Identification and characterization of the Escherichia coli gene dsbB, whose product is involved in the formation of disulfide bonds in vivo.

Abstract

We have identified and characterized the Escherichia coli gene dsbB, whose product is required for diaulfide bond formation of periplasmic proteins, by using two different approaches: (i) screening of a multicopy plasmid library for clones which protect E. coli from the lethal effects of dithiothreitol (DTT), and (ii) screening of insertion libraries of E. coli for DTT-sensitive mutants. Mapping and characterization of mutations conferring a DTT-sensitive phenotype also identified the dsbA, trxA, and trxB genes, whose products are involved in different oxidation-reduction pathways. Null mutations in dsbB conferred pleiotropic phenotypes such as sensitivity to benzylpenicillin and inability to support plaque formation of filamentous phages, and they were shown to severely affect disulfide bond oxidation of secreted proteins such as OmpA and &3lactamase. These phenotypes resemble the phenotype of bacteria carrying either a null mutation in the dsbA gene or the double mutation dsbA dsbB. Sequencing and expression of the dsbB gene revealed that it encodes a 20-kDa protein predicted to possess an "exchangeable" disulfide bond in -Cys-Val-Leu-Cys-. The dsbB gene maps at 26.5 min on the genetic map of the E. coil chromosome, and its transcription is directed from two promoters, neither of which resembles the canonical Eo7'-recognized promoter.

Characterization of the Menaquinone-dependent Disulfide Bond Formation Pathway of Escherichia coli

“…DsbA, a periplasmic enzyme of Escherichia coli, introduces disulfide bonds into newly exported proteins using its Cys 30 -Cys 33 active-site disulfide bond. DsbB, a plasma membrane protein, re-oxidizes the DsbA cysteines to enable catalytic functioning of DsbA (3)(4)(5)(6). The ultimate source of an oxidizing equivalent for this system is oxygen under aerobic conditions (7,8), in which respiratory chain components mediate electron transport (9).…”

A Genetic Screen for the Identification of Thiamin Metabolic Genes

“…Increased levels of prolipoprotein signal peptidase rendered cells resistant to globomycin, a specific inhibitor of that enzyme (10). Other selections have been used to identify proteins that could either detoxify an antibiotic or reverse the effects of the toxin (11,12).…”

Characterization of new DsbB-like thiol-oxidoreductases of Campylobacter jejuni and Helicobacter pylori and classification of the DsbB family based on phylogenomic, structural and functional criteria

“…Ellman's assay is a colorimetric reaction between Ellman's reagent [DTNB, 5,59-dithiobis(2-nitrobenzoic acid)] and the free thiol groups of proteins (Missiakas et al, 1993). Periplasmic protein samples from bacteria in the exponential phase of growth were isolated by a chloroform method (Ames et al, 1984) with a small modification.…”

“…To determine the effect of disruption of the dsbI gene in C. jejuni and H. pylori on the formation of disulfide bonds in the extracytoplasmic proteins, a quantitative assay of the free thiol groups in the periplasm was performed (Missiakas et al, 1993). The periplasmic protein samples from the wild-type and mutated strains were incubated with an excess of Ellman's reagent.…”

“…sgmjournals.org). It was shown that mutations in dsb genes of E. coli lead to an increase in sensitivity for reducing agents such as DTT (Missiakas et al, 1993). The result that C. jejuni dsbB is able to complement a defect in disulfide bond formation in E. coli was further supported by the observation that introducing pUWM602 into E. coli JCB656 restored its ability to grow in the presence of 15 mM DTT (DTT sensitivity data are available as supplementary data with the online version of this paper at http:// mic.sgmjournals.org).…”