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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 σE-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. [ABSTRACT FROM AUTHOR]

Escherichia coli mutants, (verA, dilA) specifically resistant to the Ca2+ channel inhibitors verapamil and diltiazem, respectively, are hypersensitive to EGTA and BAPTA. We have shown, using 1-D and 2-D gel electrophoresis, that the synthesis of at least 25 polypeptides in the mutants was enhanced by treatment with Ca2+ chelators and the synthesis of at least 11 polypeptides was repressed. This pattern of induction was not observed in heat- or SDS-treated cells and therefore does not appear to be a general stress response. The majority of the induced proteins are low molecular weight, extremely heat stable and acidic, characteristic properties of caimodulin. Moreover, of the major induced species, three with apparent molecular masses of 12, 18, and 34kDa all cross-reacted with polydonal and monoclonal anti-bodies to eukaryote calmodulins or calerythrin, a heat-resistant Ca2+-binding protein from Saccharo-polyspora erythraea. The verA, dilA mutants. In being hypersensitive to EGTA and to the Ca2+ ionophore A23187 + Ca2+, may be defective in the regulation of the level of free intracellular Ca2+. [ABSTRACT FROM AUTHOR]