Culture- and metagenomics-enabled analyses of the Methanosphaera genus reveals their monophyletic origin and differentiation according to genome size

Abstract

The genus Methanosphaera is a well-recognized but poorly characterized member of the mammalian gut microbiome, and distinctive from Methanobrevibacter smithii for its ability to induce a pro-inflammatory response in humans. Here we have used a combination of culture- and metagenomics-based approaches to expand the representation and information for the genus, which has supported the examination of their phylogeny and physiological capacity. Novel isolates of the genus Methanosphaera were recovered from bovine rumen digesta and human stool, with the bovine isolate remarkable for its large genome size relative to other Methanosphaera isolates from monogastric hosts. To substantiate this observation, we then recovered seven high-quality Methanosphaera-affiliated population genomes from ruminant and human gut metagenomic datasets. Our analyses confirm a monophyletic origin of Methanosphaera spp. and that the colonization of monogastric and ruminant hosts favors representatives of the genus with different genome sizes, reflecting differences in the genome content needed to persist in these different habitats.

Fig. 1

Longitudinal monitoring of the growth of Methanosphaera stadtmanae DSMZ3091^T (a), strain BMS (b) and strain WGK6 when provided with either methanol:H₂ (c) or methanol:ethanol (d). The colored lines and symbols denote different concentrations of either H₂ (0 , 1.3 , 1.8 , 2.6 , 3.5 and 4.6 mM) or in the instance of (d), ethanol (0 , 3 , 10 , 30 , 50 and 171 mM). The growth of the individual cultures is plotted until the time point at which maximal OD₆₀₀ was measured. Individual values represent the mean (±SD) produced from triplicate cultures

Fig. 2

The degree of genome synteny evident between the Methanosphaera stadtmanae DSMZ3091^T, WGK6, and BMS genomes using Mauve. In brief detail, the alignment algorithm facilitates the depiction of genome synteny (illustrated by the size and the coloring scheme used for the local collinear blocks identified by Mauve), regions of genome rearrangement (illustrated via the lines connecting locally collinear blocks between the two genome models) and any xenologous regions (illustrated by blank spaces). The xenologous regions present in the BMS genome are not ubiquitous, as illustrated by the high degree of synteny between all three genomes

Fig. 3

Maximum-likelihood phylogenetic analysis of Methanosphaera spp. isolate and rrs gene clone sequences recovered from NCBI. There is a clear separation of the available Methanosphaera spp. rrs sequences between monogastric and ruminant hosts. Bootstrap values are shown and the scale bar represents 1% sequence divergence, with Methanobrevibacter ruminantium M1 used as the outgroup

Fig. 4

a The genome based phylogenetic tree of the isolate and population genomes recovered for Methanosphaera spp. inferred from the concatenation of 293 genes from the core genome and using EDGAR. Those representatives with “small” genomes (<1.8 Mbp; blue/purple circles) appear to have evolved more recently and were more readily found in monogastric hosts, whereas representatives with “large” genomes (>2.0 Mbp; red circles) appear restricted to ruminant hosts and the deeper branching suggestive of their ancestral age. b Estimates of the core, non-core, and singleton genes present in the isolate and population genomes with >90% completeness, when expressed as a per genome equivalent. The total number of core genes is relatively stable between the “large” (red) and “small” (blue) genome groups, with the key differences between groups represented by the substantially larger counts of predicted non-core and singleton genes

Fig. 5

Growth of human Methanosphaera stadtmanae DSMZ3091^T and bovine Methanosphaera sp. BMS when cultured using BRN basal medium supplemented with either 5 or 10% (vol/vol) rumen fluid (RF5 and RF10, respectively) or 1 and 5% (vol/vol) human faecal water (FW1 and FW5, respectively). Notably, the growth rate and yield of strain DSMZ3091^T was only marginally affected by the different RF and FW concentrations; however, both the growth rate and yield of strain BMS was reduced in the presence of FW in comparison to RF-containing cultures. Individual values represent the mean (±SD) produced from triplicate cultures