Particulate methane monooxygenase genes in methanotrophs

Abstract

A 45-kDa membrane polypeptide that is associated with activity of the particulate methane monooxygenase (pMMO) has been purified from three methanotrophic bacteria, and the N-terminal amino acid sequence was found to be identical in 17 of 20 positions for all three polypeptides and identical in 14 of 20 positions for the N terminus of AmoB, the 43-kDa subunit of ammonia monooxygenase. DNA from a variety of methanotrophs was screened with two probes, an oligonucleotide designed from the N-terminal sequence of the 45-kDa polypeptide from Methylococcus capsulatus Bath and an internal fragment of amoA, which encodes the 27-kDa subunit of ammonia monooxygenase. In most cases, two hybridizing fragments were identified with each probe. Three overlapping DNA fragments containing one of the copies of the gene encoding the 45-kDa pMMO polypeptide (pmoB) were cloned from Methylococcus capsulatus Bath. A 2.1-kb region was sequenced and found to contain both pmoB and a second gene, pmoA. The predicted amino acid sequences of these genes revealed high identity with those of the gene products of amoB and amoA, respectively. Further hybridization experiments with DNA from Methylococcus capsulatus Bath and Methylobacter albus BG8 confirmed the presence of two copies of pmoB in both strains. These results suggest that the 45-and 27-kDa pMMO-associated polypeptides of methanotrophs are subunits of the pMMO and are present in duplicate gene copies in methanotrophs.Methanotrophs are a group of gram-negative bacteria that utilize methane as their sole source of carbon and energy. The initial transformation involves the conversion of methane into methanol by methane monooxygenase (MMO):

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily

“…CuMMO genes are notoriously difficult to clone in heterologous hosts (Semrau et al, 1995;Balasubramanian et al, 2010) and our initial attempts using standard E. coli vectors or E. coli-Mycobacterium shuttle vectors were unsuccessful (data not shown). To overcome these difficulties, we developed a novel fosmid-based shuttle vector, with copy number and expression control in E. coli and acetamide-inducible expression of cloned genes in Mycobacterium-this enabled the stable cloning of the hmoCAB operon of strain NBB4 yielding the plasmid pHMO ( Figure 5).…”

One millimetre makes the difference: high-resolution analysis of methane-oxidizing bacteria and their specific activity at the oxic–anoxic interface in a flooded paddy soil

“…The maximum transcript:gene ratio coincided with the highest respiration rate, but was localized slightly deeper in the soil than the maximum pmoA copy number (Figure 3). If we consider two pmoCAB operons per cell (Semrau et al, 1995), the Methylobacter-affiliated OTU had up to 18 transcripts per cell (Figure 3). The half-life of pmoA mRNA is unknown.…”

Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis

“…MmoB has no metal or prosthetic groups, but is essential for activity and electron transfer from the reductase to the hydroxylase (Green & Dalton, 1985). The genes composing the sMMO operon (mmoXYBZDC) have been cloned and sequenced from several methanotrophs, and are present as a single copy in the chromosome , whereas the genes encoding pMMO, pmoCAB, can be present in multiple copies (Semrau, 1995;Stolyar et al, 1999).…”