Transcription of Nitrogen Fixation Genes Is Enhanced at Unfavorably High Oxygen Concentrations for Diazotrophic Growth in a Methane-oxidizing Bacterium

Since nitrogenase is intrinsically sensitive to oxygen (O₂), diverse aerobic diazotrophs need strategies to cope with nitrogenase damage by O₂. In the present study, we investigated the mechanisms by which aerobic methane-oxidizing bacteria (methanotrophs) enable the concurrent activities of methane monooxygenase, which uses O₂, and nitrogenase in the cytoplasm of the same cell. By using ¹⁵N labeling, we confirmed the capacity of alphaproteobacterial methanotroph Methylosinus sp. 3S-1 for nitrogen fixation and diazotrophic growth across a wide range of O₂ concentrations <20%. When the initial O₂ concentration was increased from 2 to 20% in a diazotrophic culture, similar decreases were observed in fixed nitrogen and NifH protein levels. In contrast, the mRNA levels of nitrogen fixation genes (nif genes) markedly increased and remained elevated for the duration of slow growth at high O₂ concentrations. This pattern of nif expression in response to O₂ may be attributed to the properties of the nif-specific transcriptional regulator NifA. The present results suggest that the increase in nif transcription is one of the strategies by which this methanotroph maintains nitrogen fixation on the background of aerobic methane oxidation.