A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae.
A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.
Yu
J
J
Biological Laboratory, University of Kent, Canterbury, UK.
Webb
H
H
Hirst
T R
TR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000502:physiology; D004220:Disulfides / Q000378:metabolism; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics / Q000502:physiology*; D007535:Isomerases / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins / Q000096:biosynthesis; D012689:Sequence Homology, Nucleic Acid; D014734:Vibrio cholerae / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004251:DNA Transposable Elements; D004220:Disulfides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C027538:heat-labile enterotoxin, E coli; D007535:Isomerases; D019704:Protein Disulfide-Isomerases