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

Mol Microbiol. 1997 Oct;26(1):121-32. doi: 10.1046/j.1365-2958.1997.5581925.x.

Abstract

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 sigmaE-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 25.7kDa 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase / metabolism
  • Amino Acid Sequence
  • Bacterial Proteins / metabolism*
  • Chromosome Mapping
  • Cloning, Molecular
  • Disulfides / metabolism*
  • Dithiothreitol / metabolism
  • Dithiothreitol / pharmacology
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Escherichia coli Proteins*
  • Gene Expression Regulation, Bacterial
  • Insulin / metabolism
  • Membrane Proteins / metabolism*
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Oxidoreductases / chemistry
  • Oxidoreductases / genetics*
  • Oxidoreductases / metabolism
  • Periplasmic Proteins*
  • Point Mutation
  • Protein Disulfide-Isomerases / genetics
  • Protein Disulfide-Isomerases / metabolism*
  • Protein Folding
  • Restriction Mapping
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid

Substances

  • Bacterial Proteins
  • Disulfides
  • DsbB protein, Bacteria
  • Escherichia coli Proteins
  • Insulin
  • Membrane Proteins
  • Periplasmic Proteins
  • Oxidoreductases
  • DsbG protein, E coli
  • Alkaline Phosphatase
  • Protein Disulfide-Isomerases
  • Dithiothreitol

Associated data

  • GENBANK/AF000956
  • SWISSPROT/P77202