dsbA Mutant Research Articles

The Periplasmic Oxidoreductase DsbA Is Required for Virulence of the Phytopathogen Dickeya solani.

In bacteria, the DsbA oxidoreductase is a crucial factor responsible for the introduction of disulfide bonds to extracytoplasmic proteins, which include important virulence factors. A lack of proper disulfide bonds frequently leads to instability and/or loss of protein function; therefore, improper disulfide bonding may lead to avirulent phenotypes. The importance of the DsbA function in phytopathogens has not been extensively studied yet. Dickeya solani is a bacterium from the Soft Rot Pectobacteriaceae family which is responsible for very high economic losses mainly in potato. In this work, we constructed a D. solani dsbA mutant and demonstrated that a lack of DsbA caused a loss of virulence. The mutant bacteria showed lower activities of secreted virulence determinants and were unable to develop disease symptoms in a potato plant. The SWATH-MS-based proteomic analysis revealed that the dsbA mutation led to multifaceted effects in the D. solani cells, including not only lower levels of secreted virulence factors, but also the induction of stress responses. Finally, the outer membrane barrier seemed to be disturbed by the mutation. Our results clearly demonstrate that the function played by the DsbA oxidoreductase is crucial for D. solani virulence, and a lack of DsbA significantly disturbs cellular physiology.

Envelope Stress and Regulation of the Salmonella Pathogenicity Island 1 Type III Secretion System.

Salmonella enterica serovar Typhimurium uses a type three secretion system (T3SS) encoded on the Salmonella pathogenicity island 1 (SPI1) to invade intestinal epithelial cells and induce inflammatory diarrhea. The SPI1 T3SS is regulated by numerous environmental and physiological signals, integrated to either activate or repress invasion. Transcription of hilA, encoding the transcriptional activator of the SPI1 structural genes, is activated by three AraC-like regulators, HilD, HilC, and RtsA, that act in a complex feed-forward loop. Deletion of bamB, encoding a component of the β-barrel assembly machinery, causes a dramatic repression of SPI1, but the mechanism was unknown. Here, we show that partially defective β-barrel assembly activates the RcsCDB regulon, leading to decreased hilA transcription. This regulation is independent of RpoE activation. Though Rcs has been previously shown to repress SPI1 when disulfide bond formation is impaired, we show that activation of Rcs in a bamB background is dependent on the sensor protein RcsF, whereas disulfide bond status is sensed independently. Rcs decreases transcription of the flagellar regulon, including fliZ, the product of which indirectly activates HilD protein activity. Rcs also represses hilD, hilC, and rtsA promoters by an unknown mechanism. Both dsbA and bamB mutants have motility defects, though this is simply regulatory in a bamB background; motility is restored in the absence of Rcs. Effector secretion assays show that repression of SPI1 in a bamB background is also regulatory; if expressed, the SPI1 T3SS is functional in a bamB background. This emphasizes the sensitivity of SPI1 regulation to overall envelope homeostasis.IMPORTANCESalmonella causes worldwide foodborne illness, leading to massive disease burden and an estimated 600,000 deaths per year. Salmonella infects orally and invades intestinal epithelial cells using a type 3 secretion system that directly injects effector proteins into host cells. This first step in invasion is tightly regulated by a variety of inputs. In this work, we demonstrate that Salmonella senses the functionality of outer membrane assembly in determining regulation of invasion machinery, and we show that Salmonella uses distinct mechanisms to detect specific perturbations in envelope assembly.

Redox-tuning of oxidizing disulfide oxidoreductase generates a potent disulfide isomerase

Oxidative folding of extracellular proteins is pivotal for the biogenesis of bacterial virulence factors. Escherichia coli DsbA catalyzes disulfide bond formation in extracellular proteins and in multicomponent architectures on the cell surface. The present study assessed the significance of the redox properties of DsbA by exploiting the plaque-forming ability of bacteriophage M13, which specifically recognizes F-pili during infection of the host cell. A library of mutant dsbA genes was constructed by randomizing the dipeptide XX sequence in the active-site redox motif CXXC and then screened for mutants that altered plaque yield and appearance. In total, 24 dsbA mutant alleles produced substantially different degrees of complementation, and one mutant dsbA gene that encodes a CDIC sequence produced over 40-fold more clear plaques than wild type dsbA. The redox potential of purified DsbA [CDIC] was −172 mV, representing a less-oxidizing catalysis than the wild type DsbA (−122 mV), but one that is closer to yeast protein disulfide isomerase (−175 mV). DsbA [CDIC] exhibited a greater ability to refold fully denatured glutathionylated ribonuclease A than the wild type enzyme and a DsbA [CRIC] mutant, which has the same redox potential of −172 mV. Homology modeling and molecular dynamics simulation suggest that the CDIC mutant may have an enlarged substrate-binding cleft near the redox center, which confers kinetic advantages when acting on protein substrates.

The Multiple Localized Glyceraldehyde-3-Phosphate Dehydrogenase Contributes to the Attenuation of the Francisella tularensis dsbA Deletion Mutant.

The DsbA homolog of Francisella tularensis was previously demonstrated to be required for intracellular replication and animal death. Disruption of the dsbA gene leads to a pleiotropic phenotype that could indirectly affect a number of different cellular pathways. To reveal the broad effects of DsbA, we compared fractions enriched in membrane proteins of the wild-type FSC200 strain with the dsbA deletion strain using a SILAC-based quantitative proteomic analysis. This analysis enabled identification of 63 proteins with significantly altered amounts in the dsbA mutant strain compared to the wild-type strain. These proteins comprise a quite heterogeneous group including hypothetical proteins, proteins associated with membrane structures, and potential secreted proteins. Many of them are known to be associated with F. tularensis virulence. Several proteins were selected for further studies focused on their potential role in tularemia's pathogenesis. Of them, only the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme of glycolytic pathway, was found to be important for full virulence manifestations both in vivo and in vitro. We next created a viable mutant strain with deleted gapA gene and analyzed its phenotype. The gapA mutant is characterized by reduced virulence in mice, defective replication inside macrophages, and its ability to induce a protective immune response against systemic challenge with parental wild-type strain. We also demonstrate the multiple localization sites of this protein: In addition to within the cytosol, it was found on the cell surface, outside the cells, and in the culture medium. Recombinant GapA was successfully obtained, and it was shown that it binds host extracellular serum proteins like plasminogen, fibrinogen, and fibronectin.

HmsC Controls Yersinia pestis Biofilm Formation in Response to Redox Environment.

Yersinia pestis biofilm formation, controlled by intracellular levels of the second messenger molecule cyclic diguanylate (c-di-GMP), is important for blockage-dependent plague transmission from fleas to mammals. HmsCDE is a tripartite signaling system that modulates intracellular c-di-GMP levels to regulate biofilm formation in Y. pestis. Previously, we found that Y. pestis biofilm formation is stimulated in reducing environments in an hmsCDE-dependent manner. However, the mechanism by which HmsCDE senses the redox state remains elusive. Using a dsbA mutant and the addition of Cu2+ to simulate reducing and oxidizing periplasmic environments, we found that HmsC protein levels are decreased and the HmsC-HmsD protein-protein interaction is weakened in a reducing environment. In addition, we revealed that intraprotein disulphide bonds are critical for HmsC since breakage lowers protein stability and diminishes the interaction with HmsD. Our results suggest that HmsC might play a major role in sensing the environmental changes.

The Aeromonas dsbA mutation decreased their virulence by triggering type III secretion system but not flagella production

Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Dsb ( Di sulfide bond) proteins play an important role in catalyzing disulfide bond formation in proteins within the periplasmic space. An A. hydrophila dsbA mutant with attenuated virulence using Dictyostelium amoebae as an alternative host model was identified. The attenuated virulence was tested in other animal models (by intraperitoneal injection in fish and mice) and was correlated with the presence of a defective type III secretion system for the first time in non enteric bacteria. The dsbA mutation was shown in several enteric bacteria to involve the outer membrane secretin. The defect in Aeromonas also seems to involve the outer membrane secretin homologue named AscC. However, unlike what happen in Escherichia coli, no changes in motility or flagella expression were observed for A. hydrophila dsbA mutants. The loss of E. coli motility caused by deletion of dsbA is likely due to defective disulfide bond formation in FlgI, a component of the flagella. No disulfide bond formation in FlgI homologues in Aeromonas flagella biogenesis, either polar or lateral, could be expected according to their amino acid residues sequences.

Molecular characterization of the extracellular matrix in a Pseudomonas putida dsbA mutant: implications for acidic stress defense and plant growth promotion

The Pseudomonas putida dsbA mutant displays enhanced extracellular matrix production, which promotes biofilm formation. Here we confirmed that the extracellular matrix consists of both capsular polysaccharides and exopolysaccharides. However, the carbohydrate composition of the P. putida dsbA mutant matrix was shown to be similar to that of the wild-type strain. Our data indicate that the overproduced matrix itself, rather than alterations in the matrix composition, promotes biofilm formation in the P. putida dsbA mutant. Moreover, the mutant was more sensitive than the wild-type to alkali stress (pH 9.0 to 10.0), but not to acidic stress (pH 5.0). Interestingly, acidic stress stimulated polysaccharide production and pellicle formation, while these changes were recovered to the level of the wild-type under alkali conditions in the P. putida dsbA mutant. Enhanced biofilm formation of the dsbA mutant increased the efficiency with which P. putida attached to tomato and pepper seeds, which have longer germinated roots than the wild-type strain. This phenomenon could not be observed in the cucumber plant, which suggests that each plant seed has a different effect on the attachment of P. putida. Interestingly, this increased attachment to plant seeds resulted in more root colonization and plant growth promotion. The findings of this study suggested that the overproduced extracellular matrix caused by deletion of the dsbA gene could have pleiotropic effect on P. putida phenotypes, including acidic stress defense and plant growth promotion.

The Protein-disulfide Isomerase DsbC Cooperates with SurA and DsbA in the Assembly of the Essential β-Barrel Protein LptD

The assembly of the β-barrel proteins present in the outer membrane (OM) of Gram-negative bacteria is poorly characterized. After translocation across the inner membrane, unfolded β-barrel proteins are escorted across the periplasm by chaperones that reside within this compartment. Two partially redundant chaperones, SurA and Skp, are considered to transport the bulk mass of β-barrel proteins. We found that the periplasmic disulfide isomerase DsbC cooperates with SurA and the thiol oxidase DsbA in the folding of the essential β-barrel protein LptD. LptD inserts lipopolysaccharides in the OM. It is also the only β-barrel protein with more than two cysteine residues. We found that surAdsbC mutants, but not skpdsbC mutants, exhibit a synthetic phenotype. They have a decreased OM integrity, which is due to the lack of the isomerase activity of DsbC. We also isolated DsbC in a mixed disulfide complex with LptD. As such, LptD is identified as the first substrate of DsbC that is localized in the OM. Thus, electrons flowing from the cytoplasmic thioredoxin system maintain the integrity of the OM by assisting the folding of one of the most important β-barrel proteins.

Proteome Analysis of an Attenuated Francisella tularensis dsbA Mutant: Identification of Potential DsbA Substrate Proteins

Francisella tularensis (F. tularensis) is highly infectious for humans via aerosol route and untreated infections with the highly virulent subsp. tularensis can be fatal. Our knowledge regarding key virulence determinants has increased recently but is still somewhat limited. Surface proteins are potential virulence factors and therapeutic targets, and in this study, we decided to target three genes encoding putative membrane lipoproteins in F. tularensis LVS. One of the genes encoded a protein with high homology to the protein family of disulfide oxidoreductases DsbA. The two other genes encoded proteins with homology to the VacJ, a virulence determinant of Shigella flexneri. The gene encoding the DsbA homologue was verified to be required for survival and replication in macrophages and importantly also for in vivo virulence in the mouse infection model for tularemia. Using a combination of classical and shotgun proteome analyses, we were able to identify several proteins that accumulated in fractions enriched for membrane-associated proteins in the dsbA mutant. These proteins are substrate candidates for the DsbA disulfide oxidoreductase as well as being responsible for the virulence attenuation of the dsbA mutant.

Inactivation of thePseudomonas putidaKT2440dsbAgene promotes extracellular matrix production and biofilm formation

To identify genes essential to biofilm formation in Pseudomonas putida KT2440, 12 mutants defective in oxidative stress-related or metabolic pathway-related genes were evaluated. Of them, only the dsbA mutant lacking the disulfide bond isomerase exhibited significantly increased attachment to the polystyrene surface. Visual evaluation by extracellular matrix staining and scanning electron microscopy indicated that the KT2440-DeltadsbA strain displays enhanced extracellular matrix production, rugose colony morphology on agar plates and floating pellicles in static culture. Accordingly, we propose that deletion of the dsbA gene may stimulate production of the extracellular matrix, resulting in those phenotypes. In addition, the lack of detectable fluorescence in the KT2440-DeltadsbA under UV light as well as in both the wild type and the KT2440-DeltadsbA when grown on Luria-Bertani plates containing ferrous iron suggests that the fluorescent molecule may be a fluorescent siderophore with its synthesis/secretion controlled by DsbA in KT2440. These phenotypic defects observed in the dsbA mutant were complemented by the full-length KT2440 and Escherichia coli dsbA genes. In contrast to the role of DsbA in other bacteria, our results provide the first evidence that disruption of P. putida KT2440 dsbA gene overproduces the extracellular matrix and thus promotes biofilm formation.

Genetic Suppressors and Recovery of Repressed Biochemical Memory

Many of the Reflections articles in this journal have focused on particular biological problems that the authors have devoted their careers to or on particular techniques that have facilitated their biological studies. Although I began my career as a biochemist, one of the most common threads to my research has been the exploitation of a genetic approach, the analysis of suppressor mutations. Particularly in the last 20 years, the success of this approach has led me into biological problems that required a remembering or relearning of my early training in biochemistry. This article will describe the voyage from chemistry and biochemistry to genetics and then to a recovery of remnants of my biochemical memory. As a graduate student working under Lowell Hager at Harvard in the late 1950s, my thesis research was chemical and biochemical, as I performed the chemical synthesis and studied the biosynthesis of the chlorinated mold product caldariomycin (2,2-dichloro-1,3-cyclopentanediol) (1–3). With his postdoctoral fellow Paul Shaw, Lowell had discovered an enzyme, chloroperoxidase, that was capable of utilizing chloride ion to chlorinate organic compounds (4). Part of my work was to determine how this enzyme might be involved in the biosynthesis of caldariomycin. Apparently, the work became somewhat well known. This I learned when, toward the end of my Ph.D. work, I attended a FASEB meeting in Atlantic City to give a short presentation. As I walked into the conference center on the first day, a scientist passing by me stopped to look at my badge, hesitated a moment, and then commented “Beckwith... hmmm, oh yes, I remember... chlorination!” Despite this really quite limited chemical/biochemical notoriety, I became more and more fascinated by bacterial genetics in graduate school. I proceeded to do postdoctoral work in several bacterial genetics laboratories.

Engineering antibody fragments to fold in the absence of disulfide bonds

Disulfide bonds play a critical role in the stabilization of the immunoglobulin beta-sandwich sandwich. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment (intrabodies) accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm, which leads to the accumulation of scFvs and other disulfide-containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193-9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues.

Structural and Biochemical Characterization of the Oxidoreductase NmDsbA3 from Neisseria meningitidis

DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyzes the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Although most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whereas NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3-A resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidized by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.

Functional Analysis of the Thioredoxin Domain inPorphyromonas gingivalisHBP35

Periodontitis is one of the most common oral diseases in humans. This caused by infection by the oral bacterium Porphyromonas gingivalis. Our strategy to prevent this infection is to establish a passive immunization system in which endogenous antibodies can be applied directly to neutralize virulent factors associated with this bacterium. We focused our attention on the P. gingivalis 35 kDa surface protein, or HBP35, since this protein is involved not only in the coaggregation with oral miroflora but also in hemin binding. In addition, nucleotide sequencing of the gene, hbp35, coding for this protein revealed the presence of a catalytic center for thioredoxin, and we further attempted to characterized the protein by amino acid substitution. A total of four Cys residues were substituted for Ser residues by combining the simple method for site-directed mutagenesis and the heterodimer system, an approach designed to construct chimeric plasmids readily. Native and mutagenized hbp35 were introduced into the Eschericha coli dsbA mutant strain, JCB 572, defective in both alkaline phosphatase and motile activities due to inefficient disulfide bond formation. Transformant harboring the native hbp35 could complement the dsbA mutation, suggesting a role of disulfide bond formation of this protein in P. gingivalis cells. Possible roles of the Cys residues in complementation are discussed.

DsbA directs efficient expression of outer membrane secretin EscC of the enteropathogenic Escherichia coli type III secretion apparatus

The formation of disulfide bond is essential for the folding, activity, and stability of many secreted proteins of Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into exported proteins from the cytoplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we investigated the role of DsbA in enteropathogenic Escherichia coli. Here, we show that the DsbA is required for stable expression of outer membrane secretin EscC. DsbA has no effect on LEE transcription as measured with LEE-lacZ fusions. Replacement of either cysteine residue 136 or 155 of EscC with a serine resulted in reduced level of EscC, similar to the effect of the dsbA mutation. These results demonstrate the role of DsbA in assembly of the type III secretion apparatus.

A bacterial two‐hybrid system based on the twin‐arginine transporter pathway of E. coli

We have developed a bacterial two-hybrid system for the detection of interacting proteins that capitalizes on the folding quality control mechanism of the Twin Arginine Transporter (Tat) pathway. The Tat export pathway is responsible for the membrane translocation of folded proteins, including proteins consisting of more than one polypeptide, only one of which contains a signal peptide ("hitchhiker export"). Here, one protein (bait) is expressed as a fusion to a Tat signal peptide, whereas the second protein (prey) is fused to a protein reporter that can confer a phenotype only after export into the bacterial periplasmic space. Since the prey-reporter fusion lacks a signal peptide, it can only be exported as a complex with the bait-signal peptide fusion that is capable of targeting the Tat translocon. Using maltose-binding protein as a reporter, clones expressing interacting proteins can be grown on maltose minimal media or on MacConkey plates. In addition, we introduce the use of the cysteine disulfide oxidase DsbA as a reporter. Export of a signal peptide-prey:bait-DsbA complex into the periplasm allows complementation of dsbA(-) mutants and restores the formation of active alkaline phosphatase, which in turn can be detected by a chromogenic assay.

The role of dsbA in colonization of the wheat rhizosphere by Pseudomonas fluorescens Q8r1-96

Certain well-conserved genes in fluorescent Pseudomonas spp. are involved in pathogenic interactions between the bacteria and evolutionarily diverse hosts including plants, insects and vertebrate animals. One such gene, dsbA, encodes a periplasmic disulfide-bond-forming enzyme implicated in the biogenesis of exported proteins and cell surface structures. This study focused on the role of dsbA in Pseudomonas fluorescens Q8r1-96, a biological control strain that produces the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) and is known for its exceptional ability to colonize the roots of wheat and pea. The deduced DsbA protein from Q8r1-96 is similar to other predicted thiol : disulfide interchange proteins and contains a conserved DsbA catalytic site, a pattern associated with the thioredoxin family active site, and a signal peptide and cleavage site. A dsbA mutant of Q8r1-96 exhibited decreased motility and fluorescence, and altered colony morphology; however, it produced more 2,4-DAPG and total phloroglucinol-related compounds and was more inhibitory in vitro to the fungal root pathogen Gaeumannomyces graminis var. tritici than was the parental strain. When introduced separately into a natural soil, Q8r1-96 and the dsbA mutant did not differ in their ability to colonize the rhizosphere of wheat in greenhouse experiments lasting 12 weeks. However, when the two strains were co-inoculated, the parental strain consistently out-competed the dsbA mutant. It was concluded that dsbA does not contribute to the exceptional rhizosphere competence of Q8r1-96, although the dsbA mutation reduces competitiveness when the mutant competes with the parental strain in the same niche in the rhizosphere. The results also suggest that exoenzymes and multimeric cell surface structures are unlikely to have a critical role in root colonization by this strain.

The role of F plasmid TraF and TrbB in stabilizing the conjugative apparatus

The role of two proteins, TrbB and TraF, encoded by the F plasmid, were investigated for their ability to affect F transfer protein localization and stability using cell fractionation by sucrose gradient sedimentation and immunoblot analyses. Bacterial conjugation mediates the horizontal transfer of genetic material throughout the microbial world. The F plasmid contains a transfer region that encodes a type IV secretion system (T4SS) necessary for conjugative pilus assembly and DNA transfer. The F-type T4SS are considerably more complex than the P-type systems because of the properties of pilus retraction and stable mating pair formation. The proteins that are specific to F-T4SS are TraF, -G (C-terminal domain), TraH, -N, -U, -W, TrbB, -C and-I. TraG, -N and -U are important for mating pair stabilization and the remaining proteins are required for pilus assembly and retraction. Several F-T4SS proteins are remarkable for their high cysteine content (TraN, TraU and TraH have 22, 10 and 6 cysteines, respectively). TraF and TrbB, have thioredoxin-folds with TrbB, but not TraF, having a characteristic CxxC motif. Previously, we have shown TrbB has DsbC-like activity and acts as a disulfide bond isomerase since, like DsbC, it can partially complement a dsbA mutation. Whereas TrbB is not essential for conjugation, TraF is required for pilus assembly and transfer, suggesting that it has a role in construction of the T4SS conjugative pore. Using antisera to the F-T4SS proteins we demonstrate that traF and trbB mutations affect the stability of the T4SS complex, particularly TraH and TraV.

Conserved Role of the Linker α-Helix of the Bacterial Disulfide Isomerase DsbC in the Avoidance of Misoxidation by DsbB

In the bacterial periplasm the co-existence of a catalyst of disulfide bond formation (DsbA) that is maintained in an oxidized state and of a reduced enzyme that catalyzes the rearrangement of mispaired cysteine residues (DsbC) is important for the folding of proteins containing multiple disulfide bonds. The kinetic partitioning of the DsbA/DsbB and DsbC/DsbD pathways partly depends on the ability of DsbB to oxidize DsbA at rates >1000 times greater than DsbC. We show that the resistance of DsbC to oxidation by DsbB is abolished by deletions of one or more amino acids within the alpha-helix that connects the N-terminal dimerization domain with the C-terminal thioredoxin domain. As a result, mutant DsbC carrying alpha-helix deletions could catalyze disulfide bond formation and complemented the phenotypes of dsbA cells. Examination of DsbC homologues from Haemophilus influenzae, Pseudomonas aeruginosa, Erwinia chrysanthemi, Yersinia pseudotuberculosis, Vibrio cholerae (30-70% sequence identity with the Escherichia coli enzyme) revealed that the mechanism responsible for avoiding oxidation by DsbB is a general property of DsbC family enzymes. In addition we found that deletions in the linker region reduced, but did not abolish, the ability of DsbC to assist the formation of active vtPA and phytase in vivo, in a DsbD-dependent manner, revealing that interactions between DsbD and DsbC are also conserved.

Defects in a quinol oxidase lead to loss of KatC catalase activity in Pseudomonas aeruginosa: KatC activity is temperature dependent and it requires an intact disulphide bond formation system

Mutation or overexpression of the cyanide-insensitive terminal oxidase (CIO) of Pseudomonas aeruginosa leads to temperature-sensitivity, multiple antibiotic sensitivity, and abnormal cell division and failure to produce a temperature-inducible catalase [G.R. Tavankar, D. Mossialos, H.D. Williams, Mutation or overexpression of a terminal oxidase leads to a cell division defect and multiple antibiotic sensitivity in Pseudomonas aeruginosa, J. Biol. Chem. 278 (2003) 4524–4530]. We identify this enzyme as KatC, a newly described catalase from P. aeruginosa. Loss of KatC activity leads to temperature-dependent hydrogen peroxide sensitivity, which correlates with its temperature-inducible expression pattern. This is the first description, to our knowledge, of a temperature-inducible bacterial catalase. The transcription of katC is not affected in strains lacking or overexpressing the CIO, indicating that a post-transcriptional effect leads to loss of KatC activity. Disulphide bond formation is affected in strains lacking or overexpressing the CIO. This is shown by reduced activity of the extracellular enzymes lipase and elastase, and an altered pattern of redox states of DsbA, a key protein in disulphide bond formation in P. aeruginosa, in these strains. Moreover, a dsbA mutant had no detectable KatC activity, demonstrating that an intact disulphide bond formation system is required for KatC activity and thus explaining the loss of this catalase in the cio mutant and overexpressing strains.