Diversity and Evolution of Methane-Related Pathways in Archaea.

Methane is one of the most important greenhouse gases on Earth and holds an important place in the global carbon cycle. Archaea are the only organisms that use methanogenesis to produce energy and rely on the methyl-coenzyme M reductase complex (Mcr). Over the last decade, new results have significantly reshaped our view of the diversity of methane-related pathways in the Archaea. Many new lineages that synthesize or use methane have been identified across the whole archaeal tree, leading to a greatly expanded diversity of substrates and mechanisms. In this review, we present the state of the art of these advances and how they challenge established scenarios of the origin and evolution of methanogenesis, and we discuss the potential trajectories that may have led to this strikingly wide range of metabolisms.

Evolutionary history of carbon monoxide dehydrogenase/acetyl-CoA synthase, one of the oldest enzymatic complexes.

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a five-subunit enzyme complex responsible for the carbonyl branch of the Wood-Ljungdahl (WL) pathway, considered one of the most ancient metabolisms for anaerobic carbon fixation, but its origin and evolutionary history have been unclear. While traditionally associated with methanogens and acetogens, the presence of CODH/ACS homologs has been reported in a large number of uncultured anaerobic lineages. Here, we have carried out an exhaustive phylogenomic study of CODH/ACS in over 6,400 archaeal and bacterial genomes. The identification of complete and likely functional CODH/ACS complexes in these genomes significantly expands its distribution in microbial lineages. The CODH/ACS complex displays astounding conservation and vertical inheritance over geological times. Rare intradomain and interdomain transfer events might tie into important functional transitions, including the acquisition of CODH/ACS in some archaeal methanogens not known to fix carbon, the tinkering of the complex in a clade of model bacterial acetogens, or emergence of archaeal-bacterial hybrid complexes. Once these transfers were clearly identified, our results allowed us to infer the presence of a CODH/ACS complex with at least four subunits in the last universal common ancestor (LUCA). Different scenarios on the possible role of ancestral CODH/ACS are discussed. Despite common assumptions, all are equally compatible with an autotrophic, mixotrophic, or heterotrophic LUCA. Functional characterization of CODH/ACS from a larger spectrum of bacterial and archaeal lineages and detailed evolutionary analysis of the WL methyl branch will help resolve this issue.

Simulated drought regimes reveal community resilience and hydrological thresholds for altered decomposition.

Future climate scenarios forecast a 10-50% decline in rainfall in Eastern Amazonia. Altered precipitation patterns may change important ecosystem functions like decomposition through either changes in physical and chemical processes or shifts in the activity and/or composition of species. We experimentally manipulated hydroperiods (length of wet:dry cycles) in a tank bromeliad ecosystem to examine impacts on leaf litter decomposition. Gross loss of litter mass over 112 days was greatest in continuously submersed litter, lowest in continuously dry litter, and intermediate over a range of hydroperiods ranging from eight cycles of 7 wet:7 dry days to one cycle of 56 wet:56 dry days. The resilience of litter mass loss to hydroperiod length is due to a shift from biologically assisted decomposition (mostly microbial) at short wet:dry hydroperiods to physicochemical release of dissolved organic matter at longer wet:dry hydroperiods. Biologically assisted decomposition was maximized at wet:dry hydroperiods falling within the range of ambient conditions (12-22 consecutive dry days) but then declined under prolonged wet:dry hydroperiods (28 and 56 dry days. Fungal:bacterial ratios showed a similar pattern as biologically assisted decomposition to hydroperiod length. Our results suggest that microbial communities confer functional resilience to altered hydroperiod in tank bromeliad ecosystems. We predict a substantial decrease in biological activity relevant to decomposition under climate scenarios that increase consecutive dry days by 1.6- to 3.2-fold in our study area, whereas decreased frequency of dry periods will tend to increase the physicochemical component of decomposition.

Corrigendum to "unique characteristics of the pyrrolysine system in the 7th order of methanogens: implications for the evolution of a genetic code expansion cassette".

[This corrects the article DOI: 10.1155/2014/374146.].

Molecular methods for studying methanogens of the human gastrointestinal tract: current status and future directions.

Until recently, human gut microbiota was believed to be colonized by few methanogenic archaeal species. Much higher microbial diversity within the human gut was revealed by the use of molecular approaches as compared to routine microbiological techniques, but still, a lot remains unknown. Molecular techniques has the advantage of being rapid, reproducible, and can be highly discriminative as compared to conventional culturing methods. Some of them provide both qualitative and quantitative information. However, the choice of method should be taken with care to avoid biases. The advent of next-generation sequencing gives much deeper information from which functional and ecological hypotheses can be drawn. In this review, molecular techniques that are currently used together with their possible future developments to study gut methanogenic communities are indicated along with their limitations and difficulties that are encountered during their implementation. Moreover, the high amount of metagenomics data from the human gut microbiome indicate that this environment could be a paradigm for new directions in methanogen diversity studies and help to develop new approaches for other environments as well. Concerning humans, this should help us to better understand the possible association of methanogens with some of the diseased conditions and their peculiar distribution among age groups in human.

In-vitro model for studying methanogens in human gut microbiota.

Reported failures with gnotobiotic animal models led us to establish an in-vitro model of reciprocal conversion of methanogenic and non methanogenic microbiota from human fecal samples. Consequences on gas and microbiota compositions are reported. This should facilitate the study of the controversial role of gut methanogens in human health.

Comparative genomics highlights the unique biology of Methanomassiliicoccales, a Thermoplasmatales-related seventh order of methanogenic archaea that encodes pyrrolysine.

BACKGROUND: A seventh order of methanogens, the Methanomassiliicoccales, has been identified in diverse anaerobic environments including the gastrointestinal tracts (GIT) of humans and other animals and may contribute significantly to methane emission and global warming. Methanomassiliicoccales are phylogenetically distant from all other orders of methanogens and belong to a large evolutionary branch composed by lineages of non-methanogenic archaea such as Thermoplasmatales, the Deep Hydrothermal Vent Euryarchaeota-2 (DHVE-2, Aciduliprofundum boonei) and the Marine Group-II (MG-II). To better understand this new order and its relationship to other archaea, we manually curated and extensively compared the genome sequences of three Methanomassiliicoccales representatives derived from human GIT microbiota, "Candidatus Methanomethylophilus alvus", "Candidatus Methanomassiliicoccus intestinalis" and Methanomassiliicoccus luminyensis. RESULTS: Comparative analyses revealed atypical features, such as the scattering of the ribosomal RNA genes in the genome and the absence of eukaryotic-like histone gene otherwise present in most of Euryarchaeota genomes. Previously identified in Thermoplasmatales genomes, these features are presently extended to several completely sequenced genomes of this large evolutionary branch, including MG-II and DHVE2. The three Methanomassiliicoccales genomes share a unique composition of genes involved in energy conservation suggesting an original combination of two main energy conservation processes previously described in other methanogens. They also display substantial differences with each other, such as their codon usage, the nature and origin of their CRISPRs systems and the genes possibly involved in particular environmental adaptations. The genome of M. luminyensis encodes several features to thrive in soil and sediment conditions suggesting its larger environmental distribution than GIT. Conversely, "Ca. M. alvus" and "Ca. M. intestinalis" do not present these features and could be more restricted and specialized on GIT. Prediction of the amber codon usage, either as a termination signal of translation or coding for pyrrolysine revealed contrasted patterns among the three genomes and suggests a different handling of the Pyl-encoding capacity. CONCLUSIONS: This study represents the first insights into the genomic organization and metabolic traits of the seventh order of methanogens. It suggests contrasted evolutionary history among the three analyzed Methanomassiliicoccales representatives and provides information on conserved characteristics among the overall methanogens and among Thermoplasmata.

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 tRNA(Pyl) with an imperfect anticodon stem and a shortened tRNA(Pyl) 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.

Anaerobic hexadecane degradation by a thermophilic Hadarchaeon from Guaymas Basin.

Hadarchaeota inhabit subsurface and hydrothermally heated environments, but previous to this study, they had not been cultured. Based on metagenome-assembled genomes, most Hadarchaeota are heterotrophs that grow on sugars and amino acids, or oxidize carbon monoxide or reduce nitrite to ammonium. A few other metagenome-assembled genomes encode alkyl-coenzyme M reductases (Acrs), ß-oxidation, and Wood-Ljungdahl pathways, pointing toward multicarbon alkane metabolism. To identify the organisms involved in thermophilic oil degradation, we established anaerobic sulfate-reducing hexadecane-degrading cultures from hydrothermally heated sediments of the Guaymas Basin. Cultures at 70°C were enriched in one Hadarchaeon that we propose as Candidatus Cerberiarchaeum oleivorans. Genomic and chemical analyses indicate that Ca. C. oleivorans uses an Acr to activate hexadecane to hexadecyl-coenzyme M. A ß-oxidation pathway and a tetrahydromethanopterin methyl branch Wood-Ljungdahl (mWL) pathway allow the complete oxidation of hexadecane to CO2. Our results suggest a syntrophic lifestyle with sulfate reducers, as Ca. C. oleivorans lacks a sulfate respiration pathway. Comparative genomics show that Acr, mWL, and ß-oxidation are restricted to one family of Hadarchaeota, which we propose as Ca. Cerberiarchaeaceae. Phylogenetic analyses further indicate that the mWL pathway is basal to all Hadarchaeota. By contrast, the carbon monoxide dehydrogenase/acetyl-coenzyme A synthase complex in Ca. Cerberiarchaeaceae was horizontally acquired from Bathyarchaeia. The Acr and ß-oxidation genes of Ca. Cerberiarchaeaceae are highly similar to those of other alkane-oxidizing archaea such as Ca. Methanoliparia and Ca. Helarchaeales. Our results support the use of Acrs in the degradation of petroleum alkanes and suggest a role of Hadarchaeota in oil-rich environments.

A compendium of viruses from methanogenic archaea reveals their diversity and adaptations to the gut environment.

Methanogenic archaea are major producers of methane, a potent greenhouse gas and biofuel, and are widespread in diverse environments, including the animal gut. The ecophysiology of methanogens is likely impacted by viruses, which remain, however, largely uncharacterized. Here we carried out a global investigation of viruses associated with all current diversity of methanogens by assembling an extensive CRISPR database consisting of 156,000 spacers. We report 282 high-quality (pro)viral and 205 virus-like/plasmid sequences assigned to hosts belonging to ten main orders of methanogenic archaea. Viruses of methanogens can be classified into 87 families, underscoring a still largely undiscovered genetic diversity. Viruses infecting gut-associated archaea provide evidence of convergence in adaptation with viruses infecting gut-associated bacteria. These viruses contain a large repertoire of lysin proteins that cleave archaeal pseudomurein and are enriched in glycan-binding domains (Ig-like/Flg_new) and diversity-generating retroelements. The characterization of this vast repertoire of viruses paves the way towards a better understanding of their role in regulating methanogen communities globally, as well as the development of much-needed genetic tools.

Metagenomics uncovers dietary adaptations for chitin digestion in the gut microbiota of convergent myrmecophagous mammals.

IMPORTANCE: Myrmecophagous mammals are specialized in the consumption of ants and/or termites. They do not share a direct common ancestor and evolved convergently in five distinct placental orders raising questions about the underlying adaptive mechanisms involved and the relative contribution of natural selection and phylogenetic constraints. Understanding how these species digest their prey can help answer these questions. More specifically, the role of their gut microbial symbionts in the digestion of the insect chitinous exoskeleton has not been investigated in all myrmecophagous orders. We generated 29 new gut metagenomes from nine myrmecophagous species to reconstruct more than 300 bacterial genomes in which we identified chitin-degrading enzymes. Studying the distribution of these chitinolytic bacteria among hosts revealed both shared and specific bacteria between ant-eating species. Overall, our results highlight the potential role of gut symbionts in the convergent dietary adaptation of myrmecophagous mammals and the evolutionary mechanisms shaping their gut microbiota.

Methanomethylophilus alvi gen. nov., sp. nov., a Novel Hydrogenotrophic Methyl-Reducing Methanogenic Archaea of the Order Methanomassiliicoccales Isolated from the Human Gut and Proposal of the Novel Family Methanomethylophilaceae fam. nov.

The methanogenic strain Mx-05T was isolated from the human fecal microbiome. A phylogenetic analysis based on the 16S rRNA gene and protein marker genes indicated that the strain is affiliated with the order Methanomassiliicoccales. It shares 86.9% 16S rRNA gene sequence identity with Methanomassiliicoccus luminyensis, the only member of this order previously isolated. The cells of Mx-05T were non-motile cocci, with a diameter range of 0.4-0.7 µm. They grew anaerobically and reduced methanol, monomethylamine, dimethylamine, and trimethylamine into methane, using H2 as an electron donor. H2/CO2, formate, ethanol, and acetate were not used as energy sources. The growth of Mx-05T required an unknown medium factor(s) provided by Eggerthella lenta and present in rumen fluid. Mx-05T grew between 30 °C and 40 °C (optimum 37 °C), over a pH range of 6.9-8.3 (optimum pH 7.5), and between 0.02 and 0.34 mol.L-1 NaCl (optimum 0.12 mol.L-1 NaCl). The genome is 1.67 Mbp with a G+C content of 55.5 mol%. Genome sequence annotation confirmed the absence of the methyl branch of the H4MPT Wood-Ljungdahl pathway, as described for other Methanomassiliicoccales members. Based on an average nucleotide identity analysis, we propose strain Mx-05T as being a novel representative of the order Methanomassiliicoccales, within the novel family Methanomethylophilaceae, for which the name Methanomethylophilus alvi gen. nov, sp. nov. is proposed. The type strain is Mx-05T (JCM 31474T).

Genome sequence of "Candidatus Methanomethylophilus alvus" Mx1201, a methanogenic archaeon from the human gut belonging to a seventh order of methanogens.

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.

Methanobacterium lacus sp. nov., isolated from the profundal sediment of a freshwater meromictic lake.

An autotrophic, hydrogenotrophic methanogen, designated strain 17A1(T), was isolated from the profundal sediment of the meromictic Lake Pavin, France. The cells of the novel strain, which were non-motile, Gram-staining-negative rods that measured 2-15 µm in length and 0.2-0.4 µm in width, grew as filaments. Strain 17A1(T) grew in a mineral medium and its growth was stimulated by the addition of yeast extract, vitamins, acetate or rumen fluid. Penicillin, vancomycin and kanamycin reduced growth but did not completely inhibit it. Growth occurred at 14-41 °C (optimum 30 °C), at pH 5.0-8.5 (optimum pH 6.5) and with 0-0.4 M NaCl (optimum 0.1 M). The novel strain utilized H(2)/CO(2) and methanol/H(2) as substrates but not formate, acetate, methylamine/H(2), isobutanol or 2-propanol. Its genomic DNA G+C content was 37.0 mol%. In phylogenetic analyses based on 16S rRNA gene sequences, strain 17A1(T) appeared to be a member of the genus Methanobacterium, with Methanobacterium beijingense 8-2(T) (96.3% sequence similarity) identified as the most closely related established species. Based on phenotypic and phylogenetic data, strain 17A1(T) represents a novel species of methanogen within the genus Methanobacterium, for which the name Methanobacterium lacus sp. nov. is proposed. The type strain is 17A1(T) (=DSM 24406(T)=JCM 17760(T)).

Factors shaping the abundance and diversity of the gut archaeome across the animal kingdom.

Archaea are common constituents of the gut microbiome of humans, ruminants, and termites but little is known about their diversity and abundance in other animals. Here, we analyse sequencing and quantification data of archaeal and bacterial 16S rRNA genes from 250 species of animals covering a large taxonomic spectrum. We detect the presence of archaea in 175 animal species belonging to invertebrates, fish, amphibians, birds, reptiles and mammals. We identify five dominant gut lineages, corresponding to Methanobrevibacter, Methanosphaera, Methanocorpusculum, Methanimicrococcus and "Ca. Methanomethylophilaceae". Some archaeal clades, notably within Methanobrevibacter, are associated to certain hosts, suggesting specific adaptations. The non-methanogenic lineage Nitrososphaeraceae (Thaumarchaeota) is frequently present in animal samples, although at low abundance, but may have also adapted to the gut environment. Host phylogeny, diet type, fibre content, and intestinal tract physiology are major drivers of the diversity and abundance of the archaeome in mammals. The overall abundance of archaea is more influenced by these factors than that of bacteria. Methanogens reducing methyl-compounds with H2 can represent an important fraction of the overall methanogens in many animals. Together with CO2-reducing methanogens, they are influenced by diet and composition of gut bacteria. Our results provide key elements toward our understanding of the ecology of archaea in the gut, an emerging and important field of investigation.

Growth temperature and chromatinization in archaea.

DNA in cells is associated with proteins that constrain its structure and affect DNA-templated processes including transcription and replication. HU and histones are the main constituents of chromatin in bacteria and eukaryotes, respectively, with few exceptions. Archaea, in contrast, have diverse repertoires of nucleoid-associated proteins (NAPs). To analyse the evolutionary and ecological drivers of this diversity, we combined a phylogenomic survey of known and predicted NAPs with quantitative proteomic data. We identify the Diaforarchaea as a hotbed of NAP gain and loss, and experimentally validate candidate NAPs in two members of this clade, Thermoplasma volcanium and Methanomassiliicoccus luminyensis. Proteomic analysis across a diverse sample of 19 archaea revealed that NAP investment varies from <0.03% to >5% of total protein. This variation is predicted by growth temperature. We propose that high levels of chromatinization have evolved as a mechanism to prevent uncontrolled helix denaturation at higher temperatures, with implications for the origin of chromatin in both archaea and eukaryotes.

A catalogue of 1,167 genomes from the human gut archaeome.

The human gut microbiome plays an important role in health, but its archaeal diversity remains largely unexplored. In the present study, we report the analysis of 1,167 nonredundant archaeal genomes (608 high-quality genomes) recovered from human gastrointestinal tract, sampled across 24 countries and rural and urban populations. We identified previously undescribed taxa including 3 genera, 15 species and 52 strains. Based on distinct genomic features, we justify the split of the Methanobrevibacter smithii clade into two separate species, with one represented by the previously undescribed 'Candidatus Methanobrevibacter intestini'. Patterns derived from 28,581 protein clusters showed significant associations with sociodemographic characteristics such as age groups and lifestyle. We additionally show that archaea are characterized by specific genomic and functional adaptations to the host and carry a complex virome. Our work expands our current understanding of the human archaeome and provides a large genome catalogue for future analyses to decipher its impact on human physiology.

Identification of sulfur-cycle prokaryotes in a low-sulfate lake (Lake Pavin) using aprA and 16S rRNA gene markers.

Geochemical researches at Lake Pavin, a low-sulfate-containing freshwater lake, suggest that the dominant biogeochemical processes are iron and sulfate reduction, and methanogenesis. Although the sulfur cycle is one of the main active element cycles in this lake, little is known about the sulfate-reducer and sulfur-oxidizing bacteria. The aim of this study was to assess the vertical distribution of these microbes and their diversities and to test the hypothesis suggesting that only few SRP populations are involved in dissimilatory sulfate reduction and that Epsilonproteobacteria are the likely key players in the oxidative phase of sulfur cycle by using a PCR aprA gene-based approach in comparison with a 16S rRNA gene-based analysis. The results support this hypothesis. Finally, this preliminary work points strongly the likelihood of novel metabolic processes upon the availability of sulfate and other electron acceptors.

Comparative genomic analysis of Methanimicrococcus blatticola provides insights into host adaptation in archaea and the evolution of methanogenesis.

Other than the Methanobacteriales and Methanomassiliicoccales, the characteristics of archaea that inhabit the animal microbiome are largely unknown. Methanimicrococcus blatticola, a member of the Methanosarcinales, currently reunites two unique features within this order: it is a colonizer of the animal digestive tract and can only reduce methyl compounds with H2 for methanogenesis, a increasingly recognized metabolism in the archaea and whose origin remains debated. To understand the origin of these characteristics, we have carried out a large-scale comparative genomic analysis. We infer the loss of more than a thousand genes in M. blatticola, by far the largest genome reduction across all Methanosarcinales. These include numerous elements for sensing the environment and adapting to more stable gut conditions, as well as a significant remodeling of the cell surface components likely involved in host and gut microbiota interactions. Several of these modifications parallel those previously observed in phylogenetically distant archaea and bacteria from the animal microbiome, suggesting large-scale convergent mechanisms of adaptation to the gut. Strikingly, M. blatticola has lost almost all genes coding for the H4MPT methyl branch of the Wood-Ljungdahl pathway (to the exception of mer), a phenomenon never reported before in any member of Class I or Class II methanogens. The loss of this pathway illustrates one of the evolutionary processes that may have led to the emergence of methyl-reducing hydrogenotrophic methanogens, possibly linked to the colonization of organic-rich environments (including the animal gut) where both methyl compounds and hydrogen are abundant.