Cloning and expression of the gene for a protein disulfide oxidoreductase from Azotobacter vinelandii: complementation of an Escherichia coli dsbA mutant strain

Ng, Thomas C.N; Kwik, Jeanne F; Maier, Robert J.

doi:10.1016/s0378-1119(96)00792-5

Cited by 13 publications

(16 citation statements)

References 20 publications

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“…Finally, it was noted that R. capsulatus DsbA-null mutants are Res Ϫ , especially on enriched media, and similar growth defects have also been reported for Azotobacter vinelandii mutants (37). The more pronounced respiratory growth defect on enriched media might be due to the accumulation of excess reduced thiol compounds that could aggravate the loss of an oxidizing protein like DsbA.…”

Section: Fig 4 C-type Cyt Profiles Of the Psmentioning

confidence: 64%

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

et al. 2003

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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...

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Section: Fig 4 C-type Cyt Profiles Of the Psmentioning

confidence: 64%

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

et al. 2003

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“…1A). The highest homology score (68% identity, 82% similarity) was obtained with Azotobacter vinelandii DsbA (42). A putative signal sequence consisting of 22 amino acids was identified at the amino terminus of DsbA, suggesting a periplasmic location of the mature protein, which has calculated molecular mass of 21 kDa.…”

Section: Resultsmentioning

confidence: 92%

DsbA and DsbC Affect Extracellular Enzyme Formation in Pseudomonas aeruginosa

et al. 2001

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DsbA and DsbC proteins involved in the periplasmic formation of disulfide bonds in Pseudomonas aeruginosawere identified and shown to play an important role for the formation of extracellular enzymes. Mutants deficient in either dsbA or dsbC or both genes were constructed, and extracellular elastase, alkaline phosphatase, and lipase activities were determined. The dsbA mutant no longer produced these enzymes, whereas the lipase activity was doubled in the dsbC mutant. Also, extracellar lipase production was severely reduced in a P. aeruginosa dsbA mutant in which an inactive DsbA variant carrying the mutation C34S was expressed. Even when the lipase gene lipA was constitutively expressed in trans in a lipA dsbA double mutant, lipase activity in cell extracts and culture supernatants was still reduced to about 25%. Interestingly, the presence of dithiothreitol in the growth medium completely inhibited the formation of extracellular lipase whereas the addition of dithiothreitol to a cell-free culture supernatant did not affect lipase activity. We conclude that the correct formation of the disulfide bond catalyzed in vivo by DsbA is necessary to stabilize periplasmic lipase. Such a stabilization is the prerequisite for efficient secretion using the type II pathway.Disulfide bonds are important for the structure and stability of numerous proteins. For Escherichia coli it is now well established that the formation of disulfide bonds is an assisted process which occurs in the periplasm and is catalyzed by the thiol-disulfide oxidoreductase DsbA (7, 34). DsbA acts as a donor of disulfides to newly synthesized periplasmic proteins (23) and is reoxidized by DsbB, a second protein located in the inner membrane (5, 6). DsbA and DsbB are members of the Dsb system (the system for disulfide bond formation) which consists of at least six redox proteins belonging to the thioredoxin superfamily. These proteins contain a canonical C-X-X-C motif in the dithiol active site and seem to be conserved throughout the gram-negative bacteria (49). DsbC is another member which is suggested to act as a disulfide isomerase (56, 67). DsbD (39) keeps DsbC in a reduced state (57). More recently, DsbG was described as a novel member of the Dsb family in E. coli which oxidizes so far unknown substrates (4,8).Undoubtedly, the process of protein secretion in gram-negative bacteria is related to the function of the Dsb system. However, the results obtained so far are contradictory. Four major secretion pathways exist in gram-negative bacteria to direct proteins into the extracellular medium (37). In type I and type III pathways the secreted proteins directly pass both the inner and the outer membrane using a machinery formed by either three or more than 20 different proteins, respectively. In the type II pathway, which is also called the general secretory pathway, secretion occurs in two consecutive steps (48), with an intermediate state in the periplasm where the formation of disulfide bonds can take place. Since E. coli does not secrete exoprotein...

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“…DsbA of E. chrysanthemi is needed for full pectate lyase and cellulase activities,29 while DsbA of Shigella flexneri facilitates release of invasions into the external medium 30. Bdb, a secreted protein that can complement E. coli DsbA, is found in the gram‐positive bacterium Bacillus brevis ,31 while another DsbA homologue is found in Azotobacter vinelandii 32. Virulence proteins, Yops, in Yersinia pestis , need to be secreted by a secretion apparatus made of many proteins, one of which is YscC, and DsbA is required for disulfide‐bond formation in YscC 33.…”

Section: Dsba Homologuesmentioning

confidence: 99%

Aerobic Oxidation of Alcohols with Bifunctional Transition‐Metal Catalysts Bearing C–N Chelate Ligands

Arita

Koike

Kayaki

et al. 2008

Chemistry An Asian Journal

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The aerobic oxidation of alcohols with a family of bifunctional Ir, Rh, and Ru complexes bearing C-N chelating ligands derived from primary benzylic amines was investigated. The isolable amido-Ir complexes [Cp*Ir{kappa(2)(N,C)-(NHCR(2)-2-C(6)H(4))}] (R=C(6)H(5), CH(3); Cp*=1,2,3,4,5-pentamethylcyclopentadienyl) effected the oxidation of secondary alcohols smoothly under atmospheric pressure of air at 30 degrees C in THF to give the corresponding ketones in good yields. The hydrido(amine)-Ir complexes [Cp*IrH{kappa(2)(N,C)-(NH(2)CR(2)-2-C(6)H(4))}] and the combined catalyst system involving the chloro(amine)-Ir complex [Cp*IrCl{kappa(2)(N,C)-(NH(2)CR(2)-2-C(6)H(4))}] and KOC(CH(3))(3) were also found to be effective catalysts, whereas the tertiary amine complex [Cp*IrCl{kappa(2)(N,C)-(N(CH(3))(2)CH(2)-2-C(6)H(4))}], which does not have a metal/NH moiety, did not show catalytic activity. The employment of primary alcohols in the aerobic reaction with the Cp*IrCl complex and KOC(CH(3))(3) resulted in the formation of esters through oxidative dimerization.

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Cloning and expression of the gene for a protein disulfide oxidoreductase from Azotobacter vinelandii: complementation of an Escherichia coli dsbA mutant strain

Cited by 13 publications

References 20 publications

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 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

DsbA and DsbC Affect Extracellular Enzyme Formation in Pseudomonas aeruginosa

Aerobic Oxidation of Alcohols with Bifunctional Transition‐Metal Catalysts Bearing C–N Chelate Ligands

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