Genomic, physiological, and transcriptomic mechanisms of pH stress adaptation and response in Methylocystis sumavensis, a novel peatland methanotroph

Abstract

Methanotrophic bacteria in peatlands mitigate emissions of methane (CH4), a potent greenhouse gas, yet the mechanisms enabling them to remain active under the acidic conditions typical of many peatlands remain poorly understood. Using enrichment cultivation and single-cell sorting, we isolated a novel peatland methanotroph from Czech soil, Methylocystis sumavensis. This species is moderately acidotolerant and active across broad pH and temperature ranges, with growth optima at pH 6.8 and 24-37 {degrees}C. The genome of M. sumavensis encodes two particulate methane monooxygenase isozymes, a clade I nitrous oxide reductase, multiple terminal oxidases, and two [NiFe]-hydrogenases, including a complete complex of the previously uncharacterised membrane-bound hydrogenase group 4f, indicating substantial metabolic versatility. To link genomic potential with physiological function, we compared transcriptomes under acidic (pH 5.0) and alkaline (pH 9.0) conditions relative to the optimum pH (6.8). Under both stresses, M. sumavensis increased transcription of genes involved in membrane remodelling, ion transport across both membranes, and stress response and repair, while reducing transcription of genes associated with methane oxidation and cell division. Alkaline stress additionally suppressed growth through reduced transcription of the carbon-assimilating Serine cycle. In contrast, acidic stress triggered a coordinated response requiring greater energetic investment. Among the most strongly upregulated genes were those encoding two formate dehydrogenases, the branched-chain alpha-keto-acid dehydrogenase complex, and all seven group 4f [NiFe]-hydrogenase-related genes, suggesting enhanced respiratory redox balancing under elevated external proton concentrations. These results reveal key mechanisms of pH-stress adaptation and highlight metabolic plasticity as a major determinant of methanotroph resilience in ecosystems.