We report the draft genome sequence of "Candidatus Methanomethylophilus alvus" Mx1201, a methanogen present in the human gut. It was enriched from human feces under anaerobic conditions with methanol as the substrate. Its circular genome, of around 1.7 Mb, contains genes needed for methylotrophic methanogenesis from methanol and tri-, di-, and monomethylamine. Methanogenic archaea play a crucial role in the functioning of digestive ecosystems through the consumption of the hydrogen generated during anaerobic fermentations (3). The first archaeal species associated to the human gut, Methanobrevibacter smithii and Methanosphaera stadtmanae (order Methanobacteriales), were cultured and described 30 years ago (12, 13). During the last decade, the diversity of the human-associated archaea was progressively enlarged by the detection of 16S rRNA genes affiliated to the Crenarchaeota (15), to the Halobacteriales (14), and to a Thermoplasmatales-related lineage (10,11,16). A species of the latter lineage, Methanomassiliicoccus luminyensis, was recently isolated and described as a methanogen growing on methanol and hydrogen only (5). More recently, its genome was sequenced (6).To allow genomic characterization of novel methanogens, we performed enrichment cultures targeting methanogens in feces of elderly people (68 to 93 years old), selecting for the presence of a Thermoplasmatales-related lineage. Enrichment cultures carried out with methanol as the substrate led to the progressive dominance of "Candidatus Methanomethylophilus alvus" over the microbial community. This archaeon is distantly related to its closest related species, M. luminyensis and Aciduliprofundum boonei, with 87% and 80% 16S rRNA gene sequence identity, respectively. Sequences of 16S rRNA genes of uncultured archaea with more than 97% identity to "Ca. Methanomethylophilus alvus" have previously been recurrently retrieved in the digestive tract of various animals, suggesting that these archaea are specifically associated to these environments. Those sequences form a portion of the previously described rumen cluster C (8).A 3-kb mate-paired library was constructed and sequenced from a quarter plate of a 454 GS FLX Titanium run (Macrogen, Republic of Korea). A total of 347,052 reads corresponding to 158 Mb were obtained. The reads were assembled with a GS De Novo assembler (version 2.6), first in 15 contigs (average depth coverage of 36.9-fold) and then in a unique scaffold of 1,664,410 bp. The gaps between the contigs were partially closed using GapFiller (2) and by sequencing of PCR products (4 remaining gaps). Around 1,800 bp was lacking between the two scaffold termini, as shown by PCR amplification. The open reading frames were predicted with Glimmer 3 (4) and annotated using RAST (1).The genome of "Ca. Methanomethylophilus alvus" has a GϩC content of 55.6%, 44 usual tRNA genes, a single copy of each of the 23S and 16S rRNA genes, and 2 noncontiguous copies of 5S rRNA genes that were distant in the genome from the 23S and 16S rRNA genes. A total of 1...

The methanogenic diversity of the human intestinal microbiota has long been viewed as composed of two Methanobacteriales: Methanobrevibacter smithii and Methanosphaera stadtmanae. Recently, Mx-01, a new phylotype hypothesized to belong to a putative sixth methanogenic order, was recovered from human faeces. Here we examined the diversity and the distribution of methanogens among healthy people of three age groups by analysing mcrA and 16S rDNA clones. The mcrA analysis of ∼1200 clones revealed that the usual Methanobacteriales were present without any significant difference among adults and elderly (respectively 60% and 80% of carriers, n = 40, P = 0.3). In addition, four new phylotypes that grouped with Mx-01 in the same monophyletic clade were recovered. These phylotypes were significantly more frequently detected in elderly people (40%, n = 20) than in adults (10%, n = 20, P = 0.065). In parallel, new 16S rDNA phylotypes affiliated near or within Thermoplasmatales were recovered. Altogether, these results indicate an age-related apparition of Mx-phylotypes, putatively methanogenic, which are formed of several species carrying a mcrA gene and that are not related to any of the five methanogenic orders. These species may be related to Thermoplasmatales or may cohabit with archaeal species related to Thermoplasmatales.

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“Candidatus Methanomassiliicoccus intestinalis” Issoire-Mx1 is a methanogenic archaeon found in the human gut and is a representative of the novel order of methanogens related to Thermoplasmatales. Its complete genome sequence is presented here.