1887

Abstract

The polar flagella of have sodium-driven motors, and four membrane proteins, PomA, PomB, MotX and MotY, are essential for torque generation of the motor. PomA and PomB are believed to form a sodium-conducting channel. This paper reports the purification of the motor complex by using sucrose monocaprate, a non-ionic detergent, to solubilize the complex. Plasmid pKJ301, which encodes intact PomA, and PomB tagged with a C-terminal hexahistidine that does not interfere with PomB function, was constructed. The membrane fraction of cells transformed with pKJ301 was solubilized with sucrose monocaprate, and the solubilized materials were applied to a Ni-NTA column. The imidazole eluate contained both PomA and PomB, which were further purified by anion-exchange chromatography. Gel-filtration chromatography was used to investigate the apparent molecular size of the complex; the PomA/PomB complex was eluted as approx. 900 kDa and PomB alone was eluted as approx. 260 kDa. These findings suggest that the motor complex may have a larger structure than previously assumed.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26577-0
2004-04-01
2020-08-07
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/4/mic1500911.html?itemId=/content/journal/micro/10.1099/mic.0.26577-0&mimeType=html&fmt=ahah

References

  1. Asai Y., Kojima S., Kato H., Nishioka N., Kawagishi I., Homma M.. 1997; Putative channel components for the fast-rotating sodium-driven flagellar motor of a marine bacterium. J Bacteriol179:5104–5110
    [Google Scholar]
  2. Asai Y., Kawagishi I., Sockett E., Homma M.. 1999; Hybrid motor with the H+- and Na+-driven components can rotate Vibrio polar flagella by using sodium ions. J Bacteriol181:6322–6338
    [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[CrossRef]
    [Google Scholar]
  4. Atsumi T., Sugiyama S., Cragoe E. J., Jr, Imae Y.. 1990; Specific inhibition of the Na+-driven flagellar motors of alkalophilic Bacillus strains by the amiloride analog phenamil. J Bacteriol172:1634–1639
    [Google Scholar]
  5. Atsumi T., McCarter L., Imae Y.. 1992; Polar and lateral flagellar motors of marine Vibrio are driven by different ion-motive forces. Nature355:182–184[CrossRef]
    [Google Scholar]
  6. Bakkeva L. E., Chumakov K. M., Drachev A. L., Metlina A. L., Skulachev V. P.. 1986; The sodium cycle. III. Vibrio alginolyticus resembles Vibrio cholerae and some other vibriones by flagellar motor and ribosomal 5S-RNA structures. Biochim Biophys Acta850:466–472[CrossRef]
    [Google Scholar]
  7. Bartolome B., Jubete Y., Martinez E., Cruz F.. 1991; Construction and properties of a family of pACY184-derived cloning vectors compatible with pBR322 and its derivatives. Gene102:75–78[CrossRef]
    [Google Scholar]
  8. Berry R. M., Armitage J. P.. 1999; The bacterial flagella motor. Adv Microb Physiol41:291–337
    [Google Scholar]
  9. Blair D. F.. 1995; How bacteria sense and swim. Annu Rev Microbiol49:489–522[CrossRef]
    [Google Scholar]
  10. Blair D. F.. 2003; Flagellar movement driven by proton translocation. FEBS Lett545:86–95[CrossRef]
    [Google Scholar]
  11. 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[CrossRef]
    [Google Scholar]
  12. Braun T. F., Blair D. F.. 2001; Targeted disulfide cross-linking of the MotB protein of Escherichia coli: evidence for two H+ channels in the stator complex. Biochemistry40:13051–13059[CrossRef]
    [Google Scholar]
  13. Chun S. Y., Parkinson J. S.. 1988; Bacterial motility: membrane topology of the Escherichia coli. MotB protein. Science239:276–278[CrossRef]
    [Google Scholar]
  14. DeRosier D. J.. 1998; The turn of the screw: the bacterial flagellar motor. Cell93:17–20[CrossRef]
    [Google Scholar]
  15. Driks A., DeRosier D. J.. 1990; Additional structures associated with bacterial flagellar basal body. J Mol Biol211:669–672[CrossRef]
    [Google Scholar]
  16. Furuno M., Nishioka N., Kawagishi I., Homma M.. 1999; Suppression by the DNA fragment of the motX promoter region on long flagellar mutants of Vibrio alginolyticus. Microbiol Immunol43:39–43[CrossRef]
    [Google Scholar]
  17. Garza A. G., Harrishaller L. W., Stoebner R. A., Manson M. D.. 1995; Motility protein interactions in the bacterial flagellar motor. Proc Natl Acad Sci U S A92:1970–1974[CrossRef]
    [Google Scholar]
  18. Garza A. G., Biran R., Wohlschlegel J. A., Manson M. D.. 1996; Mutations in motB suppressible by changes in stator or rotor components of the bacterial flagellar motor. J Mol Biol258:270–285[CrossRef]
    [Google Scholar]
  19. Gosink K. K., Häse C. C.. 2000; Requirements for conversion of the Na+-driven flagellar motor of Vibrio cholerae to the H+-driven motor ofEscherichia coli. J Bacteriol182:4234–4240[CrossRef]
    [Google Scholar]
  20. Homma M., Aizawa S.-I., Dean G. E., Macnab R. M.. 1987; Identification of the M-ring protein of the flagellar motor of Salmonella typhimurium. Proc Natl Acad Sci U S A84:7483–7487[CrossRef]
    [Google Scholar]
  21. Jaques S., Kim Y. K., McCarter L. L.. 1999; Mutations conferring resistance to phenamil and amiloride, inhibitors of sodium-driven motility of Vibrio parahaemolyticus. Proc Natl Acad Sci U S A96:5740–5745[CrossRef]
    [Google Scholar]
  22. Kawagishi I., Maekawa Y., Atsumi T., Homma M., Imae Y.. 1995; Isolation of the polar and lateral flagellum-defective mutants in Vibrio alginolyticus and identification of their flagellar driving energy sources. J Bacteriol177:5158–5160
    [Google Scholar]
  23. Kojima S., Blair D. F.. 2001; Conformational change in the stator of the bacterial flagellar motor. Biochemistry40:13041–13050[CrossRef]
    [Google Scholar]
  24. Kojima S., Atsumi T., Muramoto K., Kudo S., Kawagishi I., Homma M.. 1997; Vibrio alginolyticus mutants resistant to phenamil, a specific inhibitor of the sodium-driven flagellar motor. J Mol Biol265:310–318[CrossRef]
    [Google Scholar]
  25. Kojima S., Asai Y., Atsumi T., Kawagishi I., Homma M.. 1999; Na+-driven flagellar motor resistant to phenamil, an amiloride analog, caused by mutations of putative channel components. J Mol Biol285:1537–1547[CrossRef]
    [Google Scholar]
  26. Macnab R. M.. 1999; The bacterial flagellum: reversible rotary propellor and type III export apparatus. J Bacteriol181:7149–7153
    [Google Scholar]
  27. McCarter L. L.. 1994a; MotY, a component of the sodium-type flagellar motor. J Bacteriol176:4219–4225
    [Google Scholar]
  28. McCarter L. L.. 1994b; MotX, the channel component of the sodium-type flagellar motor. J Bacteriol176:5988–5998
    [Google Scholar]
  29. McCarter L. L.. 2001; Polar flagellar motility of the Vibrionaceae. Microbiol Mol Biol Rev65:445–462[CrossRef]
    [Google Scholar]
  30. Morales B. M., Backman A., Bagdasarian M.. 1991; A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene97:39–47[CrossRef]
    [Google Scholar]
  31. Okabe M., Yakushi T., Asai Y., Homma M.. 2001; Cloning and characterization of motX, a Vibrio alginolyticus sodium-driven flagellar motor gene. J Biochem130:879–884[CrossRef]
    [Google Scholar]
  32. Okabe M., Yakushi T., Kojima M., Homma M.. 2002; MotX and MotY, specific components of the sodium-driven flagellar motor, colocalize to the outer membrane in Vibrio alginolyticus. Mol Microbiol46:125–134[CrossRef]
    [Google Scholar]
  33. Okunishi I., Kawagishi I., Homma M.. 1996; Cloning and characterization of motY, a gene coding for a component of the sodium-driven flagellar motor inVibrio alginolyticus. J Bacteriol178:2409–2415
    [Google Scholar]
  34. Sato K., Homma M.. 2000a; Functional reconstitution of the Na+-driven polar flagellar motor component ofVibrio alginolyticus. J Biol Chem275:5718–5722[CrossRef]
    [Google Scholar]
  35. Sato K., Homma M.. 2000b; Multimeric structure of PomA, the Na+-driven polar flagellar motor component of Vibrio alginolyticus. J Biol Chem275:20223–20228[CrossRef]
    [Google Scholar]
  36. Sharp L. L., Zhou J. D., Blair D. F.. 1995a; Tryptophan-scanning mutagenesis of MotB, an integral membrane protein essential for flagellar rotation in Escherichia coli. Biochemistry34:9166–9171[CrossRef]
    [Google Scholar]
  37. Sharp L. L., Zhou J. D., Blair D. F.. 1995b; Features of MotA proton channel structure revealed by tryptophan-scanning mutagenesis. Proc Natl Acad Sci U S A92:7946–7950[CrossRef]
    [Google Scholar]
  38. 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]
  39. Sugiyama S., Imae Y., Cragoe E. J., Jr. 1988; Amiloride, a specific inhibitor for the Na+-driven flagellar motors of alkalophilicBacillus. J Biol Chem263:8215–8219
    [Google Scholar]
  40. Tang H., Braun T. F., Blair D. F.. 1996; Motility protein complexes in the bacterial flagellar motor. J Mol Biol261:209–221[CrossRef]
    [Google Scholar]
  41. Wilson M. L., Macnab R. M.. 1990; Co-overproduction and localization of the Escherichia coli motility proteins MotA and MotB. J Bacteriol173:3932–3939
    [Google Scholar]
  42. Yorimitsu T., Homma M.. 2001; Na+-driven flagellar motor of Vibrio. Biochim Biophys Acta 1505;82–93[CrossRef]
    [Google Scholar]
  43. Yorimitsu T., Sato K., Asai Y., Kawagishi I., Homma M.. 1999; Functional interaction between PomA and PomB, the Na+-driven flagellar motor components ofVibrio alginolyticus. J Bacteriol181:5103–5106
    [Google Scholar]
  44. Yorimitsu T., Kojima M., Yakukshi T., Homma M.. 2004; Multimeric structure of the PomA/PomB channel complex in the Na+-driven flagellar motor ofVibrio alginolyticus. J Biochem185:43–45
    [Google Scholar]
  45. Zhou J. D., Fazzio R. T., Blair D. F.. 1995; Membrane topology of the MotA protein of Escherichia coli. J Mol Biol251:237–242[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26577-0
Loading
/content/journal/micro/10.1099/mic.0.26577-0
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