Cloning and sequence analysis of cycH gene from Paracoccus denitrificans: the cycH gene product is required for assembly of all c-type cytochromes, including cytochrome c1

Abstract

A transposon Tn5 mutant of Paracoccus denitrificans, DP108, was incapable of anaerobic or methylotrophic growth and scored negative in the Nadi cytochrome c oxidase test. P. denitrificans DP108 grown aerobically on succinate or choline was devoid of soluble c-type cytochromes and accumulated periplasmic apocytochrome c550, but the membrane-bound holocytochromes c1 and c552 were present at 5-10% of the levels observed in wild-type cells. DP108 genomic DNA flanking the site of Tn5 insertion was cloned by marker rescue and used to probe a P. denitrificans wild-type DNA library. A hybridizing 3.05 kb BamHI fragment capable of complementing the DP108 mutation was isolated and a 2.05 kb region of this was sequenced. One major open reading frame equivalent to 413 amino acids was identified, the predicted product of which was similar (33% identity, 55% similarity) to the predicted product of the cycH gene previously identified in Bradyrhizobium japonicum. Similarity of the two cycH gene products to the predicted products of two Escherichia coli genes, nrfG and yejP, was also detected. Significant differences between the phenotypes of P. denitrificans DP108 and the B. japonicum cycH mutant COX3, especially with respect to cytochrome c1 synthesis, suggest that the cycH gene product may be an assembly factor.