Mutagenesis of the gene encoding cytochrome c550 of Paracoccus denitrificans and analysis of the resultant physiological effects

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Abstract
By using synthetic oligonucleotides, the gene encoding soluble cytochrome c550 was isolated from a genomic bank of Paracoccus denitrificans. The nucleotide sequence of the gene was determined, and the deduced amino acid sequence of the mature protein was found to be similar to the primary structure of purified cytochrome c550 except for the presence of seven additional amino acid residues at the C terminus. At the N terminus of the primary structure was found an additional stretch of 19 amino acid residues that had the typical features of the signal sequence of the cytochrome. Comparison of the nucleotide sequences of the upstream regions of the P. denitrificans cytochrome c550 gene and bc1 operon revealed three regions with a distinct organization that showed strong similarity. Downstream of the c550 gene was found part of another gene, the deduced amino acid sequence of which showed strong homology with subunit 1 of the cytochrome aa3 oxidase. For gene replacement experiments, the suicide vector pGRPd1 was constructed. The cytochrome c550 gene was inactivated by insertion of a kanamycin resistance gene, and the mutated gene was cloned into this vector. Recombination with the wild-type gene resulted in a mutant strain with an inactivated cytochrome gene. Isolated mutant strains were unable to synthesize the soluble cytochrome, as judged by spectrum analysis and analysis of periplasmic proteins by gel electrophoresis and heme staining. The mutation resulted in a 14% decrease in the growth yield during aerobic heterotrophic growth and in a 40% decrease in the maximum specific growth rate during growth on methylamine. Furthermore, a longer lag phase was observed under both growth conditions. The mutation had no effect on growth yield, maximum specific growth rate, and duration of the lag phase during anaerobic growth in the presence of nitrate. In addition, there was no accumulation of nitrite and nitrous oxide.