Isolation and characterization of the moxJ, moxG, moxI, and moxR genes of Paracoccus denitrificans: inactivation of moxJ, moxG, and moxR and the resultant effect on methylotrophic growth.

By using the moxF gene encoding the large fragment of methanol dehydrogenase as a probe, a downstream linked chromosomal fragment was isolated from a genomic bank of Paracoccus denitrificans. The nucleotide sequence of the fragment was determined and revealed the 3' part of moxF, four additional open reading frames, and the 5' part of a sixth one. The organization and deduced amino acid sequences of the first three frames downstream from moxF were found to be largely homologous to the moxJ, moxG, and moxI gene products of Methylobacterium extorquens AM1. Directly downstream from these three genes, a new mox gene was identified. The gene is designated moxR. By using the suicide vector pGRPd1, the moxJ, moxG, and moxR genes were inactivated by the insertion of a kanamycin resistance gene. Subsequently, suicide vector pRVS1 was used to replace the marker genes in moxJ and moxG for unmarked deletions made in vitro. As a result, the three insertion strains as well as the two unmarked mutant strains were unable to grow on methanol, even in the presence of pyrroloquinoline quinone. Growth on succinate and on methylamine was not affected. In all five mutant strains, synthesis of the large subunit of methanol dehydrogenase and of inducible cytochrome c553i was observed. The moxJ and moxG insertion mutant strains were unable to synthesize both the cytochrome c551i and the small subunit of methanol dehydrogenase, and this lack of synthesis was attended by the loss of methanol dehydrogenase activity. The moxJ deletion mutant strain partly synthesized the latter two proteins, cytochrome c551i. Partial synthesis of the small subunit of methanol dehydrogenase observed with the latter strain was attended by a corresponding extent of methanol dehydrogenase activity. The moxR insertion mutant strain was shown to synthesize cytochrome c551i as well as the large and small subunits of methanol dehydrogenase, but no methanol dehydrogenase activity was observed. The results show that periplasmic cytochrome c551i is the moxG gene product and the natural electron acceptor of methanol dehydrogenase in P. denitrificans. In contrast to earlier suggestions, this cytochrome was found to be different from membrane-bound cytochrome c552. In addition, it is demonstrated that moxI encodes the small subunit of methanol dehydrogenase. It is suggested that MoxJ is involved in the assemblage of active methanol dehydrogenase in the periplasm and, in addition, that MoxR is involved in the regulation of formation of active methanol dehydrogenase.

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.

Mutagenesis of the gene encoding amicyanin of Paracoccus denitrificans and the resultant effect on methylamine oxidation.

The gene encoding the blue-copper protein amicyanin was isolated from a genomic bank of Paracoccus denitrificans by using a synthetic oligonucleotide. It is located directly downstream of the gene encoding the small subunit of methylamine dehydrogenase. Amicyanin is transcribed as a precursor protein with a signal sequence, typical for periplasmic proteins. Specific inactivation of amicyanin by means of gene replacement techniques resulted in the complete loss of the ability to grow on methylamine.