title: 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: 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.
authors.affiliation: Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Meenal
authors.initials: M
authors.lastname: Deshmukh
authors.forename: Serdar
authors.initials: S
authors.lastname: Turkarslan
authors.forename: Donniel
authors.initials: D
authors.lastname: Astor
authors.forename: Maria
authors.initials: M
authors.lastname: Valkova-Valchanova
authors.forename: Fevzi
authors.initials: F
authors.lastname: Daldal
mesh_terms: 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
chemical_list: 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