Phylogenomic data support a seventh order of Methylotrophic methanogens and provide insights into the evolution of Methanogenesis

Abstract

Increasing evidence from sequence data from various environments, including the human gut, suggests the existence of a previously unknown putative seventh order of methanogens. The first genomic data from members of this lineage, Methanomassiliicoccus luminyensis and "Candidatus Methanomethylophilus alvus," provide insights into its evolutionary history and metabolic features. Phylogenetic analysis of ribosomal proteins robustly indicates a monophyletic group independent of any previously known methanogenic order, which shares ancestry with the Marine Benthic Group D, the Marine Group II, the DHVE2 group, and the Thermoplasmatales. This phylogenetic position, along with the analysis of enzymes involved in core methanogenesis, strengthens a single ancient origin of methanogenesis in the Euryarchaeota and indicates further multiple independent losses of this metabolism in nonmethanogenic lineages than previously suggested. Genomic analysis revealed an unprecedented loss of the genes coding for the first six steps of methanogenesis from H₂/CO₂ and the oxidative part of methylotrophic methanogenesis, consistent with the fact that M. luminyensis and "Ca. M. alvus" are obligate H₂-dependent methylotrophic methanogens. Genomic data also suggest that these methanogens may use a large panel of methylated compounds. Phylogenetic analysis including homologs retrieved from environmental samples indicates that methylotrophic methanogenesis (regardless of dependency on H₂) is not restricted to gut representatives but may be an ancestral characteristic of the whole order, and possibly also of ancient origin in the Euryarchaeota. 16S rRNA and McrA trees show that this new order of methanogens is very diverse and occupies environments highly relevant for methane production, therefore representing a key lineage to fully understand the diversity and evolution of methanogenesis.

Keywords: Archaea; Methanomassiliicoccales; Methanoplasmatales; evolution; genomics; methanogenesis.

Fig. 1.—

Main methanogenic pathways and associated genes. The blue box highlights the first six steps of methanogenesis from H₂/CO₂ (downward arrows) and the production of reducing equivalents during methylotrophic methanogenesis without external H₂ source (upward arrows), while the dotted line in the blue box indicates the entry of the methyl group in the acetoclastic pathway. The green box highlights the first steps of methylotrophic methanogenesis and H₂-dependent methylotrophic methanogenesis. The red box highlights the final step of methanogenesis in all three pathways. The genes absent in the Mx lineage are indicated in gray. The mtaA and mtaB genes are marked with an asterisk to signify that the homologs present in Mx cannot be presently assigned to one or the other enzyme category (see text for details). Dotted arrows designate the presence of steps not detailed on the figure. The oxidation of the carbonyl group of the acetate in the acetoclastic methanogenesis is not apparent on the figure. MFR, methanofuran; H₄MPT, tetrahydromethanopterin; HS-CoM, coenzyme M; HS-CoB, coenzyme B, CoM-S-S-CoB, heterodisulfide of HS-CoM and HS-CoB; F₄₂₀H₂, reduced coenzyme F₄₂₀; Fd_red, reduced ferredoxin; Fd_ox, oxidized ferredoxin. For simplicity, tetrahydrosarcinapterin (H₄SPT, an analog of H₄MPT) is not displayed.

Fig. 2.—

Phylogenetic position of the Mx order and inferred losses of methanogenesis. (a) Bayesian phylogeny of Euryarchaeota based on a concatenation of 57 ribosomal proteins comprising 7,472 amino acid positions. Values at nodes represent Bayesian posterior probabilities and bootstrap values based on maximum likelihood analysis and 100 resamplings of the original data set. For clarity, only the values corresponding to the monophyly of orders and their evolutionary relationships are shown. “../” symbols indicate that the corresponding node was not recovered in the maximum likelihood phylogeny. The scale bar represents the average number of substitutions per site. For details on analyses, see Materials and Methods. Red crosses indicate complete loss of methanogenesis. Colored spots indicate presence of the genes of the respective methanogenic pathways/reactions as indicated by the colored boxes in figure 1. Crosses within spots indicate that the enzymes are present but not used to perform methanogenesis from H₂/CO₂ and/or from methylated compounds. Genomes with a green spot harbor the genes needed to use at least one type of methyl compound. (b) Maximum likelihood phylogeny of methanogens based on a concatenation of McrA-B-C-D-G protein sequences comprising 1,159 amino acid positions. Values at nodes represent bootstrap proportions calculated based on 100 resamplings of the original alignment and Bayesian posterior probabilities. For clarity, only the values corresponding to the monophyly of orders and their evolutionary relationships are shown. The scale bar represents the average number of substitutions per site. For details on analyses, see Materials and Methods.

Fig. 3.—

Diversity of the Mx order and its evolutionary relationships with other uncultured lineages. (a) Maximum likelihood McrA phylogeny of the candidate Mx order. (b) Maximum likelihood 16S rRNA phylogeny showing the uncultured lineages branching between the Mx lineage and the Thermoplasmatales/DVHE2, MG-II, and MBG-D lineages. Both trees were rooted by using six representatives of Methanobacteriales and Methanococcales that are not shown for clarity. Trees were calculated from a data set of 138 unambiguously aligned amino acid positions (McrA) and 1,113 unambiguously aligned nucleic acid positions (16S rRNA). Values at nodes represent bootstrap support calculated on 100 resamplings of the original data set and Bayesian posterior probabilities calculated by Bayesian analysis. For clarity, only the values corresponding to the monophyly of lineages and their evolutionary relationships are shown. “../” symbols indicate that the corresponding node was not recovered in the Bayesian phylogeny. The scale bar represents the average number of substitutions per site. For details on data set construction and tree calculation, see Materials and Methods. The dotted box delineates the candidate Mx order. Sequences from the complete genomes of “Candidatus Methanomethylophilus alvus” and Methanomassiliicoccus luminyensis are indicated in red; other colored sequences indicate McrA and 16S rRNA sequences retrieved from the same sample in previous studies. Sequences corresponding to the Lake Pavin sample from which we retrieved Mt coding gene sequences are underlined. Other than the already discussed losses of methanogenesis in the Thermoplasmatales/DVHE2, MG-II lineages, and MBG-D, additional putative losses can be inferred (red crosses with a question mark) in two uncultured lineages based on the environment from which the corresponding sequences were retrieved: oxygenated water column for MG-III; oxygenated and extremely acidic environments (pH <3) for TAC. Lineages 20c4, VC-21Arc6, CCA-47 (divided in two subgroup -1 and -2), ANT06-05, ASC21, F2apm1A36, and MKCST-A3 were derived from the July 2012 version (v.111) of the Silva Database (http://www.arb-silva.de/) and are named according to the clone definition. TMEG, Terrestrial Miscellaneous Euryarchaeotal Group; MBG-D, Marine Benthic Group D; MG-III, Marine Group III; MG-II, Marine Group II; TAC, Thermoplasmatales-related Acidic Cluster.

Fig. 4.—

Physical maps of mtaA/mtbA, mtaB, and mtaC. Physical maps of mtaA/mtbA, mtaB, and mtaC genes in “Candidatus Methanomethylophilus alvus” Mx1201, Methanomassiliicoccus luminyensis B10, in comparison with Methanosarcina acetivorans CA2 as a representative of Methanosarcinales, and with Methanosphaera stadtmanae DSM 3091 as a representative of Methanobacteriales. The contigs of M. luminyensis (the assembled genome is not yet available) are separated by slashes. MAP, Methyltransferase Activation Protein.

Fig. 5.—

Phylogeny of MtaA/MtbA, MtaB, and MtaC. Unrooted maximum likelihood phylogenies of (a) MtaA/MtbA, (b) MtaB, and (c) MtaC. The Methanosarcinales are indicated in green, the Methanobacteriales in blue, and the Mx lineage in red. Sequences in bold belong to uncultivated microorganisms. Of the environmental sequences from Lake Pavin obtained in this study, only MtaB could be included in phylogenetic analysis because the others were too partial. Also, the MtaC homolog from the bioreactor metagenome was too partial to be included in the tree. However, these are clearly affiliated with the Mx lineage. Values at nodes represent bootstrap support calculated based on 100 resamplings of the original data set. For clarity, only the values corresponding to the monophyly of lineages and their evolutionary relationships are shown. The scale bar represents the average number of substitutions per site. For details on data set construction and tree calculation, see Materials and Methods.