The Evolution of Reverse Gyrase Suggests a Nonhyperthermophilic Last Universal Common Ancestor

Abstract

Reverse gyrase (RG) is the only protein found ubiquitously in hyperthermophilic organisms, but absent from mesophiles. As such, its simple presence or absence allows us to deduce information about the optimal growth temperature of long-extinct organisms, even as far as the last universal common ancestor of extant life (LUCA). The growth environment and gene content of the LUCA has long been a source of debate in which RG often features. In an attempt to settle this debate, we carried out an exhaustive search for RG proteins, generating the largest RG data set to date. Comprising 376 sequences, our data set allows for phylogenetic reconstructions of RG with unprecedented size and detail. These RG phylogenies are strikingly different from those of universal proteins inferred to be present in the LUCA, even when using the same set of species. Unlike such proteins, RG does not form monophyletic archaeal and bacterial clades, suggesting RG emergence after the formation of these domains, and/or significant horizontal gene transfer. Additionally, the branch lengths separating archaeal and bacterial groups are very short, inconsistent with the tempo of evolution from the time of the LUCA. Despite this, phylogenies limited to archaeal RG resolve most archaeal phyla, suggesting predominantly vertical evolution since the time of the last archaeal ancestor. In contrast, bacterial RG indicates emergence after the last bacterial ancestor followed by significant horizontal transfer. Taken together, these results suggest a nonhyperthermophilic LUCA and bacterial ancestor, with hyperthermophily emerging early in the evolution of the archaeal and bacterial domains.

Keywords: LUCA; evolution; hyperthermophiles; phylogeny; reverse gyrase.

Fig. 1.

Sequence conservation recovered from alignment of RG data set mapped onto RG structure from Thermotoga maritima (pdb: 4DDT). Conserved residues indicated in red shades, less conserved residues indicated in blue shades.

Fig. 2.

Schematic representation of phylogenetic tree generated using entire RG data set. Archaeal clades colored in blue, Bacterial clades in red, with phyla indicated. Clades formed inside canonical phyla are indicated in darker shades, and labeled with an asterisk. Clades labeled with italicized text indicate ≤2 sequences present. Crenarchaeal TopRG1-like and TopRG2-like paralogues indicated in pale blue. Ultrafast bootstrap values for major bipartitions are indicated on branches. Detailed tree is available in supplementary figure 3 (Supplementary Material online).

Fig. 3.

Phylogenetic trees generated using universal proteins from RG-encoding species, compared with RG itself. First panel, RNA Polymerase subunit β; second panel, reverse gyrase; third panel, Elongation Factor G. In all trees, sequences encoded by Archaeal species are indicated in blue, Bacterial species in red.

Fig. 4.

Schematic representation of phylogenetic trees generated using only Archaeal RG sequences (blue), or Bacterial RG sequences (red). Clades formed inside canonical phyla are indicated in darker shades, and labeled with an asterisk. Clades labeled with italicized text indicate ≤2 sequences present. Aquificae and Thermotoga clades are labeled with arrow and cross, respectively, to highlight their paraphyletic nature.

Fig. 5.

Schematic representation of RG phylogenetic trees rooted using either Topoisomerase I or Helicase sequences. Archaeal outgroup sequences are colored purple, bacterial outgroup sequences in green. Archaeal RG sequences are colored in blue and bacterial RG in red. In both cases, the outgroup is extremely distant from RG and results in different rooting (within the large bacterial clade for Topoisomerase; between a bacterial and archaeal clade for Helicase).