It is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that in Methylosinus trichosporium OB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced by M. trichosporium OB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RTqPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and active in situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.T he increased availability of methane through industrial practices such as hydraulic fracturing has enhanced interest in using methanotrophs, prokaryotes that thrive on methane as their sole source of carbon and energy, to convert methane to more valuable products, e.g., liquid biofuels, plastics, and protein to supplement animal feed (1-7). Methanotrophs have also received increasing attention given that methane is a very potent greenhouse gas, with a global warming potential 28 to 34 times that of carbon dioxide over a 100-year period (8), and methanotrophs are estimated to remove up to 90% of the methane produced in anaerobic soils (9). Methanotrophs are widespread in the environment, found in diverse locations such as landfill cover, forest, agricultural, and volcanic soils; freshwater and marine sediments; and sewage sludge (6, 10, 11). Many are amenable to genetic manipulation (12-18).An important issue, however, in the use of methanotrophy for any purpose is that the first step in methane oxidation, the conversion of methane to methanol, is performed by two different forms of methane monooxygenase (MMO) with different oxidation kinetics and affinities for methane. One form, the soluble methane monooxygenase (sMMO), is found in the cytoplasm and has relatively high turnover (maximal whole-cell methane oxidation rate [V max ] of ϳ730 nmol · min Ϫ...