| In this study, dsbA from Salmonella typhimurium was cloned and sequenced. It shares 87% amino acid identity, including the active site and the proposed substrate-binding domains, with the Escherichia coli version of the same protein. This is confirmed by the strong cross-reactivity of S. typhimurium DsbA with antibodies raised against E. coli DsbA. PefS has only 33% identity with E. coli DsbA at the amino acid level and does not cross react with the DsbA antisera.; By diverse functional analyses, such as motility, {dollar}beta{dollar}-galactosidase and alkaline phosphatase assays, we demonstrate that in an E. coli dsbA null background strain, DsbA from S. typhimurium can functionally replace the E. coli DsbA. PefS is less active than the E. coli and S. typhimurium DsbA's in the oxidation of the same cysteine-containing substrates.; To determine the contribution of DsbA to the proper folding and assembly of several proteins of S. typhimurium, deletion mutants were created in the avirulent strain SARA2 as well as in the virulent strain SL1344. These null alleles were constructed by partial deletion of the dsbA coding region and insertion of an antibiotic resistance marker in the gene. A counter-selectable suicide vector was used to recombine the null allele back into S. typhimurium. Mutants no longer express a functional disulfide oxidoreductase and affect disulfide bond formation in a number of proteins in S. typhimurium. In DsbA mutants, colony morphology is changed, motility is affected, and susceptibility to some antimicrobial agents including the cationic peptide protamine sulphate is increased. In addition, the disruption of disulfide bond formation affects the protein profile of the {dollar}dsbAsp-{dollar} strains. (Abstract shortened by UMI.)... |
