Periplasmic and fimbrial SefA from Salmonella enteritidis.
Salmonella enteritidis produces thin, filamentous fimbriae composed of the fimbrin subunit SefA. Although insoluble in most detergents and chaotropic agents, these fimbriae were soluble at pH 10.5. Furthermore, in sodium dodecyl sulfate, these fibers depolymerized into monomers, dimers and other multimers of SefA, which precipitated on removal of the detergent. In contrast, unassembled periplasmic SefA fimbrins purified from Escherichia coli expressing cloned sefA and sefB were readily soluble in aqueous solution. Fimbrial and periplasmic SefA also differed in their reaction with an anti-SEF14 monoclonal antibody and in their surface hydrophobicity, indicating that the two forms had different properties. Precise mass measurements of periplasmic and fimbrial SefA by mass spectroscopy showed that these variations were not due to post-translational modifications. Periplasmic SefA consisted primarily of intact as well as some N-terminally truncated forms. The main 24 amino acid, N-terminally truncated form of periplasmic SefA was present as a 12.2 kDa monomer which had a low tendency to dimerize whereas intact periplasmic SefA was present as a 34.1 kDa homodimer. Intact periplasmic SefA also formed stable multimers at low concentrations of chemical cross-linker but multimerization of the truncated form required high concentrations of protein or cross-linker. Thus, SefA fimbrins appear to multimerize through their N-termini and undergo a conformational change prior to assembly into fibers. Within these fibers, subunit-subunit contact is maintained through strong hydrophobic interactions.
Clouthier
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Department of Biochemistry and Microbiology, Petch Building, University of Victoria, P.O. Box 3055, Victoria, B.C. V8W 3P6, Canada.
Collinson
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Lippert
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Ausio
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White
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Kay
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D001426:Bacterial Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000378:metabolism; D036781:Fimbriae Proteins; D019897:Periplasm / Q000737:chemistry; D018933:Pili, Sex / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012477:Salmonella enteritidis / Q000737:chemistry*; D013388:Succinimides / Q000378:metabolism; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D013388:Succinimides; C080931:sefA protein, Salmonella enteritidis; D036781:Fimbriae Proteins; C035760:bis(sulfosuccinimidyl)suberate