Functional Insights of Salinity Stress-Related Pathways in Metagenome-Resolved Methanothrix Genomes

Recently, methanogenic archaea belonging to the genus Methanothrix were reported to have a fundamental role in maintaining stable ecosystem functioning in anaerobic bioreactors under different configurations/conditions. In this study, we reconstructed three Methanothrix metagenome-assembled genomes (MAGs) from granular sludge collected from saline upflow anaerobic sludge blanket (UASB) reactors, where Methanothrix harundinacea was previously implicated with the formation of compact and stable granules under elevated salinity levels (up to 20 g/L Na⁺). Genome annotation and pathway analysis of the Methanothrix MAGs revealed a genetic repertoire supporting their growth under high salinity. Specifically, the most dominant Methanothrix (MAG_279), classified as a subspecies of Methanothrix_A harundinacea_D, had the potential to augment its salinity resistance through the production of different glycoconjugates via the N-glycosylation process, and via the production of compatible solutes as N^ε-acetyl-β-lysine and ectoine. The stabilization and reinforcement of the cell membrane via the production of isoprenoids was identified as an additional stress-related pathway in this microorganism. The improved understanding of the salinity stress-related mechanisms of M. harundinacea highlights its ecological niche in extreme conditions, opening new perspectives for high-efficiency methanisation of organic waste at high salinities, as well as the possible persistence of this methanogen in highly-saline natural anaerobic environments. IMPORTANCE Using genome-centric metagenomics, we discovered a new Methanothrix harundinacea subspecies that appears to be a halotolerant acetoclastic methanogen with the flexibility for adaptation in the anaerobic digestion process both at low (5 g/L Na⁺) and high salinity conditions (20 g/L Na⁺). Annotation of the recovered M. harundinacea genome revealed salinity stress-related functions, including the modification of EPS glycoconjugates and the production of compatible solutes. This is the first study reporting these genomic features within a Methanothrix sp., a milestone further supporting previous studies that identified M. harundinacea as a key-driver in anaerobic granulation under high salinity stress.