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.
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.
Deshmukh
Meenal
M
Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Turkarslan
Serdar
S
Astor
Donniel
D
Valkova-Valchanova
Maria
M
Daldal
Fevzi
F
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010088:Oxidoreductases / Q000378:metabolism; D010788:Photosynthesis; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D017422:Sequence Analysis, DNA; D014050:Toluene / Q000031:analogs & derivatives* / Q000378:metabolism
D001426:Bacterial Proteins; C068355:CcdA protein, Bacteria; D003574:Cytochrome c Group; D004220:Disulfides; C079503:DsbB protein, Bacteria; D008565:Membrane Proteins; D014050:Toluene; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases; C004848:dithiol