Methanogenesis and Salt Tolerance Genes of a Novel Halophilic Methanosarcinaceae Metagenome-Assembled Genome from a Former Solar Saltern

Abstract

Anaerobic archaeal methanogens are key players in the global carbon cycle due to their role in the final stages of organic matter decomposition in anaerobic environments such as wetland sediments. Here we present the first draft metagenome-assembled genome (MAG) sequence of an unclassified Methanosarcinaceae methanogen phylogenetically placed adjacent to the Methanolobus and Methanomethylovorans genera that appears to be a distinct genus and species. The genome is derived from sediments of a hypersaline (97-148 ppt chloride) unrestored industrial saltern that has been observed to be a significant methane source. The source sediment is more saline than previous sources of Methanolobus and Methanomethylovorans. We propose a new genus name, Methanosalis, to house this genome, which we designate with the strain name SBSPR1A. The MAG was binned with CONCOCT and then improved via scaffold extension and reassembly. The genome contains pathways for methylotrophic methanogenesis from trimethylamine and dimethylamine, as well as genes for the synthesis and transport of compatible solutes. Some genes involved in acetoclastic and hydrogenotrophic methanogenesis are present, but those pathways appear incomplete in the genome. The MAG was more abundant in two former industrial salterns than in a nearby reference wetland and a restored wetland, both of which have much lower salinity levels, as well as significantly lower methane emissions than the salterns.

Keywords: anaerobic; archaea; methanogenesis; phylogenomics; salt tolerance.

Figure 1

RAxML phylogenetic tree of the 11 genomes in this study using a concatenated alignment of 122 single copy archaeal genes and the PROTGAMMALG model of amino acid substitution. Branch labels show the bootstrap support, calculated with 1000 bootstraps. Also shown are the genome size, number of protein-coding genes, % G + C content, mean isoelectric point of protein-coding genes (pI), and percent completeness and contamination estimates from CheckM. The greatest values in each column are bolded. The tree is rooted with Nitrosopumilus adriaticus (Thaumarchaeota) as an outgroup (not shown).

Figure 2

Shared orthologous gene groups among different combinations of genomes as calculated by (a) proteinortho, and (b) KEGG orthology profiles. Shown here are intersections between all 11 genomes, genes unique to Ms. sp. SBSPR1A (only in panel b), intersections between 10 other genomes but not Methanosalis sp. SBSPR1A, and pairwise comparisons between Ms. sp. SBSPR1A and the other genomes. Numbers above the columns state the number of shared orthogroups or shared KOs. For strain names of all of the species, see the methods section or Figure 1.

Figure 3

Presence (red) or absence (blue) of genes involved in methanogenesis. Shown here are six different pathways for methyl-CoM formation (Figure S1), the single methane production step of methyl-CoM reduction, and four pathways for coenzyme B (CoB) and coenzyme M (CoM) regeneration. The names of the genes and their KEGG K number assignments are shown. Columns are clustered by KO presence/absence rather than phylogeny.

Figure 4

Abundance of Methanosalis sp. SBSPR1A, expressed as counts per million assembled reads, across 24 metagenomic samples from Zhou et al. (2021) [30], organized by (a) site, showing the four wetland sites sampled, (b) methane flux (µmol/m²/day), and (c) salinity (ppt chloride). Samples were taken from two unrestored salterns, a restored saltern, and a reference wetland. Lines in (b,c) were fitted with loess functions.

Figure 5

Presence (red) or absence (blue) of genes involved in compatible solute or salt biosynthesis and transport, for salt tolerance. Compounds include betaine, cations (K⁺ or Na⁺), ectoine, glutamate, glutamine, proline, and sucrose. The gene names and their KEGG K number assignments are shown. Columns were clustered according to the phylogeny in Figure 1.