Investigating novel methylotrophic archaea in Yellowstone hot springs through meta-omics and targeted cultivation

Abstract

In the past two decades, metagenomic sequencing of environmental samples has led to a substantial expansion in our understanding of the domain Archaea. In particular, several newly discovered lineages encode the potential for anaerobic methylotrophy, in either a non- methanogenic or methanogenic fashion. However, due to the absence of cultures, many lineages are only hypothesized to perform methylotrophy based on metagenomic analysis and lack experimental support of their proposed physiology. Furthermore, given the large diversity of uncultivated taxa, there are likely additional groups of methylotrophic archaea that are not yet characterized. Methyl groups are ubiquitous in nature and thus are important in fueling many metabolic lifestyles and impacting global carbon cycling. Non-methanogenic methylotrophs can convert methyl groups to carbon dioxide and may compete with methanogens for substrates. Methylotrophic methanogens are capable of producing methane, a climate active gas, from methyl groups. For decades methanogens were thought to be restricted to a single lineage "Euryarchaeota". However, in the last decade, culture-independent metagenomic sequencing has revealed the presence of marker genes for methanogenesis in several uncultivated groups within another archaeal phylum, Thermoproteota. This thesis presents results on three class-level lineages of methylotrophic archaea within the Thermoproteota phylum identified in Yellowstone National Park hot springs. The first is a metagenomic description of a new uncultivated class-level lineage, termed Culexarchaeia, that encodes a versatile metabolism, including the capacity for non-methanogenic anaerobic methylotrophy. The second lineage discussed is "Methanomethylicia", whose members are predicted methylotrophic methanogens. Using hot spring sediments, we obtained an enrichment culture containing Candidatus Methanosuratincola verstraetei and demonstrated that it produces methane from methanol and hydrogen. The third lineage discussed is Korarchaeia, an enigmatic lineage commonly found in terrestrial hot springs. We obtained an enrichment culture containing Candidatus Methanodesulfokora washburnensis, an organism that also makes methane from methanol and hydrogen. These represent the first cultured methanogens outside the Euryarchaeota, and set the stage for future detailed studies on the physiology and biochemistry of these organisms. Together, these results highlight Yellowstone as a natural laboratory for the investigation of poorly understood archaeal lineages through metagenomics and cultivation.