habitats (38).This raises the possibility that CO2 may be channeled into thereductive TCA cycle, thus making CO oxidation deep in theocean an important component in fixing carbon.

Phylogenetic affiliation of the fosmids. The phylogeneticrelations shown by the CoxL subunits of different microbeshave been shown to be largely consistent with the 16S rRNAphylogeny (20). Our sequences appear to be associated toothers from the Bacteroidetes/Chlorobi or Chloroflexi group.Actually, the only housekeeping gene found in our fosmids thatallows an easy taxonomic placement was the ribosomal proteinS1 of KM3-60-B01, which had the highest BLAST hit to theDeltaproteobacteria Stigmatella aurantiaca DW4/3-1, but with arelatively low (66%) similarity. However, the genes of thefosmids gave overall highest similarities to the Chloroflexi andBacteroidetes genomes (Fig. 1; see also Table S1 in the supple-mental material), supporting the classification indicated by thecoxL genes. This also suggests that the CODH cluster genesfound in our Km3 fosmids may belong to related microbes or

are transferred horizontally so often that their sequences ap-pear independently from the phylogenetic affiliation of the restof the genome.

Representation of CODH genes in metagenomic and metatran-scriptomic marine collections. Although the presence of coxL-related genes in superficial metagenomic collections has beenalready established (26), the presence of similar genes deep inthe ocean has not been previously investigated.

Besides the sequences deep in the Mediterranean Sea, theonly deep-water column DNA sequence databases available arethe HOT database in the subtropical Pacific (HF10, HF70,HF130, HF200, HF500, HF770, and HF4000; 63.95 Mb se-quenced) (7) and the recently published whole-genome shot-gun (WGS) library from the microbial community at a 4,000-mdepth from the same sample and the same DNA preparationas the 4,000-m-depth fosmid library (HF4000) (77.43 Mb) (21).First, we have searched for the presence of the CoxL, CoxM,and CoxS proteins in these databases by using BLAST andcorroborated these results, looking for their characteristic do-mains (see Materials and Methods). Surprisingly, the resultsalong the water column showed that putative coxL genes weremore abundant below 200 m than in superficial waters (Fig. 2aand b). The presence of one or even two of the domains onlyis not enough to confirm that the sequences found code fortrue CoxL subunits, but at least 4 sequences have the form Icatalytic center below 200 m depth (3 sequences in the fosmidlibraries and 1 in the 4,000-m-depth WGS library), and another26 sequences have form II (9 in the fosmid libraries and 17 inthe 4,000-m-depth WGS library) (Fig. 2a and b). For compar-ison, a similar search detected three form II CoxL proteins atKm3 (3,000 m), the sample from which our sequences arederived. The relative abundance (normalized to database size;see Materials and Methods) of the coxL-like genes in thesesamples and Km3 is shown in Fig. 2a. The data show that,contrary to what we expected, these genes were relatively morefrequent in deeper waters. If we assume an average genomesize of 3.5 Mb and consider the size of the metagenomic li-braries used, the data obtained would imply that, at the least,there must be one coxL gene per genome below 200 m. This isa very high number compared with the results of the previouswork with the Sargasso database, in which a ratio of one coxLgene per 14 genomes was estimated (26). We searched forCODH genes in the more recent GOS (25) surface collections(16.96 Gbp) (34), and we could find 103 form I genes thatcould be confirmed by their catalytic domain and 847 form IIgenes. The distribution in the different collections was nothomogeneous, with a higher concentration in coastal waters(see Fig. S1 in the supplemental material). But still, normaliz-ing to the size of the database, the frequency of CODH genesappears smaller than that in the deep-water sequence collec-tions. The deep-water CoxL from the Pacific Ocean appearedrelated to those from the Mediterranean Sea in the case ofform I, with three of the four Pacific CoxL proteins also havingtheir best similarities with R. marinus DSM 4252 (similaritiesover 89%) and KM3-41-E12 CoxL (similarities near 60%).However, regarding form II, things were not so clear, as onlyone Pacific fosmid end from a 4,000-m depth had 80% simi-larity to KM3-60-B01, and the taxonomic affiliation of theremaining Pacific form II sequences seemed different, beingmore related to alphaproteobacterial genes.

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Also, since several complete fosmids from HOT metagenomicsamples have been fully sequenced, we searched for the pres-ence of CODH gene subunits. We detected up to four coxL-like genes in fosmids from the 4,000-m collection, but only oneof them, HF4000-APKG5H11, contains a CoxL form II proteinwith the catalytic site conserved together with the CoxM sub-unit. In this case, its highest similarity was also to T. roseumDSM 5159 (CoxL, 75%; CoxM, 56%). On the other hand,aside from the CODH subunits, none of these genomic clones

from the HOT station have conserved synteny or any othersimilarities with the fosmids of the Mediterranean Sea.

We have also analyzed the presence of coxL, coxM, or coxSin the metatranscriptomic collection from biomass recoveredat 25, 75, 125, and 500 m of depth in the central North PacificGyre (48 Mbp) (37). Presumably due to the difficulties inher-ent to obtaining fresh deep-ocean samples, there are nometatranscriptomic data sets from deep in the ocean. How-ever, we found that the number of transcripts similar to coxL

FIG. 2. (a and b) Distribution of genes coxL, coxM, and coxS found in the environmental sequence collections of the HOT (subtropical Pacific)(7) and Km3 (Ionian Sea) stations (24) (a) and in the WGS library at a 4,000-m depth in the ALOHA station (21) (b). Numbers in the coxL columnsindicate the number of CoxL proteins found in each collection that have one or two domains characteristic of this protein. Bullets show the numberof these proteins that conserved the catalytic domain of form I and form II CoxL. (c) Distribution of cDNA-like coxL, coxM, and coxS sequencesfound in the marine metatranscriptome of the subtropical Pacific (37).

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increased with depth from 75 m, and at 500 m, the number ofcDNA sequences obtained was the same as that obtained at25 m (Fig. 2c). This fact is a clear indicator that the CODHgenes found in the metagenomic collection at depths down toat least 500 m are transcribed and might be functional.

Finally, we have analyzed the recruitment of the fosmids inthe HOT and GOS metagenomic collections to assess the pres-ence of similar microbes there (Fig. 3; see also Fig. S2 in thesupplemental material). Focusing on the fosmid containingform I coxL, KM3-41-E12, we found 1,473 sequences in theGOS collection with over 60% similarity, not a very high num-ber considering the database size (1 sequence per 11.5 Mb).On the other hand, the aphotic HOT database contained rel-atively more sequences with higher similarity (19 sequences, 1per 3.36 Mb); despite the much smaller size of the database,most of them belonged to the deeper-ocean collections (Fig.3). This suggests that the genes found deep in the Mediterra-nean Sea are more frequently found in the aphotic zone, al-though they might also be present at the surface. In this fos-mid, it was also observable that form I coxL recruits at highersimilarity than form II, not surprising considering that form IIis known to have a much wider diversity of sequences evenamong cultivated microbes. Similar analysis was done with therest of the fosmids with similar results (see Fig. S2 in thesupplemental material).

DISCUSSION

The finding of many genes with best hits to putative CODHcoding sequences in a relatively small fosmid library from deepin the Mediterranean Sea was unexpected, since CO oxidationis known to be a common process in superficial waters but notin the abyssal region. In the photic zone, CO is known to begenerated by organic matter photolysis (20, 44, 49), which isassumed to be the major source for this reduced compound.

Previous work on the same deep Mediterranean Sea metage-nome already hinted at the existence of CO oxidation in thebathypelagic habitat, but the evidence for a widespread occur-rence of this metabolic strategy was much weaker, providingonly short sequences that are not enough to diagnose this com-plex metabolic activity that requires the contributions from alarge gene cluster. The evidence presented here indicates thatbona fide CODH genes with a high probability of correspond-ing to CO-oxidizing microbes are at least found deep in theMediterranean Sea but also probably in other deep oceanicenvironments. The fact that the form I cluster has been foundin a screening of only about 7 Mb shows that indeed the presenceof CODH genes is very common among organisms found deepin the Mediterranean Sea. Moreover, the same probabilityapplies at least to the deep North Pacific, as shown by ouranalysis of the HOT database. That the potential for this me-tabolism is very widespread was confirmed by the fact of find-ing cDNA of sequences very similar to those of some of itscomponents at 500 m. This is particularly remarkable, consid-ering that form I CODHs are low-affinity enzymes that requirehigh partial pressure of CO to be active. These findings raisethe question of what could be the origin of CO in the bathy-pelagic habitat. In general, CO concentration decreases atdeeper waters, at least for the 200-m upper layer (14). Unfor-tunately, there is very little information about CO concentra-tions and biogeochemistry in the mesopelagic and bathypelagiczones. Considering that both Km3 and HOT stations are lo-cated in tectonically active areas rich in submarine volcanoes,the geothermal origins appear to be the most likely. However,we have not found evidence for other chemolithotrophic en-ergy generation mechanisms in the same screening, while COis considered a rather minor product of geothermal emissions(33, 41). More data from other oceanic regions with morestable geologic settings might help prove this hypothesis. An

FIG. 3. Metagenome recruitment of the KM3-41-E12 fosmid in the metagenomic libraries of the GOS and HOT stations. TBLASTXcomparisons were done (cutoff, 30% similarity in 50% of the length of the environmental sequence), and individual fosmid end sequences werealigned along their homologous regions. The alignment sequence conservation was visualized in the form of percent identity plot. Shadowed in blueand yellow are the recruitment of the subunits of the CODH that exist in this fosmid. The number of sequences over a similarity of 60% is indicatedin the upper right corner of each graphic. The pie chart inset to the right of HOT sequence recruitment shows the distribution in percentages ofthe sequences found at each depth.

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alternative might be that the anaerobic metabolism takes placein the water column within large particles or aggregates oreven in the sediment (although the mesopelagic samples fromthe HOT database also had potential for CODH presence).There have been observations of a strong “dark production,”which contributed a significant fraction of the diurnal COsource within the top 17 m of the ocean (5, 50). The dark COsource correlated strongly with biological oxidation rates andorganic carbon, suggesting that it is the result of incompleterespiration of biologically labile organic matter. There is al-ways the possibility that fosmid cloning might bias the results,enriching the microbes that carry CODH genes by some mo-lecular selection. However, the fact that they have been foundin independent libraries and origins and, in the case of form II,that they seem to have different biological origins (taxonomy)support the presence of real carboxydovores deep in the ocean.How relevant they are for the ecosystem functioning and itsbiogeochemistry remains an open question that more extensivesampling from deep-ocean waters might help to answer.

As for the identities of the microbes carrying out the pro-cess, we cannot advance a definitive answer either. However,the repetitive finding of very high similarity hits to Bacte-roidetes and, more specifically, to R. marinus DSM4252 mightindicate that the microbes involved belong to a psychrophilicrelative of this microbe and belong to this phylum, at least theones possessing the form I CODH homologs found in KM3-41-E12. The next phylum represented in the hits for this fosmidis the Chloroflexi; both phyla have been found to have otherconverging features such as the possession of similar carote-noids (44), and both groups have been found in significantnumbers in deep oceanic samples by PCR 16S rRNA amplifi-cation and metagenomic studies (7, 24, 48). The repeated find-ing of highest similarities with genes from cultivated thermo-philes in this rather cold environment is intriguing. It is evenmore remarkable considering the relatively high GC contentcharacteristic of most thermophiles not found in our fosmids,and that would tend to decrease nonfunctional similarity. Inany case, the taxonomic affiliation of the microbes carrying outthis process deep in the ocean remains elusive, and the evi-dence presented is significant only to point out that the mi-crobes that carry out the CO oxidation in the bathypelagiccompartment are probably different from those possessing thismetabolism in the photic zone.

ACKNOWLEDGMENTS

This work was supported by projects GEN2007-30014-E andBIO2008-02444, and A.-B.M.-C. was supported by a Juan de la Ciervascholarship, all from the Spanish Ministerio de Ciencia e Innovacion.

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