RESULTS:

An anaerobic mesophilic syntrophic archaeon strain MK-D1T was isolated as a pure co-culture with Methanogenium sp. strain MK-MG from deep-sea methane seep sediment. This organism is to our knowledge the first cultured representative of ‘Asgard’ archaea an archaeal group closely related to eukaryotes. Here we describe the detailed physiology and phylogeny of MK-D1T and propose Promethearchaeum syntrophicum gen. nov. sp. nov. to accommodate this strain. Cells were non-motile small cocci approximately 300–750 nm in diameter and produced membrane vesicles chains of blebs and membrane-based protrusions. MK-D1T grew at 4–30 °C with optimum growth at 20 °C. The strain grew chemoorganotrophically with amino acids peptides and yeast extract with obligate dependence on syntrophy with H2-/formate-utilizing organisms. MK-D1T showed the fastest growth and highest maximum cell yield when grown with yeast extract as the substrate: approximately 3 months to full growth reaching up to 6.7×106 16S rRNA gene copies ml−1. MK-D1T had a circular 4.32 Mb chromosome with a DNA G+C content of 31.1 mol%. The results of phylogenetic analyses of the 16S rRNA gene and conserved marker proteins indicated that the strain is affiliated with ‘Asgard’ archaea and more specifically DHVC1/DSAG/MBG-B and ‘Lokiarchaeota’/‘Lokiarchaeia’. On the basis of the results of 16S rRNA gene sequence analysis the most closely related isolated relatives were Infirmifilum lucidum 3507LTT (76.09 %) and Methanothermobacter tenebrarum RMAST (77.45 %) and the closest relative in enrichment culture was Candidatus ‘Lokiarchaeum ossiferum’ (95.39 %). The type strain of the type species is MK-D1T (JCM 39240T and JAMSTEC no. 115508). We propose the associated family order class phylum and kingdom as Promethearchaeaceae fam. nov. Promethearchaeales ord. nov. Promethearchaeia class. nov. Promethearchaeota phyl. nov. and Promethearchaeati regn. nov. respectively. These are in accordance with ICNP Rules 8 and 22 for nomenclature Rule 30(3)(b) for validation and maintenance of the type strain and Rule 31a for description as a member of an unambiguous syntrophic association.

Methanogenic archaea are a diverse polyphyletic group of strictly anaerobic prokaryotes capable of producing methane as their primary metabolic product. It has been over three decades since minimal standards for their taxonomic description have been proposed. In light of advancements in technology and amendments in systematic microbiology revision of the older criteria for taxonomic description is essential. Most of the previously recommended minimum standards regarding phenotypic characterization of pure cultures are maintained. Electron microscopy and chemotaxonomic methods like whole-cell protein and lipid analysis are desirable but not required. Because of advancements in DNA sequencing technologies obtaining a complete or draft whole genome sequence for type strains and its deposition in a public database are now mandatory. Genomic data should be used for rigorous comparison to close relatives using overall genome related indices such as average nucleotide identity and digital DNA–DNA hybridization. Phylogenetic analysis of the 16S rRNA gene is also required and can be supplemented by phylogenies of the mcrA gene and phylogenomic analysis using multiple conserved single-copy marker genes. Additionally it is now established that culture purity is not essential for studying prokaryotes and description of Candidatus methanogenic taxa using single-cell or metagenomics along with other appropriate criteria is a viable alternative. The revisions to the minimal criteria proposed here by the members of the Subcommittee on the Taxonomy of Methanogenic Archaea of the International Committee on Systematics of Prokaryotes should allow for rigorous yet practical taxonomic description of these important and diverse microbes.