Isolation and characterization of a chromosomally encoded disulphide oxidoreductase from Salmonella enterica serovar Typhimurium.
In this study, the chromosomally encoded disulphide oxidoreductase dsbA from Salmonella typhimurium was cloned and characterized. A survey of a number of serovars of Salmonella subspecies I showed that dsbA is highly conserved in most, but not all members of this subclass of Salmonella species. Using motility, beta-galactosidase, and alkaline phosphatase assays as indirect indicators of disulphide oxidoreductase activity, we demonstrated that DsbA from S. typhimurium LT2 can only partially complement an Escherichia coli dsbA-null strain. This is surprising considering the high degree of conservation between these two DsbA proteins (87% amino acid identity). To determine the contribution of DsbA to the proper folding and assembly of proteins of S. typhimurium, deletion mutants were created in the avirulent strain LT2 and in the virulent strain SL1344. These null alleles were constructed by partial deletion of the dsbA-coding region and then insertion of an antibiotic resistance marker in the gene. Mutants no longer expressing a functional disulphide oxidoreductase exhibit pleitropic effects, including an increase in colony mucoidy, a dramatic decrease in motility, and an increased susceptibility to the cationic peptide protamine sulphate. The disruption of disulphide bond formation was also shown to specifically affect the stability of several proteins secreted into the extracellular environment.
Turcot
I
I
Department of Microbiology and Immunology, Queen's University, Kingston, ON, Canada.
Ponnampalam
T V
TV
Bouwman
C W
CW
Martin
N L
NL
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011479:Protamines / Q000494:pharmacology; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017510:Protein Folding; D012486:Salmonella typhimurium / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D011479:Protamines; D019704:Protein Disulfide-Isomerases