A method for introduction of unmarked mutations in the genome of Paracoccus denitrificans: construction of strains with multiple mutations in the genes encoding periplasmic cytochromes c550, c551i, and c553i.

A new suicide vector, pRVS1, was constructed to facilitate the site-directed introduction of unmarked mutations in the chromosome of Paracoccus denitrificans. The vector was derived from suicide vector pGRPd1, which was equipped with the lacZ gene encoding beta-galactosidase. The reporter gene was found to be a successful screening marker for the discrimination between plasmid integrant strains and mutant strains which had lost the plasmid after homologous recombination. Suicide vectors pGRPd1 and pRVS1 were used in gene replacement techniques for the construction of mutant strains with multiple mutations in the cycA, moxG, and cycB genes encoding the periplasmic cytochromes c550, c551i, and c553i, respectively. Southern analyses of the DNA and protein analyses of the resultant single, double, and triple mutant strains confirmed the correctness of the mutations. The wild type and mutant strains were all able to grow on succinate and choline chloride. In addition, all strains grew on methylamine and displayed wild-type levels of methylamine dehydrogenase activities. cycA mutant strains, however, showed a decreased maximum specific growth rate on the methylamine substrate. The wild-type strain, cycA and cycB mutant strains, and the cycA cycB double mutant strain were able to grow on methanol and showed wild-type levels of methanol dehydrogenase activities. moxG mutant strains failed to grow on methanol and had low levels of methanol dehydrogenase activities. The maximum specific growth rate of the cycA mutant strain on methanol was comparable with that of the wild-type strain. The data indicate the involvement of the soluble cytochromes c in clearly defined electron transport routes.