Role of multiple gene copies in particulate methane monooxygenase activityin the methane-oxidizing bacterium Methylococcus capsulatus Bath

Genes for the subunits of particulate methane monooxygenase, PmoABC, have b een sequenced from the gamma-proteobacterial methanotroph Methylococcus cap sulatus 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 b p. At the amino acid level, each translated gene product contained only one differing residue in each copy. However, the pmoC3 sequence was more diver gent from the two other pmoC copies at both the far N-terminus and far C-te rminus, Chromosomal insertion mutations were generated in all seven genes. Null mutants could not be obtained for pmoC3, suggesting that it may play a n essential role in growth on methane. Null mutants were obtained for pmoC1 , pmoC2, pmoA1, pmoA2, pmoB1 and pmoB2, All of these mutants grew on methan e, demonstrating that both gene copies were functional. Copy 1 mutants show ed about two-thirds of the wild-type whole-cell methane oxidation rate, whi le copy 2 mutants showed only about one-third of the wildtype rate, indicat ing that both gene copies were necessary for wild-type particulate methane monooxygenase activity. It was not possible to obtain double null mutants t hat were defective in both pmo copies, which may indicate that some express ion of pMMO is important for growth.