1887

Abstract

The bacterial flagellar motor is embedded in the cytoplasmic membrane, and penetrates the peptidoglycan layer and the outer membrane. A ring structure of the basal body called the P ring, which is located in the peptidoglycan layer, is thought to be required for smooth rotation and to function as a bushing. In this work, we characterized 32 cysteine-substituted P-ring protein FlgI variants which were designed to substitute every 10th residue in the 346 aa mature form of FlgI. Immunoblot analysis against FlgI protein revealed that the cellular amounts of five FlgI variants were significantly decreased. Swarm assays showed that almost all of the variants had nearly wild-type function, but five variants significantly reduced the motility of the cells, and one of them in particular, FlgI G21C, completely disrupted FlgI function. The five residues that impaired motility of the cells were localized in the N terminus of FlgI. To demonstrate which residue(s) of FlgI is exposed to solvent on the surface of the protein, we examined cysteine modification by using the thiol-specific reagent methoxypolyethylene glycol 5000 maleimide, and classified the FlgI Cys variants into three groups: well-, moderately and less-labelled. Interestingly, the well- and moderately labelled residues of FlgI never overlapped with the residues known to be important for protein amount or motility. From these results and multiple alignments of amino acid sequences of various FlgI proteins, the highly conserved region in the N terminus, residues 1–120, of FlgI is speculated to play important roles in the stabilization of FlgI structure and the formation of the P ring by interacting with FlgI molecules and/or other flagellar components.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/013854-0
2008-03-01
2020-08-11
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/3/810.html?itemId=/content/journal/micro/10.1099/mic.0.2007/013854-0&mimeType=html&fmt=ahah

References

  1. Akiba T., Yoshimura H., Namba K.. 1991; Monolayer crystallization of flagellar L-P rings by sequential addition and depletion of lipid. Science252:1544–1546
    [Google Scholar]
  2. Akiyama Y., Kanehara K., Ito K.. 2004; RseP (YaeL), an Escherichia coli RIP protease, cleaves transmembrane sequences. EMBO J23:4434–4442
    [Google Scholar]
  3. Asai Y., Yakushi T., Kawagishi I., Homma M.. 2003; Ion-coupling determinants of Na+-driven and H+-driven flagellar motors. J Mol Biol327:453–463
    [Google Scholar]
  4. Bartolome B., Jubete Y., Martinez E., de la Cruz F.. 1991; Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives. Gene102:75–78
    [Google Scholar]
  5. Blair D. F., Berg H. C.. 1990; The MotA protein of E. coli is a proton-conducting component of the flagellar motor. Cell60:439–449
    [Google Scholar]
  6. Datsenko K. A., Wanner B. L.. 2000; One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A97:6640–6645
    [Google Scholar]
  7. DeRosier D. J.. 1998; The turn of the screw: the bacterial flagellar motor. Cell93:17–20
    [Google Scholar]
  8. Guzman L. M., Belin D., Carson M. J., Beckwith J.. 1995; Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol177:4121–4130
    [Google Scholar]
  9. Hizukuri Y., Yakushi T., Kawagishi I., Homma M.. 2006; Role of the intramolecular disulfide bond in FlgI, the flagellar P-ring component of Escherichia coli . J Bacteriol188:4190–4197
    [Google Scholar]
  10. Homma M., Komeda Y., Iino T., Macnab R. M.. 1987; The flaFIX gene product of Salmonella typhimurium is a flagellar basal body component with a signal peptide for export. J Bacteriol169:1493–1498
    [Google Scholar]
  11. Jones C. J., Homma M., Macnab R. M.. 1989; L-, P-, and M-ring proteins of the flagellar basal body of Salmonella typhimurium : gene sequences and deduced protein sequences. J Bacteriol171:3890–3900
    [Google Scholar]
  12. Jones C. J., Macnab R. M., Okino H., Aizawa S.. 1990; Stoichiometric analysis of the flagellar hook-(basal-body) complex of Salmonella typhimurium . J Mol Biol212:377–387
    [Google Scholar]
  13. Kojima S., Blair D. F.. 2004; Solubilization and purification of the MotA/MotB complex of Escherichia coli . Biochemistry43:26–34
    [Google Scholar]
  14. Kubori T., Shimamoto N., Yamaguchi S., Namba K., Aizawa S.. 1992; Morphological pathway of flagellar assembly in Salmonella typhimurium . J Mol Biol226:433–446
    [Google Scholar]
  15. Leake M. C., Chandler J. H., Wadhams G. H., Bai F., Berry R. M., Armitage J. P.. 2006; Stoichiometry and turnover in single, functioning membrane protein complexes. Nature443:355–358
    [Google Scholar]
  16. Okabe M., Yakushi T., Homma M.. 2005; Interactions of MotX with MotY and with the PomA/PomB sodium ion channel complex of the Vibrio alginolyticus polar flagellum. J Biol Chem280:25659–25664
    [Google Scholar]
  17. Parkinson J. S., Houts S. E.. 1982; Isolation and behavior of Escherichia coli deletion mutants lacking chemotaxis functions. J Bacteriol151:106–113
    [Google Scholar]
  18. Reid S. W., Leake M. C., Chandler J. H., Lo C. J., Armitage J. P., Berry R. M.. 2006; The maximum number of torque-generating units in the flagellar motor of Escherichia coli is at least 11. Proc Natl Acad Sci U S A103:8066–8071
    [Google Scholar]
  19. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  20. Silhavy T. J., Berman M. L., Enquist L. W.. 1984; Experiments with Gene Fusions Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  21. Sosinsky G. E., Francis N. R., DeRosier D. J., Wall J. S., Simon M. N., Hainfeld J.. 1992; Mass determination and estimation of subunit stoichiometry of the bacterial hook–basal body flagellar complex of Salmonella typhimurium by scanning transmission electron microscopy. Proc Natl Acad Sci U S A89:4801–4805
    [Google Scholar]
  22. Stolz B., Berg H. C.. 1991; Evidence for interactions between MotA and MotB, torque-generating elements of the flagellar motor of Escherichia coli . J Bacteriol173:7033–7037
    [Google Scholar]
  23. Terashima H., Fukuoka H., Yakushi T., Kojima S., Homma M.. 2006; The Vibrio motor proteins, MotX and MotY, are associated with the basal body of Na+-driven flagella and required for stator formation. Mol Microbiol62:1170–1180
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/013854-0
Loading
/content/journal/micro/10.1099/mic.0.2007/013854-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error