Figure 1.
Phylogenetic tree of Thermococcales based on 16S rRNA gene sequences.
The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The bootstrap consensus tree inferred from 100 replicates. A discrete Gamma distribution was used to model evolutionary rate differences among sites (4 categories (+G, parameter = 0.1068)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 75.0448% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 45 nucleotide sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 1232 positions in the final dataset. Strains carrying sequenced plasmids are indicated by bold characters. The five plasmid-carrying Thermococcus strains isolated and characterized in this study are underlined.
Figure 2.
Comparative genomic analysis of the five novel thermococcal plasmids.
(A) pTN2-like and (B) pEXT9a-like plasmids Genes absolutely conserved in all eight plasmids are shown in red; the semi-conserved genes present in some members of both plasmid groups are in green; conserved genes restricted to either one of the two groups are depicted in blue. Abbreviations: SFI, superfamily I; wHTH, winged helix-turn-helix motif; ABC, ATP-binding cassette; RHH, ribbon-helix-helix motif; Prim-pol, primase-polymerase; TAS, toxin-antitoxin system. General characteristics of the plasmids depicted in this Figure can be found in Table 1.
Table 1.
General characteristics of the plasmids analysed in this study.
Figure 3.
Modular relationship among plasmid-encoded replication proteins.
Horizontal bars represent replication proteins with distinct domains indicated with different colours – the primase-polymerase domain is shown in blue, the C-terminal domain of pTN2 replicase is in red, and the winged helix-turn-helix domain is in green. The numbers above the bars represent the amino acid coordinates of the depicted domains in the replicases of respective plasmids (plasmid names are on the right). The numbers next to the plasmid names denote how many additional plasmids encode the specific type of a replication protein.
Figure 4.
Phylogenetic analysis of the plasmid-encoded superfamily I helicases.
The plasmid names are coloured according to the geographical origin of the strains from which they were isolated: blue, East Pacific Ocean ridge; red, Mid-Atlantic Ocean ridge; green, Indian Ocean triple junction. Replicase–helicase (rep and hel) gene cassettes are shown on the right; domain organizations of the replication proteins encoded by plasmids within the three phylogenetic clades (1–3) are indicated. The plasmid of Methanocaldococcus vulcanius M7 is boxed. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT model (+I, +G [4 categories]). Numbers at the branch-points represent bootstrap values (100 replicates). The scale bar represents the number of substitutions per site. The analysis involved 12 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 398 positions in the final dataset. The tree was rooted with helicases from haloarchaea, based on previous phylogenetic analysis [11]. GenBank accession numbers: Halorubrum lacusprofundi ATCC 49239, YP_002564878; Halogeometricum borinquense DSM 11551, YP_004037766; Thermococcus onnurineus NA1, YP_002307767; Thermococcus gammatolerans EJ3 integrating element TGV2, YP_002959996; pMETVU01, YP_003248093; pTN2, YP_003603582; pP12-1, YP_003603439.
Figure 5.
Relationship between thermococcal plasmids and virus PAV1.
Homologous genes are coloured similarly. PAV1 ORF528, which has homologues in haloarchaeal plasmids, is shaded grey.