On the chimeric nature, thermophilic origin, and phylogenetic placement of the Thermotogales

Abstract

Since publication of the first Thermotogales genome, Thermotoga maritima strain MSB8, single- and multi-gene analyses have disagreed on the phylogenetic position of this order of Bacteria. Here we present the genome sequences of 4 additional members of the Thermotogales (Tt. petrophila, Tt. lettingae, Thermosipho melanesiensis, and Fervidobacterium nodosum) and a comprehensive comparative analysis including the original T. maritima genome. While ribosomal protein genes strongly place Thermotogales as a sister group to Aquificales, the majority of genes with sufficient phylogenetic signal show affinities to Archaea and Firmicutes, especially Clostridia. Indeed, on the basis of the majority of genes in their genomes (including genes that are also found in Aquificales), Thermotogales should be considered members of the Firmicutes. This result highlights the conflict between the taxonomic goal of assigning every species to a unique position in an inclusive Linnaean hierarchy and the evolutionary goal of understanding phylogenesis in the presence of pervasive horizontal gene transfer (HGT) within prokaryotes. Amino acid compositions of reconstructed ancestral sequences from 423 gene families suggest an origin of this gene pool even more thermophilic than extant members of this order, followed by adaptation to lower growth temperatures within the Thermotogales.

Fig. 1.

Pan-genome of 5 members of Thermotogales. The Venn diagram shows the number of genes shared by different subsets of genomes, as determined by BLAST+BRANCHCLUST (see SI Methods for details). Pie charts show the distribution of genes unique for each genome across functional supercategories. The size of a pie chart is proportional to the number of genes.

Fig. 2.

The ferredoxin:NAD(P)H oxidioreductase operon shared by Thermotogales and Thermococcales species. The homologous genes in P. furiosus compose the mbx operon. The promoter-proximal and -distal locus names are indicated.

Fig. 3.

Examination of evolutionary relationships between genes in the Thermotogales genomes and their homologs in the Archaea, Aquificales, and Clostridia. For each scenario, all embedded quartets in a tree made from Thermotogales, 2 Archaea, 2 Aquificales, and 2 Clostridia homologs were evaluated. A score per scenario was calculated as the ratio of the number of embedded quartets significantly supporting the scenario to the total number of evaluated quartets (for more details see SI Methods). Numbers in parentheses refer to subsets of data from the “Information Storage and Processing” functional supercategory. A quartet topology is unrooted and does not allow distinguishing which 2 taxa are responsible for the observed relationship (i.e., the inferred topology could be because of gene flow either to/from Thermotogales or to/from Aquificales); analyses in Table 1 provide some evidence for directionality. Abbreviations: T, Thermotogales genome; C, Clostridiales; Aq, Aquificales; Ar, Archaea.

Fig. 4.

CvP values indicate the ancestor of the Thermotogales was an extreme thermophile. (A) Distribution of CvP values for predicted proteins of Thermotogales' ancestral proteome. The results of reconstruction with 2 different programs are shown (see Methods). (B) Extrapolation of optimal growth temperature for the ancestral Thermotogales proteome. Red points represent median CvP values and optimal growth temperature of 5 contemporary Thermotogales genomes. A strong linear correlation is observed between CvP values and optimal growth temperature. The median CvP value of the ancestral proteome is based on 423 inferred ancestral protein sequences.