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.The c-type cytochromes (Cyts) are electron transport proteins that contain heme molecules as prosthetic groups. In these proteins, heme is covalently attached to the polypeptide via thioether linkages formed between the heme vinyl groups and the thiol side chains of the cysteine residues in the conserved CXXCH heme-binding motif of the apoprotein. In gram-negative bacteria, synthesis of holo-Cyt c (heme-bound Cyt c) is an elaborate process that occurs on the periplasmic face of the cytoplasmic membrane and requires multiple membrane-associated components (e.g., products of ccmAB...

The Acidic Nature of the CcmG Redox-Active Center Is Important for CytochromecMaturation inEscherichia coli

“…Furthermore, recent evidence has shown that the Cys-X-X-Cys-His motif in apocytochrome c is capable of forming a disulfide bond and that this disulfide prevents the formation of mature cytochrome c in vitro (5). However, DsbA may not be involved directly in the maturation of cytochromes c (1,6).…”

Extracytoplasmic prosthetic group ligation to apoproteins: maturation of c‐type cytochromes

“…These questions raise a more general issue, namely, to what extent apocytochrome c thio-redox processing is required for c-type cytochromes maturation? Although DsbA and DsbD mutants of E. coli were initially reported to be unable to produce c cytochromes, recent work with other SI and SII organisms (Rhodobacter capsulatus and Bacillus subtilis respectively) indicate that DsbA-null mutants do so, and more strikingly, they also bypass the lack of a DsbD/CcdA function (Erlendsson and Hederstedt, 2002;Deshmukh et al, 2003). More recent work also showed that DsbA is not essential for c cytochromes production per se, even in E. coli (Allen et al, 2003).…”

“…1D, right). Electrons initiating at the cytoplasmic thioredoxin A are shuttled across the cytoplasmic membrane by DsbD (or its orthologue CcdA in other SI organisms) to CcmG, then to the N-terminal portion of CcmH (see Figure legend), ultimately to reduce the disulphide bonds at the haem ligation sites, and to allow haem ligation (Thony-Meyer, 2002;Deshmukh et al, 2003). The discovery of the CcsAB-dependent system lacking a CcmG (or CcsX) component raises an important question about the formation, and eventually subsequent reduction, of such disulphide bonds.…”

Overproduction or Absence of the Periplasmic Protease DegP Severely Compromises Bacterial Growth in the Absence of the Dithiol: Disulfide Oxidoreductase DsbA

“…Hg 2ϩ , Cd 2ϩ , and TeO 3 2Ϫ ), biofilm formation, and reduced virulence (18). In an earlier study we found that, unlike in E. coli (23)(24)(25), DsbA-null mutants of R. capsulatus produce c-type cytochromes, indicating that DsbA is not essential for the cytochrome c maturation process (16). However, we observed that DsbA-null mutants of R. capsulatus are severely impaired for respiratory (Res) growth, especially in enriched growth medium (mineralpeptone-yeast extract (MPYE)), although they are proficient for photosynthetic (Ps) growth.…”

“…DsbA homologues have been found in many bacteria (11)(12)(13)(14)(15), including the purple, non-sulfur, facultative phototrophic bacterium Rhodobacter capsulatus (16), and disulfide bond formation has been studied extensively in Escherichia coli. Upon translocation of a polypeptide into the periplasm, DsbA rapidly and nonspecifically oxidizes pairs of cysteine thiols into disulfide bonds by transferring its active site disulfide (7,17).…”