Molecular charachterization of alkane-metabolizing archaea

Alkanes are carbon compounds composed exclusively of carbon and hydrogen. They are the main constituents of natural gas, petroleum, and fossil fuels and, thus, influence both the climate and human activity. Alkanes play an important role in anoxic ocean sediments and hydrothermal systems, where they can leak into the ocean. In these environments, microbes modulate both the release and the consumption of alkanes: either through the biogenic formation of methane by methane-producing archaea (methanogens), or the biological consumption of alkanes by anaerobic alkane-oxidizing archaea (ANKA). Methanogens and ANKA share evolutionary history and metabolic features, but they function differently. Unlike methanogens, ANKA depend on sulfate-reducing bacteria (SRB) or other extracellular electron acceptors to oxidize alkanes, in part by the reversal of the metabolism of methanogens. Both methanogens and ANKA play an important role in carbon cycling in energy-limited ecosystems. However, little is known about their anabolic pathways or how electrons and carbon flow from catabolic substrates to anabolic products. In this thesis, I describe a novel ethane-oxidizing archaeon and investigate the anabolism of select ANKA and methanogens. In Chapter 2 (Manuscript 1), I describe Candidatus Ethanoperedens ambientalis, the first ethane oxidizer known to grow at room temperature. The ethane oxidizer forms consortia with a member of the Desulfobacteriota, Cand. Desulfosymmachos locustemperatii, of the Seep-SRB1d clade. Their syntrophic interaction likely relies on direct interspecies electron transfer. Both partners assimilate dissolved inorganic carbon as a carbon source. I also found that the disaccharide trehalose was by far the most abundant sugar in enrichments of Cand. E. ambientalis. In Chapter 3 (Manuscript 2), I expanded the search for disaccharides across multiple ANKA enrichments and demonstrated that trehalose and other disaccharides are present at similar abundances to those found in Cand. E. ambientalis enrichments. I developed a bioinformatic pipeline that demonstrated that trehalose metabolism was common across the partner bacteria of ANKA. However, metabolomics showed substantial amounts of trehalose only in ANKA enrichments where the ANKA had substantial trehalose metabolism. I also show that ANKA and their partner bacteria readily share trehalose metabolism through horizontal gene transfer. In Chapter 4 (Manuscript 3), I explore electron flow and anabolism in pure cultures of Methanothermococcus thermolithotrophicus, currently the only methanogen known to grow on sulfate. I examine how M. thermolithotrophicus remodels its transcriptome in response to sulfur sources, and how this in turn affects how M. thermolithotrophicus shuttles electrons from catabolic substrates to anabolic products. In this study, we also observed the overexpression of a putative viral-like element and identified the likely capsid protein using artificial intelligence–based protein modeling. In Chapter 5, I synthesize the findings of my doctoral research. I discuss the implications of my findings on the energy landscape of ethane oxidation, the potential roles and sources of disaccharides in ANKA consortia, and how sulfur acts as an environmental modulator of transcriptional resources in M. thermolithotrophicus.