The Dithiol:Disulfide Oxidoreductases DsbA and DsbB of Rhodobacter capsulatus Are Not Directly Involved in Cytochrome c Biogenesis, but Their Inactivation Restores the Cytochrome c Biogenesis Defect of CcdA-Null Mutants

Abstract

ABSTRACT The cytoplasmic membrane protein CcdA and its homologues in other species, such as DsbD of Escherichia coli , are thought to supply the reducing equivalents required for the biogenesis of c -type cytochromes that occurs in the periplasm of gram-negative bacteria. CcdA - null mutants of the facultative phototroph Rhodobacter capsulatus are unable to grow under photosynthetic conditions (Ps − ) and do not produce any active cytochrome c oxidase (Nadi − ) due to a pleiotropic cytochrome c deficiency. However, under photosynthetic or respiratory growth conditions, these mutants revert frequently to yield Ps + Nadi + colonies that produce c -type cytochromes despite the absence of CcdA. Complementation of a CcdA-null mutant for the Ps + growth phenotype was attempted by using a genomic library constructed with chromosomal DNA from a revertant. No complementation was observed, but plasmids that rescued a CcdA-null mutant for photosynthetic growth by homologous recombination were recovered. Analysis of one such plasmid revealed that the rescue ability was mediated by open reading frame 3149, encoding the dithiol:disulfide oxidoreductase DsbA. DNA sequence data revealed that the dsbA allele on the rescuing plasmid contained a frameshift mutation expected to produce a truncated, nonfunctional DsbA. Indeed, a dsbA ccdA double mutant was shown to be Ps + Nadi + , establishing that in R. capsulatus the inactivation of dsbA suppresses the c -type cytochrome deficiency due to the absence of ccdA . Next, the ability of the wild-type dsbA allele to suppress the Ps + growth phenotype of the dsbA ccdA double mutant was exploited to isolate dsbA -independent ccdA revertants. Sequence analysis revealed that these revertants carried mutations in dsbB and that their Ps + phenotypes could be suppressed by the wild-type allele of dsbB . As with dsbA , a dsbB ccdA double mutant was also Ps + Nadi + and produced c -type cytochromes. Therefore, the absence of either DsbA or DsbB restores c -type cytochrome biogenesis in the absence of CcdA. Finally, it was also found that the DsbA-null and DsbB-null single mutants of R. capsulatus are Ps + and produce c -type cytochromes, unlike their E. coli counterparts, but are impaired for growth under respiratory conditions. This finding demonstrates that in R. capsulatus the dithiol:disulfide oxidoreductases DsbA and DsbB are not essential for cytochrome c biogenesis even though they are important for respiration under certain conditions.