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A proteomic approach to study Salmonella typhi periplasmic proteins altered by a lack of the DsbA thiol: Disulfide isomerase
Author(s) -
Agudo David,
Mendoza María Teresa,
Castañares Cristina,
Nombela César,
Rotger Rafael
Publication year - 2004
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200300554
Subject(s) - dsba , periplasmic space , protein disulfide isomerase , mutant , biology , escherichia coli , biochemistry , microbiology and biotechnology , chemistry , gene , enzyme
Two‐dimensional electrophoresis (2‐DE) was used to analyze the pleiotropic effects of a deficiency in DsbA, a periplasmic disulfide‐bond oxidoreductase, in Salmonella typhi . With this aim, the dsbA gene was cloned and assayed for activity in a dsbA ‐null mutant of Escherichia coli . A dsbA/chloramphenicol acetylase construct was then used to disrupt the wild‐type gene of S. typhi . The resultant dsbA ‐null mutant of S. typhi , like the E. coli mutant, exhibited a lack of flagellation and of glucose‐1‐phosphatase activity. Periplasmic extracts from the parental and mutant strains were analyzed by 2‐DE using standard denaturing and nondenaturing conditions. Differences in protein expression were more marked in nondenaturing conditions. Ninety‐nine protein spots were analyzed by peptide mass fingerprinting, and 65 spots were identified by searching a S. typhi database. Twenty‐five spots were exclusively detected in the wild‐type strain, 10 were found only in the mutant strain, and 21 were common to both strains. We observed a lack of DsbA, glucose‐1‐phosphatase and flagellin in the dsbA ‐null mutant, which explains two of the observed phenotypes. The AI‐2 autoinducer‐producing protein LuxS, which is involved in quorum‐sensing signalling was also absent.