1887

Abstract

In enterobacteria such as and species, flagellar biogenesis is strictly dependent upon the master regulator . Here, we demonstrate that in enterohaemorrhagic (EHEC), the flagellar regulon is controlled by ClpXP, a member of the ATP-dependent protease family, through two pathways: (i) post-translational control of the FlhD/FlhC master regulator and (ii) transcriptional control of the operon. Both FlhD and FlhC proteins accumulated markedly following ClpXP depletion, and their half-lives were significantly longer in the mutant cells, suggesting that ClpXP is responsible for degrading FlhD and FlhC proteins, leading to downregulation of flagellar expression. ClpXP was involved in regulating the transcription of the promoter only when the cells had entered stationary phase in a culture medium that markedly induced expression of the locus of enterocyte effacement (LEE). Comparative analyses of transcription from the promoter in EHEC cells with different genetic backgrounds suggested that the downregulation of expression by ClpXP is dependent on the LEE-encoded GrlR–GrlA system. We have also shown that the degradation of FlhD and FlhC by ClpXP is responsible for downregulating flagellar expression even when LEE expression is induced.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.051151-0
2011-11-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/157/11/3094.html?itemId=/content/journal/micro/10.1099/mic.0.051151-0&mimeType=html&fmt=ahah

References

  1. Abe H., Tatsuno I., Tobe T., Okutani A., Sasakawa C. ( 2002). Bicarbonate ion stimulates the expression of locus of enterocyte effacement-encoded genes in enterohemorrhagic Escherichia coli O157:H7. Infect Immun 70:3500–3509 [View Article][PubMed]
    [Google Scholar]
  2. Akashi S., Joh K., Mori T., Tsuji A., Ito H., Hoshi H., Hayakawa T., Ihara J., Abe T. et al. & other authors ( 1994). A severe outbreak of haemorrhagic colitis and haemolytic uraemic syndrome associated with Escherichia coli O157:H7 in Japan. Eur J Pediatr 153:650–655 [View Article][PubMed]
    [Google Scholar]
  3. Berin M. C., Darfeuille-Michaud A., Egan L. J., Miyamoto Y., Kagnoff M. F. ( 2002). Role of EHEC O157 : H7 virulence factors in the activation of intestinal epithelial cell NF-κB and MAP kinase pathways and the upregulated expression of interleukin 8. Cell Microbiol 4:635–648 [View Article][PubMed]
    [Google Scholar]
  4. Bustamante V. H., Santana F. J., Calva E., Puente J. L. ( 2001). Transcriptional regulation of type III secretion genes in enteropathogenic Escherichia coli: Ler antagonizes H-NS-dependent repression. Mol Microbiol 39:664–678 [View Article][PubMed]
    [Google Scholar]
  5. Cherepanov P. P., Wackernagel W. ( 1995). Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158:9–14 [View Article][PubMed]
    [Google Scholar]
  6. Cummings L. A., Wilkerson W. D., Bergsbaken T., Cookson B. T. ( 2006). In vivo, fliC expression by Salmonella enterica serovar Typhimurium is heterogeneous, regulated by ClpX, and anatomically restricted. Mol Microbiol 61:795–809 [View Article][PubMed]
    [Google Scholar]
  7. Datsenko K. A., Wanner B. L. ( 2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645 [View Article][PubMed]
    [Google Scholar]
  8. Deng W., Puente J. L., Gruenheid S., Li Y., Vallance B. A., Vázquez A., Barba J., Ibarra J. A., O’Donnell P. et al. & other authors ( 2004). Dissecting virulence: systematic and functional analyses of a pathogenicity island. Proc Natl Acad Sci U S A 101:3597–3602 [View Article][PubMed]
    [Google Scholar]
  9. Donnenberg M. S., Kaper J. B. ( 1991). Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun 59:4310–4317[PubMed]
    [Google Scholar]
  10. Fontaine F., Stewart E. J., Lindner A. B., Taddei F. ( 2008). Mutations in two global regulators lower individual mortality in Escherichia coli. Mol Microbiol 67:2–14[PubMed]
    [Google Scholar]
  11. Francez-Charlot A., Laugel B., Van Gemert A., Dubarry N., Wiorowski F., Castanié-Cornet M. P., Gutierrez C., Cam K. ( 2003). RcsCDB His–Asp phosphorelay system negatively regulates the flhDC operon in Escherichia coli. Mol Microbiol 49:823–832 [View Article][PubMed]
    [Google Scholar]
  12. Friedberg D., Umanski T., Fang Y., Rosenshine I. ( 1999). Hierarchy in the expression of the locus of enterocyte effacement genes of enteropathogenic Escherichia coli. Mol Microbiol 34:941–952 [View Article][PubMed]
    [Google Scholar]
  13. Gamer J., Bujard H., Bukau B. ( 1992). Physical interaction between heat shock proteins DnaK, DnaJ, and GrpE and the bacterial heat shock transcription factor σ32. Cell 69:833–842 [View Article][PubMed]
    [Google Scholar]
  14. Gottesman S., Roche E., Zhou Y., Sauer R. T. ( 1998). The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. Genes Dev 12:1338–1347 [View Article][PubMed]
    [Google Scholar]
  15. Grant A. J., Farris M., Alefounder P., Williams P. H., Woodward M. J., O’Connor C. D. ( 2003). Co-ordination of pathogenicity island expression by the BipA GTPase in enteropathogenic Escherichia coli (EPEC). Mol Microbiol 48:507–521 [View Article][PubMed]
    [Google Scholar]
  16. Habdas B. J., Smart J., Kaper J. B., Sperandio V. ( 2010). The LysR-type transcriptional regulator QseD alters type three secretion in enterohemorrhagic Escherichia coli and motility in K-12 Escherichia coli. J Bacteriol 192:3699–3712 [View Article][PubMed]
    [Google Scholar]
  17. Iyoda S., Watanabe H. ( 2005). ClpXP protease controls expression of the type III protein secretion system through regulation of RpoS and GrlR levels in enterohemorrhagic Escherichia coli. J Bacteriol 187:4086–4094 [View Article][PubMed]
    [Google Scholar]
  18. Iyoda S., Koizumi N., Satou H., Lu Y., Saitoh T., Ohnishi M., Watanabe H. ( 2006). The GrlR–GrlA regulatory system coordinately controls the expression of flagellar and LEE-encoded type III protein secretion systems in enterohemorrhagic Escherichia coli. J Bacteriol 188:5682–5692 [View Article][PubMed]
    [Google Scholar]
  19. Lehnen D., Blumer C., Polen T., Wackwitz B., Wendisch V. F., Unden G. ( 2002). LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli. Mol Microbiol 45:521–532 [View Article][PubMed]
    [Google Scholar]
  20. Macnab R. M. ( 1996). Flagella and motility. Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn.123–145 Neidhardt F. C., Curtiss R. III, Ingraham H. L., Lin E. C. C., Low K. B., Magasanik B., Reznikoff W. S., Riley M., Schaechter M., Umbarger H. E. Washington, D.C.: American Society for Microbiology;
    [Google Scholar]
  21. Macnab R. M. ( 2003). How bacteria assemble flagella. Annu Rev Microbiol 57:77–100 [View Article][PubMed]
    [Google Scholar]
  22. Mahajan A., Currie C. G., Mackie S., Tree J., McAteer S., McKendrick I., McNeilly T. N., Roe A., La Ragione R. M. et al. & other authors ( 2009). An investigation of the expression and adhesin function of H7 flagella in the interaction of Escherichia coli O157 : H7 with bovine intestinal epithelium. Cell Microbiol 11:121–137 [View Article][PubMed]
    [Google Scholar]
  23. Ortega J., Singh S. K., Ishikawa T., Maurizi M. R., Steven A. C. ( 2000). Visualization of substrate binding and translocation by the ATP-dependent protease, ClpXP. Mol Cell 6:1515–1521 [View Article][PubMed]
    [Google Scholar]
  24. Schneider K., Beck C. F. ( 1986). Promoter-probe vectors for the analysis of divergently arranged promoters. Gene 42:37–48 [View Article][PubMed]
    [Google Scholar]
  25. Shi W., Li C., Louise C. J., Adler J. ( 1993). Mechanism of adverse conditions causing lack of flagella in Escherichia coli. J Bacteriol 175:2236–2240[PubMed]
    [Google Scholar]
  26. Shin S., Park C. ( 1995). Modulation of flagellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR. J Bacteriol 177:4696–4702[PubMed]
    [Google Scholar]
  27. Soutourina O., Kolb A., Krin E., Laurent-Winter C., Rimsky S., Danchin A., Bertin P. ( 1999). Multiple control of flagellum biosynthesis in Escherichia coli: role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon. J Bacteriol 181:7500–7508[PubMed]
    [Google Scholar]
  28. Soutourina O. A., Krin E., Laurent-Winter C., Hommais F., Danchin A., Bertin P. N. ( 2002). Regulation of bacterial motility in response to low pH in Escherichia coli: the role of H-NS protein. Microbiology 148:1543–1551[PubMed]
    [Google Scholar]
  29. Sperandio V., Torres A. G., Kaper J. B. ( 2002). Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli. Mol Microbiol 43:809–821 [View Article][PubMed]
    [Google Scholar]
  30. Takaya A., Matsui M., Tomoyasu T., Kaya M., Yamamoto T. ( 2006). The DnaK chaperone machinery converts the native FlhD2C2 hetero-tetramer into a functional transcriptional regulator of flagellar regulon expression in Salmonella. Mol Microbiol 59:1327–1340 [View Article][PubMed]
    [Google Scholar]
  31. Tomoyasu T., Ohkishi T., Ukyo Y., Tokumitsu A., Takaya A., Suzuki M., Sekiya K., Matsui H., Kutsukake K., Yamamoto T. ( 2002). The ClpXP ATP-dependent protease regulates flagellum synthesis in Salmonella enterica serovar typhimurium. J Bacteriol 184:645–653 [View Article][PubMed]
    [Google Scholar]
  32. Tomoyasu T., Takaya A., Isogai E., Yamamoto T. ( 2003). Turnover of FlhD and FlhC, master regulator proteins for Salmonella flagellum biogenesis, by the ATP-dependent ClpXP protease. Mol Microbiol 48:443–452 [View Article][PubMed]
    [Google Scholar]
  33. Tomoyasu T., Takaya A., Handa Y., Karata K., Yamamoto T. ( 2005). ClpXP controls the expression of LEE genes in enterohaemorrhagic Escherichia coli. FEMS Microbiol Lett 253:59–66 [View Article][PubMed]
    [Google Scholar]
  34. Umanski T., Rosenshine I., Friedberg D. ( 2002). Thermoregulated expression of virulence genes in enteropathogenic Escherichia coli. Microbiology 148:2735–2744[PubMed]
    [Google Scholar]
  35. Vazquez-Juarez R. C., Kuriakose J. A., Rasko D. A., Ritchie J. M., Kendall M. M., Slater T. M., Sinha M., Luxon B. A., Popov V. L. et al. & other authors ( 2008). CadA negatively regulates Escherichia coli O157 : H7 adherence and intestinal colonization. Infect Immun 76:5072–5081 [View Article][PubMed]
    [Google Scholar]
  36. Wada T., Morizane T., Abo T., Tominaga A., Inoue-Tanaka K., Kutsukake K. ( 2011). EAL domain protein YdiV acts as an anti-FlhD4C2 factor responsible for nutritional control of the flagellar regulon in Salmonella enterica serovar Typhimurium. J Bacteriol 193:1600–1611 [View Article][PubMed]
    [Google Scholar]
  37. Wei B. L., Brun-Zinkernagel A. M., Simecka J. W., Prüß B. M., Babitzke P., Romeo T. ( 2001). Positive regulation of motility and flhDC expression by the RNA-binding protein CsrA of Escherichia coli. Mol Microbiol 40:245–256 [View Article][PubMed]
    [Google Scholar]
  38. Wozniak C. E., Lee C., Hughes K. T. ( 2009). T-POP array identifies EcnR and PefI–SrgD as novel regulators of flagellar gene expression. J Bacteriol 191:1498–1508 [View Article][PubMed]
    [Google Scholar]
  39. Yona-Nadler C., Umanski T., Aizawa S., Friedberg D., Rosenshine I. ( 2003). Integration host factor (IHF) mediates repression of flagella in enteropathogenic and enterohaemorrhagic Escherichia coli. Microbiology 149:877–884 [View Article][PubMed]
    [Google Scholar]
  40. Zhou Y., Gottesman S., Hoskins J. R., Maurizi M. R., Wickner S. ( 2001). The RssB response regulator directly targets σS for degradation by ClpXP. Genes Dev 15:627–637 [View Article][PubMed]
    [Google Scholar]
  41. Zhou X., Girón J. A., Torres A. G., Crawford J. A., Negrete E., Vogel S. N., Kaper J. B. ( 2003). Flagellin of enteropathogenic Escherichia coli stimulates interleukin-8 production in T84 cells. Infect Immun 71:2120–2129 [View Article][PubMed]
    [Google Scholar]
  42. Zhou X., Meng X., Sun B. ( 2008). An EAL domain protein and cyclic AMP contribute to the interaction between the two quorum sensing systems in Escherichia coli. Cell Res 18:937–948 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.051151-0
Loading
/content/journal/micro/10.1099/mic.0.051151-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

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