New Insights into the Ecology and Physiology of Methanomassiliicoccales from Terrestrial and Aquatic Environments

Abstract

Members of the archaeal order Methanomassiliicoccales are methanogens mainly associated with animal digestive tracts. However, environmental members remain poorly characterized as no representatives not associated with a host have been cultivated so far. In this study, metabarcoding screening combined with quantitative PCR analyses on a collection of diverse non-host-associated environmental samples revealed that Methanomassiliicoccales were very scarce in most terrestrial and aquatic ecosystems. Relative abundance of Methanomassiliicoccales and substrates/products of methanogenesis were monitored during incubation of environmental slurries. A sediment slurry enriched in Methanomassiliicoccales was obtained from a freshwater sample. It allowed the reconstruction of a high-quality metagenome-assembled genome (MAG) corresponding to a new candidate species, for which we propose the name of Candidatus 'Methanomassiliicoccus armoricus MXMAG1'. Comparison of the annotated genome of MXMAG1 with the published genomes and MAGs from Methanomassiliicoccales belonging to the 2 known clades ('free-living'/non-host-associated environmental clade and 'host-associated'/digestive clade) allowed us to explore the putative physiological traits of Candidatus 'M. armoricus MXMAG1'. As expected, Ca. 'Methanomassiliicoccus armoricus MXMAG1' had the genetic potential to produce methane by reduction of methyl compounds and dihydrogen oxidation. This MAG encodes for several putative physiological and stress response adaptations, including biosynthesis of trehalose (osmotic and temperature regulations), agmatine production (pH regulation), and arsenic detoxication, by reduction and excretion of arsenite, a mechanism that was only present in the 'free-living' clade. An analysis of co-occurrence networks carried out on environmental samples and slurries also showed that Methanomassiliicoccales detected in terrestrial and aquatic ecosystems were strongly associated with acetate and dihydrogen producing bacteria commonly found in digestive habitats and which have been reported to form syntrophic relationships with methanogens.

Keywords: Methanomassiliicoccales; cultivation; environmental cluster; methyl-compounds; networks.

Figure 1

Distribution of environmental samples screened for Methanomassiliicoccales. On the map, green dots indicate the presence of Operational taxonomic units (OTUs) assigned to Methanomassiliicoccales in the sample; black diamonds indicate samples in which no Methanomassiliicoccales was detected by PCR with primers targeting Methanomassiliicoccales. Green and black pentagons show the position of nearby sites characterized by the presence of Methanomassiliicoccales for some, and their absence for others. The Network based on Bray-Curtis index shows the dissimilarity in the composition of total microbial communities between samples; pictures on the right represent, respectively, the MOUG2, DOUD, and PENF sampling sites.

Figure 2

Dendrogram showing the relationships between 16S rRNA gene sequences (253 bp) detected in environmental samples, in substrate-amended slurry (PENF slurry) and inferred to metagenome-assembled genomes (MAGs), among Methanomassiliicoccales [22]. The 85 OTUs of Methanomassiliicoccales detected in this study are shown on this dendrogram. OTUs detected only in environmental samples are indicated by a black star. OTUs without a symbol were detected in the culture-based experiment performed with the sediment PENF. OTUs indicated by a circle were detected both in bulk samples and in the culture-based experiment. The 16S rRNA gene sequences k119_35148 and k119_91804 were extracted from the metagenome sequences of the culture-based experiment PENF, after 8 weeks of incubation. Sequences discussed in the text are indicated by black arrows. The Methanomassiliicoccales-related sequences encompassed representative 16S rRNA gene sequences of Ca. ‘Lunaplasmatales’, UBA10834 and SG8-5 [80]. This reconstruction was performed on partial 16S rRNA gene sequences (253 bp), using the BIONJ method [81], with the modifications of Jukes and Cantor and using 1000 bootstrap replicates. Black-filled dots indicate nodes with bootstrap supports <50%, grey-filled dots 50–75%, and white-filled dots show support values between 75 and 100%. The top-right insert indicates the relative abundance of the main Methanomassiliicoccales-affiliated OTUs in the substrate-amended slurry PENF, after 8 weeks of incubation.

Figure 3

(A) Consumption kinetics of methanogenesis substrates during the weeks of incubation of the substrate-amended slurry performed with the PENF sample; (B) Consumption kinetics of some metabolic products (methane, ammonia, propionate) during the weeks of incubation of the substrate-amended slurry performed with the PENF sample. Concentrations of propionate are indicated on the right vertical axis of the figure and other microbial products (CH₄ and NH₄⁺) are indicate on the left vertical axis. The vertical red line indicates a novel addition of trimethylamine (TMA), methanol and H₂/CO₂ at T6 to stimulate methanogenesis; (C) Trends in microbial diversity revealed by metabarcoding; (D) Methanomassiliicoccales abundances determined by qPCR (logarithmic scale). Methanomassiliicoccales could not be quantified by qPCR in 4 samples (T0, T1, T6, and T7). Average copy numbers are given on the chart bars. Renewal of methanogenesis substrates (H₂, CO₂, MeOH, TMA) after six weeks of incubation is represented by the vertical red lines. Peptostreptococcales-T.: Peptostreptococcales-Tissierellales.

Figure 4

Maximum Likelihood phylogenomic tree (LG + F + G4) showing the position of the two Methanomassiliicoccales MAGs obtained in this study (in green) with respect to closely related MAGs and taxa. This phylogeny is based on a concatenation of 40 conserved phylogenetic markers (10,111 positions). The scale bar represents the average number of substitutions per site, and numbers indicate bootstrap replicates.

Figure 5

Predicted metabolic pathways for methanogenesis, carbon assimilation, and energy conservation in Ca. ‘Methanomassiliicoccus armoricus MXMAG1’, belonging to the ‘free-living clade’ of Methanomassiliicoccales. Genes encoding the MtmBC and MtaBC enzymes were found in two copies in the MAG. The “*” symbol indicates the presence of enzyme homologous to the MtaA/MtbA which are found in other Methanomassiliicoccales for MeOH and MMA incorporation, as described elsewhere [22]. Legend: CdhDE, Acetyl-CoA decarbonylase/synthase complex subunits; DMS, dimethylsulfide; MeOH, methanol; MeSH, methanethiol, MMA, monomethylamine.

Figure 6

Co-occurrence network reconstructed based on 16S rRNA gene-sequencing data from substrate-amended slurries (DOUR, PENF, and MOUG2, respectively), based on a Spearman rank’s correlation between OTUs and calculated with a percolation threshold of 0.79. (A) Clusters in slurries DOUR, PENF, and MOUG2, respectively. Modules in PENF network are colored to highlight them; (B) modules (M-6, module 6; M-198, module 198; M-232, module 232; M-234, module 234 and M-245, module 245, respectively) containing Methanomassiliicoccales in the cluster from the PENF culture-based incubation experiment. Modules consisting of a single Methanomassiliicoccales (modules 22 and 68, respectively) were excluded. The number of Methanomassiliicoccales in each module is shown in brackets; (C) non-random co-occurrences between Methanomassiliicoccales and other prokaryotes in module 6 showing co-occurrences of Methanomassiliicoccales (OTU21 and OTU101, respectively) with putative acetate- and H₂-producing bacteria. Black arrows followed by a plus and a number indicate the number of taxa non-randomly co-occurring with other prokaryotes outside the groups represented. The weights of the co-occurrences are written above the link (OTU101) or in the insert below the cluster of the OTU21.

Figure 7

Schematic diagram representing the substrates amended to the original slurry in the culture-based incubation experiment PENF (1) which led to the reconstruction of two Methanomassiliicoccales MAGs (Ca. ‘M. armoricus MXMAG1′ and MXMAG2) affiliated to the ‘free-living’ clade (2). Tentative reconstruction of the microbial metabolic interactions and substrate functions in this culture-based incubation experiment (3). The purple frame represents the module 6 of the co-occurrence network analysis, composed of reported fermentative Bacteria which maintained strong non-random co-occurrences with the dominant Methanomassiliicoccales OTUs sequences; FL: ‘Free-living’ clade; HA: ‘Host-associated’ clade; numbers in green rings indicate the completeness of the two Methanomassiliicoccales MAGs; dark blue arrows indicate fermentative products; grey arrows indicate the methanogenesis pathways probably occurring in this slurry; brown arrow indicates a fermentative substrate found in the enrichment slurry; light blue arrow indicates that acetate is potentially consume for methanogens cell growth. The colored circles indicate the dominant methanogenic taxa during the enrichment process and their known metabolic properties. The hatched color indicates that this property is present in only a part of the taxa of the lineage.