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Applied and Environmental Microbiology, March 2000, p. 966-975, Vol. 66, No. 3
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Bettina Gilbert, Ian R. McDonald, Ruth Finch,dagger Graham P. Stafford, Allan K. Nielsen,Dagger and J. Colin Murrell*

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom

Received 11 October 1999/Accepted 14 December 1999

The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II methanotrophs of the alpha -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 sigma 70 promoters were identified, suggesting that these pmo genes are recognized by sigma 70 and negatively regulated under low-copper conditions. The pmo genes were cloned in several overlapping fragments, since parts of these genes appeared to be toxic to the Escherichia coli host. Methanotrophs contain two virtually identical copies of pmo 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 pmo genes 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 gamma -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 of pmoC in both M. trichosporium OB3b and Methylocystis sp. strain M, and not three copies as found in M. capsulatus Bath.

* Corresponding author. Mailing address: Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, United Kingdom. Phone: 44 (0) 2476 523553. Fax: 44 (0) 2476 523568. E-mail: cm{at}dna.bio.warwick.ac.uk.

dagger Present address: Horticulture Research International, Wellesbourne, Warwick CV35 9EF, United Kingdom.

Dagger Present address: Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark.

Applied and Environmental Microbiology, March 2000, p. 966-975, Vol. 66, No. 3
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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