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J OURNAL OF B ACTERIOLOGY , Apr. 2009, p. 2371–2379 Vol. 191, No. 70021-9193/09/$08.00 ϩ 0 doi:10.1128/JB.01525-08Copyright © 2009, American Society for Microbiology. All Rights Reserved. Complete Genome Sequence of the Anaerobic, Protein-DegradingHyperthermophilic Crenarchaeon Desulfurococcus kamchatkensis ᰔ † Nikolai V. Ravin, 1 ‡ Andrey V. Mardanov, 1 ‡ Alexey V. Beletsky, 1 Ilya V. Kublanov, 2 Tatiana V. Kolganova, 1 Alexander V. Lebedinsky, 2 Nikolai A. Chernyh, 2 Elizaveta A. Bonch-Osmolovskaya, 2 and Konstantin G. Skryabin 1 * Centre “Bioengineering,” Russian Academy of Sciences, Moscow 117312, Russia, 1 and Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow 117312, Russia 2 Received 28 October 2008/Accepted 23 December 2008 Desulfurococcus kamchatkensis is an anaerobic organotrophic hyperthermophilic crenarchaeon isolated froma terrestrial hot spring. Its genome consists of a single circular chromosome of 1,365,223 bp with no extra-chromosomal elements. A total of 1,474 protein-encoding genes were annotated, among which 205 are exclusivefor D. kamchatkensis . The search for a replication srcin site revealed a single region coinciding with a globalextreme of the nucleotide composition disparity curve and containing a set of crenarchaeon-type srcinrecognition boxes. Unlike in most archaea, two genes encoding homologs of the eukaryotic initiator proteinsOrc1 and Cdc6 are located distantly from this site. A number of mobile elements are present in the genome,including seven transposons representing IS 607 and IS 200 /IS 605 families and multiple copies of miniatureinverted repeat transposable elements. Two large clusters of regularly interspaced repeats are present; noneof the spacer sequences matches known archaeal extrachromosomal elements, except one spacer matches thesequence of a resident gene of D. kamchatkensis . Many of the predicted metabolic enzymes are associated withthe fermentation of peptides and sugars, including more than 30 peptidases with diverse specificities, a numberof polysaccharide degradation enzymes, and many transporters. Consistently, the genome encodes both en-zymes of the modified Embden-Meyerhof pathway of glucose oxidation and a set of enzymes needed forgluconeogenesis. The genome structure and content reflect the organism’s nutritionally diverse, competitivenatural environment, which is periodically invaded by viruses and other mobile elements. Desulfurococcus kamchatkensis strain 1221n T (also known asDSMZ 18924 T or VKM B-2413 T ) was isolated from a hotspring of Uzon Caldera (Kamchatka Peninsula, Russia). Theorganism is an obligately anaerobic, heterotrophic archaeonable to grow at temperatures between 65 and 87°C, with theoptimum at 85°C. Its cells are nonmotile regular cocci of 0.6 to1 m in diameter. D. kamchatkensis utilizes a wide range of proteinaceous substrates, including ␣ -keratin, albumin, andgelatin, as carbon and energy sources. It is also able to fermentsome monosugars (hexoses), disaccharides, and oligosaccha-rides. Elemental sulfur is not essential but stimulates growth.Products of glucose fermentation in the absence of sulfur in-clude CO 2 , H 2 , acetate, and a minor amount of propionate. Inthe presence of elemental sulfur, H 2 S is formed instead of H 2 . A detailed phenotypic description of D. kamchatkensis waspresented previously (21). D. kamchatkensis is a member of the archaeal phylum Cre- narchaeota . To date, more than 700 bacterial and 50 archaealgenomes have been sequenced completely, and 15 of themrepresent thermophilic crenarchaea (Fig. 1). In addition, thecomplete genome sequences of two mesophilic crenarchaea, Nitrosopumilus maritimus and Cenarchaeum symbiosum , havebeen determined, although the phylogenetic position of thisgroup is under discussion.Hyperthermophilic archaea of the genus Desulfurococcus are anaerobic organotrophs inhabiting terrestrial hot springs of Iceland, Kamchatka, Kurils, and Yellowstone National Park(56). Members of the genus Desulfurococcus are able to hy-drolyze diverse substrates, including proteins and polysaccha-rides, obtaining energy for growth from fermentation of themonomers formed. Their genomes bear genes encoding di- verse thermostable hydrolases that could find application indifferent fields of industry (9). The ability of these organisms toform molecular hydrogen as the end product of biopolymerdegradation could be used in the process of biofuel production.The D. kamchatkensis genome presented here is the first to bereported for the genus Desulfurococcus and constitutes an im-portant step in extending our knowledge of crenarchaeal di- versity. MATERIALS AND METHODSCultivation of D. kamchatkensis . D. kamchatkensis 1221n T was obtained fromthe culture collection of the Laboratory of Hyperthermophilic Microbial Com-munities, Winogradsky Institute of Microbiology, Russian Academy of Sciences.Cells were grown in anaerobically prepared medium consisting of mineral back-ground to which peptone (2 g liter Ϫ 1 ), yeast extract (0.2 g liter Ϫ 1 ), and elementalsulfur (10 g liter Ϫ 1 ) were added. The headspace of the bottle was filled with anN 2 -CO 2 (80:20) mixture. The pH of the medium was 6.5, and the incubationtemperature was 85°C. Cells were harvested in the early exponential phase bycentrifugation at 8,000 rpm for 15 min. Genome sequencing. A combined strategy including Sanger sequencing of aclonal shotgun library and 454 pyrosequencing was used. For Sanger sequencing, * Corresponding author. Mailing address: Centre “Bioengineering,”Russian Academy of Sciences, Prosp. 60-Let Oktyabrya, Bld. 7-1, Mos-cow 117312, Russia. Phone: (7) 499 7833264. Fax: (7) 499 1357319.E-mail:
[email protected].† Supplemental material for this article may be found at http://jb.asm.org/.‡ N.V.R. and A.V.M. contributed equally to this work. ᰔ Published ahead of print on 29 December 2008.2371 the shotgun library was constructed in the plasmid vector pUC19 (average insertfragment size, 2 kb), followed by sequencing of 5,000 clones from both ends. Theresulting sequences were assembled into about 200 large contigs with the Consedsoftware package (14). The 454 pyrosequencing part of the project was per-formed on a GS FLX sequencer and resulted in the generation of about 50 Mbof sequences, with an average read length of 220 bp. The GS FLX reads wereassembled into 13 large contigs ( Ͼ 2,000 bp) by use of a GS de novo assem-bler. The 454 contigs were oriented into scaffolds, and the complete genomesequence was generated upon adding the set of Sanger contigs to the assem-bly. Several sequence ambiguities were resolved by generating and sequenc-ing appropriate PCR fragments. The assembly of the genome at sites withinsertion sequence (IS) elements was verified by PCR amplification andsequencing of these regions. Genome annotation and analysis. The rRNA genes were identified by a searchagainst the Rfam database (15). tRNA genes were located with tRNAscan-SE(26). Protein coding genes were identified with the GLIMMER gene finder (11).Whole-genome annotation and analysis were performed with the AutoFACTannotation tool (20). Frameshifts or premature stop codons within coding se-quences were identified by comparison to other species and confirmed to be“authentic” by either their high-quality sequencing reads or resequencing. Clus-ters of regularly interspaced repeats (CRISPR) were identified using CRISPRFinder (16); putative transposon-related proteins were found by a search againstthe IS database (http://www-is.biotoul.fr/is.html). Signal peptides were predicted with SignalP v. 3.0 (http://www.cbs.dtu.dk/services/SignalP/), using the HMMalgorithm. Transmembrane protein topology was predicted with TMHMMServer v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Nucleotide sequence accession number. The annotated genome sequence hasbeen deposited in GenBank under accession no. CP001140. RESULTS AND DISCUSSIONGeneral features. D. kamchatkensis has a single circularchromosome of 1,365,223 bp with no extrachromosomal ele-ments (Fig. 2). There is a single copy of the 16S-23S rRNA operon and a single distantly located 5S rRNA gene. A total of 47 tRNA genes are scattered over the genome. By a combina-tion of coding potential prediction and similarity searches,1,474 potential protein-encoding genes were identified, with anaverage length of 818 bp, covering 88.4% of the genome. These values are in good accordance with the general correlation FIG. 1. 16S rRNA-based phylogenetic tree showing representatives of all validly described genera of the phylum Crenarchaeota and two genera of mesophilic crenarchaea still lacking valid description ( N. maritimus and C. symbiosum ). Underlined are organisms for which complete genome sequenceshave been determined. The genera Sulfolobus and Pyrobaculum (genomes of three species were sequenced for each genus) are exemplified by S. acidocaldarius and P. islandicum . Caldisphaera lagunensis and Acidilobus aceticus are shown within the order Desulfurococcales ; inclusion of these generain the new order Acidilobales is under discussion. Sequence alignment was performed using ClustalX, version 1.81, and the tree was constructed by theneighbor-joining method, using the TREECON program (48). The euryarchaeon Thermococcus kodakaraensis was used as the outgroup.FIG. 2. Circular representation of the D. kamchatkensis genome.Circles, from outer to inner, display a physical map scaled to kb(outermost circle), predicted protein-encoding genes in the clockwisedirection (second circle) and in the counterclockwise direction (thirdcircle), predicted tRNA and rRNA genes (fourth circle), and predictedmobile elements ISDka1, ISDka2, MITE-1, and MITE-2 (innermostcircle). oriC , the putative replication srcin.2372 RAVIN ET AL. J. B ACTERIOL . between the microbial genome size and the predicted genenumbers.Through similarity and domain searches for the predictedprotein products, functions of 75% (1,111 genes) of them maybe predicted with different degrees of confidence and gener-alization. The functions of the remaining 363 genes (25%)cannot be predicted from the deduced amino acid sequences;among them, 205 genes are unique to D. kamchatkensis , withno significant similarity to any known sequences.The numbers of predicted protein genes that are homolo-gous with genes from the other sequenced representatives of the family Desulfurococcaceae , namely, Staphylothermus mari- nus (NC_009033) and Ignicoccus hospitalis (NC_009776), areillustrated in Fig. 3 (left panel). The data show that D. kam- chatkensis and S. marinus share about 55 to 58% of the totalgene pool, but only about half of these common genes are alsopresent in the I. hospitalis genome. Consistent with this obser- vation, fewer than 5% of D. kamchatkensis genes have ho-mologs in I. hospitalis but not in the S. marinus genome; thesame is true for S. marinus . In this trio, each genome carries alarge number of genes exclusive to it, about 40% for D. kam- chatkensis and S. marinus and 63% for the genome of I. hos- pitalis . These results serve to underline the considerable diver-sity even among members of one family of hyperthermophiliccrenarchaea.The second plot (Fig. 3, right panel) demonstrates the num-bers of homologs shared between D. kamchatkensis , S. mari- nus , and a representative of the evolutionary distant eury-archaeal order Thermococcales , Thermococcus kodakaraensis. Interestingly, T. kodakaraensis carries even more homologs of D. kamchatkensis and S. marinus genes than does I. hospitalis ,a member of the same crenarchaeal family, Desulfurococ- caceae . This may be attributed partly to the larger number of genes in the T. kodakaraensis genome, but the primary reasonis probably the similarity of the main metabolic pathways of D. kamchatkensis and T. kodakaraensis : both organisms are fer-menters growing on proteinaceous substrates, while I. hospi-talis grows chemolithoautotrophically (37). These results showthat genome diversity does not directly correlate with evolu-tionary distances between organisms, as measured by 16SrRNA sequence divergence, but may be influenced strongly byenvironmental adaptation requirements.Genes required for protein export systems are present in D. kamchatkensis , and the SignalP algorithm predicts a total of 97proteins carrying N-terminal signal sequences. Most of themhave been annotated as transporters, exported binding pro-teins, proteinases, or hypothetical proteins. Mobile elements. Analysis of the repeated sequences and asearch against the IS database revealed seven putative trans-posons belonging to two IS families. We found six almostidentical complete copies of an ISC1913-like transposon of theIS 607 family, which was named ISDka1. This 1,992-bp trans-poson contains two genes, orfA and orfB , encoding homologsof resolvase and transposase of ISC1913. In addition, we foundeight almost identical copies of 267-bp miniature inverted re-peat transposable elements (MITEs) derived from ISDka1.Such MITEs often occur in crenarchaeal genomes where ISelements are present (3). The almost identical sequences of different copies of ISDka1 and MITE-1, derived from it, sug-gest that this IS element invaded the D. kamchatkensis genomerecently on the evolutionary time scale and probably remainsfunctional. Another IS family, IS 200 /IS 605 , is represented by asingle copy of an ISSis2-like transposon. This IS element,named ISDka2, is 1,890 bp long and also carry two genesencoding homologs of transposase and resolvase. Unlike mostIS elements, the transposons present in D. kamchatkensis haveno inverted repeats in their terminal regions, as has beenreported for the IS 200 /IS 605 family (30).The third set of IS elements comprise eight copies of an-other MITE. These repeats (MITE-2) are more divergent inboth sequence and length, and some of them contain internaldeletions; the lengths of individual repeats are between 225and 260 bp, and the identities of sequences are in the range of 74 to 98%. The parental IS element cannot be identified in the D. kamchatkensis genome. Probably, these MITEs are rem-nants of an ancient invasion of an IS sequence whose full copyhas already been eliminated, while its MITE derivatives are indifferent stages of decay.Searches against the Sulfolobus database (5) yielded no clearevidence for the presence of integrated archaeal viruses andplasmids in the genome. We found, however, two phage-re-lated integrase genes (Dkam_1130 and Dkam_1004) that couldrepresent ancient integration events. Introns and inteins. The D. kamchatkensis genome contains47 tRNA genes, and 16 of them contain introns. No introns arepresent in the rRNA genes. Until now, only one intron hasbeen found to occur within an archaeal protein-encoding gene(52), but several others have been predicted (4). This intron islocated in homologs of the eukaryotic cbf5 gene, encodingRNA pseudouridine synthase. Its excision from mRNAs in Aeropyrum pernix , Sulfolobus solfataricus , and Sulfolobus toko- daii restores the integrity of the coding region of the cbf5 gene(52). The D. kamchatkensis cbf5 gene (Dkam_1147) also con-tains an internal stop codon, suggesting the presence of a 22-bpintron whose excision would restore the full coding potential of the cbf5 gene (see Fig. S1 in the supplemental material). The FIG. 3. Homologous protein coding genes of D. kamchatkensis andother hyperthermophilic archaea. The overlapping circle plots showthe numbers of homologs shared between the genomes of three mem-bers of the family Desulfurococcaceae ( D. kamchatkensis , S. marinus ,and I. hospitalis ) and the genomes of D. kamchatkensis , S. marinus , and T. kodakaraensis , representing hyperthermophilic euryarchaea. Num-bers of D. kamchatkensis genes are shown in bold, and numbers of S. marinus genes are underlined. Genes were considered present in a pairof genomes if the region of similarity covered Ͼ 70% of the corre-sponding protein, with Ͼ 50% identity of the amino acid sequences.V OL . 191, 2009 DESULFUROCOCCUS KAMCHATKENSIS GENOME SEQUENCE 2373 search for other “split” genes whose coding potential could berestored by excision of hypothetical introns revealed only ei-ther potential points of programmed frameshifts, known tooccur in some archaea (8), or genes broken by insertions of ISelements. Archaeal genomes often contain inteins, in-frame sequencesexcised by a self-catalytic mechanism after translation.Searches against the intein database (38) gave only one signif-icant hit, a 201-amino-acid intein in a predicted anaerobicribonucleoside triphosphate reductase (Dkam_1118). This in-tein contains a self-splicing domain but not a homing endonu-clease domain and, consequently, is not able to spread further.Its closest homolog is an intein located within the gene encodingthe large subunit of DNA polymerase II of the euryarchaeon Pyrococcus abyssi . Genome defense systems. The search for potential restric-tion enzymes identified an unusual hybrid endonuclease-meth- yltransferase fusion protein (Dkam_1203) similar to enzymesfound in T. kodakaraensis (TK1158 and TK1460). Both ade-nine- and cytosine-specific DNA methylases are present (en-coded by genes Dkam_0485 and Dkam_1265, respectively). Another gene, Dkam_0306, is similar to the adenine-specificDNA methylase gene of the Sulfolobus virus STSV1 and mayhave appeared in the genome as a result of a horizontal genetransfer event. D. kamchatkensis contains two CRISPR located close toeach other and containing 8 and 85 repeat spacer units. Thespacer regions are supposed to be derived from mobile ele-ments, such as viruses (34), and their spacer transcripts mayinactivate mobile element propagation by a mechanism some- what similar to eukaryotic RNA interference (2, 24). We foundno matches between the spacer sequences and any knownarchaeal extrachromosomal genetic elements, but this maymerely reflect the unavailability in public databases of se-quences of viruses and plasmids that can propagate in D. ka- mchatkensis . There was also no CRISPR spacer match in thesequences of the IS elements present in the D. kamchatkensis genome.The only match of spacer sequences of the CRISPR locus isin another region within the D. kamchatkensis genome itself.One 41-bp spacer sequence matches the 3 Ј region of theDkam_1260 gene, so the predicted mRNA of the spacer pro-duced by transcription of the CRISPR locus is complementaryto the mRNA of Dkam_1260. The function of Dkam_1260 isunknown, although homologous genes are present in the S. marinus (Smar_0909) and Hyperthermus butylicus (Hbut_0467)genomes. Matching of a spacer sequence of the CRISPR locusand a sequence of an endogenous gene opens the question of whether CRISPR activity may be a mechanism for controllingexpression of the organism’s resident genes, similar to RNA interference in eukaryotes. A single superoperon containing a set of cas and csa genesadjoins a larger repeat cluster. These proteins are supposed tobe involved in the development and activity of the CRISPRlocus (19, 24). The gene order is crenarchaeon specific (24),except for the absence of apparent homologs of the cas4 and cas6 genes. DNA replication. The archaeal chromosomal replication ini-tiation site ( oriC ) was first identified in P. abyssi within thenoncoding region located upstream of a gene encoding a ho-molog of eukaryotic Orc1/Cdc6 cell division control proteins(36). Some archaea carry multiple cdc6 genes, and for Sulfolo- bus and Pyrococcus species, multiple chromosomal replicationsrcins were found to be located close to them (27, 33, 40).Z-curve analysis (55) of the D. kamchatkensis genome showedone major peak, at around 1,150 kb, where a nucleotide com-positional deviation change occurred (Fig. 4). This peak wasfound for the Y component of the Z curve (MK disparity), asobserved for Sulfolobus acidocaldarius (6) and Pyrobaculum aerophilum (55) chromosomal ori sites. The search for crenar-chaeal srcin recognition box (ORB) sequences, the bindingsites for Orc1/Cdc6 proteins (40, 41), revealed that the non-coding region located between genes Dkam_1234 (putativeDNA binding protein Alba2) and Dkam_1235 (unknown func-tion) contains four copies of the ORB motif (Fig. 4). Theposition of this region coincides with the location of the MK disparity peak (Fig. 4), further supporting the hypothesis thatthe ori site is located at this point. This site also contains twocopies of another ori -specific motif, UCM (41). Anotherknown ori -binding protein is the archaeal WhiP initiator pro-tein, a homolog of the eukaryotic protein Cdt1 (41). The whiP genes are present in genomes of Sulfolobus species, A. pernix ,and H. butylicus (41), but we found no apparent homolog of this protein in D. kamchatkensis . Two orc1 / cdc6 homologs,encoding replication initiation proteins, are present in the D. kamchatkensis genome (Dkam_1377 and Dkam_1427), butboth are located distantly from the potential oriC site, and noORB-like sequences were found around these genes.The normal set of genes associated with the crenarchaealreplication apparatus is present in the D. kamchatkensis ge-nome (32), as well as two homologs (Dkam_1233 and Dkam_0826) of reverse gyrase, the hyperthermophile-specific enzymeresponsible for introduction of positive supercoils in the DNA molecule (13). Transcription and translation. The full set of crenarchaealRNA polymerase subunits (53) is present, as are archaealtranscription factors, including a single TATA-box-bindingprotein (Dkam_0275) and two transcription initiation factorsIIB (Dkam_0056 and Dkam_0922). A single copy of the 16S/ 23S rRNA gene operon and a single distantly located 5S rRNA gene are present, as well as the usual set of crenarchaealribosomal protein genes (25).The D. kamchatkensis genome contains 47 tRNA genes car-rying 43 different anticodons coding for 19 amino acids. We were not able to identify a gene for tRNA Trp (CCT), althoughthe corresponding tryptophanyl-tRNA synthetase gene ispresent (Dkam_1457). As in most other archaeal genomes,three tRNA Met (CAT) genes are present, and one of themcontains an intron. A selenocysteine incorporation system isabsent, as in other crenarchaeal genomes. Searches for otheruntranslated RNAs revealed a gene encoding the RNA com-ponent of RNase P. Aminoacyl tRNA synthetases were identified for all aminoacids except glutamine. Two genes encoding subunits of glu-tamyl-tRNA Gln amidotransferase were found; thus, as in manyother archaea and gram-positive bacteria (10), glutamine-tRNAs are probably aminoacylated with glutamic acid, fol-lowed by transamidation to yield the glutaminyl-tRNA. Twogenes were found for methionyl-tRNA synthetase: the first oneencodes the full-size enzyme of 499 amino acids (Dkam_0547), 2374 RAVIN ET AL. J. B ACTERIOL . while the second gene (Dkam_0847) encodes a shorter, 109-amino-acid protein corresponding to the C-terminal part of thelong methionyl-tRNA synthetase gene. Close homologs of thefirst enzyme are present in the genomes of S. marinus (Smar_1511) and many thermophilic bacteria ( Aquifex , Thermus , Pet- rotoga , Thermoanaerobacter , etc.) but not in other archaea.This gene was probably horizontally transferred from a ther-mophilic bacterium to a common progenitor of D. kamchat- kensis and S. marinus . The short variant of methionyl-tRNA synthetase is of archaeal srcin. Peptidases. The particular property of D. kamchatkensis thatprompted us to sequence its genome is the ability of thisarchaeon to grow on a variety of proteinaceous substrates,including peptone, tryptone, beef extract, albumin, gelatin, andsuch resistant protein as ␣ -keratin. This suggests that D. kam- chatkensis should produce heat-stable extracellular peptidases. A number of peptidases have been isolated from crenarchaeonsof the genera Desulfurococcus , Sulfolobus , Staphylothermus , Py- robaculum , and Aeropyrum (reviewed in reference 51).The genome of D. kamchatkensis contains more than 30different endo- and exopeptidases that belong to different fam-ilies, and genes for peptide transport into the cell. At least fiveproteases carry putative N-terminal signal peptides, suggestingthat they may be active extracellularly: 70-kDa serine protease-like protein Dkam_0359, 49-kDa putative membrane-boundserine protease Dkam_0447, 17-kDa signal peptidase Dkam_1437, and two subtilisin-like serine proteases, Dkam_1274 (141kDa) and Dkam_1144 (44 kDa). Intracellular proteolysis con-sists in joint action of several intracellular proteases, includingaminopeptidases of different families, Dkam_0225, Dkam_0465, Dkam_1239, and others; metallopeptidases, Dkam_0358,Dkam_1341, and Dkam_1138; trypsin-like serine protease,Dkam_0433; and intracellular proteasome subunits, Dkam_1288 and Dkam_1278.The comparison of amino acid sequences of putative extra-cellular proteases from D. kamchatkensis with those availablein GenBank showed that their closest homologs are peptidasesinferred from genome sequences of Crenarchaeota species.Dkam_0359 belongs to S16-type family of serine proteasesubiquitously present in cren- and euryarchaeal genomes(COG1750). Protease Dkam_0447 belongs to NfeD-like mem-brane-bound serine proteases; its amino acid sequence waspredicted to contain six transmembrane domains. ProteasesDkam_1274 and Dkam_1144 belong to subtilisin-like family of serine proteases (COG1404). The closest homologs of Dkam_1274 are protease Tpen_1714 from T. pendens and a surfacelayer-associated stable protease from P. aerophilum (30 to 38%identity). Protease Dkam_1144 has more than 50% identity FIG. 4. Z-curve analysis of the D. kamchatkensis genome (only the MK disparity component is presented) showing the major peaks wherenucleotide compositional deviations occur. On the genome map, cdc6 genes are shown. In the bottom part of the figure, the structure of thepredicted oriC region is shown. Open arrows indicate the position and direction of ORB elements; black arrowheads show the positions of theUCM motif. ORB and UCM nucleotide sequences are presented. Open rectangles show the open reading frames flanking the ori site, with arrowsindicating the direction of transcription.V OL . 191, 2009 DESULFUROCOCCUS KAMCHATKENSIS GENOME SEQUENCE 2375
