Genes involved in the copper-dependent regulation of soluble methane monooxygenase of Methylococcus capsulatus (Bath): cloning, sequencing and mutational analysis

Abstract

The key enzyme in methane metabolism is methane monooxygenase (MMO), which catalyses the oxidation of methane to methanol. Some methanotrophs, includingMethylococcus capsulatus(Bath), possess two distinct MMOs. The level of copper in the environment regulates the biosynthesis of the MMO enzymes in these methanotrophs. Under low-copper conditions, soluble MMO (sMMO) is expressed and regulation takes place at the level of transcription. The structural genes of sMMO were previously identified asmmoXYBZ,mmoDandmmoC. Putative transcriptional start sites, containing aσ70- and aσN-dependent motif, were identified in the 5′ region ofmmoX. The promoter region ofmmoXwas mapped using truncated 5′ end regions fused to a promoterless green fluorescent protein gene. A 9·5 kb region, adjacent to the sMMO structural gene cluster, was analysed. Downstream (3′) from the last gene of the operon,mmoC, four ORFs were found,mmoG,mmoQ,mmoSandmmoR.mmoGshows significant identity to the large subunit of the bacterial chaperonin gene,groEL. In the opposite orientation, two genes,mmoQandmmoS, showed significant identity to two-component sensor–regulator system genes. Next tommoS, a gene encoding a putativeσN-dependent transcriptional activator,mmoRwas identified. ThemmoGandmmoRgenes were mutated by marker-exchange mutagenesis and the effects of these mutations on the expression of sMMO was investigated. sMMO transcription was impaired in both mutants. These results indicate thatmmoGandmmoRare essential for the expression of sMMO inMc. capsulatus(Bath).