bMethanotrophs have multiple methane monooxygenases that are well known to be regulated by copper, i.e., a "copper switch." At low copper/biomass ratios the soluble methane monooxygenase (sMMO) is expressed while expression and activity of the particulate methane monooxygenase (pMMO) increases with increasing availability of copper. In many methanotrophs there are also multiple methanol dehydrogenases (MeDHs), one based on Mxa and another based on Xox. Mxa-MeDH is known to have calcium in its active site, while Xox-MeDHs have been shown to have rare earth elements in their active site. We show here that the expression levels of Mxa-MeDH and Xox-MeDH in Methylosinus trichosporium OB3b significantly decreased and increased, respectively, when grown in the presence of cerium but the absence of copper compared to the absence of both metals. Expression of sMMO and pMMO was not affected. In the presence of copper, the effect of cerium on gene expression was less significant, i.e., expression of Mxa-MeDH in the presence of copper and cerium was slightly lower than in the presence of copper alone, but Xox-MeDH was again found to increase significantly. As expected, the addition of copper caused sMMO and pMMO expression levels to significantly decrease and increase, respectively, but the simultaneous addition of cerium had no discernible effect on MMO expression. As a result, it appears Mxa-MeDH can be uncoupled from methane oxidation by sMMO in M. trichosporium OB3b but not from pMMO. It is well known that microorganisms have diverse mechanisms to sense and respond to metals in their environment. These mechanisms typically include strategies to regulate gene expression in response to the presence or absence of metals such as copper, zinc, iron, manganese, arsenic, and mercury (see, for example, references 1, 2, 3 and 4). One such phenomenon is the "copper switch" in methanotrophs. That is, these microbes utilize methane as their sole growth substrate but have two different monooxygenases for the initial oxidation of methane to methanol. One, the particulate methane monooxygenase (pMMO), is found in the intracytoplasmic membranes of these microbes, and its expression and activity increases with increasing availability of copper. The second, the soluble methane monooxygenase (sMMO), is found in the cytoplasm and is only expressed when copper is unavailable (5). These two forms of MMO have very different structures, activities, and substrate ranges (5-14), and so careful consideration of the form of MMO expressed is critical for understanding methanotrophic ecology, as well for various applications of methanotrophy, including the removal of chlorinated solvents and methane, a potent greenhouse gas (5, 10, 12, 15).Further, interest in the commercial application of methanotrophy has dramatically accelerated in recent years, in part due to increased methane supplies given advances in hydraulic fracturing of shale formations. As a result, methane prices have become quite low, with the wellhead price of natural gas dropping fro...

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...

bMethanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 M CuCl 2 , expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl 2 , the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl 2 . mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl 2 . These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin "piracy" may be commonplace. Methanotrophs are distinguished from other microorganisms by their ability to utilize methane as a sole carbon and energy source yet are phylogenetically and physiologically diverse. Microbial methane oxidation can be coupled to a variety of terminal electron acceptors, including oxygen, sulfate, nitrate, and nitrite (1-4). The aerobic methanotrophs are typically mesophilic and group phylogenetically within the Gammaproteobacteria and Alphaproteobacteria (1). Thermo-and meso-acidophilic aerobic methanotrophs, however, that grow at pH Ͻ3 and at optimal temperatures ranging from 35°C to greater than 50°C have also been discovered in the phylum Verrucomicrobia (5-9). Further, novel oxygenic methanotrophs that couple methane oxidation to nitrite reduction have been reported, e.g., "Candidatus Methylomirabilis oxyfera" that generates oxygen from a unique denitrification pathway, which is then used for methane oxidation (2). Aerobic methanotrophs are found in many environments, e.g., freshwater and marine sediments, bogs, forest, and agricultural soils, among other locations (1, 2, 5-11).These microorganisms have been extensively studied for many different reasons, including the fact that they play a key role in the global carbon cycle. All aerobic methanotrophs employ the enzyme methane monooxygenase (MMO) to co...