1887

Abstract

is a leading cause of bacterial gastroenteritis in the developed world. The role of a homologue of the negative transcriptional regulatory protein HspR, which in other organisms participates in the control of the heat-shock response, was investigated. Following inactivation of in , members of the HspR regulon were identified by DNA microarray transcript profiling. In agreement with the predicted role of HspR as a negative regulator of genes involved in the heat-shock response, it was observed that the transcript amounts of 13 genes were increased in the mutant, including the chaperone genes , and , and a gene encoding the heat-shock regulator HrcA. Proteomic analysis also revealed increased synthesis of the heat-shock proteins DnaK, GrpE, GroEL and GroES in the absence of HspR. The altered expression of chaperones was accompanied by heat sensitivity, as the mutant was unable to form colonies at 44 °C. Surprisingly, transcriptome analysis also revealed a group of 17 genes with lower transcript levels in the mutant. Of these, eight were predicted to be involved in the formation of the flagella apparatus, and the decreased expression is likely to be responsible for the reduced motility and ability to autoagglutinate that was observed for mutant cells. Electron micrographs showed that mutant cells were spiral-shaped and carried intact flagella, but were elongated compared to wild-type cells. The inactivation of also reduced the ability of to adhere to and invade human epithelial INT-407 cells , possibly as a consequence of the reduced motility or lower expression of the flagellar export apparatus in mutant cells. It was concluded that, in , HspR influences the expression of several genes that are likely to have an impact on the ability of the bacterium to successfully survive in food products and subsequently infect the consumer.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27513-0
2005-03-01
2019-10-23
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/3/mic1510905.html?itemId=/content/journal/micro/10.1099/mic.0.27513-0&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. Bucca, G., Hindle, Z. & Smith, C. P. ( 1997; ). Regulation of the dnaK operon of Streptomyces coelicolor A3(2) is governed by HspR, an autoregulatory repressor protein. J Bacteriol 179, 5999–6004.
    [Google Scholar]
  3. Bucca, G., Brassington, A. M., Hotchkiss, G., Mersinias, V. & Smith, C. P. ( 2003; ). Negative feedback regulation of dnaK, clpB and lon expression by the DnaK chaperone machine in Streptomyces coelicolor, identified by transcriptome and in vivo DnaK-depletion analysis. Mol Microbiol 50, 153–166.[CrossRef]
    [Google Scholar]
  4. Carrillo, C. D., Taboada, E., Nash, J. H. & 15 other authors ( 2004; ). Genome-wide expression analyses of Campylobacter jejuni NCTC 11168 reveals coordinate regulation of motility and virulence by flhA. J Biol Chem 279, 20327–20338.[CrossRef]
    [Google Scholar]
  5. Chastanet, A., Fert, J. & Msadek, T. ( 2003; ). Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria. Mol Microbiol 47, 1061–1073.[CrossRef]
    [Google Scholar]
  6. Cohen, S. N., Chang, A. C. & Hsu, L. ( 1972; ). Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A 69, 2110–2114.[CrossRef]
    [Google Scholar]
  7. Colegio, O. R., Griffin, T. J., Grindley, N. D. & Galan, J. E. ( 2001; ). In vitro transposition system for efficient generation of random mutants of Campylobacter jejuni. J Bacteriol 183, 2384–2388.[CrossRef]
    [Google Scholar]
  8. Delany, I., Spohn, G., Rappuoli, R. & Scarlato, V. ( 2002; ). In vitro selection of high affinity HspR-binding sites within the genome of Helicobacter pylori. Gene 283, 63–69.[CrossRef]
    [Google Scholar]
  9. Goldberg, A. L. ( 1972; ). Degradation of abnormal proteins in Escherichia coli (protein breakdown-protein structure-mistranslation-amino acid analogs-puromycin). Proc Natl Acad Sci U S A 69, 422–426.[CrossRef]
    [Google Scholar]
  10. Golden, N. J. & Acheson, D. W. ( 2002; ). Identification of motility and autoagglutination Campylobacter jejuni mutants by random transposon mutagenesis. Infect Immun 70, 1761–1771.[CrossRef]
    [Google Scholar]
  11. Goloubinoff, P., Mogk, A., Zvi, A. P., Tomoyasu, T. & Bukau, B. ( 1999; ). Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network. Proc Natl Acad Sci U S A 96, 13732–13737.[CrossRef]
    [Google Scholar]
  12. Grandvalet, C., Servant, P. & Mazodier, P. ( 1997; ). Disruption of hspR, the repressor gene of the dnaK operon in Streptomyces albus G. Mol Microbiol 23, 77–84.[CrossRef]
    [Google Scholar]
  13. Grandvalet, C., Crecy-Lagard, V. & Mazodier, P. ( 1999; ). The ClpB ATPase of Streptomyces albus G belongs to the HspR heat shock regulon. Mol Microbiol 31, 521–532.[CrossRef]
    [Google Scholar]
  14. Guerry, P., Alm, R. A., Power, M. E., Logan, S. M. & Trust, T. J. ( 1991; ). Role of two flagellin genes in Campylobacter motility. J Bacteriol 173, 4757–4764.
    [Google Scholar]
  15. Guerry, P., Alm, R., Szymanski, C. M. & Trust, T. J. ( 2000; ). Structure, function, and antigenicity of Campylobacter flagella. In Campylobacter, pp. 405–422. Edited by I. Nachamkin & M. J. Blaser. Washington, DC: American Society for Microbiology.
  16. Hendrixson, D. R. & DiRita, V. J. ( 2003; ). Transcription of σ 54-dependent but not σ 28-dependent flagellar genes in Campylobacter jejuni is associated with formation of the flagellar secretory apparatus. Mol Microbiol 50, 687–702.[CrossRef]
    [Google Scholar]
  17. Hendrixson, D. R., Akerley, B. J. & DiRita, V. J. ( 2001; ). Transposon mutagenesis of Campylobacter jejuni identifies a bipartite energy taxis system required for motility. Mol Microbiol 40, 214–224.[CrossRef]
    [Google Scholar]
  18. Holmes, K., Mulholland, F., Pearson, B. M., Pin, C., McNicholl-Kennedy, J., Ketley, J. & Wells, J. ( 2005; ). Campylobacter jejuni gene expression in response to iron limitation and the role of Fur. Microbiology 151, 243–257.[CrossRef]
    [Google Scholar]
  19. Horton, R. M., Ho, S. N., Pullen, J. K., Hunt, H. D., Cai, Z. & Pease, L. R. ( 1993; ). Gene splicing by overlap extension. Methods Enzymol 217, 270–279.
    [Google Scholar]
  20. Hu, L. & Kopecko, D. J. ( 1999; ). Campylobacter jejuni 81-176 associates with microtubules and dynein during invasion of human intestinal cells. Infect Immun 67, 4171–4182.
    [Google Scholar]
  21. Jagannathan, A., Constantinidou, C. & Penn, C. W. ( 2001; ). Roles of rpoN, fliA, and flgR in expression of flagella in Campylobacter jejuni. J Bacteriol 183, 2937–2942.[CrossRef]
    [Google Scholar]
  22. Ketley, J. M. ( 1997; ). Pathogenesis of enteric infection by Campylobacter. Microbiology 143, 5–21.[CrossRef]
    [Google Scholar]
  23. Kinsella, N., Guerry, P., Cooney, J. & Trust, T. J. ( 1997; ). The flgE gene of Campylobacter coli is under the control of the alternative sigma factor σ 54. J Bacteriol 179, 4647–4653.
    [Google Scholar]
  24. Konkel, M. E., Klena, J. D., Rivera-Amill, V., Monteville, M. R., Biswas, D., Raphael, B. & Mickelson, J. ( 2004; ). Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus. J Bacteriol 186, 3296–3303.[CrossRef]
    [Google Scholar]
  25. Lüneberg, E., Glenn-Calvo, E., Hartmann, M., Bar, W. & Frosch, M. ( 1998; ). The central, surface-exposed region of the flagellar hook protein FlgE of Campylobacter jejuni shows hypervariability among strains. J Bacteriol 180, 3711–3714.
    [Google Scholar]
  26. Misawa, N. & Blaser, M. J. ( 2000; ). Detection and characterization of autoagglutination activity by Campylobacter jejuni. Infect Immun 68, 6168–6175.[CrossRef]
    [Google Scholar]
  27. Mogk, A., Völker, A., Engelmann, S., Hecker, M., Schumann, W. & Völker, U. ( 1998; ). Nonnative proteins induce expression of the Bacillus subtilis CIRCE regulon. J Bacteriol 180, 2895–2900.
    [Google Scholar]
  28. Monteville, M. R., Yoon, J. E. & Konkel, M. E. ( 2003; ). Maximal adherence and invasion of INT 407 cells by Campylobacter jejuni requires the CadF outer-membrane protein and microfilament reorganization. Microbiology 149, 153–165.[CrossRef]
    [Google Scholar]
  29. Morooka, T., Umeda, A. & Amako, K. ( 1985; ). Motility as an intestinal colonization factor for Campylobacter jejuni. J Gen Microbiol 131, 1973–1980.
    [Google Scholar]
  30. Nachamkin, I., Yang, X. H. & Stern, N. J. ( 1993; ). Role of Campylobacter jejuni flagella as colonization factors for three-day-old chicks: analysis with flagellar mutants. Appl Environ Microbiol 59, 1269–1273.
    [Google Scholar]
  31. Nachamkin, I., Allos, B. M. & Ho, T. ( 1998; ). Campylobacter species and Guillain-Barré syndrome. Clin Microbiol Rev 11, 555–567.
    [Google Scholar]
  32. Narberhaus, F. ( 1999; ). Negative regulation of bacterial heat shock genes. Mol Microbiol 31, 1–8.[CrossRef]
    [Google Scholar]
  33. Nuijten, P. J., van Asten, F. J., Gaastra, W. & van der Zeijst, B. A. ( 1990; ). Structural and functional analysis of two Campylobacter jejuni flagellin genes. J Biol Chem 265, 17798–17804.
    [Google Scholar]
  34. Parkhill, J. B. W., Wren, K., Mungall, J. M. & 18 other authors ( 2000; ). The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403, 665–668.[CrossRef]
    [Google Scholar]
  35. Pearson, B. M., Pin, C., Wright, J., I'Anson, K., Humphrey, T. & Wells, J. M. ( 2003; ). Comparative genome analysis of Campylobacter jejuni using whole genome DNA microarrays. FEBS Lett 554, 224–230.[CrossRef]
    [Google Scholar]
  36. Reischl, S., Wiegert, T. & Schumann, W. ( 2002; ). Isolation and analysis of mutant alleles of the Bacillus subtilis HrcA repressor with reduced dependency on GroE function. J Biol Chem 277, 32659–32667.[CrossRef]
    [Google Scholar]
  37. Schulz, A. & Schumann, W. ( 1996; ). hrcA, the first gene of the Bacillus subtilis dnaK operon encodes a negative regulator of class I heat shock genes. J Bacteriol 178, 1088–1093.
    [Google Scholar]
  38. Servant, P. & Mazodier, P. ( 2001; ). Negative regulation of the heat shock response in Streptomyces. Arch Microbiol 176, 237–242.[CrossRef]
    [Google Scholar]
  39. Song, Y. C., Jin, S., Louie, H. & 8 other authors ( 2004; ). FlaC, a protein of Campylobacter jejuni TGH9011 (ATCC43431) secreted through the flagellar apparatus, binds epithelial cells and influences cell invasion. Mol Microbiol 53, 541–553.[CrossRef]
    [Google Scholar]
  40. Spohn, G. & Scarlato, V. ( 1999; ). The autoregulatory HspR repressor protein governs chaperone gene transcription in Helicobacter pylori. Mol Microbiol 34, 663–674.[CrossRef]
    [Google Scholar]
  41. Spohn, G., Danielli, A., Roncarati, D., Delany, I., Rappuoli, R. & Scarlato, V. ( 2004; ). Dual control of Helicobacter pylori heat shock gene transcription by HspR and HrcA. J Bacteriol 186, 2956–2965.[CrossRef]
    [Google Scholar]
  42. Stewart, G. R., Snewin, V. A., Walzl, G. & 7 other authors ( 2001; ). Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection. Nat Med 7, 732–737.[CrossRef]
    [Google Scholar]
  43. Stewart, G. R., Wernisch, L., Stabler, R., Mangan, J. A., Hinds, J., Laing, K. G., Young, D. B. & Butcher, P. D. ( 2002; ). Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 148, 3129–3138.
    [Google Scholar]
  44. Stintzi, A. ( 2003; ). Gene expression profile of Campylobacter jejuni in response to growth temperature variation. J Bacteriol 185, 2009–2016.[CrossRef]
    [Google Scholar]
  45. Szymanski, C. M., King, M., Haardt, M. & Armstrong, G. D. ( 1995; ). Campylobacter jejuni motility and invasion of Caco-2 cells. Infect Immun 63, 4295–4300.
    [Google Scholar]
  46. Tomb, J.-F., White, O., Kerlavage, A. R. & 39 other authors ( 1997; ). The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539–547.[CrossRef]
    [Google Scholar]
  47. Ueguchi, C., Kakeda, M., Yamada, H. & Mizuno, T. ( 1994; ). An analogue of the DnaJ molecular chaperone in Escherichia coli. Proc Natl Acad Sci U S A 91, 1054–1058.[CrossRef]
    [Google Scholar]
  48. VanBogelen, R. A., Kelley, P. M. & Neidhardt, F. C. ( 1987; ). Differential induction of heat shock, SOS, and oxidation stress regulons and accumulation of nucleotides in Escherichia coli. J Bacteriol 169, 26–32.
    [Google Scholar]
  49. Wang, W. L., Luechtefeld, N. W., Blaser, M. J. & Reller, L. B. ( 1983; ). Effect of incubation atmosphere and temperature on isolation of Campylobacter jejuni from human stools. Can J Microbiol 29, 468–470.[CrossRef]
    [Google Scholar]
  50. Wassenaar, T. M. ( 1997; ). Toxin production by Campylobacter spp. Clin Microbiol Rev 10, 466–476.
    [Google Scholar]
  51. Wassenaar, T. M., Fry, B. N. & van der Zeijst, B. A. ( 1993a; ). Genetic manipulation of Campylobacter: evaluation of natural transformation and electro-transformation. Gene 132, 131–135.[CrossRef]
    [Google Scholar]
  52. Wassenaar, T. M., van der Zeijst, B. A., Ayling, R. & Newell, D. G. ( 1993b; ). Colonization of chicks by motility mutants of Campylobacter jejuni demonstrates the importance of flagellin A expression. J Gen Microbiol 139, 1171–1175.[CrossRef]
    [Google Scholar]
  53. Wooldridge, K. G. & Ketley, J. M. ( 1997; ). Campylobacter-host cell interactions. Trends Microbiol 5, 96–102.[CrossRef]
    [Google Scholar]
  54. Wösten, M. M., Wagenaar, J. A. & van Putten, J. P. ( 2004; ). The FlgS/FlgR two-component signal transduction system regulates the fla regulon in Campylobacter jejuni. J Biol Chem 279, 16214–16222.[CrossRef]
    [Google Scholar]
  55. Yao, R., Alm, R. A., Trust, T. J. & Guerry, P. ( 1993; ). Construction of new Campylobacter cloning vectors and a new mutational cat cassette. Gene 130, 127–130.[CrossRef]
    [Google Scholar]
  56. Yao, R., Burr, D. H., Doig, P., Trust, T. J., Niu, H. & Guerry, P. ( 1994; ). Isolation of motile and non-motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cells. Mol Microbiol 14, 883–893.[CrossRef]
    [Google Scholar]
  57. Zuber, U. & Schumann, W. ( 1994; ). CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis. J Bacteriol 176, 1359–1363.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27513-0
Loading
/content/journal/micro/10.1099/mic.0.27513-0
Loading

Data & Media loading...

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