The dsbA-dsbB disulfide bond formation system of Burkholderia cepacia is involved in the production of protease and alkaline phosphatase, motility, metal resistance, and multi-drug resistance

In a previous study, we isolated a dsbB mutant of Burkholderia cepacia KF1 and showed that phenotypes of protease production and motility are dependent on DsbB, a membrane-bound disulfide bond oxidoreductase. We have now isolated a dsbA mutant by transposon mutagenesis, cloned the dsbA gene encoding a periplasmic disulfide bond oxidoreductase, and characterized the function of the DsbA-DsbB disulfide bond formation system in B. cepacia. The complementing DNA fragment had an open reading frame for a 212-amino acid polypeptide with a potential redox-active site sequence of Cys-Pro-His-Cys that is homologous to Escherichia coli DsbA. The dsbA mutant, as well as the previously isolated dsbB mutant, was defective in the production of extracellular protease and alkaline phosphatase, as well as in motility. In addition, mutation in the DsbA-DsbB system resulted in an increase in sensitivity to Cd2+ and Zn2+ as well as a variety of antibiotics including beta-lactams, kanamycin, erythromycin, novobiocin, ofloxacin and sodium dodecyl sulfate. These results suggested that the DsbA-DsbB system might be involved in the formation of a metal efflux system as well as a multi-drug resistance system.     

A mutation in either dsbA or dsbB, a gene encoding a component of a periplasmic disulfide bond-catalyzing system, is required for high-level expression of the Bacteroides fragilis metallo-beta-lactamase, CcrA, in Escherichia coli.

The metallo-beta-lactamase gene, ccrA, from Bacteroides fragilis is functionally expressed in Escherichia coli only in the presence of a genomic mutation in iarA or iarB (increased ampicillin resistance), identified in this study as dsbA or dsbB, respectively. DsbA and DsbB are components of a periplasmic protein disulfide bond-catalyzing system. Data indicated that DsbA interacted with CcrA, creating aberrant disulfide bond linkages that render CcrA proteolytically unstable. Mutations in dsbA or dsbB permissive for CcrA expression eliminated or greatly reduced DsbA activity, allowing CcrA to assume a disulfide bond-free and proteolytically stable conformation.     

Characterization of DsbC, a periplasmic protein of Erwinia chrysanthemi and Escherichia coli with disulfide isomerase activity.

We identified and characterized an Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation that prevents disulfide bond formation in periplasmic proteins. This gene, dsbC, codes for a 24 kDa periplasmic protein that contains a characteristic active site sequence of disulfide isomerases, Phe-X-X-X-X-Cys-X-X-Cys. Besides the active site, DsbC has no homology with DsbA, thioredoxin or eukaryotic protein disulfide isomerase and it could define a new subfamily of disulfide isomerases. Purified DsbC protein is able to catalyse insulin oxidation in a dithiothreitol dependent manner. The E.coli gene xprA codes for a protein functionally equivalent to DsbC. The in vivo function of DsbC seems to be the formation of disulfide bonds in proteins. The presence of XprA could explain the residual disulfide isomerase activity existing in dsbA mutants. Re-oxidation of XprA does not seem to occur through DsbB, the protein that probably re-oxidizes DsbA.     

F-like type IV secretion systems encode proteins with thioredoxin folds that are putative DsbC homologues

F and R27 are conjugative plasmids of enteric bacteria belonging to the IncF and IncHI1 plasmid incompatibility groups, respectively. Based on sequence analysis, two genes of the F transfer region, traF and trbB, and three genes of the R27 transfer region, trhF, dsbC, and htdT, are predicted to encode periplasmic proteins containing a C-terminal thioredoxin fold. The C-X-X-C active-site motif of thioredoxins is present in all of these proteins except TraF(F). Escherichia coli carrying a dsbA mutation, which is deficient in disulfide bond formation, cannot synthesize pili and exhibits hypersensitivity to dithiothreitol (DTT) as monitored by mating ability. Overproduction of the E. coli disulfide bond isomerase DsbC, TrbB(F), DsbC(R27), or HtdT(R27), but not TraF(F) or TrhF(R27), reverses this hypersensitivity to DTT. Site-directed mutagenesis established that the C-X-X-C motif was necessary for this activity. Secretion into the periplasm of the C-terminal regions of TrbB(F) and DsbC(R27), containing putative thioredoxin folds, but not TrhF(R27), partially complemented the host dsbA mutation. A trbB(F) deletion mutant showed a 10-fold-lower mating efficiency in an E. coli dsbC null strain but had no phenotype in wild-type E. coli, suggesting redundancy in function between TrbB(F) and E. coli DsbC. Our results indicate that TrbB(F), DsbC(R27), and HtdT(R27) are putative disulfide bond isomerases for their respective transfer systems. TraF(F) is essential for conjugation but appears to have a function other than disulfide bond chemistry.     

The Escherichia coli dsbC (xprA) gene encodes a periplasmic protein involved in disulfide bond formation.

We have identified and functionally characterized a new Escherichia coli gene, dsbC, whose product is involved in disulfide bond formation in the periplasmic space. It corresponds to a previously sequenced open reading frame mapping upstream of recJ with no previously assigned function. Null mutations in dsbC were obtained using a screen for dithiothreitol (DTT)-sensitive mutants and were shown to result in the accumulation of reduced forms of a variety of disulfide bond-containing periplasmic proteins. This defect could be rescued by the addition of either oxidized DTT or cystine or by multicopy expression of dsbA, a known periplasmic disulfide oxidase. The DsbC protein is synthesized as a precursor form of 25.5 kDa which is processed to a 23.3 kDa mature species located in the periplasmic space. The DsbC protein was overexpressed, purified to homogeneity and shown to catalyse the reduction of insulin in a DTT-dependent manner at levels comparable with those of purified DsbA. The replacement of either cysteine residue of the predicted active site, F-(X4)-C-G-Y-C, completely inactivates DsbC protein function. We have further shown that in vivo overexpression of DsbC can functionally substitute for a loss of DsbA function. Taken together, all of our results demonstrate that DsbC acts in vivo as a disulfide oxidase.     

Mutations in cytochrome assembly and periplasmic redox pathways in Bordetella pertussis

Transposon mutagenesis of Bordetella pertussis was used to discover mutations in the cytochrome c biogenesis pathway called system II. Using a tetramethyl-p-phenylenediamine cytochrome c oxidase screen, 27 oxidase-negative mutants were isolated and characterized. Nine mutants were still able to synthesize c-type cytochromes and possessed insertions in the genes for cytochrome c oxidase subunits (ctaC, -D, and -E), heme a biosynthesis (ctaB), assembly of cytochrome c oxidase (sco2), or ferrochelatase (hemZ). Eighteen mutants were unable to synthesize all c-type cytochromes. Seven of these had transposons in dipZ (dsbD), encoding the transmembrane thioreduction protein, and all seven mutants were corrected for cytochrome c assembly by exogenous dithiothreitol, which was consistent with the cytochrome c cysteinyl residues of the CXXCH motif requiring periplasmic reduction. The remaining 11 insertions were located in the ccsBA operon, suggesting that with the appropriate thiol-reducing environment, the CcsB and CcsA proteins comprise the entire system II biosynthetic pathway. Antiserum to CcsB was used to show that CcsB is absent in ccsA mutants, providing evidence for a stable CcsA-CcsB complex. No mutations were found in the genes necessary for disulfide bond formation (dsbA or dsbB). To examine whether the periplasmic disulfide bond pathway is required for cytochrome c biogenesis in B. pertussis, a targeted knockout was made in dsbB. The DsbB- mutant makes holocytochromes c like the wild type does and secretes and assembles the active periplasmic alkaline phosphatase. A dipZ mutant is not corrected by a dsbB mutation. Alternative mechanisms to oxidize disulfides in B. pertussis are analyzed and discussed.     

Dsb-insensitive expression of CcrA, a metallo-beta-lactamase from Bacteroides fragilis, in Escherichia coli after amino acid substitution at two cysteine residues within CcrA.

It has previously been shown that functional expression of CcrA, a metallo-beta-lactamase from Bacteroides fragilis, in Escherichia coli requires a mutation in either dsbA or dsbB, components of a periplasmic disulfide bond-catalyzing system. Site-directed mutagenesis resulting in the substitution of various amino acids for two of the three cysteine residues within CcrA allowed the expression of CcrA in a dsb+ background. This finding supports the hypothesis that DsbA creates aberrant disulfide bonds involving the Cys residues of CcrA.     

Extracellular DsbA-insensitive folding of Escherichia coli heat-stable enterotoxin STa in vitro

To study the folding of human Escherichia coli heat-stable enterotoxin STh, we used the major protein subunit of CS31A fimbriae (ClpG) as a marker of STh secretion and a provider of a signal peptide. We established that STh genetically fused to the N or C terminus of ClpG was able to mobilize ClpG to the culture supernatant while still retaining full enterotoxicity. These features indicate that the STh activity was not altered by the chimeric structure and suggest that spatial conformation of STh in the fusion is close to that of the native toxin, thus permitting recognition and activation of the intestinal STh receptor in vivo. In contrast to other studies, we showed that disulfide bond formation did not occur in the periplasm through the DsbA pathway and that there was no correlation between DsbA and secretion, folding, or activity. This discrepancy was not attributable to the chimeric nature of STh since there was no effect of dsbA or dsbB mutations on secretion and activity of recombinant STh from which ClpG had been deleted. Periplasmic and lysate fractions of dsbA(+) and dsbA(-) cells did not have any STh activity. In addition, the STh chimera was exclusively found in an inactive reduced form intracellularly and in an active oxidized form extracellularly, irrespective of the dsbA background. Subsequently, a time course experiment in regard to the secretion of STh from both dsbA(+) and dsbA(-) cells indicated that the enterotoxin activity (proper folding) in the extracellular milieu increased with time. Overall, these findings provide evidence that STa toxins can be cell-released in an unfolded state before being completely disulfide-bonded outside the cell.     

The disulphide isomerase DsbC cooperates with the oxidase DsbA in a DsbD-independent manner

In Escherichia coli, DsbA introduces disulphide bonds into secreted proteins. DsbA is recycled by DsbB, which generates disulphides from quinone reduction. DsbA is not known to have any proofreading activity and can form incorrect disulphides in proteins with multiple cysteines. These incorrect disulphides are thought to be corrected by a protein disulphide isomerase, DsbC, which is kept in the reduced and active configuration by DsbD. The DsbC/DsbD isomerization pathway is considered to be isolated from the DsbA/DsbB pathway. We show that the DsbC and DsbA pathways are more intimately connected than previously thought. dsbA(-)dsbC(-) mutants have a number of phenotypes not exhibited by either dsbA(-), dsbC(-) or dsbA(-)dsbD(-) mutations: they exhibit an increased permeability of the outer membrane, are resistant to the lambdoid phage Phi80, and are unable to assemble the maltoporin LamB. Using differential two-dimensional liquid chromatographic tandem mass spectrometry/mass spectrometry analysis, we estimated the abundance of about 130 secreted proteins in various dsb(-) strains. dsbA(-)dsbC(-) mutants exhibit unique changes at the protein level that are not exhibited by dsbA(-)dsbD(-) mutants. Our data indicate that DsbC can assist DsbA in a DsbD-independent manner to oxidatively fold envelope proteins. The view that DsbC's function is limited to the disulphide isomerization pathway should therefore be reinterpreted.     

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.     

Overexpression of the rhodanese PspE, a single cysteine-containing protein, restores disulphide bond formation to an Escherichia coli strain lacking DsbA

Escherichia coli uses the DsbA/DsbB system for introducing disulphide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulphide bond formation but does not entirely eliminate it. Whether such background disulphide bond forming activity is enzyme-catalysed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulphide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulphide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulphide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulphenic acid form and reacts with substrate proteins to form mixed disulphide adducts. DsbC either prevents the formation of these mixed disulphides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyse disulphide bond formation. Although this PspE/DsbC system is not responsible for the background disulphide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system.     

Turning a disulfide isomerase into an oxidase: DsbC mutants that imitate DsbA

There are two distinct pathways for disulfide formation in prokaryotes. The DsbA-DsbB pathway introduces disulfide bonds de novo, while the DsbC-DsbD pathway functions to isomerize disulfides. One of the key questions in disulfide biology is how the isomerase pathway is kept separate from the oxidase pathway in vivo. Cross-talk between these two systems would be mutually destructive. To force communication between these two systems we have selected dsbC mutants that complement a dsbA null mutation. In these mutants, DsbC is present as a monomer as compared with dimeric wild-type DsbC. Based on these findings we rationally designed DsbC mutants in the dimerization domain. All of these mutants are able to rescue the dsbA null phenotype. Rescue depends on the presence of DsbB, the native re-oxidant of DsbA, both in vivo and in vitro. Our results suggest that dimerization acts to protect DsbC's active sites from DsbB-mediated oxidation. These results explain how oxidative and reductive pathways can co-exist in the periplasm of Escherichia coli.     

Insertional inactivation of dsbA produces sensitivity to cadmium and zinc in Escherichia coli.

In a search for genes that produce hypersensitivity to cadmium salts in Escherichia coli, random transposon mutagenesis with TnphoA was used. One of the mutant strains obtained was sensitive to Cd2+ and Zn2+. Sequence analysis showed that the TnphoA insertion was located in the dsbA gene coding for a periplasmic protein required for disulfide bond formation.     

Molecular aspects of complement-mediated bacterial killing. Periplasmic conversion of C9 from a protoxin to a toxin

As part of the membrane attack complex complement protein C9 is responsible for direct killing of bacteria. Here we show that in the periplasmic space of an Escherichia coli cell C9 is converted from a protoxin to a toxin by periplasmic conditions missing in spheroplasts. This conversion is independent of the pathway by which C9 enters the periplasm. Both, C9 shocked into the periplasm and plasmid-expressed C9 targeted to the periplasm via a signal sequence are toxic. Toxicity requires disulfide-linked C9 because export into the periplasm of cells defective in disulfide bond synthesis (dsbA and dsbB mutants) is not toxic unless N-acetylcysteine is added externally to promote cystines. A N-terminal fragment, C9[1-144], is not toxic nor is cytoplasmically expressed C9, even in trxB mutants that are able to form disulfide bonds in the cytoplasm. Importantly, expression of full-length C9 in complement-resistant cells has no effect on their viability. Expression and translocation into the periplasm may provide a novel model to identify molecular mechanisms of other bactericidal disulfide-linked proteins and to investigate the nature of bacterial complement resistance.     

Two periplasmic disulfide oxidoreductases, DsbA and SrgA, target outer membrane protein SpiA, a component of the Salmonella pathogenicity island 2 type III secretion system

The formation of disulfide is essential for the folding, activity, and stability of many proteins secreted by Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into proteins exported from the cytoplasm to periplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we examined the role of the Dsb enzymes in Salmonella pathogenesis, and we demonstrated that DsbA, but not DsbC, is required for the full expression of virulence in a mouse infection model of Salmonella enterica serovar Typhimurium. Salmonella strains carrying a dsbA mutation showed reduced function mediated by type III secretion systems (TTSSs) encoded on Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). To obtain a more detailed understanding of the contribution of DsbA to both SPI-1 and SPI-2 TTSS function, we identified a protein component of the SPI-2 TTSS apparatus affected by DsbA. Although we found no substrate protein for DsbA in the SPI-1 TTSS apparatus, we identified SpiA (SsaC), an outer membrane protein of SPI-2 TTSS, as a DsbA substrate. Site-directed mutagenesis of the two cysteine residues present in the SpiA protein resulted in the loss of SPI-2 function in vitro and in vivo. Furthermore, we provided evidence that a second disulfide oxidoreductase, SrgA, also oxidizes SpiA. Analysis of in vivo mixed infections demonstrated that a Salmonella dsbA srgA double mutant strain was more attenuated than either single mutant, suggesting that DsbA acts in concert with SrgA in vivo.     

Identification of a protein required for disulfide bond formation in vivo

We describe a mutation (dsbA) that renders Escherichia coli severely defective in disulfide bond formation. In dsbA mutant cells, pulse-labeled beta-lactamase, alkaline phosphatase, and OmpA are secreted but largely lack disulfide bonds. These disulfideless proteins may represent in vivo folding intermediates, since they are protease sensitive and chase slowly into stable oxidized forms. The dsbA gene codes for a 21,000 Mr periplasmic protein containing the sequence cys-pro-his-cys, which resembles the active sites of certain disulfide oxidoreductases. The purified DsbA protein is capable of reducing the disulfide bonds of insulin, an activity that it shares with these disulfide oxidoreductases. Our results suggest that disulfide bond formation is facilitated by DsbA in vivo.     

A new Escherichia coli gene, dsbG, encodes a periplasmic protein involved in disulphide bond formation, required for recycling DsbA/DsbB and DsbC redox proteins

We have identified and functionally characterized a new Escherichia coli gene, dsbG , whose product is involved in disulphide bond formation in the periplasm. The dsbG gene was cloned from a multicopy plasmid library lacking the dsbB redox protein-encoding gene. Multicopy dsbG -carrying clones were selected, since they allowed E. coli to grow at lethal concentrations of dithiothreitol. In a complementary genetic approach, point mutations were independently obtained and mapped to the dsbG gene. Such mutations led simultaneously to a dithiothreitol-sensitive phenotype and an increased σ^E-dependent heat shock response, which reflects the presence of misfolded proteins in the extracytoplasm. In agreement with these observations, dsbG mutants were shown to accumulate reduced forms of a variety of disulphide bond-containing proteins in the periplasm. This DsbG defect could be rescued by addition to the growth medium of either oxidized dithiothreitol or cystine, or by overexpression of the dsbA or dsbB genes. DsbG is synthesized as a precursor form of 27.5 kDa and processed to a 25.7 kDa mature species located in the periplasm. DsbG was overproduced, purified to homogeneity and shown to have redox properties of thiol–disulphide oxidoreductases in vitro . Replacement of the first Cys residue of the predicted active site, Phe–(Xaa)₄–Cys–Pro–Tyr–Cys by Ala, completely inactivated DsbG protein function. Taken together, all our results demonstrate that DsbG acts in vivo as an efficient thiol–disulphide oxidase. In addition, dsbG is the first member of the dsb family for which null mutations are conditionally lethal and can be propagated only if supplemented with oxidants in the growth medium. We propose that the main role of DsbG is to maintain the proper redox balance between the DsbA/DsbB and DsbC systems.     

DsbB is required for the pathogenesis process of Xanthomonas campestris pv. campestris

The DsbA/DsbB oxidation pathway is one of the two pathways that catalyze disulfide bond formation of proteins in the periplasm of gram-negative bacteria. It has been demonstrated that DsbA is essential for multiple virulence factors of several animal bacterial pathogens. In this article, we present genetic evidence to show that the open reading frame XC_3314 encodes a DsbB protein that is involved in disulfide bond formation in periplasm of Xanthomonas campestris pv. campestris, the causative agent of crucifer black rot disease. The dsbB mutant of X. campestris pv. campestris exhibited attenuation in virulence, hypersensitive response, cell motility, and bacterial growth in planta. Furthermore, mutation in the dsbB gene resulted in ineffective type II and type III secretion systems as well as flagellar assembly. These findings reveal that DsbB is required for the pathogenesis process of X. campestris pv. campestris.