Molecular Analysis of the pmo (Particulate Methane Monooxygenase) Operons from Two Type II Methanotrophs

Abstract

The particulate methane monooxygenase gene clusters,pmoCAB, from two representative type II methanotrophs of the α-Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp. strain M, have been cloned and sequenced. Primer extension experiments revealed that the pmo cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, for Methylocystis sp. strain M. Immediately upstream of the putative start site, consensus sequences for ς⁷⁰ promoters were identified, suggesting that thesepmo genes are recognized by ς⁷⁰ and negatively regulated under low-copper conditions. The pmogenes were cloned in several overlapping fragments, since parts of these genes appeared to be toxic to the Escherichia colihost. Methanotrophs contain two virtually identical copies ofpmo genes, and it was necessary to use Southern blotting and probing with pmo gene fragments in order to differentiate between the two pmoCAB clusters in both methanotrophs. The complete DNA sequence of one copy of pmogenes from each organism is reported here. The gene sequences are 84% similar to each other and 75% similar to that of a type I methanotroph of the γ-Proteobacteria, Methylococcus capsulatus Bath. The derived proteins PmoC and PmoA are predicted to be highly hydrophobic and consist mainly of transmembrane-spanning regions, whereas PmoB has only two putative transmembrane-spanning helices. Hybridization experiments showed that there are two copies ofpmoC in both M. trichosporium OB3b andMethylocystis sp. strain M, and not three copies as found in M. capsulatus Bath.