Methylotrophic methanogenesis in the Archaeoglobi: Cultivation ofCa.Methanoglobus hypatiae from a Yellowstone hot spring

Abstract Over the past decade, environmental metagenomics and PCR-based marker gene surveys have revealed that several lineages beyond just a few well-established groups within the Euryarchaeota superphylum harbor the genetic potential for methanogenesis. One of these groups are the Archaeoglobi, a class of obligately thermophilic Euryarchaeotes that have long been considered to live a non-methanogenic lifestyle. Here, we enriched Candidatus Methanoglobus hypatiae, a methanogen affiliated with the family Archaeoglobaceae, from a hot spring in Yellowstone National Park. The enrichment is sediment-free, grows at 64-70 °C and a pH of 7.8, and produces methane from mono-, di-, and tri-methylamine. Ca. M. hypatiae is represented by a 1.62 Mb metagenome-assembled genome with an estimated completeness of 100% and accounts for up to 67% of cells in the culture according to fluorescence in situ hybridization. Via genome-resolved metatranscriptomics and stable isotope tracing, we demonstrate that Ca. M. hypatiae expresses methylotrophic methanogenesis and energy-conserving pathways for reducing monomethylamine to methane. The detection of Archaeoglobi populations related to Ca. M. hypatiae in 36 geochemically diverse geothermal sites within Yellowstone National Park (pH 2.61-9.35; 18.4 to 93.8 °C), as revealed through the examination of previously published gene amplicon datasets, implies a previously underestimated contribution to anaerobic carbon cycling in extreme ecosystems. Significance statement The majority of global methane emissions are attributed to the activity of methane-producing anaerobic archaea, the methanogens. Over the last decade, environmental DNA sequencing demonstrated that culture collections do not adequately represent the true taxonomic and metabolic diversity of methanogens present in nature. One group of archaea postulated to contribute to methane production in high temperature marine and terrestrial environments are the Archaeoglobi, a group of obligate thermophilic archaea within the Euryarchaeota superphylum. Here, we report the cultivation and physiological characterization of a methanogenic member of the Archaeoglobi, Ca. Methanoglobus hypatiae, from a hot spring in Yellowstone National Park. Via a combination of growth experiments, stable isotope tracing, metatranscriptomics, microscopy analyses, and re-examination of gene amplicon surveys, our study provides direct experimental evidence of methanogenesis in the Archaeoglobi and shows that closely related archaea are widely distributed in Yellowstone’s geothermal features.