Genome sequence analysis of a highly formaldehyde-resistant Methylomicrobium alcaliphilum strain.

Formaldehyde is a highly reactive and cytotoxic compound, and its efficient detoxification is essential for cellular survival. This requirement is particularly critical in methylotrophic microorganisms, where formaldehyde serves as a central intermediate in methanol assimilation. Despite the toxicity of this compound, methylotrophic and methanotrophic bacteria have evolved mechanisms that allow limited tolerance, yet the isolation of highly resistant variants remains extremely challenging due to the intrinsic difficulties of cultivating and purifying these organisms. Here, we report the isolation and complete genome sequence of a Methylomicrobium alcaliphilum 20Z derivative capable of sustained growth at 30 mM formaldehyde-representing a six-fold increase over the parental strain and positioning it among the most formaldehyde-tolerant methanotrophs described to date. The strain was obtained after prolonged adaptation and successful purification of a single resistant colony, a technically demanding process in this species. In parallel, we re-sequenced and re-annotated the wild-type genome, generating an improved genetic reference for M. alcaliphilum 20Z. Comparative genome analysis revealed 168 mutations affecting 31 open reading frames in the adapted strain. These mutations span genes involved in stress response, membrane remodeling, macromolecular repair, and regulatory functions, suggesting multifactorial adaptive strategies beyond canonical formaldehyde detoxification pathways. The genomic data provided here constitute a valuable foundation for future mechanistic studies and offer a resource for researchers aiming to understand or engineer aldehyde tolerance in methylotrophic bacteria.