A proteomic approach to study Salmonella typhi periplasmic proteins altered by a lack of the DsbA thiol:: Disulfide isomerase

被引:22
作者
Agudo, D [1 ]
Mendoza, MT [1 ]
Castañares, C [1 ]
Nombela, C [1 ]
Rotger, R [1 ]
机构
[1] Univ Complutense Madrid, Fac Farm, Dept Microbiol 2, E-28040 Madrid, Spain
关键词
disulfide bonds; DsbA; periplasm; Salmonella typhi; two-dimensional gel electrophoresis;
D O I
10.1002/pmic.200300554
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 Al-2 autoinducer-producing protein LuxS, which is involved in quorum-sensing signalling was also absent.
引用
收藏
页码:355 / 363
页数:9
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