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

Abstract

SummaryWe 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 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.

The protein disulphide-isomerase family: unravelling a string of folds

“…This large family accommodates thioredoxin-like, glutaredoxin-like and PDI-like proteins, as well as members of the bacterial Dsb family [14,26,42,43], all of which contain one or more copies of the highly conserved thioredoxin fold, i.e. a β-α-β-α-β-α-β-β-α structure (as shown in Figures 1 and 2) encompassing a reactive -Cys-Xaa-Xaa-Cys-tetrapeptide [9,34,[44][45][46].…”

In Vivo and in Vitro Function of theEscherichia coli Periplasmic Cysteine Oxidoreductase DsbG

“…Construction and Characterization of dsbG Null Mutants-In earlier studies Andersen et al (1) reported that a dsbG::⍀Tet mutation could be crossed onto the chromosome only when the cells were grown in the presence of low molecular weight oxidants such as cystine or oxidized DTT. Despite finding that a dsbG::⍀Kan mutation could be transduced without supplementation of oxidants, they nevertheless hypothesized that this observation resulted from the accumulation of second site suppressor mutations.…”

“…This is surprising, since none of the other dsb genes, including dsbA, which encodes the main catalyst of protein oxidation in the periplasm, is essential. In addition, Andersen et al (1) reported * This work was supported by the National Science Foundation and National Institutes of Health (NIH) Grant GM-47520 (to G. G.). The costs of publication of this article were defrayed in part by the payment of page charges.…”

“…The same gene (dsbG) was also isolated in a search for mutants conferring increased sensitivity to DTT and an increase in E -dependent periplasmic heat shock response. Interestingly, Andersen et al (1) reported that dsbG is required for growth unless the cells are provided with exogenous oxidants. This is surprising, since none of the other dsb genes, including dsbA, which encodes the main catalyst of protein oxidation in the periplasm, is essential.…”

“…1 One of these genes was named dsbE and has been proposed to play a role in oxidative protein folding. However, dsbE is identical to ccmG, which has been shown to be an inner membrane protein involved in cytochrome biosynthesis in Escherichia coli and other bacteria (19 -21).…”