Genome Sequence of a Hyperthermophilic Archaeon, Thermococcus nautili 30-1, That Produces Viral Vesicles

Abstract

Thermococcus nautili 30-1 (formerly Thermococcus nautilus), an anaerobic hyperthermophilic marine archaeon, was isolated in 1999 from a deep-sea hydrothermal vent during the Amistad campaign. Here, we present the complete sequence of T. nautili, which is able to produce membrane vesicles containing plasmid DNA. This property makes T. nautili a model organism to study horizontal gene transfer.

GENOME ANNOUNCEMENT

Thermococcus nautili 30-1 (formerly Thermococcus nautilus) was isolated on the East Pacific ridge near a black smoker, 2,600 m below the ocean surface (1). T. nautili qualifies as a new species and the characterization of its physiology and metabolism has been reported (2).

T. nautili was grown anaerobically in ZB medium at 85°C (1), and genomic DNA was purified and sequenced using paired-end Illumina libraries to generate 16 × 10⁶ reads assembled with Velvet (3). Genome assembly was performed with Consed (4). The remaining gaps were filled using bacterial artificial chromosome (BAC) sequencing with Thermofidelase and Fimers (5). Genes were annotated using FGENESB (Softberry). Additional functional annotations were obtained with Integrated Microbial Genomes (6). The chromosome of T. nautili is 1,976,356 bp long and harbors 2,288 genes. The noncoding genes comprise 4 rRNA genes (23S, BD01_1261, and 16S, BD01_1259), with a duplication of the 5S rRNA gene (BD01_1590 and BD01_2049), and 46 tRNA genes. The chromosomal origin of replication was identified with FITBAR (7) using published origin recognition complex (ORC) sequences (8). It is positioned upstream of the CDC6 gene (BD01_1824). A similar approach located the replication termination region at coordinate 772,784 using archaeal dif sites (9).

Several strains of Thermococcus contain either one or two plasmids (10–13). Our group previously sequenced and characterized two plasmids of 3.6 and 13 kb from T. nautili that were named pTN1 and pTN2, respectively (14–16). The rolling-circle plasmid pTN1 was used to construct the first shuttle vector able to replicate in both Escherichia coli and the model organism Thermococcus kodakaraensis (17) and contributed to the development of T. kodakaraensis as a host for genetics studies (18). Plasmid pTN2 encodes a new family of DNA primase formed by the fusion of protein domains homologous to the archaeo-eukaryotic primases PriS and PriL (19). DNA sequencing revealed the presence of an additional plasmid of 18 kb, pTN3 (16). This extrachromosomal element, related to the virus-like element TKV4 from T. kodakaraensis (20), is found as a freely replicating plasmid and also integrated at a specific position on the chromosome into a tRNA^Leu gene (BD01_0018). The plasmid attachment site (attP) is located within the integrase-encoding gene TnaP3-13 (16). The attP and attB sites share an identical stretch of 41 nucleotides (nt), as observed previously in the case of the TKV4 integron of T. kodakaraensis (20). In the chromosome of T. nautili, 6 clustered regularly interspaced short palindromic repeat (CRISPR) loci were found using CRISPRFinder (21). Interestingly, two CRISPR spacers are directed against resident plasmid pTN3.

A major interest in the study of T. nautili is its ability to generate membrane vesicles containing plasmid DNA (22), including the virus-like genome of pTN3 (viral vesicles) (16). The discovery that these vesicles can transfer genetic information between related Thermococcales (23) provides an important tool for the study of horizontal gene transfer across species or even domains, from which genomic evolution models can be inferred. The genetic determinism of the biosynthesis of these viral vesicles is presently being investigated and relies primarily on the T. nautili genome sequence presented here.

Nucleotide sequence accession number.

The T. nautili genome sequence has been deposited in the NCBI repository under the accession number CP007264.