Molecular genetics of methane oxidation.

Biological methane oxidation is carried out by methanotrophs, bacteria that utilize methane as their sole carbon and energy source. The enzyme they contain that is responsible for methane oxidation is methane monooxygenase, the most well studied being the soluble methane monooxygenase enzyme complexes from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. In both organisms, the genes encoding soluble methane monooxygenase have been found to be clustered on the chromosome in the order mmoX, mmoY, mmoB, mmoZ, orfY and mmoC. These genes encode the alpha and beta subunits of Protein A, Protein B, the gamma subunit of Protein A, a protein of unknown function and Protein C respectively of the soluble methane monooxygenase complex. The complete DNA sequences of both gene clusters have been determined and they show considerable homology. Expression of soluble methane monooxygenase genes occurs under growth conditions where the copper-to-biomass ratio is low. Transcriptional regulation of the gene cluster from Methylosinus occurred at an RpoN-like promoter, 5' of the mmoX gene. mmoB and mmoC of Methylococcus have been expressed in E. coli and the proteins obtained were functionally active. Soluble methane monooxygenase mutants have been constructed by marker-exchange mutagenesis. They were found to be more stable than those generated using the suicide substrate dichloromethane. Soluble methane monooxygenase probes have been used to detect both methane monooxygenase gene-specific DNA and methanotrophs in natural environmental samples.