Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1985 Mar;161(3):836–849. doi: 10.1128/jb.161.3.836-849.1985

Purification and characterization of the flagellar hook-basal body complex of Salmonella typhimurium.

S I Aizawa, G E Dean, C J Jones, R M Macnab, S Yamaguchi
PMCID: PMC214974  PMID: 2982790

Abstract

The hook-basal body complex of Salmonella typhimurium, a major component of its flagellar apparatus, was subjected to detailed analysis by electron microscopy and gel electrophoresis. The study was facilitated by the development of an improved protocol for isolation of the complexes in high yield and purity. Nine proteins were identified with the structure. These proteins had apparent molecular weights of 65,000 (65K), 60K, 42K, 38K, 32K, 30K, 27K, 16K, and 14K. Small but reproducible shifts in the apparent molecular weights of specific proteins from conditionally nonflagellate mutants indicated the following gene-polypeptide correspondences: flaFV, 42K; flaFVI, 32K; flaFVII, 30K; flaFIX, 38K; flaAII.1, 65K. Several new morphological features of hook-basal body complexes were recognized, including a clawlike structure on the cytoplasm-proximal M ring and additional material at the cytoplasmic face of the M ring. Based on this study and the work of others, we suggest that the morphological features of the hook-basal body complex correspond to the following proteins: hook-filament junction, 60K; hook, 42K; rod, 30K and 32K; L ring and outer cylinder wall, 27K; P ring, 38K; S ring, unknown; M ring 65K.

Full text

PDF
836

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ASAKURA S., EGUCHI G., IINO T. RECONSTITUTION OF BACTERIAL FLAGELLA IN VITRO. J Mol Biol. 1964 Oct;10:42–56. doi: 10.1016/s0022-2836(64)80026-7. [DOI] [PubMed] [Google Scholar]
  2. Aizawa S. I., Kato S., Asakura S., Kagawa H., Yamaguchi S. In vitro polymerization of polyhook protein from Salmonella SJW880. Biochim Biophys Acta. 1980 Oct 21;625(2):291–303. doi: 10.1016/0005-2795(80)90293-7. [DOI] [PubMed] [Google Scholar]
  3. Aizawa S., Maéda Y. A new method for determination of parity in optical diffraction patterns from the structures with helical symmetry. J Mol Biol. 1980 Mar 15;137(4):437–442. doi: 10.1016/0022-2836(80)90168-0. [DOI] [PubMed] [Google Scholar]
  4. Ames G. F., Nikaido K. Two-dimensional gel electrophoresis of membrane proteins. Biochemistry. 1976 Feb 10;15(3):616–623. doi: 10.1021/bi00648a026. [DOI] [PubMed] [Google Scholar]
  5. Asakura S. Polymerization of flagellin and polymorphism of flagella. Adv Biophys. 1970;1:99–155. [PubMed] [Google Scholar]
  6. Aswad D., Koshland D. E., Jr Isolation, characterization and complementation of Salmonella typhimurium chemotaxis mutants. J Mol Biol. 1975 Sep 15;97(2):225–235. doi: 10.1016/s0022-2836(75)80036-2. [DOI] [PubMed] [Google Scholar]
  7. Bartlett D. H., Matsumura P. Identification of Escherichia coli region III flagellar gene products and description of two new flagellar genes. J Bacteriol. 1984 Nov;160(2):577–585. doi: 10.1128/jb.160.2.577-585.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Berg H. C. Dynamic properties of bacterial flagellar motors. Nature. 1974 May 3;249(452):77–79. doi: 10.1038/249077a0. [DOI] [PubMed] [Google Scholar]
  9. Block S. M., Berg H. C. Successive incorporation of force-generating units in the bacterial rotary motor. 1984 May 31-Jun 6Nature. 309(5967):470–472. doi: 10.1038/309470a0. [DOI] [PubMed] [Google Scholar]
  10. Collins A. L., Stocker B. A. Salmonella typhimurium mutants generally defective in chemotaxis. J Bacteriol. 1976 Dec;128(3):754–765. doi: 10.1128/jb.128.3.754-765.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DePamphilis M. L., Adler J. Attachment of flagellar basal bodies to the cell envelope: specific attachment to the outer, lipopolysaccharide membrane and the cyoplasmic membrane. J Bacteriol. 1971 Jan;105(1):396–407. doi: 10.1128/jb.105.1.396-407.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. DePamphilis M. L., Adler J. Fine structure and isolation of the hook-basal body complex of flagella from Escherichia coli and Bacillus subtilis. J Bacteriol. 1971 Jan;105(1):384–395. doi: 10.1128/jb.105.1.384-395.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. DePamphilis M. L., Adler J. Purification of intact flagella from Escherichia coli and Bacillus subtilis. J Bacteriol. 1971 Jan;105(1):376–383. doi: 10.1128/jb.105.1.376-383.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. DePamphilis M. L. Dissociation and reassembly of Escherichia coli outer membrane and of lipopolysaccharide, and their reassembly onto flagellar basal bodies. J Bacteriol. 1971 Mar;105(3):1184–1199. doi: 10.1128/jb.105.3.1184-1199.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dean G. E., Aizawa S. I., Macnab R. M. flaAII (motC, cheV) of Salmonella typhimurium is a structural gene involved in energization and switching of the flagellar motor. J Bacteriol. 1983 Apr;154(1):84–91. doi: 10.1128/jb.154.1.84-91.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dean G. E., Macnab R. M., Stader J., Matsumura P., Burks C. Gene sequence and predicted amino acid sequence of the motA protein, a membrane-associated protein required for flagellar rotation in Escherichia coli. J Bacteriol. 1984 Sep;159(3):991–999. doi: 10.1128/jb.159.3.991-999.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dimmitt K., Simon M. Purification and thermal stability of intact Bacillus subtilis flagella. J Bacteriol. 1971 Jan;105(1):369–375. doi: 10.1128/jb.105.1.369-375.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Enomoto M. Genetic studies of paralyzed mutant in Salmonella. I. Genetic fine structure of the mot loci in Salmonella typhimurium. Genetics. 1966 Sep;54(3):715–726. doi: 10.1093/genetics/54.3.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fujita H., Yamaguchi S., Taira T., Iino T. A simple method for the isolation of flagellar shape mutants in Salmonella. J Gen Microbiol. 1981 Jul;125(1):213–216. doi: 10.1099/00221287-125-1-213. [DOI] [PubMed] [Google Scholar]
  20. Hilmen M., Silverman M., Simon M. The regulation of flagellar formation and function. J Supramol Struct. 1974;2(2-4):360–371. doi: 10.1002/jss.400020225. [DOI] [PubMed] [Google Scholar]
  21. Homma M., Fujita H., Yamaguchi S., Iino T. Excretion of unassembled flagellin by Salmonella typhimurium mutants deficient in hook-associated proteins. J Bacteriol. 1984 Sep;159(3):1056–1059. doi: 10.1128/jb.159.3.1056-1059.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Homma M., Kutsukake K., Iino T., Yamaguchi S. Hook-associated proteins essential for flagellar filament formation in Salmonella typhimurium. J Bacteriol. 1984 Jan;157(1):100–108. doi: 10.1128/jb.157.1.100-108.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Iino T. Genetics of structure and function of bacterial flagella. Annu Rev Genet. 1977;11:161–182. doi: 10.1146/annurev.ge.11.120177.001113. [DOI] [PubMed] [Google Scholar]
  24. Kagawa H., Aizawa S. I., Asakura S. Transformations in isolated polyhooks. J Mol Biol. 1979 Apr 5;129(2):333–336. doi: 10.1016/0022-2836(79)90286-9. [DOI] [PubMed] [Google Scholar]
  25. Kagawa H., Owaribe K., Asakura S., Takahashi N. Flagellar hook protein from Salmonella SJ25. J Bacteriol. 1976 Jan;125(1):68–73. doi: 10.1128/jb.125.1.68-73.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kamiya R., Asakura S. Flagellar transformations at alkaline pH. J Mol Biol. 1976 Dec;108(2):513–518. doi: 10.1016/s0022-2836(76)80133-7. [DOI] [PubMed] [Google Scholar]
  27. Komeda Y., Silverman M., Matsumura P., Simon M. Genes for the hook-basal body proteins of the flagellar apparatus in Escherichia coli. J Bacteriol. 1978 May;134(2):655–667. doi: 10.1128/jb.134.2.655-667.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kutsukake K., Iino T., Komeda Y., Yamaguchi S. Functional homology of fla genes between Salmonella typhimurium and Escherichia coli. Mol Gen Genet. 1980 Apr;178(1):59–67. doi: 10.1007/BF00267213. [DOI] [PubMed] [Google Scholar]
  29. Kutsukake K., Suzuki T., Yamaguchi S., Iino T. Role of gene flaFV on flagellar hook formation in Salmonella typhimurium. J Bacteriol. 1979 Oct;140(1):267–275. doi: 10.1128/jb.140.1.267-275.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  31. Larsen S. H., Reader R. W., Kort E. N., Tso W. W., Adler J. Change in direction of flagellar rotation is the basis of the chemotactic response in Escherichia coli. Nature. 1974 May 3;249(452):74–77. doi: 10.1038/249074a0. [DOI] [PubMed] [Google Scholar]
  32. MEYNELL E. W. A phage, phi chi, which attacks motile bacteria. J Gen Microbiol. 1961 Jun;25:253–290. doi: 10.1099/00221287-25-2-253. [DOI] [PubMed] [Google Scholar]
  33. Macnab R. M., Aizawa S. Bacterial motility and the bacterial flagellar motor. Annu Rev Biophys Bioeng. 1984;13:51–83. doi: 10.1146/annurev.bb.13.060184.000411. [DOI] [PubMed] [Google Scholar]
  34. Macnab R. M. Examination of bacterial flagellation by dark-field microscopy. J Clin Microbiol. 1976 Sep;4(3):258–265. doi: 10.1128/jcm.4.3.258-265.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Macnab R. M., Ornston M. K. Normal-to-curly flagellar transitions and their role in bacterial tumbling. Stabilization of an alternative quaternary structure by mechanical force. J Mol Biol. 1977 May 5;112(1):1–30. doi: 10.1016/s0022-2836(77)80153-8. [DOI] [PubMed] [Google Scholar]
  36. Macnab R., Koshland D. E., Jr Bacterial motility and chemotaxis: light-induced tumbling response and visualization of individual flagella. J Mol Biol. 1974 Apr 15;84(3):399–406. doi: 10.1016/0022-2836(74)90448-3. [DOI] [PubMed] [Google Scholar]
  37. Manson M. D., Tedesco P., Berg H. C., Harold F. M., Van der Drift C. A protonmotive force drives bacterial flagella. Proc Natl Acad Sci U S A. 1977 Jul;74(7):3060–3064. doi: 10.1073/pnas.74.7.3060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Matsumura P., Silverman M., Simon M. Cloning and expression of the flagellar hook gene on hybird plasmids in minicells. Nature. 1977 Feb 24;265(5596):758–760. doi: 10.1038/265758a0. [DOI] [PubMed] [Google Scholar]
  39. Matsura S., Shioi J., Imae Y. Motility in Bacillus subtilis driven by an artificial protonmotive force. FEBS Lett. 1977 Oct 15;82(2):187–190. doi: 10.1016/0014-5793(77)80581-4. [DOI] [PubMed] [Google Scholar]
  40. Merril C. R., Switzer R. C., Van Keuren M. L. Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4335–4339. doi: 10.1073/pnas.76.9.4335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  42. O'Brien E. J., Bennett P. M. Structure of straight flagella from a mutant Salmonella. J Mol Biol. 1972 Sep 14;70(1):133–152. doi: 10.1016/0022-2836(72)90168-4. [DOI] [PubMed] [Google Scholar]
  43. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  44. Ochs D. Protein contaminants of sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem. 1983 Dec;135(2):470–474. doi: 10.1016/0003-2697(83)90714-5. [DOI] [PubMed] [Google Scholar]
  45. Parkinson J. S., Parker S. R., Talbert P. B., Houts S. E. Interactions between chemotaxis genes and flagellar genes in Escherichia coli. J Bacteriol. 1983 Jul;155(1):265–274. doi: 10.1128/jb.155.1.265-274.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Patterson-Delafield J., Martinez R. J., Stocker B. A., Yamaguchi S. A new fla gene in Salmonella typhimurium--flaR--and its mutant phenotype-superhooks. Arch Mikrobiol. 1973 Mar 26;90(2):107–120. doi: 10.1007/BF00414513. [DOI] [PubMed] [Google Scholar]
  47. Rosenbusch J. P. Characterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptidoglycan and unusual dodecyl sulfate binding. J Biol Chem. 1974 Dec 25;249(24):8019–8029. [PubMed] [Google Scholar]
  48. Silverman M., Simon M. Flagellar rotation and the mechanism of bacterial motility. Nature. 1974 May 3;249(452):73–74. doi: 10.1038/249073a0. [DOI] [PubMed] [Google Scholar]
  49. Suzuki T., Iino T., Horiguchi T., Yamaguchi S. Incomplete flagellar structures in nonflagellate mutants of Salmonella typhimurium. J Bacteriol. 1978 Feb;133(2):904–915. doi: 10.1128/jb.133.2.904-915.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Suzuki T., Komeda Y. Incomplete flagellar structures in Escherichia coli mutants. J Bacteriol. 1981 Feb;145(2):1036–1041. doi: 10.1128/jb.145.2.1036-1041.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tasheva B., Dessev G. Artifacts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to 2-mercaptoethanol. Anal Biochem. 1983 Feb 15;129(1):98–102. doi: 10.1016/0003-2697(83)90057-x. [DOI] [PubMed] [Google Scholar]
  52. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  53. Wagenknecht T., DeRosier D. J., Aizawa S., Macnab R. M. Flagellar hook structures of Caulobacter and Salmonella and their relationship to filament structure. J Mol Biol. 1982 Nov 25;162(1):69–87. doi: 10.1016/0022-2836(82)90162-0. [DOI] [PubMed] [Google Scholar]
  54. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  55. Yamaguchi S., Fujita H., Sugata K., Taira T., Iino T. Genetic analysis of H2, the structural gene for phase-2 flagellin in Salmonella. J Gen Microbiol. 1984 Feb;130(2):255–265. doi: 10.1099/00221287-130-2-255. [DOI] [PubMed] [Google Scholar]
  56. Zieg J., Hilmen M., Simon M. Regulation of gene expression by site-specific inversion. Cell. 1978 Sep;15(1):237–244. doi: 10.1016/0092-8674(78)90098-3. [DOI] [PubMed] [Google Scholar]
  57. de Jong W. W., Zweers A., Cohen L. H. Influence of single amino acid substitutions on electrophoretic mobility of sodium dodecyl sulfate-protein complexes. Biochem Biophys Res Commun. 1978 May 30;82(2):532–539. doi: 10.1016/0006-291x(78)90907-5. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES