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Journal of Bacteriology, November 2008, p. 7491-7499, Vol. 190, No. 22
0021-9193/08/$08.00+0     doi:10.1128/JB.00746-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Complete Genome Sequence of Thermococcus onnurineus NA1 Reveals a Mixed Heterotrophic and Carboxydotrophic Metabolism ^,

Hyun Sook Lee,^1, Sung Gyun Kang,^1, Seung Seob Bae,^1, Jae Kyu Lim,¹ Yona Cho,¹ Yun Jae Kim,¹ Jeong Ho Jeon,¹ Sun-Shin Cha,¹ Kae Kyoung Kwon,¹ Hyung-Tae Kim,² Cheol-Joo Park,² Hee-Wook Lee,² Seung Il Kim,³ Jongsik Chun,⁴ Rita R. Colwell,⁵ Sang-Jin Kim,¹ and Jung-Hyun Lee^1*

Korea Ocean Research and Development Institute, Ansan, P.O. Box 29, Seoul 425-600, South Korea,¹ Macrogen, Inc., Gasan-dong, Seoul 153-781, South Korea,² Korea Basic Science Institute, Daejeon 305-806, South Korea,³ School of Biological Sciences, Seoul National University, Seoul 151-742, South Korea,⁴ Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland⁵

Received 26 May 2008/ Accepted 29 August 2008

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Members of the genus Thermococcus, sulfur-reducing hyperthermophilic archaea, are ubiquitously present in various deep-sea hydrothermal vent systems and are considered to play a significant role in the microbial consortia. We present the complete genome sequence and feature analysis of Thermococcus onnurineus NA1 isolated from a deep-sea hydrothermal vent area, which reveal clues to its physiology. Based on results of genomic analysis, T. onnurineus NA1 possesses the metabolic pathways for organotrophic growth on peptides, amino acids, or sugars. More interesting was the discovery that the genome encoded unique proteins that are involved in carboxydotrophy to generate energy by oxidation of CO to CO₂, thereby providing a mechanistic basis for growth with CO as a substrate. This lithotrophic feature in combination with carbon fixation via RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) introduces a new strategy with a complementing energy supply for T. onnurineus NA1 potentially allowing it to cope with nutrient stress in the surrounding of hydrothermal vents, providing the first genomic evidence for the carboxydotrophy in Thermococcus.

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Deep-sea hydrothermal vents comprise a plethora of potential habitats, with gradients of nutrient and extreme physicochemical conditions that vary from high to low with respect to temperature (350 to 2°C), oxygenation states, and fluid velocities (13). Many multidisciplinary studies have been carried out to understand the complexities of hydrothermal vent systems. Biological studies have also been accomplished using samples collected from hydrothermal vent areas and culture-dependent and culture-independent techniques, revealing the presence of physiologically, metabolically, and phylogenetically diverse microorganisms (15). These findings have been followed by characterization of many bacterial and archaeal thermophiles (and hyperthermophiles), including both chemolithoautotrophic and chemoorganoheterotrophic strains. Among representative species of the Archaea, sulfur-reducing heterotrophs belonging to the order Thermococcales (encompassing the genera Thermococcus, Pyrococcus, and Palaeococcus) have been reported to be one of the predominant groups (20, 25). Notably, members of the species of Thermococcus were found to be more abundant in the vent ecosystem, with such isolates more frequently reported than the Pyrococcus species (9, 11, 23, 24). Such large populations indicate some significance for the presence of Thermococcus in the microbial consortia that make up the microbial ecology of hydrothermal vent systems.

In addition to ecological significance, the hyperthermophilic feature of Thermococcales has fascinated microbiologists interested in fundamental and/or application-based research. To date, the complete genome sequences of three Pyrococcus species, i.e., Pyrococcus horikoshii (16), Pyrococcus furiosus (26), and Pyrococcus abyssi (5), and a Thermococcus strain, Thermococcus kodakaraensis KOD1 (7), have been determined. Analysis of the sequences and the physiological insight provided by these analyses have led to the conclusion that members of the Thermococcales obtain energy via fermentation of peptides, amino acids, and sugars, with the process yielding organic acids, CO₂, and H₂ (1, 10). Comparative genomic studies of the Thermococcales showed that T. kodakaraensis KOD1 and Pyrococcus spp. share approximately one-half of their proteins, including those involved in information processing and basic metabolism, even though the genomes have undergone extensive genetic rearrangements (7). Several cellular functions unique in T. kodakaraensis have been proposed to explain the ubiquitous distribution and large populations of Thermococcus spp. compared to Pyrococcus (7). Considering the information available to date, it was hypothesized that the genome of an additional Thermococcus isolate could provide some insight into the apparent successful competition of Thermococcus spp. and factors that contribute to their ubiquity in hydrothermal vent fields.

Thermococcus onnurineus NA1 was recently isolated from a deep-sea hydrothermal vent region in the PACMANUS field (3°14'S, 151°42'E) of the East Manus Basin (2). This isolate requires elemental sulfur as a terminal electron acceptor for heterotrophic growth on peptides or an amino acid mixture and exhibits optimum growth at 80°C and pH 8.5 (2). In this study, by analyzing the genome sequence we show that T. onnurineus NA1 retains the metabolic pathways not only for organotrophic growth but also for carboxydotrophic growth and thus provide a mechanistic basis for experimental growth utilizing CO.

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Cultivation. For routine cultivation, cells were grown anaerobically at 80°C in a yeast extract-peptone-sulfur (YPS) medium (9). Physiological tests were performed using modified medium 1 (33) with the addition of 1 ml of a trace element mixture, 1 ml of vitamin solution (3), NaCl (30 g liter^–1), and yeast extract (0.5 g liter^–1). The pH was adjusted to 8.0 using NaOH. The anaerobically prepared medium was dispensed into 25-ml serum bottles, and the gas phase (15 ml) was changed to 80% N₂-20% CO₂ (10⁵ Pa) or 100% CO.

Genome sequencing and assembly. The genome sequence of T. onnurineus NA1 was determined by whole-genome shotgun sequencing and pyrosequencing. For capillary sequencing, a 2- to 3-kb insert library (11,028 clones), 40-kb insert library (1,870 clones), and 35-kb insert library (288 clones) were constructed and sequenced using an ABI 3730XL sequencer (Applied Biosystems, CA). For pyrosequencing, 581,990 fragments of DNA were sequenced using a GS-20 sequencer (454 Life Sciences, CT). The contigs generated by both sequencers were combined, and closure of the sequencing gap was performed by clone walking and PCR sequencing. The program used for open reading frame (ORF) prediction was Glimmer, which uses Markov's interpolated models and GS-Finder. The RBSfinder program (www.tigr.org), which searches for ribosome-binding sites in the extragenic regions, was also used in order to increase the reliability of the Glimmer results, followed by a manual ORF fitting process. After all the ORFs had been determined, further analysis of the protein sequence was performed by BLASTP searches against the nonredundant protein sequences of the National Center for Biotechnology Information (NCBI), Kyoto Encyclopedia of Genes and Genomes, and COG (clusters of orthologous groups of proteins) databases (35). tRNAScan-SE was used for the tRNA predictions (18).

Comparative genome analysis. Briefly, an all-versus-all BLASTP search was performed using the protein sets in genomes to identify pairwise matches and an E-value lower than 1 x 10^–10 with over 80% coverage. Subsequent single-linkage and complete-linkage hierarchical clustering was also done. To obtain an integrated view of the genomic location of homologous coding DNA sequences (CDSs) in T. onnurineus NA1 versus other strains belonging to the Thermococcales, pairs of best matches in the all-versus-all BLASTP search were marked in each set of comparisons. For each protein in seven hydrogenase gene clusters in T. onnurineus NA1, putative orthologues among 31 archaeal genomes were searched with BLASTP, and the number of identical amino acids in a match was normalized by the total length of the CDSs. The resulting 107-by-31 matrix was then grouped and visualized by the distribution pattern across species using GeneSpring GX, version 7.3.1 (Silicon Genetics, CA). Protein sequences of carbon monoxide dehydrogenase (CODH) and the subunit of F420 hydrogenase were aligned by ClustalX, and the alignments were refined using the MEGA program, version 3.1. The phylogeny was constructed with the neighbor-joining method using PHYLIP, version 3.65, and was visualized using TREEVIEW software. The distance method for the neighbor-joining algorithm was generated by a Dayhoff PAM matrix. The robustness of the topology in the phylogenetic trees was evaluated by bootstrap analyses of the neighbor-joining algorithms based on 1,000 resamples.

Proteome analysis. T. onnurineus NA1 cells were suspended in 100 mM Tris-HCl buffer (pH 6.8) containing 4% sodium dodecyl sulfate and 4 mM EDTA and boiled for 10 min, followed by centrifugation at 22,000 x g for 20 min. The cell lysate was separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 30 fractions were obtained based on molecular size. They were then in-gel digested using trypsin (Promega, WI) (17), and tryptic digests were dissolved in 0.5% trifluoroacetic acid solution to be analyzed by mass spectrometry (Thermo Finnigan LTQ, CA). The identities of peptides were determined by using the Sequest program (Thermo Finnigan, CA).

PCR and reverse transcription-PCR analyses. PCR of the CODH gene was performed using genomic DNAs of seven Thermococcus spp. (T. onnurineus NA1, Thermococcus acidoaminovorans, Thermococcus peptonophilus, Thermococcus gorgonarius, Thermococcus stetter, Thermococcus fumicolans, and Thermococcus litoralis) as templates and degenerate primers, two forward primers (5'-GGACCATGTAGAATCGAYCCGTTY-3' and 5'-TGCTGTACSGGAAAYGAA-3') and two reverse primers (5'-TTCRTTTCCGGTACAGCA-3' and 5'-RTCMACACAGCTTCCCATGTG-3') in combination (standard International Union of Biochemistry-IUPAC symbols are used to indicate the nucleotide mixtures), which were designed based on regions conserved in the CODH amino acid sequences.

A 50-ml culture of T. onnurineus NA1 was grown to early to mid-exponential growth phase on modified medium 1 with the addition of various concentrations of yeast extract under the gas phase of 80% N₂-20% CO₂ (10⁵ Pa) or 100% CO. Cells were harvested by centrifugation at 6,000 x g for 30 min. The pellet was resuspended in 50 µl of 50 mM Tris-HCl buffer (pH 7.5), with the addition 500 µl of Trizol reagent (Invitrogen, CA). The cells were lysed by freezing and thawing, and then the samples were extracted with 200 µl of chloroform. The aqueous phase containing total RNA was further processed by ethanol precipitation and then resuspended in distilled water. RNA concentration and integrity were determined by measuring the absorbance at 260 and 280 nm, as well as by 0.8% agarose gel analysis. Reverse transcription and PCR amplification were carried out using SuperScript II reverse transcriptase (Invitrogen, CA), according to the manufacturer's instructions. Two sets of primers were used for amplification of the CODH gene and the Hsp60 gene (control): the pair codh-f (5'-GGACCATGTAGAATCGAYCCGTTY-3') and codh-r (5'-TTCRTTTCCGGTACAGCA-3') and the pair and hsp-f (5'-ATGGCACAGCTTAGTGGACAG-3') and hsp-r (5'-CAAGGATTTCCTGGGCTTTCTC-3').

Nucleotide sequence accession number. The sequence of the complete T. onnurineus NA1 genome has been deposited in the GenBank database under accession number CP000855.

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General genome features. To provide some insight into factors that contributed to the apparent successful competition of Thermococcus spp. in hydrothermal vent fields, the genome sequence of T. onnurineus NA1 was determined by combining random whole-genome shotgun sequencing with pyrosequencing. As a result, it was revealed that T. onnurineus NA1 has a single circular chromosome (1,847,607 bp) without any exochromosomal elements, and a total of 1,976 CDSs were identified (Table 1). Of these, 1,104 CDSs (55.8%) were annotatable by homology and domain searches, but the function of the residual 872 CDSs could not be predicted from the primary structure. When protein similarity was searched on a genome-wide scale, 82.7% of the T. onnurineus NA1 proteins showed similarity to those of other members of the Thermococcales (Fig. 1). Information processing and the basic metabolism of the Thermococcales were conserved (see Table S2 in the supplemental material), and essential genes for organotrophy were well conserved in T. onnurineus NA1 (see Table S6 in the supplemental material). Proteomic analysis of whole proteins showed that enzymes involved in organotrophy were expressed (data not shown). The presence on the T. onnurineus NA1 genome of Thermococcales glycolytic motif (TGM) sequences, which are potential cis-regulatory elements, in promoter regions and of an orthologue (TON_1797) of Tgr, a global transcriptional regulator in T. kodakaraensis KOD1 that controls the expression levels of both glycolytic and gluconeogenic enzyme-encoding genes, suggests that a similar regulon is also present in T. onnurineus NA1 (14). Synteny comparison by all-versus-all BLASTP searches between T. onnurineus NA1 and other members of the Thermococcales confirmed the high frequency of DNA rearrangement (Fig. 2). The gene rearrangement between T. onnurineus NA1 and T. kodakaraensis KOD1 seemed to take place at the level of chromosomal segments, confirming a closer phylogenetic relationship than to Pyrococcus strains.

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TABLE 1. General features of the T. onnurineus NA1 genome and T. kodakaraensis KOD1 and Pyrococcus strains

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FIG. 1. A Venn diagram of the shared and unique portions of the proteome of four Thermococcales strains, T. onnurineus NA1 (NA1), T. kodakaraensis, P. furiosus, and P. abyssi. The protein sets for the strains were obtained from the RefSeq collection in NCBI.

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FIG. 2. Synteny comparison between T. onnurineus NA1 and other Thermococcales strains: T. onnurineus NA1 (NA1) versus T. kodakaraensis KOD1 (KOD1) (a); T. onnurineus NA1 versus P. furiosus (Furiosus) (b); T. onnurineus NA1 versus P. abyssi (Abyssi) (c); T. onnurineus NA1 versus P. horikoshii (Horikoshii) (d).

CODH gene cluster and carboxydotrophic growth. During the genome analysis, it was discovered that T. onnurineus NA1 possessed a unique gene cluster (Hyg4-II, encoded by TON_1016 to TON_1020 [TON_1016-1020]) that was comprised of a putative transcriptional regulator (TON_1016), a CODH accessory protein (CooC; TON_1019), a catalytic subunit of CODH (CooS; TON_1018), and an electron transfer protein (CooF; TON_1017), along with the hydrogenase of the Hyg4-II gene cluster (TON_1021-1031) (Fig. 3). CooS (TON_1018), a central enzyme in microbial carbon monooxide (CO) metabolism, showed significant similarities with CODHs from CO-oxidizing methanogenic archaea such as CODH (AAM06652) from Methanosarcina acetivorans C2A (60%) and CODH (AAM29817) from Methanosarcina mazei Go1 (59%) (Fig. 4 and 5) and seemed to be a monofunctional CODH (4, 37), lacking the acetyl coenzyme A synthesis/cleavage activity of the bifunctional CODH/acetyl coenzyme A synthetase enzyme. According to Fox et al. (6), the monofunctional CODH/hydrogenase complex from Rhodospirillum rubrum participated in CO-driven proton respiration, whereby energy is conserved in the form of a proton gradient generated across the cell membrane. In this sense, to address the issue that the CODH cluster could play a similar role in energy conservation by oxidizing CO, we tested whether T. onnurineus NA1 could utilize CO and found that the strain, indeed, was able to grow better in medium 1 under a CO atmosphere, in both the absence and the presence of sulfur, than in the basal medium (Fig. 6a and b), even though the growth yield was still lower than that in the YPS medium (Fig. 6a). The growth under CO atmosphere was associated with the transcription of the CooS gene, indicating that the gene could be induced by the presence of CO (Fig. 6c). It is noteworthy that the addition of sulfur decreased the transcriptional level of the CooS gene. These results, taken together, support the hypothesis that T. onnurineus NA1 most likely uses CO as an energy and/or carbon source.

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FIG. 3. Representative map of hydrogenase gene clusters in T. onnurineus NA1. A, B, C, and D represent membrane-bound hydrogenases and cytoplasmic NiFe-hydrogenases. S1, S2, and S3 represent hydrogenases unique to T. onnurineus NA1, i.e., Hyg4-I, Hyg4-II, and Hyg4-III, respectively. Genes are colored according to COG functional categories.

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FIG. 4. Distribution and conservation patterns of hydrogenase gene clusters in 31 archaeal genomes. Blue brackets encompass CDSs showing low similarities (<25%) to any CDSs from 31 archaeal genomes. The black brackets indicate CDSs similar to hydrogenase 4 from P. abyssi.

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FIG. 5. Phylogenetic analysis of CODH (TON_1018). Protein alignment of 33 amino acid sequences was performed by ClustalX, and the phylogeny was constructed using PHYLIP. Bootstrap values are indicated at the branch of the tree. Proteins are represented by the gene identification (gi) numbers in the NCBI/GenBank protein sequence database, followed by the species names, except CooS proteins from Carboxydothermus hydrogenoformans, which are denoted by their corresponding gene names.

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FIG. 6. Growth profile of T. onnurineus NA1 depending on CO. T. onnurineus NA1 was grown in medium 1 supplemented with CO (lane 2 in a and c; up triangles in b), sulfur (lane 3; squares) or both (lane 4; triangles down). Controls without supplement (lane 1; circles) and culture in YPS medium (C) were included. DAPI-stained cells were directly counted on filters by fluorescence microscopy. (a) Effect of medium composition under the various concentrations of yeast extract (YE). (b) Growth curves of T. onnurineus NA1 in medium 1 with other supplements. (c) Analysis of the transcription of the CODH gene.

The carboxydotrophic pathway appears not to be conserved in T. kodakaraensis KOD1 as previously demonstrated by genomic analysis (7). According to Sokolova et al. (33), the Thermococcus sp. strain AM4 from an active chimney in the East Pacific Rise at 13°N also coupled CO oxidation with H₂ production anaerobically, indicating that members of the Thermococcus genus can use carboxydotrophy in geologically distinct hydrothermal vents. To determine how widely carboxydotrophy was distributed within the genus Thermococcus growing in hydrothermal vents, the presence of the CODH-encoding gene in seven Thermococcus species isolated from a solfataric acid field or hydrothermal vent was investigated. PCR products of CODH were not detected in any of the strains tested (data not shown). It is therefore suggested that CODH is restricted to only a few Thermococcus spp. It is possible, of course, that amplification was not successful because of differences in gene sequence variation and codon usage. When the genome sequencing data of Thermococcus sp. strain AM4 is completed, it will be possible to determine whether CODH is present in that strain. Sequences of additional Thermococcus spp. coupled with growth studies will provide an understanding of the distribution of genes controlling this new important metabolic pathway in hydrothermal vent microbial systems.

Carbon fixation. A gene (TON_1234) encoding type III ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39), which was identified in T. kodakaraensis KOD1 (29), was also found in T. onnurineus NA1, suggesting the possibility that the strain gains both energy and carbon via carboxydotrophy. T. onnurineus NA1 harbors both homologs for AMP phosphorylase (TON_1062) and ribose-1,5-bisphosphate isomerase (TON_1296), enzymes required to supply the RuBisCO substrate. When heterotrophic substrates are not available and CO₂ formed by catabolism is limited, the CO₂ yield from CO oxidation would be a suitable substrate for the RuBisCO enzyme. Key enzymes for other known carbon fixation pathways such as the reverse tricarboxylic acid and the 3-hydroxypropionate cycles were not found in this genome. It seems likely that carbon fixation in concert with carboxydotrophy by T. onnurineus NA1 contributes to its survival and persistence in hydrothermal vents.

Hydrogenase clusters. Besides evidence of carbon fixation and carboxydotrophy, an extraordinary proportion of hydrogenases and related proteins was detected in the T. onnurineus NA1 genome (5.5%), reflecting enhanced conservation or recycling of reducing potentials in association with oxidoreductases, including CO dehydrogenase and formate dehydrogenases (Fig. 3; see also Table S3 in the supplemental material). As shown in Fig. 3, three additional hydrogenase clusters (Hyg4-I, Hyg4-II, and Hyg4-III) were found in the T. onnurineus NA1 genome along with two membrane-bound hydrogenases (Mbh, TON_1583-1595; Mbx, TON_0486-0498) and two cytoplasmic NiFe-hydrogenases (Sulf-I, TON_0533-0544; Sulf-II, TON_0051-0055) reported in Pyrococcus spp. and T. kodakaraensis KOD1. It is known that in P. furiosus, when oxidation of reduced ferredoxin generated by the glycolytic pathway is coupled to H₂ production by Mbh, protons are pumped across the membrane, and energy is conserved in the form of ATP (27, 28). Mbx, which is highly similar to Mbh, is very likely involved in oxidizing reduced ferredoxin to ultimately transfer these electrons to elemental sulfur (30). The functions of Sulf-I and Sulf-II in P. furiosus are unclear, but it is possible that they serve to recycle H₂, providing NADPH for biosynthesis (19). Gene cluster analysis of hydrogenases with CDSs from 31 archaeal genomes clearly showed that Hyg4-I, Hyg4-II, and Hyg4-III were unique in primary sequence, showing low similarities to hydrogenase 4 components from P. abyssi and R. rubrum (Fig. 4; see also Table S4 in the supplemental material). Along with the additional hydrogenases, the Hyg4-III cluster (TON_1559-1582) included /β/ subunits of F420 hydrogenase (TON_1559-1561) in the genome. The /β/ subunits of F420 hydrogenase had unique primary sequences, showing similarities to the coenzyme F420-reducing hydrogenase (YP_001097176) from Methanococcus maripaludis (33%) and coenzyme F420-reducing hydrogenase (NP_987940) from M. maripaludis S2 (33%) (Fig. 4 and 7). No F420-hydrogenase homologues from the Thermococcales have been reported, to our knowledge. It is not certain what the functions are of these additional hydrogenases in T. onnurineus NA1, and further analysis will be necessary.

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FIG. 7. Phylogenetic analysis of the subunit of F420 hydrogenase (TON_1559). Protein alignment of 20 amino acid sequences was performed by ClustalX, and the phylogeny was constructed using PHYLIP. Bootstrap values are indicated at the branch of the tree. Proteins are represented by the gene identification (gi) numbers in the NCBI/GenBank protein sequence database, followed by the species names.

Based on a proteomic analysis of the whole cell, Hyg4-I and the /β/ subunits of F420 hydrogenase as well as Mbh, Mbx, and Sulf-I were present, implying that the Hyg4-I and F420 hydrogenases may be functional (see Table S3 in the supplemental material) even in heterotrophic growth of T. onnurineus NA1. The Hyg4-I gene cluster (TON_0261-0289) was found to be associated with genes encoding auxiliary proteins involved in hydrogenase maturation, perhaps working in trans for maturation of hydrogenases, similar to the three hydrogenases in Escherichia coli (12).

Biosynthetic pathways. The genome size of T. onnurineus NA1 was similar to that of the Pyrococcus strains sequenced to date but 10% smaller than that of T. kodakaraensis KOD1. The smaller size of T. onnurineus NA1 may be the result of a strategy of high coding density (90%) (Table 1), with genes essential for free living retained and the optimization of a variety of biosynthetic pathways. That is, since T. onnurineus NA1 does not have enzymes needed early in the 5'-phosphoribosyl-4-carboxamide-5'-aminoimidazole purine biosynthetic pathway, the purine building blocks may be derived as by-products of histidine biosynthesis (TON_0882 and TON_0884). The loss of a de novo biosynthetic pathway, therefore, is compensated for by adding unique enzymes through efficient recycling of deoxynucleotides, e.g., by the uridine phosphorylase (TON_0131), purine nucleoside phosphorylase (TON_0128 and TON_1651), thymidine kinase (TON_1233), and thymidylate synthase (TON_0437). Amino acid biosynthetic pathways are also minimized in the T. onnurineus NA1 genome since the complete pathways for amino acids, except tyrosine (TON_1130-1142) and histidine (TON_0879-0887), are absent (see Table S5 in the supplemental material). The lack of de novo pathways for amino acid biosynthesis can be compensated by the addition of unique amino acid transporters for the branched amino acids (TON_0158-0162) and cationic amino acids (TON_0783). According to Haberstroh and Karl (8), amino acids are detectable in vent fluids at micromolar concentrations, which are sufficient to support growth of heterotrophic hyperthermophiles.

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CO is ubiquitous in marine surface waters (38) and is also a natural component of volcanic exhalations (34). Carboxydotrophic microorganisms capable of utilizing CO as an energy and primary carbon source have been reported after Uffen (36) showed that a Rhodopseudomonas sp. was able to grow anaerobically in a CO atmosphere. Recently, a group of thermophilic microorganisms, termed hydrogenogens, isolated from hot springs or hydrothermal vents were found to fix CO and generate H₂ (31-33), showing CO serving as an energy and carbon source in a thermophilic environment. Even though most of the carboxydotrophs are autotrophic, Moran et al. (21) demonstrated that Silicibacter, a heterotrophic microorganism, could also employ carboxydotrophy under conditions of nutrient restriction; this is critical for microorganisms in hydrothermal vents since fluid discharge in hydrothermal vents that sustain biological communities can vary by hours, days, and years, as well as by distances of centimeters to kilometers (15). These changes expose vent communities to limitations of essential nutrients, inducing nutrient stress upon the ecosystem. As suggested by the findings for Silicibacter (21), a supplemental lithotrophic pathway, combined with organotrophic metabolism, can make it possible for organotrophs to cope with such nutrient stress and generate sufficient energy from inorganic compounds to survive and at least maintain viability until necessary organic nutrients are replenished. Unlike lithoheterotrophic bacteria at the ocean surface (22), T. onnurineus NA1 contains genes required for both CO oxidation and CO₂ fixation, and a study of how the two processes can be coupled during CO oxidation is necessary. Nonetheless, the power to utilize CO and the experimental proof of growth under a CO atmosphere lead to the conclusion that T. onnurineus NA1 has a significant advantage in its competition within the hydrothermal vent community.

Genomic analysis of T. onnurineus NA1 has proven to be critical in demonstrating the presence of the carboxydotrophic pathway in combination with heterotrophy and offers the first evidence through discovery of a gene cluster that the Thermococcales are lithotrophic. This lithotrophic feature in combination with carbon fixation via RuBisCO introduces a new strategy with a complementing energy supply for T. onnurineus NA1, potentially allowing it to cope with nutrient stress in the environment of hydrothermal vents.

 
We thank HoJin Jung from Ensoltek Co. for bioinformatics support.

This work was supported by KORDI in-house program (PE98210) and the Marine and Extreme Genome Research Center program, Ministry of Land, Transport and Maritime Affairs, Republic of Korea.

 
* Corresponding author. Mailing address: Korea Ocean Research and Development Institute, Ansan, P.O. Box 29, Seoul 425-600, South Korea. Phone: 82 31 400 6243. Fax: 82 31 406 2495. E-mail: jlee{at}kordi.re.kr

Published ahead of print on 12 September 2008.

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

H.S.L., S.G.K., and S.S.B. contributed equally to this study.

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Journal of Bacteriology, November 2008, p. 7491-7499, Vol. 190, No. 22
0021-9193/08/$08.00+0     doi:10.1128/JB.00746-08
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