1887

Abstract

We describe here the functional characterization of the gene in . Direct interaction of FlgM with the alternative sigma factor (FliA) was first confirmed. A conserved region in the C-terminus of FlgM was found which included the binding domain. By site-directed mutagenesis, bacterial two-hybrid analysis and Western blotting, the primary FlgM binding sites with were shown to be Ile85, Ala86 and Leu89. A role for FlgM in swimming motility was demonstrated by inactivation of and subsequent complementation . Transcriptional fusion analyses showed differential gene expression of , , and in the and mutants compared with the wild-type. expression was not influenced by or FlgM while expression was abolished in the mutant and considerably reduced in the mutant when compared to the wild-type, indicating that both FliA and FlgM can activate transcription. Conversely, transcription was higher in the mutant when compared to the wild-type, suggesting that transcription was repressed by . Interestingly, expression was markedly increased in the mutant, suggesting a negative regulatory role for FlgM in expression. The transcription of other -dependent genes (, , , and ) was also examined in and mutant backgrounds and this revealed that other -factors apart from may be involved in flagellar biogenesis in . Taking together the motility phenotypes and effects of mutation on the regulation of these key motility genes, we propose that the mechanisms regulating flagellar biogenesis in may differ from those described for other bacteria.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.026294-0
2009-06-01
2019-10-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/6/1890.html?itemId=/content/journal/micro/10.1099/mic.0.026294-0&mimeType=html&fmt=ahah

References

  1. Aldridge, P. & Hughes, K. T. ( 2002; ). Regulation of flagellar assembly. Curr Opin Microbiol 5, 160–165.[CrossRef]
    [Google Scholar]
  2. Aldridge, P. D., Karlinsey, J. E., Aldridge, C., Birchall, C., Thompson, D., Yagasaki, J. & Hughes, K. T. ( 2006; ). The flagellar-specific transcription factor, σ 28, is the Type III secretion chaperone for the flagellar-specific anti-σ 28 factor FlgM. Genes Dev 20, 2315–2326.[CrossRef]
    [Google Scholar]
  3. Atkinson, S., Throup, J. P., Stewart, G. S. & Williams, P. ( 1999; ). A hierarchical quorum-sensing system in Yersinia pseudotuberculosis is involved in the regulation of motility and clumping. Mol Microbiol 33, 1267–1277.
    [Google Scholar]
  4. Barrios, H., Valderrama, B. & Morett, E. ( 1999; ). Compilation and analysis of σ 54-dependent promoter sequences. Nucleic Acids Res 27, 4305–4313.[CrossRef]
    [Google Scholar]
  5. Blair, D. F. ( 1995; ). How bacteria sense and swim. Annu Rev Microbiol 49, 489–522.[CrossRef]
    [Google Scholar]
  6. Bleves, S., Marenne, M. N., Detry, G. & Cornelis, G. R. ( 2002; ). Up-regulation of the Yersinia enterocolitica yop regulon by deletion of the flagellum master operon flhDC. J Bacteriol 184, 3214–3223.[CrossRef]
    [Google Scholar]
  7. Carniel, E. & Hinnebusch, B. J. (editors) ( 2004; ). Yersinia: Molecular and Cellular Biology. Norwich, UK: Horizon Bioscience Press.
  8. Chadsey, M. S. & Hughes, K. T. ( 2001; ). A multipartite interaction between Salmonella transcription factor σ 28 and its anti-sigma factor FlgM: implications for σ 28 holoenzyme destabilization through stepwise binding. J Mol Biol 306, 915–929.[CrossRef]
    [Google Scholar]
  9. Chadsey, M. S., Karlinsey, J. E. & Hughes, K. T. ( 1998; ). The flagellar anti-sigma factor FlgM actively dissociates Salmonella typhimurium sigma28 RNA polymerase holoenzyme. Genes Dev 12, 3123–3136.[CrossRef]
    [Google Scholar]
  10. Chatterjee, B. D. & Neogy, K. N. ( 1972; ). Detection of capsule in Vibrio parahaemolyticus. Experientia 28, 103–104.[CrossRef]
    [Google Scholar]
  11. Chilcott, G. S. & Hughes, K. T. ( 2000; ). Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar Typhimurium and Escherichia coli. Microbiol Mol Biol Rev 64, 694–708.[CrossRef]
    [Google Scholar]
  12. Clarke, M. B. & Sperandio, V. ( 2005; ). Transcriptional regulation of flhDC by QseBC and sigma (FliA) in enterohaemorrhagic Escherichia coli. Mol Microbiol 57, 1734–1749.[CrossRef]
    [Google Scholar]
  13. Colland, F., Rain, J. C., Gounon, P., Labigne, A., Legrain, P. & De Reuse, H. ( 2001; ). Identification of the Helicobacter pylori anti-σ 28 factor. Mol Microbiol 41, 477–487.[CrossRef]
    [Google Scholar]
  14. Correa, N. E., Barker, J. R. & Klose, K. E. ( 2004; ). The Vibrio cholerae FlgM homologue is an anti-σ 28 factor that is secreted through the sheathed polar flagellum. J Bacteriol 186, 4613–4619.[CrossRef]
    [Google Scholar]
  15. Daughdrill, G. W., Chadsey, M. S., Karlinsey, J. E., Hughes, K. T. & Dahlquist, F. W. ( 1997; ). The C-terminal half of the anti-sigma factor, FlgM, becomes structured when bound to its target, σ 28. Nat Struct Biol 4, 285–291.[CrossRef]
    [Google Scholar]
  16. Frisk, A., Jyot, J., Arora, S. K. & Ramphal, R. ( 2002; ). Identification and functional characterization of flgM, a gene encoding the anti-σ 28 factor in Pseudomonas aeruginosa. J Bacteriol 184, 1514–1521.[CrossRef]
    [Google Scholar]
  17. Gillen, K. L. & Hughes, K. T. ( 1991a; ). Molecular characterization of flgM, a gene encoding a negative regulator of flagellin synthesis in Salmonella typhimurium. J Bacteriol 173, 6453–6459.
    [Google Scholar]
  18. Gillen, K. L. & Hughes, K. T. ( 1991b; ). Negative regulatory loci coupling flagellin synthesis to flagellar assembly in Salmonella typhimurium. J Bacteriol 173, 2301–2310.
    [Google Scholar]
  19. Gillen, K. L. & Hughes, K. T. ( 1993; ). Transcription from two promoters and autoregulation contribute to the control of expression of the Salmonella typhimurium flagellar regulatory gene flgM. J Bacteriol 175, 7006–7015.
    [Google Scholar]
  20. Givaudan, A. & Lanois, A. ( 2000; ). flhDC, the flagellar master operon of Xenorhabdus nematophilus: requirement for motility, lipolysis, extracellular hemolysis, and full virulence in insects. J Bacteriol 182, 107–115.[CrossRef]
    [Google Scholar]
  21. Gunton, J. E., Gilmour, M. W., Baptista, K. P., Lawley, T. D. & Taylor, D. E. ( 2007; ). Interaction between the co-inherited TraG coupling protein and the TraJ membrane-associated protein of the H-plasmid conjugative DNA transfer system resembles chromosomal DNA translocases. Microbiology 153, 428–441.[CrossRef]
    [Google Scholar]
  22. Hughes, K. T., Gillen, K. L., Semon, M. J. & Karlinsey, J. E. ( 1993; ). Sensing structural intermediates in bacterial flagellar assembly by export of a negative regulator. Science 262, 1277–1280.[CrossRef]
    [Google Scholar]
  23. Josenhans, C. & Suerbaum, S. ( 2002; ). The role of motility as a virulence factor in bacteria. Int J Med Microbiol 291, 605–614.[CrossRef]
    [Google Scholar]
  24. Josenhans, C., Niehus, E., Amersbach, S., Horster, A., Betz, C., Drescher, B., Hughes, K. T. & Suerbaum, S. ( 2002; ). Functional characterization of the antagonistic flagellar late regulators FliA and FlgM of Helicobacter pylori and their effects on the H. pylori transcriptome. Mol Microbiol 43, 307–322.[CrossRef]
    [Google Scholar]
  25. Karimova, G., Ullmann, A. & Ladant, D. ( 2000; ). A bacterial two-hybrid system that exploits a cAMP signaling cascade in Escherichia coli. Methods Enzymol 328, 59–73.
    [Google Scholar]
  26. Karlinsey, J. E. & Hughes, K. T. ( 2006; ). Genetic transplantation: Salmonella enterica serovar Typhimurium as a host to study sigma factor and anti-sigma factor interactions in genetically intractable systems. J Bacteriol 188, 103–114.[CrossRef]
    [Google Scholar]
  27. Karlinsey, J. E., Tanaka, S., Bettenworth, V., Yamaguchi, S., Boos, W., Aizawa, S. I. & Hughes, K. T. ( 2000; ). Completion of the hook-basal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription. Mol Microbiol 37, 1220–1231.[CrossRef]
    [Google Scholar]
  28. Kirov, S. M. ( 2003; ). Bacteria that express lateral flagella enable dissection of the multifunctional roles of flagella in pathogenesis. FEMS Microbiol Lett 224, 151–159.[CrossRef]
    [Google Scholar]
  29. Kutsukake, K., Ohya, Y. & Iino, T. ( 1990; ). Transcriptional analysis of the flagellar regulon of Salmonella typhimurium. J Bacteriol 172, 741–747.
    [Google Scholar]
  30. Lee, S. H., Butler, S. M. & Camilli, A. ( 2001; ). Selection for in vivo regulators of bacterial virulence. Proc Natl Acad Sci U S A 98, 6889–6894.[CrossRef]
    [Google Scholar]
  31. Liu, X. & Matsumura, P. ( 1994; ). The FlhD/FlhC complex, a transcriptional activator of the Escherichia coli flagellar class II operons. J Bacteriol 176, 7345–7351.
    [Google Scholar]
  32. Miller, J. H. ( 1992; ). A Short Course in Bacterial Genetics: a Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  33. Milton, D. L., O'Toole, R., Horstedt, P. & Wolf-Watz, H. ( 1996; ). Flagellin A is essential for the virulence of Vibrio anguillarum. J Bacteriol 178, 1310–1319.
    [Google Scholar]
  34. Ohnishi, K., Kutsukake, K., Suzuki, H. & Lino, T. ( 1992; ). A novel transcriptional regulation mechanism in the flagellar regulon of Salmonella typhimurium: an antisigma factor inhibits the activity of the flagellum-specific sigma factor, σ F. Mol Microbiol 6, 3149–3157.[CrossRef]
    [Google Scholar]
  35. Okada, K., Ichihara, H., Takahashi, H., Fujita, N., Ishihama, A. & Hakoshima, T. ( 2007; ). Preparation and preliminary X-ray diffraction analysis of crystals of bacterial flagellar sigma factor σ 28 in complex with the σ 28-binding region of its antisigma factor, FlgM. Acta Crystallogr Sect F Struct Biol Cryst Commun 63, 196–199.[CrossRef]
    [Google Scholar]
  36. Pons, T., Gonzalez, B., Ceciliani, F. & Galizzi, A. ( 2006; ). FlgM anti-sigma factors: identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence–structure–function relationships. J Mol Model 12, 973–983.[CrossRef]
    [Google Scholar]
  37. Ramos, H. C., Rumbo, M. & Sirard, J. C. ( 2004; ). Bacterial flagellins: mediators of pathogenicity and host immune responses in mucosa. Trends Microbiol 12, 509–517.[CrossRef]
    [Google Scholar]
  38. Richardson, K. ( 1991; ). Roles of motility and flagellar structure in pathogenicity of Vibrio-Cholerae – analysis of motility mutants in three animal models. Infect Immun 59, 2727–2736.
    [Google Scholar]
  39. Rosqvist, R., Skurnik, M. & Wolf-Watz, H. ( 1988; ). Increased virulence of Yersinia pseudotuberculosis by two independent mutations. Nature 334, 522–524.[CrossRef]
    [Google Scholar]
  40. Saini, S., Brown, J. D., Aldridge, P. D. & Rao, C. V. ( 2008; ). FliZ is a post-translational activator of FlhD4C2-dependent flagellar gene expression. J Bacteriol 190, 4979–4988.[CrossRef]
    [Google Scholar]
  41. Schmitt, C. K., Darnell, S. C., Tesh, V. L., Stocker, B. A. & O'Brien, A. D. ( 1994; ). Mutation of flgM attenuates virulence of Salmonella typhimurium, and mutation of fliA represses the attenuated phenotype. J Bacteriol 176, 368–377.
    [Google Scholar]
  42. Sexton, J. A., Pinkner, J. S., Roth, R., Heuser, J. E., Hultgren, S. J. & Vogel, J. P. ( 2004; ). The Legionella pneumophila PilT homologue DotB exhibits ATPase activity that is critical for intracellular growth. J Bacteriol 186, 1658–1666.[CrossRef]
    [Google Scholar]
  43. Sorenson, M. K., Ray, S. S. & Darst, S. A. ( 2004; ). Crystal structure of the flagellar sigma/anti-sigma complex σ 28/FlgM reveals an intact sigma factor in an inactive conformation. Mol Cell 14, 127–138.[CrossRef]
    [Google Scholar]
  44. Spohn, G. & Scarlato, V. ( 1999; ). Motility of Helicobacter pylori is coordinately regulated by the transcriptional activator FlgR, an NtrC homolog. J Bacteriol 181, 593–599.
    [Google Scholar]
  45. Wadhams, G. H. & Armitage, J. P. ( 2004; ). Making sense of it all: bacterial chemotaxis. Nat Rev Mol Cell Biol 5, 1024–1037.[CrossRef]
    [Google Scholar]
  46. Watnick, P. I., Lauriano, C. M., Klose, K. E., Croal, L. & Kolter, R. ( 2001; ). The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139. Mol Microbiol 39, 223–235.[CrossRef]
    [Google Scholar]
  47. Wood, T. K., Gonzalez Barrios, A. F., Herzberg, M. & Lee, J. ( 2006; ). Motility influences biofilm architecture in Escherichia coli. Appl Microbiol Biotechnol 72, 361–367.[CrossRef]
    [Google Scholar]
  48. Young, G. M., Schmiel, D. H. & Miller, V. L. ( 1999; ). A new pathway for the secretion of virulence factors by bacteria: the flagellar export apparatus functions as a protein-secretion system. Proc Natl Acad Sci U S A 96, 6456–6461.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.026294-0
Loading
/content/journal/micro/10.1099/mic.0.026294-0
Loading

Data & Media loading...

Supplements

vol. , part 6, pp. 1890 - 1900

Oligonucleotide primers used in this study [ PDF] (17 kb)



PDF
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