Complete Genome Sequence of the Hyperthermophilic, Piezophilic, Heterotrophic, and Carboxydotrophic Archaeon Thermococcus barophilus MP

Abstract

Thermococcus barophilus is a hyperthermophilic, anaerobic, mixed heterotrophic, and carboxydotrophic euryarchaeon isolated from the deep sea hydrothermal vent Snakepit site on the mid-Atlantic ridge at a depth of 3,550 m. T. barophilus is the first true piezophilic, hyperthermophilic archaeon isolated, having an optimal growth at 40 MPa. Here we report the complete genome sequence of strain MP, the type strain of T. barophilus. The genome data reveal a close proximity with Thermococcus sibiricus, another Thermococcus isolated from the deep biosphere and a possible connection to life in the depths.

Thermococcus barophilus strain MP is the first true hyperthermophilic, piezophilic archaeon and was isolated in 1993 (8) from an enrichment of chimney samples in YPS-20 rich medium at 95°C and 40 MPa. Strain MP grows from 48°C to 100°C with an optimum at 85°C and within a pressure range of 0.1 to 85 MPa with an optimum of 40 MPa (8, 10). A phylogenetic analysis using concatenated ribosomal proteins has shown that strain MP is most closely related to Thermococcus sibiricus strain MM379 (7). These two strains define a cluster which is almost equally distant from the Pyrococcus core cluster represented by P. abyssi GE5, P. furiosus DSM 3638, P. yayanosii CH1, and P. horikoshii OT3 (1a, 2, 4, 6, 10) and the Thermococcus core cluster represented by T. gammatolerans EJ3, T. kodakaraensis KOD1, and T. onnurineus NA1 (3, 5, 11).

T. barophilus MP was accepted in the marine microbe sequencing project (www.moore.org/microgenome) for whole-genome shotgun (WGS) sequencing in 2005 by the Gordon and Betty Moore Foundation. Genomic libraries of 4 kb and 10 kb were constructed and sequenced by the Sanger method to an 8-fold level of coverage.

Sequence reads from a total of 55,488 shotgun clones were assembled and analyzed with the JCVI CONSED and Manatee packages, and 12 contigs were generated and connected by PCR. A preliminary open reading frame prediction for 2,268 genes across the genome was conducted by automated annotation with Glimmer (http://www.cbcb.umd.edu/software/glimmer) and RAST (1). The annotation was manually cured using BLAST and the nr database of the NCBI.

The T. barophilus MP genome consists of a circular chromosome of 2,010,078 bp and a circular plasmid, pTBMP1, of 54,159 bp, with average G+C contents of 41.7 and 38.3%, respectively. There are one copy of 16S-23S and two copies of 5S and 46 tRNA genes. The T. barophilus chromosome contains four CRISPR loci composed of 7 to 26 direct DNA repeats (repeat length, 30 and 31 bp) interspersed with 6 to 25 nonrepetitive sections of 34 to 51 nucleotides called spacers. Six CRISPR-associated genes (cas) that encode proteins involved in adaptation and interference were found in the vicinity of a single CRISPR locus (9), which is composed of seven repeats (30 bp) and six spacers (34 to 49 bp).

Like T. onnurineus and T. gammatolerans, T. barophilus possesses the carboxydotrophic pathway and bears seven different hydrogenase complexes (MBx, Mbh, Hyg 4-I, Hyg4-II, hyg4-III, SulFI, and SulF II). T. barophilus shares 1,089, 1,272, 1,112, and 1,236 genes (>60% identity) with T. gammatolerans, T. sibiricus, T. kodakaraensis, and T. onnurineus, respectively, while 221 genes of T. barophilus have no homologues in the above four Thermococcus species. The T. barophilus specific gene set, which bears little homology to proteins in the database, may provide clues to its adaptation to growth under the high-pressure conditions which are typical of the deep biosphere.

Nucleotide sequence accession numbers.

The final annotated genome and plasmid of T. barophilus strain MP reported in this paper are now available in GenBank under accession numbers CP002372 and CP002373, respectively.