Genomic Reconstruction of an Uncultured Hydrothermal Vent Gammaproteobacterial Methanotroph (Family Methylothermaceae) Indicates Multiple Adaptations to Oxygen Limitation

Abstract

Hydrothermal vents are an important contributor to marine biogeochemistry, producing large volumes of reduced fluids, gasses, and metals and housing unique, productive microbial and animal communities fueled by chemosynthesis. Methane is a common constituent of hydrothermal vent fluid and is frequently consumed at vent sites by methanotrophic bacteria that serve to control escape of this greenhouse gas into the atmosphere. Despite their ecological and geochemical importance, little is known about the ecophysiology of uncultured hydrothermal vent-associated methanotrophic bacteria. Using metagenomic binning techniques, we recovered and analyzed a near-complete genome from a novel gammaproteobacterial methanotroph (B42) associated with a white smoker chimney in the Southern Lau basin. B42 was the dominant methanotroph in the community, at ∼80x coverage, with only four others detected in the metagenome, all on low coverage contigs (7x-12x). Phylogenetic placement of B42 showed it is a member of the Methylothermaceae, a family currently represented by only one sequenced genome. Metabolic inferences based on the presence of known pathways in the genome showed that B42 possesses a branched respiratory chain with A- and B-family heme copper oxidases, cytochrome bd oxidase and a partial denitrification pathway. These genes could allow B42 to respire over a wide range of oxygen concentrations within the highly dynamic vent environment. Phylogenies of the denitrification genes revealed they are the result of separate horizontal gene transfer from other Proteobacteria and suggest that denitrification is a selective advantage in conditions where extremely low oxygen concentrations require all oxygen to be used for methane activation.

Keywords: Lau Basin; deep sea; denitrification; hydrothermal vent; methane; methane oxidation; nitrate; thermophile.

FIGURE 1

Maximum likelihood phylogenetic tree of the 16S rRNA gene from cultured Methanococcales and associated non-methanotrophic Gammaproteobacteria, and some related environmental clones. Gray wedges indicate monophyletic groups of sequences. Cultured isolated from the Fusobacteria were used as the outgroup. Nodes with greater than 70 or 90% bootstrap support are indicated with a gray or black circle, respectively. Scale bar indicates substitutions per site.

FIGURE 2

Maximum likelihood phylogenetic tree of PmoA from cultured Methanococcales, and some related environmental clones. PmoA sequences recovered from the metagenome are underlined and labeled with their contig names. The Nitrosopumilus maritimus ammonia monooxygenase subunit A protein was used as the outgroup of the tree. Nodes with greater than 70 or 90% bootstrap support are indicated with a gray or black circle, respectively. Scale bar indicates substitutions per site.

FIGURE 3

Heatmap of genome relatedness between B42 and other gammaproteobacterial methanotrophs. The average amino acid identity (AAI) between homologous proteins between pairs of genomes is shown in the upper triangle. The percentage of total proteins that are homologous is shown in the lower triangle.

FIGURE 4

Model predictions of the central metabolism inferred from the B42 genome sequence. Important energy generating reactions are shown expanded on the lower left corner. Carbon derived from methane is either completely oxidized to CO₂ (dashed line) or is assimilated through the RuMP pathway into the glycolysis and TCA cycle. A proposed cellular ultrastructure is shown in the top right. The genome also encodes genes to the construction of pili, flagella and for the accumulation of polyphosphate and glycogen granules. Abbreviations: G6P, glucose 6-phosphate; G1,5LP, 6-phospho D-glucono-1,5-lactone; 6GP, gluconate 6-phosphate; Ru5P, ribulose 5-phosphate; Ri5P, ribose 5-phosphate; X5P, xylulose 5-phosphate; GAP, glyceraldehyde 3-phosphate; S7P, sedoheptulose 7-phosphate; F6P, fructofuranose 6-phosphate; E4P, erythrose 4-phosphate; F1,6P, fructose 1,6-bisphosphate; 3PGA, 3-phospho-D-glycerate; 2PGA, 2-phospho-D-glycerate; PEP, phosphoenolpyruvate; CH₂ = H₄MPT, 5,10-methylene-tetrahydromethanopterin; CH₂≡H₄MPT, 5,10-methenyltetrahydromethanopterin; CHO-H₄MPT, 5-formyl-tetrahydromethanopterin.

FIGURE 5

Maximum likelihood phylogenetic tree of NarG from reference genomes and B42. Tip labels show the genome name and the IMG gene id in brackets. The tree was rooted at the mid-point node, no outgroup was included. Nodes with greater than 70 or 90% bootstrap support are indicated with a gray or black circle, respectively. Scale bar indicates substitutions per site.

FIGURE 6

Maximum likelihood phylogenetic tree of NirK from reference genomes and B42. Nodes with greater than 70 or 90% bootstrap support are indicated with a gray or black circle, respectively. Scale bar indicates substitutions per site.