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ABSTRACT Methane oxidation by aerobic methanotrophs is well known to be strongly regulated by the availability of copper, i.e., the “copper switch.” That is, there are two forms of methane monooxygenase: a cytoplasmic or soluble methane monooxygenase (sMMO) and a membrane-bound or particulate methane monooxygenase (pMMO). sMMO is only expressed and active in the absence of copper, while pMMO requires copper. Previous work has also shown that one gene in the operon of the soluble methane monooxygenase—mmoD—also plays a critical role in the “copper switch,” but its function is still vague. Herein, we show that MmoD is not needed for the expression of genes in the sMMO gene cluster but is critical for the formation of sMMO polypeptides and sMMO activity inMethylosinus trichosporiumOB3b, indicating that MmoD plays a key post-transcriptional role in the maturation of sMMO. Furthermore, data also show that MmoD controls the expression of methanobactin, a copper-binding compound used by some methanotrophs, includingM. trichosporiumOB3b, for copper sequestration. Collectively, these results provide greater insights into the components of the “copper switch” and provide new strategies to manipulate methanotrophic activity. IMPORTANCEAerobic methanotrophs play a critical role in the global carbon cycle, particularly in controlling net emissions of methane to the atmosphere. As methane is a much more potent greenhouse gas than carbon dioxide, there is increasing interest in utilizing these microbes to mitigate future climate change by increasing their ability to consume methane. Any such efforts, however, require a detailed understanding of how to manipulate methanotrophic activity. Herein, we show that methanotrophic activity is strongly controlled by MmoD, i.e., MmoD regulates methanotrophy through the post-transcriptional regulation of the soluble methane monooxygenase and controls the ability of methanotrophs to collect copper. Such data are likely to prove quite useful in future strategies to enhance the use of methanotrophs to not only reduce methane emissions but also remove methane from the atmosphere. 

The discovery that MB will couple the oxidation of H₂O to metal ion reduction and the release of O₂suggests that methanotrophs expressing MB may be able to maintain their activity under hypoxic/anoxic conditions through the “self-generation” of dioxygen required for the initial oxidation of methane to methanol. Such an ability may be an important factor in enabling methanotrophs to not only colonize the oxic-anoxic interface where methane concentrations are highest but also tolerate significant temporal fluctuations of this interface.