Abstract
Methanogenic archaea are strict anaerobes that occur in diverse environments like marine and freshwater sediments, soils, hot springs, sewage sludge and the digestive tracts of animals and humans. Methanogens belong to the phylum Euryarchaeota, which comprises both methanogenic and non-methanogenic orders and many lineages of uncultivated archaea with unknown properties. By a comprehensive phylogenetic analysis, we connected the 16S rRNA gene sequences of one of these deep-branching lineages, distantly related to Thermoplasmatales, to a large clade of unknown mcrA gene sequences, a functional marker for methanogenesis. The analysis suggested that both genes stem from the same organism, indicating the methanogenic nature of this group. This was further confirmed by our two highly enriched cultures of methanogenic archaea, Candidatus Methanoplasma termitum strain MpT1 from a higher termite and strain MpM2 from the millipede gut, which had 16S rRNA genes that fell within in this lineage. Together with the recent isolation of Methanomassiliicoccus luminyensis from human feces, the results of our study supported that the entire lineage, distantly related to the Thermoplasmatales, represents the seventh order of methanogens, the “Methanoplasmatales” (now referred to as Methanomassiliicoccales). To gain deeper insight into this novel order of methanogens, we sequenced and analyzed the genome of Ca. Mp. termitum strain MpT1, and compared it to the three other genomes of the order Methanomassiliicoccales available to date. Our results confirmed that all members of the lineage are obligately hydrogen-dependent methylotrophs that perform methanogenesis by the hydrogen-dependent reduction of methanol or methylamines and lack the entire C1 pathway for reduction CO2 to CH4. However, this raises questions concerning the mechanism of energy conservation that had so far escaped attention. Our comparative analysis revealed that energy conversion in Methanomassiliicoccales differs from those of other obligately hydrogen-dependent methylotrophs. We identified a complex encoded by all four genomes that is related to the membrane-bound F420:methanophenazine oxidoreductase (Fpo) of Methanosarcinales, but lacks the F420-oxidzing module, as in the apparently ferredoxin-dependent Fpo-like homolog in Methanosaeta thermophila. We suggests that this Fpo-like complex of the Methanomassiliicoccales uses the present D subunit of the heterodisulfide reductase as an electron acceptor to form an energy-converting ferredoxin:heterodisulfide oxidoreductase. This suggests that in Methanomassiliicoccales, the heterodisulfide serves two functions: the production of reduced ferredoxin during electron bifurcation at the cytoplasmic MvhADG/HdrABC complex, and the generation of a membrane portential during the reoxidation of ferredoxin via a membrane-bound electron transport chain. This dual function of heterodisulfide may be a unique characteristic of the entire order. Furthermore, we identified an unusual two-membrane system in Ca. Mp. termitum and strain MpM2 by transmission electron micrographs that might be typical for the complete order. While methanogenesis in insect guts has been investigated by numerous authors, almost nothing is known about methanogenesis and the methanogenic community structure in millipedes, the only other group of arthropods that emit methane. Our analysis of the phylogenetic diversity of archaea associated with tropical millipedes documented that most methanogens in their guts fall into the orders Methanobacteriales, Methanosarcinales, Methanomicrobiales and Methano-massiliicoccales. Their close relatedness to methanogens from the guts of termites, cockroaches and scarab beetle larvae suggests that methanogenic community structure in methane-emitting arthropods is not necessarily shaped by cospeciation. Recently, it has been shown that bacterial communities mirror major events in the evolutionary history of the termites and cockroaches, which leads to the speculation if this is also case for the archaeal community. Here, we present a study that consists of both clone libraries and high-throughput sequencing which concludes that the archaeal community structure and phylogeny is shaped more by the major host groups than by coevolution and diet. This indicates that the host habitat is the major driving force for the selection of the archaeal community.
