Guillaume Borrel

Production and consumption of methane in freshwater lake ecosystems

The atmospheric concentration of methane (CH(4)), a major greenhouse gas, is mainly controlled by the activities of methane-producing (methanogens) and methane-consuming (methanotrophs) microorganisms. Freshwater lakes are identified as one of the main CH(4) sources, as it was estimated that they contribute to 6-16% of natural CH(4) emissions. It is therefore critical to better understanding the biogeochemical cycling of CH(4) in these ecosystems. In this paper, the effects of environmental factors on methanogenic and methanotrophic rates are reviewed and an inventory of the methanogens and methanotrophs at the genus/species level in freshwater lakes is given. We focus on the anaerobic oxidation of methane, which is a still poorly known process but increasingly reported in freshwater lakes.

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.

Identification of microbial communities involved in the methane cycle of a freshwater meromictic lake

Lake Pavin is a meromictic crater lake located in the French Massif Central area. In this ecosystem, most methane (CH(4)) produced in high quantity in the anoxic bottom layers, and especially in sediments, is consumed in the water column, with only a small fraction of annual production reaching the atmosphere. This study assessed the diversity of methanogenic and methanotrophic populations along the water column and in sediments using PCR and reverse transcription-PCR-based approaches targeting functional genes, i.e. pmoA (α-subunit of the particulate methane monooxygenase) for methanotrophy and mcrA (α-subunit of the methyl-coenzyme M reductase) for methanogenesis as well as the phylogenetic 16S rRNA genes. Although methanogenesis rates were much higher in sediments, our results confirm that CH(4) production also occurs in the water column where methanogens were almost exclusively composed of hydrogenotrophic methanogens, whereas both hydrogenotrophs and acetotrophs were almost equivalent in the sediments. Sequence analysis of markers, pmoA and the 16S rRNA gene, suggested that Methylobacter may be an important group actively involved in CH(4) oxidation in the water column. Two main phylotypes were characterized, one of which could consume CH(4) under conditions where the oxygen amount is undetectable.

Stratification of Archaea in the Deep Sediments of a Freshwater Meromictic Lake: Vertical Shift from Methanogenic to Uncultured Archaeal Lineages

As for lineages of known methanogens, several lineages of uncultured archaea were recurrently retrieved in freshwater sediments. However, knowledge is missing about how these lineages might be affected and structured according to depth. In the present study, the vertical changes of archaeal communities were characterized in the deep sediment of the freshwater meromictic Lake Pavin. For that purpose, an integrated molecular approach was performed to gain information on the structure, composition, abundance and vertical stratification of archaeal communities thriving in anoxic freshwater sediments along a gradient of sediments encompassing 130 years of sedimentation. Huge changes occurred in the structure and composition of archaeal assemblages along the sediment core. Methanogenic taxa (i.e. Methanosaeta and Methanomicrobiales) were progressively replaced by uncultured archaeal lineages (i.e. Marine Benthic Group-D (MBG-D) and Miscellaneous Crenarchaeal Group (MCG)) which are suspected to be involved in the methane cycle.

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.

Unexpected and novel putative viruses in the sediments of a deep-dark permanently anoxic freshwater habitat.

Morphological diversity, abundance and community structure of viruses were examined in the deep and anoxic sediments of the volcanic Lake Pavin (France). The sediment core, encompassing 130 years of sedimentation, was subsampled every centimeter. High viral abundances were recorded and correlated to prokaryotic densities. Abundances of viruses and prokaryotes decreased with the depth, contrasting the pattern of virus-to-prokaryote ratio. According to fingerprint analyses, the community structure of viruses, bacteria and archaea gradually changed, and communities of the surface (0–10 cm) could be discriminated from those of the intermediate (11–27 cm) and deep (28–40 cm) sediment layers. Viral morphotypes similar to virions of ubiquitous dsDNA viruses of bacteria were observed. Exceptional morphotypes, previously never reported in freshwater systems, were also detected. Some of these resembled dsDNA viruses of hyperthermophilic and hyperhalophilic archaea. Moreover, unusual types of spherical and cubic virus-like particles (VLPs) were observed. Infected prokaryotic cells were detected in the whole sediment core, and their vertical distribution correlated with both viral and prokaryotic abundances. Pleomorphic ellipsoid VLPs were visible in filamentous cells tentatively identified as representatives of the archaeal genus Methanosaeta, a major group of methane producers on earth.