Jean-François Brugère

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

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.

Archaea and the human gut: new beginning of an old story.

Methanogenic archaea are known as human gut inhabitants since more than 30 years ago through the detection of methane in the breath and isolation of two methanogenic species belonging to the order Methanobacteriales, Methanobrevibacter smithii and Methanosphaera stadtmanae. During the last decade, diversity of archaea encountered in the human gastrointestinal tract (GIT) has been extended by sequence identification and culturing of new strains. Here we provide an updated census of the archaeal diversity associated with the human GIT and their possible role in the gut physiology and health. We particularly focus on the still poorly characterized 7th order of methanogens, the Methanomassiliicoccales, associated to aged population. While also largely distributed in non-GIT environments, our actual knowledge on this novel order of methanogens has been mainly revealed through GIT inhabitants. They enlarge the number of final electron acceptors of the gut metabolites to mono- di- and trimethylamine. Trimethylamine is exclusively a microbiota-derived product of nutrients (lecithin, choline, TMAO, L-carnitine) from normal diet, from which seems originate two diseases, trimethylaminuria (or Fish-Odor Syndrome) and cardiovascular disease through the proatherogenic property of its oxidized liver-derived form. This therefore supports interest on these methanogenic species and its use as archaebiotics, a term coined from the notion of archaea-derived probiotics.

Archaebiotics: Proposed therapeutic use of archaea to prevent trimethylaminuria and cardiovascular disease

Trimethylamine (TMA) is produced by gut bacteria from dietary ingredients. In individuals with a hereditary defect in flavin-containing monooxygenase 3, bacterial TMA production is believed to contribute to the symptoms of trimethylaminuria (TMAU; fish-odor syndrome). Intestinal microbiota TMA metabolism may also modulate atherosclerosis risk by affecting trimethylamine oxide (TMAO) production levels. We propose that reducing TMA formation in the gut by converting it to an inert molecule could be used to prevent or limit these human diseases, while avoiding the major drawbacks of other clinical interventions. Reducing TMA levels by microbiological interventions could also be helpful in some vaginoses. Particular members of a recently discovered group of methanogens, that are variably present in the human gut, are unusual in being apparently restricted to utilizing only methyl compounds including TMA as substrates. We confirmed experimentally that one of these strains tested, Methanomassiliicoccus luminyensis B10, is able to deplete TMA, by reducing it with H 2 for methanogenesis. We therefore suggest that members of this archaeal lineage could be used as treatments for metabolic disorders.

A putative new order of methanogenic Archaea inhabiting the human gut, as revealed by molecular analyses of the mcrA gene

The diversity of methanogenic Archaea from the gut of 6 humans was investigated by targeting mcrA, a molecular metabolic marker of methanogenesis. Three operational taxonomic units (OTUs) were recovered from about 400 clones analyzed, two of which were attributed to the expected Methanobacteriales Methanobrevibacter smithii (4 volunteers) and Methanosphaera stadtmanae (1 volunteer). The third OTU (1 volunteer) corresponded to a distant phylotype that does not cluster with any of the five methanogenic orders. This result, also supported by 16S archaeal sequences retrieved from the same volunteer, strongly suggests there may be a sixth order and hence potential underestimation of the role of methanogens in gut physiology.

Molecular evaluation of the human gut methanogenic archaeal microbiota reveals an age‐associated increase of the diversity

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.

The Human Gut Chip "HuGChip'', an explorative phylogenetic microarray for determining gut microbiome diversity at family level

Evaluating the composition of the human gut microbiota greatly facilitates studies on its role in human pathophysiology, and is heavily reliant on culture-independent molecular methods. A microarray designated the Human Gut Chip (HuGChip) was developed to analyze and compare human gut microbiota samples. The PhylArray software was used to design specific and sensitive probes. The DNA chip was composed of 4,441 probes (2,442 specific and 1,919 explorative probes) targeting 66 bacterial families. A mock community composed of 16S rRNA gene sequences from intestinal species was used to define the threshold criteria to be used to analyze complex samples. This was then experimentally verified with three human faecal samples and results were compared (i) with pyrosequencing of the V4 hypervariable region of the 16S rRNA gene, (ii) metagenomic data, and (iii) qPCR analysis of three phyla. When compared at both the phylum and the family level, high Pearsons correlation coefficients were obtained between data from all methods. The HuGChip development and validation showed that it is not only able to assess the known human gut microbiota but could also detect unknown species with the explorative probes to reveal the large number of bacterial sequences not yet described in the human gut microbiota, overcoming the main inconvenience encountered when developing microarrays.

Genome Sequence of "Candidatus Methanomassiliicoccus intestinalis" Issoire-Mx1, a Third Thermoplasmatales-Related Methanogenic Archaeon from Human Feces

ABSTRACT “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.

Unique Characteristics of the Pyrrolysine System in the 7th Order of Methanogens: Implications for the Evolution of a Genetic Code Expansion Cassette

Pyrrolysine (Pyl), the 22nd proteogenic amino acid, was restricted until recently to few organisms. Its translational use necessitates the presence of enzymes for synthesizing it from lysine, a dedicated amber stop codon suppressor tRNA, and a specific amino-acyl tRNA synthetase. The three genomes of the recently proposed Thermoplasmata-related 7th order of methanogens contain the complete genetic set for Pyl synthesis and its translational use. Here, we have analyzed the genomic features of the Pyl-coding system in these three genomes with those previously known from Bacteria and Archaea and analyzed the phylogeny of each component. This shows unique peculiarities, notably an amber tRNAPyl with an imperfect anticodon stem and a shortened tRNAPyl synthetase. Phylogenetic analysis indicates that a Pyl-coding system was present in the ancestor of the seventh order of methanogens and appears more closely related to Bacteria than to Methanosarcinaceae, suggesting the involvement of lateral gene transfer in the spreading of pyrrolysine between the two prokaryotic domains. We propose that the Pyl-coding system likely emerged once in Archaea, in a hydrogenotrophic and methanol-H2-dependent methylotrophic methanogen. The close relationship between methanogenesis and the Pyl system provides a possible example of expansion of a still evolving genetic code, shaped by metabolic requirements.

Tools for stools: the challenge of assessing human intestinal microbiota using molecular diagnostics

The human GI tract is inhabited by an incredibly complex and abundant microbiota, whose composition is dependent on a variety of factors. The gut microbiota has an influence in the morphological, immunological and nutritional functions of the digestive tract and may be involved in many diseases. This article proposes the rationale behind conducting in vitro diagnostics (IVDs) of the human microbiota, as well as outlining the conceptual and technical difficulties involved in IVD testing. The molecular methods that can be used according to whether the IVD tools are employed to study one individual constituent species or to determine the microbiota as a whole will also be described. In the latter case, these technologies include high-throughput sequencing for metagenomics and DNA microarrays, which can now be efficiently used to study gut ecology and are believed to represent the future of standardized diagnostics.

Three-stage continuous culture system with a self-generated anaerobia to study the regionalized metabolism of the human gut microbiota

The technical and ethical difficulties in studying the gut microbiota in vivo warrant the development and improvement of in vitro systems able to simulate and control the physicochemical factors of the gut biology. Moreover, the functional regionalization of this organ implies a model simulating these differences. Here we propose an improved and alternative three-stage continuous bioreactor called 3S-ECSIM (three-stage Environmental Control System for Intestinal Microbiota) to study the human large intestine. Its main feature compared with other in vitro systems is the anaerobic atmosphere originating directly from the microbiota metabolism, leading to different gas ratios of CO2 and H2 in each compartment. Analyses of the metabolic and microbiological profiles (LC-MS and a phylogenetic microarray) show different profiles together with a maintenance of this differentiation between the three compartments, simulating respectively a proximal, a transversal and a distal colon. Moreover, the last reactor presents a high similarity with the initial fecal sample, at the microbiological diversity level. Based on our results, this in-vitro process improvement is a valuable alternative tool to dynamically study the structure and metabolism of gut microbiota, and its response to nutrients, prebiotics, probiotics, drugs or xenobiotics.