Role of multiple gene copies in particulate methane monooxygenase activity in the methane-oxidizing bacterium Methylococcus capsulatus Bath.
Genes for the subunits of particulate methane monooxygenase, PmoABC, have been sequenced from the gamma-proteobacterial methanotroph Methylococcus capsulatus Bath. M. capsulatus Bath contains two complete copies of pmoCAB, as well as a third copy of pmoC. The two pmoCAB regions were almost identical at the nucleotide sequence level, differing in only 13 positions in 3183 bp. At the amino acid level, each translated gene product contained only one differing residue in each copy. However, the pmoC3 sequence was more divergent from the two other pmoC copies at both the far N-terminus and far C-terminus. Chromosomal insertion mutations were generated in all seven genes. Null mutants could not be obtained for pmoC3, suggesting that it may play an essential role in growth on methane. Null mutants were obtained for pmoC1, pmoC2, pmoA1, pmoA2, pmoB1 and pmoB2. All of these mutants grew on methane, demonstrating that both gene copies were functional. Copy 1 mutants showed about two-thirds of the wild-type whole-cell methane oxidation rate, while copy 2 mutants showed only about one-third of the wild-type rate, indicating that both gene copies were necessary for wild-type particulate methane monooxygenase activity. It was not possible to obtain double null mutants that were defective in both pmo copies, which may indicate that some expression of pMMO is important for growth.
Stolyar
Sergei
S
Departments of Chemical Engineering and Microbiology2, University of Washington, Box 351750, Seattle, WA 98195, USA.
Costello
Andria M
AM
Environmental Engineering Science 138-78, California Institute of Technology, Pasadena, CA 91125, USA.
Peeples
Tonya L
TL
Environmental Engineering Science 138-78, California Institute of Technology, Pasadena, CA 91125, USA.
Lidstrom
Mary E
ME
Microbiology, University of Washington, Box 351750, Seattle, WA 98195, USA.|Departments of Chemical Engineering and Microbiology2, University of Washington, Box 351750, Seattle, WA 98195, USA.
D000595:Amino Acid Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial; D020131:Genes, Duplicate; D008697:Methane / Q000378:metabolism; D008772:Methylococcaceae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D005810:Multigene Family; D009693:Nucleic Acid Hybridization; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D010105:Oxygenases; C021555:methane monooxygenase; D008697:Methane