1887

Abstract

The motor proteins FliM and FliG physically interact, presumably to control one or more of the functions of the bacterial flagellum clockwise/counterclockwise (CW/CCW) switch. We have previously demonstrated this interaction using the yeast two-hybrid system and have identified mutations in that disrupt the interaction. Starting with the most interaction-defective of these mutants, we mutagenized to identify suppressor mutations that restore the FliM/FliG two-hybrid interaction. Certain suppressor mutations exhibit allele specificity. These mutations help define a FliG-interaction surface on FliM. Moreover, the pattern of suppression suggests that two distinct sites on FliG interact with FliM, perhaps with two FliM molecules in a dimer per molecule of FliG.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/014597-0
2008-03-01
2020-07-09
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/3/714.html?itemId=/content/journal/micro/10.1099/mic.0.2007/014597-0&mimeType=html&fmt=ahah

References

  1. Armstrong J. B., Adler J., Dahl M. M.. 1967; Nonchemotactic mutants of Escherichia coli . J Bacteriol93:390–398
    [Google Scholar]
  2. Bartlett D. H., Matsumura P.. 1984; Identification of Escherichia coli region III flagellar gene products and description of two new flagellar genes. J Bacteriol160:577–585
    [Google Scholar]
  3. Berg H. C.. 1988; A physicist looks at bacterial chemotaxis. Cold Spring Harb Symp Quant Biol53:1–9
    [Google Scholar]
  4. Blomfield I. C., Vaughn V., Rest R. F., Eisenstein B. I.. 1991; Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol Microbiol5:1447–1457
    [Google Scholar]
  5. Brent R., Finley R. L. Jr. 1997; Understanding gene and allele function with two-hybrid methods. Annu Rev Genet31:663–704
    [Google Scholar]
  6. Brown P. N., Hill C. P., Blair D. F.. 2002; Crystal structure of the middle and C-terminal domains of the flagellar rotor protein FliG. EMBO J21:3225–3234
    [Google Scholar]
  7. Brown P. N., Mathews M. A. A., Joss L. A., Hill C. P., Blair D. F.. 2005; Crystal structure of the flagellar rotor protein FliN from Thermotoga maritima . J Bacteriol187:2890–2902
    [Google Scholar]
  8. Brown P. N., Terrazas M., Paul K., Blair D. F.. 2007; Mutational analysis of the flagellar protein FliG: sites of interaction with FliM and implications for organization of the switch complex. J Bacteriol189:305–312
    [Google Scholar]
  9. Chien C.-T., Bartel P. L., Sternglanz R., Fields S.. 1991; The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A88:9578–9582
    [Google Scholar]
  10. Estojak J., Brent R., Golemis E. A.. 1995; Correlation of two-hybrid affinity data with in vitro measurements. Mol Cell Biol15:5820–5829
    [Google Scholar]
  11. Fields S., Song O.-K.. 1989; A novel genetic system to detect protein–protein interactions. Nature340:245–246
    [Google Scholar]
  12. Francis N. R., Irikura V. M., Yamaguchi S., DeRosier D. J., Macnab R. M.. 1992; Localization of the Salmonella typhimurium flagellar switch protein FliG to the cytoplasmic M-ring face of the basal body. Proc Natl Acad Sci U S A89:6304–6308
    [Google Scholar]
  13. Francis N. R., Sosinsky G. E., Thomas D., DeRosier D. J.. 1994; Isolation, characterization and structure of bacterial flagellar motors containing the switch complex. J Mol Biol235:1261–1270
    [Google Scholar]
  14. Gill G., Ptashne M.. 1987; Mutants of GAL4 protein altered in activation function. Cell51:121–126
    [Google Scholar]
  15. Gillen K. L., Hughes K. T.. 1991; Negative regulatory loci coupling flagellin synthesis to flagellar assembly in Salmonella typhimurium . J Bacteriol173:6453–6459
    [Google Scholar]
  16. González-Pedrajo B., Minamino T., Kihara M., Namba K.. 2006; Interactions between C ring proteins and export apparatus components: a possible mechanism for facilitating type III protein export. Mol Microbiol60:984–998
    [Google Scholar]
  17. Grünenfelder B., Gehrig S., Jenal U.. 2003; Role of the cytoplasmic C terminus of the FliF motor protein in flagellar assembly and rotation. J Bacteriol185:1624–1633
    [Google Scholar]
  18. Heimbrook M. E., Wang W. L. L., Campbell G.. 1989; Staining bacterial flagella easily. J Clin Microbiol27:2612–2615
    [Google Scholar]
  19. Irikura V. M., Kihara M., Yamaguchi S., Sockett H., Macnab R. M.. 1993; Salmonella typhimurium fliG and fliN mutations causing defects in assembly, rotation, and switching of the flagellar motor. J Bacteriol175:802–810
    [Google Scholar]
  20. Jarvik J., Botstein D.. 1975; Conditional-lethal mutations that suppress genetic defects in morphogenesis by altering structural proteins. Proc Natl Acad Sci U S A72:2738–2742
    [Google Scholar]
  21. 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]
  22. Khan I. H., Reese T. S., Khan S.. 1992; The cytoplasmic component of the bacterial flagellar motor. Proc Natl Acad Sci U S A89:5956–5960
    [Google Scholar]
  23. Khan S., Zhao R., Reese T. S.. 1998; Architectural features of the Salmonella typhimurium flagellar motor switch revealed by disrupted C-rings. J Struct Biol122:311–319
    [Google Scholar]
  24. Kihara M., Miller G. U., Macnab R. M.. 2000; Deletion analysis of the flagellar switch protein FliG of Salmonella . J Bacteriol182:3022–3028
    [Google Scholar]
  25. Kutsukake K., Ohya Y., Iino T.. 1990; Transcriptional analysis of the flagellar regulon in Salmonella typhimurium . J Bacteriol172:741–747
    [Google Scholar]
  26. Lloyd S. A., Tang H., Wang X., Billings S., Blair D. F.. 1996; Torque generation in the flagellar motor of Escherichia coli : evidence of a direct role for FliG but not for FliM or FliN. J Bacteriol178:223–231
    [Google Scholar]
  27. Lloyd S. A., Whitby F. G., Blair D. F., Hill C. P.. 1999; Structure of the C-terminal domain of FliG, a component of the rotor in the bacterial flagellar motor. Nature400:472–475
    [Google Scholar]
  28. Magariyama Y., Yamaguchi S., Aizawa S.. 1990; Genetic and behavioral analysis of flagellar switch mutants of Salmonella typhimurium . J Bacteriol172:4359–4369
    [Google Scholar]
  29. Marykwas D. L., Berg H. C.. 1996; A mutational analysis of the interaction between FliG and FliM, two components of the flagellar motor of Escherichia coli . J Bacteriol178:1289–1294
    [Google Scholar]
  30. Marykwas D. L., Passmore S. E.. 1995; Mapping by multifragment cloning in vivo . Proc Natl Acad Sci U S A92:11701–11705
    [Google Scholar]
  31. Marykwas D. L., Schmidt S. A., Berg H. C.. 1996; Interacting components of the flagellar motor of Escherichia coli revealed by the two-hybrid system in yeast. J Mol Biol256:564–576
    [Google Scholar]
  32. Mathews M. A. A., Tang H. L., Blair D. F.. 1998; Domain analysis of the FliM protein of Escherichia coli . J Bacteriol180:5580–5590
    [Google Scholar]
  33. Miller J. H.. 1992; A Short Course in Bacterial Genetics, a Laboratory Manual for Escherichia coli and Related Bacteria Cold Spring Harbor, NY: Cold Spring Harbor Press;
    [Google Scholar]
  34. Oosawa K., Ueno T., Aizawa S.-I.. 1994; Overproduction of the bacterial flagellar switch complex proteins and their interactions with the MS ring complex in vitro . J Bacteriol176:3683–3691
    [Google Scholar]
  35. Park S. Y., Chao X., Gonzalez-Bonet G., Beel B. D., Bilwes A. M., Crane B. R.. 2004; Structure and function of an unusual family of protein phosphatases: the bacterial chemotaxis proteins CheC and CheX. Mol Cell16:563–574
    [Google Scholar]
  36. Park S. Y., Lowder B., Bilwes A. M., Blair D. F., Crane B. R.. 2006; Structure of FliM provides insight into assembly of the switch complex in the bacterial flagella motor. Proc Natl Acad Sci U S A103:11886–11891
    [Google Scholar]
  37. Parkinson J. S., Parker S. R., Talbert P. B., Houts S. E.. 1983; Interactions between chemotaxis genes and flagellar genes in Escherichia coli . J Bacteriol155:265–274
    [Google Scholar]
  38. Sandrock T. M., O'Dell J. L., Adams A. E. M.. 1997; Allele-specific suppression by formation of new protein–protein interactions in yeast. Genetics147:1635–1642
    [Google Scholar]
  39. Schwede T., Kopp J., Guex N., Peitsch M. C.. 2003; SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res31:3381–3385
    [Google Scholar]
  40. Scott W. R. P., Hünenberger P. H., Tironi I. G., Mark A. E., Billeter S. R., Fennen J., Torda A. E., Huber T., Krüger P., van Gunsteren W. F.. 1999; The GROMOS biomolecular simulation program package. J Phys Chem A103:3596–3607
    [Google Scholar]
  41. Silverman M., Simon S.. 1974; Flagellar rotation and the mechanism of bacterial motility. Nature249:73–74
    [Google Scholar]
  42. Sockett H., Yamaguchi S., Kihara M., Irikura V. M., Macnab R. M.. 1992; Molecular analysis of the flagellar switch protein FliM of Salmonella typhimurium . J Bacteriol174:793–806
    [Google Scholar]
  43. 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]
  44. Sujatha S., Chatterji D.. 2000; Understanding protein–protein interactions by genetic suppression. J Genet79:125–129
    [Google Scholar]
  45. Suzuki H., Yonekura K., Namba K.. 2004; Structure of the rotor of the bacterial flagellar motor revealed by electron cryomicroscopy and single-particle image analysis. J Mol Biol337:105–113
    [Google Scholar]
  46. Tang H., Blair D. F.. 1995; Regulated underexpression of the FliM protein of Escherichia coli and evidence for a location in the flagellar motor distinct from the MotA/MotB torque generators. J Bacteriol177:3485–3495
    [Google Scholar]
  47. Tang H., Billings S., Wang X., Sharp L., Blair D. F.. 1995; Regulated underexpression and overexpression of the FliN protein of Escherichia coli and evidence for an interaction between FliN and FliM in the flagellar motor. J Bacteriol177:3496–3503
    [Google Scholar]
  48. Tang H., Braun T. F., Blair D. F.. 1996; Motility protein complexes in the bacterial flagellar motor. J Mol Biol261:209–221
    [Google Scholar]
  49. Thomas D. R., Morgan D. G., DeRosier D. J.. 1999; Rotational symmetry of the C ring and a mechanism for the flagellar rotary motor. Proc Natl Acad Sci U S A96:10134–10139
    [Google Scholar]
  50. Thomas D., Morgan D. G., DeRosier D. J.. 2001; Structures of bacterial flagellar motors from two FliF–FliG gene fusion mutants. J Bacteriol183:6404–6412
    [Google Scholar]
  51. Thomas D. R., Francis N. R., Xu C., DeRosier D. J.. 2006; The three-dimensional structure of the flagellar rotor from a clockwise-locked mutant of Salmonella enterica serovar Typhimurium. J Bacteriol188:7039–7048
    [Google Scholar]
  52. Togashi F., Yamaguchi S., Kihara M., Aizawa S.-I., Macnab R. M.. 1997; An extreme clockwise switch bias mutation in fliG of Salmonella typhimurium and its suppression by slow-motile mutations in motA and motB . J Bacteriol179:2994–3003
    [Google Scholar]
  53. Toker A. S., Macnab R. M.. 1997; Distinct regions of bacterial flagellar switch protein FliM interact with FliG, FliN and CheY. J Mol Biol273:623–634
    [Google Scholar]
  54. Toker A. S., Kihara M., Macnab R. M.. 1996; Deletion analysis of the FliM flagellar switch protein of Salmonella typhimurium . J Bacteriol178:7069–7079
    [Google Scholar]
  55. Yamaguchi S., Aizawa S., Kihara M., Isomura M., Jones C. J., Macnab R. M.. 1986a; Genetic evidence for a switching and energy-transducing complex in the flagellar motor of Salmonella typhimurium . J Bacteriol168:1172–1179
    [Google Scholar]
  56. Yamaguchi S., Fujita H., Ishihara A., Aizawa S., Macnab R. M.. 1986b; Subdivision of flagellar genes of Salmonella typhimurium into regions responsible for assembly, rotation, and switching. J Bacteriol166:187–193
    [Google Scholar]
  57. Yolov A. A., Shabarova Z. A.. 1990; Constructing DNA by polymerase recombination. Nucleic Acids Res18:3983–3986
    [Google Scholar]
  58. Young H. S., Dang H., Lai Y., DeRosier D. J., Khan S.. 2003; Variable symmetry in Salmonella typhimurium flagellar motors. Biophys J84:571–577
    [Google Scholar]
  59. Zhao R., Schuster S. C., Khan S.. 1995; Structural effects of mutations in Salmonella typhimurium flagellar switch complex. J Mol Biol251:400–412
    [Google Scholar]
  60. Zhao R., Amsler C. D., Matsumura P., Khan S.. 1996a; FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies. J Bacteriol178:258–265
    [Google Scholar]
  61. Zhao R., Pathak N., Jaffe H., Reese T. S., Khan S.. 1996b; FliN is a major structural protein of the C-ring in the Salmonella typhimurium flagellar basal body. J Mol Biol261:195–208
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/014597-0
Loading
/content/journal/micro/10.1099/mic.0.2007/014597-0
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