A proteomic approach to study Salmonella typhi periplasmic proteins altered by a lack of the DsbA thiol: disulfide isomerase.
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.
Agudo
David
D
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Mendoza
María Teresa
MT
Castañares
Cristina
C
Nombela
César
C
Rotger
Rafael
R
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005407:Flagella / Q000473:pathology; D013058:Mass Spectrometry; D009154:Mutation; D019897:Periplasm / Q000378:metabolism*; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D012485:Salmonella typhi / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; C062887:agp protein, E coli; D010744:Phosphoric Monoester Hydrolases; D019704:Protein Disulfide-Isomerases