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 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.

FIG. 1

Southern blot of genomic DNA from M. trichosporium OB3b (a) and Methylocystis sp. strain M (b) probed with a pmoA probe. (a) Lanes: 1, BamHI; 2, BglII; 3, EcoRI; 4, HpaI; 5, HindIII; 6, KpnI; 7, PstI; 8, SalI; 9, XhoI. The blot was probed with a PCR-amplified fragment of pmoA (550 bp) from M. trichosporium OB3b at 65°C, 2× SSC. (b) Lanes: 1, molecular mass standard, 1-kb ladder (Gibco BRL); 2, PstI; 3, HindIII; 4, EcoRI; 5, BamHI; 6, KpnI; 7, XhoI; 8, BglII; 9, HpaI; 10, SalI. The blot was probed with a PCR-amplified fragment of pmoA (550 bp) from Methylocystis sp. strain M at 70°C, 2× SSC.

FIG. 2

Physical and genetic map of the pmo genes in M. trichosporium OB3b and the overlapping cloned DNA fragments. The binding regions for probes OB1, OB2, and OB3 and for primers O1, O2, O3, and O4 used in primer extension experiments are also shown. See Tables 2 and 3 for exact positions.

FIG. 3

Physical and genetic map of the pmo genes in Methylocystis sp. strain M and the overlapping cloned DNA fragments. The binding regions for probes pC1, pC59, and p286 and for primers M1, M2, M3, and M4 used in primer extension experiments are also shown. See Tables 2 and 3 for exact positions.

FIG. 4

Southern blot of genomic DNA probed with a 400-bp pmoC probe homologous to Methylocystis sp. strain M. The wash conditions were 2× SSC at 75°C. Lanes: 1, M. capsulatus Bath digested with SmaI; 2 through 5, M. trichosporium OB3b DNA digested with BclI, EcoRI, NotI, and PstI, respectively; 7 through 10, Methylocystis sp. strain M DNA digested with BclI, PstI, SalI, and XhoI, respectively.

FIG. 5

Predicted topology of derived Pmo proteins from M. trichosporium OB3b and Methylocystis sp. strain M. The protein sequences were analyzed with the TMHMM tool (Expasy website [http://www.expasy.ch/tools/#transmem]). The shaded columns depict regions of high hydrophobicity (probability of transmembrane location on the y axis) for amino acid residues (x axis) which are predicted to form transmembrane helices. See the text for the locations of transmembrane helices for M. trichosporium OB3b proteins.

FIG. 6

Primer extension analysis to identify the transcriptional start site for the pmo genes in Methylocystis sp. strain M. The positions of the −35 and −10 regions (boxed) and the transcriptional start sites (arrows) are indicated.

FIG. 7

Alignment of the promoter region in Methylocystis sp. strain M and the equivalent region in M. trichosporium OB3b. The identity is 62% over 58 bp. The −35 and −10 motifs are overlined, and the start of transcription is indicated at +1.