A complex interplay between nitric oxide, quorum sensing and a unique secondary metabolite tundrenone constitutes hypoxia response in Methylobacter

Methylobacter species, members of the Methylococcales, have recently emerged as some of the globally widespread, cosmopolitan species, appearing to play a key role in consumption of methane in a variety of environments and across gradients of dioxygen tensions. We approached the question of how Methylobacter copes with low dioxygen partial pressures that it encounters in its natural habitats, via laboratory manipulation. Through comparative transcriptomics of cultures grown under high dioxygen partial pressure and cultures starved for dioxygen, we identified a gene cluster encoding a hybrid cluster protein along with sensing and regulatory functions. Through mutant analysis we demonstrated that this gene cluster is involved in both oxidative and nitrosative stress responses and in NO reduction, while being responsive to NO, likely through direct sensing. Through additional transcriptomic analyses, we further uncovered a complex interconnection between the NO-mediated stress response and quorum sensing in turn controlling synthesis of the secondary metabolite tundrenone, these two systems being inversely regulated. Some of the key methylotrophy functions, such as the two alternative methanol dehydrogenases seem to be parts of the stress response regulatory cascade. One intriguing scenario that emerged from our analyses is that the main role of the respiratory denitrification pathway may be in producing the signaling molecule, NO, in response to hypoxia, rather than in serving as an energy-generating pathway. This novel and complex hypoxia stress response system is unique to Methylobacter tundripaludum, and it may play a role in the environmental fitness of these organisms and in their cosmopolitan environmental distribution. Overall design: 10 samples from Methylobacter tundripaludum 31/32 and 6 from M. tundripaludum 21/22 were processed with biological duplicates across a variety of conditions. In 31/32 the wild-type sequence was grown with either nitrate or ammonia as the provided nitrogen sources (2 samples). Also in 31/32, the wild-type and a deletion mutant in hcp/hcr were grown with and without low dioxygen stress, and with either nitrate or ammonia as the added nitrogen source (8 samples). In 21/22, the wild type and two mutant strains (one in the quorum sensing systema and one in the production of tundrinone) and the wild type were each grown in nitrate or ammonia as the proviced nitrogen source (6 samples).