1887

Abstract

Enteropathogenic (EPEC) expresses a type III secretion system (T3SS) required for pathogenesis. Regulation of the genes encoding the T3SS is complex; two major regulators control transcription, the silencer H-NS, and the related H-NS-like protein Ler. Our laboratory is interested in understanding the molecular differences that distinguish the anti-silencer Ler from H-NS, and how Ler differentially regulates EPEC virulence genes. Here, we demonstrate that mutated Ler proteins either containing H-NS -helices 1 and 2, missing from Ler, or truncated for the 11 aa C-terminal extension compared with the related H-NS protein, did not appreciably alter Ler function. In contrast, mutating the proline at position 92 of Ler, in the conserved C-terminal DNA binding motif, eliminated Ler activity. Inserting 11 H-NS-specific amino acids, 11 alanines or 6 alanines into the Ler linker severely impaired the ability of Ler to increase transcription. To extend our analysis, we constructed six chimeric proteins containing the N terminus, linker region or C terminus of Ler in different combinations with the complementary domains of H-NS, and monitored their activities. Replacing the Ler linker domain with that of H-NS, or replacing the Ler C-terminal, DNA binding domain with that of H-NS eliminated the ability of Ler to increase transcription at the promoter. Thus, the linker and C-terminal domains of Ler and H-NS are not functionally equivalent. Conversely, replacing the H-NS linker region with that of Ler caused increased transcription at in a strain deleted for . In summary, the interdomain linker specific to Ler is necessary for anti-silencing activity in EPEC.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2008/023382-0
2008-12-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/12/3624.html?itemId=/content/journal/micro/10.1099/mic.0.2008/023382-0&mimeType=html&fmt=ahah

References

  1. Barba J., Bustamante V. H., Flores-Valdez M. A., Deng W., Finlay B. B., Puente J. L. 2005; A positive regulatory loop controls expression of the locus of enterocyte effacement-encoded regulators Ler and GrlA. J Bacteriol 187:7918–7930
    [Google Scholar]
  2. Beltrametti F., Kresse A. U., Guzmán C. A. 1999; Transcriptional regulation of the esp genes of enterohemorrhagic Escherichia coli . J Bacteriol 181:3409–3418
    [Google Scholar]
  3. Bertin P., Benhabiles N., Krin E., Laurent-Winter C., Tendeng C., Turlin E., Thomas A., Danchin A., Brasseur R. 1999; The structural and functional organization of H-NS-like proteins is evolutionarily conserved in Gram-negative bacteria. Mol Microbiol 31:319–329
    [Google Scholar]
  4. Bloch V., Yang Y., Margeat E., Chavanieu A., Auge M. T., Robert B., Arold S., Rimsky S., Kochoyan M. 2003; The H-NS dimerization domain defines a new fold contributing to DNA recognition. Nat Struct Biol 10:212–218
    [Google Scholar]
  5. Bouffartigues E., Buckle M., Badaut C., Travers A., Rimsky S. 2007; H-NS cooperative binding to high-affinity sites in a regulatory element results in transcriptional silencing. Nat Struct Mol Biol 14:441–448
    [Google Scholar]
  6. 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
    [Google Scholar]
  7. Casadaban M. J. 1976; Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol 104:541–555
    [Google Scholar]
  8. Clarke S. C., Haigh R. D., Freestone P. P., Williams P. H. 2003; Virulence of enteropathogenic Escherichia coli, a global pathogen. Clin Microbiol Rev 16:365–378
    [Google Scholar]
  9. Colonna B., Casalino M., Fradiani P. A., Zagaglia C., Naitza S., Leoni L., Prosseda G., Coppo A., Ghelardini P., Nicoletti M. 1995; H-NS regulation of virulence gene expression in enteroinvasive Escherichia coli harboring the virulence plasmid integrated into the host chromosome. J Bacteriol 177:4703–4712
    [Google Scholar]
  10. Corbett D., Bennett H. J., Askar H., Green J., Roberts I. S. 2007; SlyA and H-NS regulate transcription of the Escherichia coli K5 capsule gene cluster, and expression of slyA in Escherichia coli is temperature-dependent, positively autoregulated, and independent of H-NS. J Biol Chem 282:33326–33335
    [Google Scholar]
  11. Delgado J., Forst S., Harlocker S., Inouye M. 1993; Identification of a phosphorylation site and functional analysis of conserved aspartic acid residues of OmpR, a transcriptional activator for ompF and ompC in Escherichia coli . Mol Microbiol 10:1037–1047
    [Google Scholar]
  12. Deng W., Puente J. L., Gruenheid S., Li Y., Vallance B. A., Vázquez A., Barba J., Ibarra J. A., O'Donnell P. other authors 2004; Dissecting virulence: systematic and functional analyses of a pathogenicity island. Proc Natl Acad Sci U S A 101:3597–3602
    [Google Scholar]
  13. Dorman C. J. 2004; H-NS: a universal regulator for a dynamic genome. Nat Rev Microbiol 2:391–400
    [Google Scholar]
  14. Dorman C. J. 2007; H-NS, the genome sentinel. Nat Rev Microbiol 5:157–161
    [Google Scholar]
  15. Dorman C. J., Hinton J. C., Free A. 1999; Domain organization and oligomerization among H-NS-like nucleoid-associated proteins in bacteria. Trends Microbiol 7:124–128
    [Google Scholar]
  16. Elliott S. J., Sperandio V., Girón J. A., Shin S., Mellies J. L., Wainwright L., Hutcheson S. W., McDaniel T. K., Kaper J. B. 2000; The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli . Infect Immun 68:6115–6126
    [Google Scholar]
  17. Ellison D. W., Miller V. L. 2006; Regulation of virulence by members of the MarR/SlyA family. Curr Opin Microbiol 9:153–159
    [Google Scholar]
  18. Esposito D., Petrovic A., Harris R., Ono S., Eccleston J. F., Mbabaali A., Haq I., Higgins C. F., Hinton J. C. other authors 2002; H-NS oligomerization domain structure reveals the mechanism for high order self-association of the intact protein. J Mol Biol 324:841–850
    [Google Scholar]
  19. Falconi M., Colonna B., Prosseda G., Micheli G., Gualerzi C. O. 1998; Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS. EMBO J 17:7033–7043
    [Google Scholar]
  20. Fang F. C., Rimsky S. 2008; New insights into transcriptional regulation by H-NS. Curr Opin Microbiol 11:113–120
    [Google Scholar]
  21. 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
    [Google Scholar]
  22. Garmendia J., Frankel G., Crepin V. F. 2005; Enteropathogenic and enterohemorrhagic Escherichia coli infections: translocation, translocation, translocation. Infect Immun 73:2573–2585
    [Google Scholar]
  23. Goldberg M. D., Johnson M., Hinton J. C., Williams P. H. 2001; Role of the nucleoid-associated protein Fis in the regulation of virulence properties of enteropathogenic Escherichia coli . Mol Microbiol 41:549–559
    [Google Scholar]
  24. Gomez-Duarte O. G., Kaper J. B. 1995; A plasmid-encoded regulatory region activates chromosomal eaeA expression in enteropathogenic Escherichia coli . Infect Immun 63:1767–1776
    [Google Scholar]
  25. 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
    [Google Scholar]
  26. Guzman L. M., Belin D., Carson M. J., Beckwith J. 1995; Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130
    [Google Scholar]
  27. Haack K. R., Robinson C. L., Miller K. J., Fowlkes J. W., Mellies J. L. 2003; Interaction of Ler at the LEE5 ( tir) operon of enteropathogenic Escherichia coli . Infect Immun 71:384–392
    [Google Scholar]
  28. Hansen A. M., Qiu Y., Yeh N., Blattner F. R., Durfee T., Jin D. J. 2005; SspA is required for acid resistance in stationary phase by downregulation of H-NS in Escherichia coli . Mol Microbiol 56:719–734
    [Google Scholar]
  29. Heroven A. K., Nagel G., Tran H. J., Parr S., Dersch P. 2004; RovA is autoregulated and antagonizes H-NS-mediated silencing of invasin and rovA expression in Yersinia pseudotuberculosis . Mol Microbiol 53:871–888
    [Google Scholar]
  30. Ide T., Michgehl S., Knappstein S., Heusipp G., Schmidt M. A. 2003; Differential modulation by Ca2+ of type III secretion of diffusely adhering enteropathogenic Escherichia coli . Infect Immun 71:1725–1732
    [Google Scholar]
  31. Iyoda S., Watanabe H. 2004; Positive effects of multiple pch genes on expression of the locus of enterocyte effacement genes and adherence of enterohaemorrhagic Escherichia coli O157 : H7 to HEp-2 cells. Microbiology 150:2357–2571
    [Google Scholar]
  32. Kanamaru K., Aiba H., Mizuno T. 1990; Transmembrane signal transduction and osmoregulation in Escherichia coli: I. Analysis by site-directed mutagenesis of the amino acid residues involved in phosphotransfer between the two regulatory components. EnvZ and OmpR. J Biochem 108:483–487
    [Google Scholar]
  33. Kaper J. B., Nataro J. P., Mobley H. L. 2004; Pathogenic Escherichia coli . Nat Rev Microbiol 2:123–140
    [Google Scholar]
  34. Kenny B., Finlay B. B. 1995; Protein secretion by enteropathogenic Escherichia coli is essential for transducing signals to epithelial cells. Proc Natl Acad Sci U S A 92:7991–7995
    [Google Scholar]
  35. Kenny B., Abe A., Stein M., Finlay B. B. 1997; Enteropathogenic Escherichia coli protein secretion is induced in response to conditions similar to those in the gastrointestinal tract. Infect Immun 65:2606–2612
    [Google Scholar]
  36. Li M., Rosenshine I., Tung S. L., Wang X. H., Friedberg D., Hew C. L., Leung K. Y. 2004; Comparative proteomic analysis of extracellular proteins of enterohemorrhagic and enteropathogenic Escherichia coli strains and their ihf and ler mutants. Appl Environ Microbiol 70:5274–5282
    [Google Scholar]
  37. Lithgow J. K., Haider F., Roberts I. S., Green J. 2007; Alternate SlyA and H-NS nucleoprotein complexes control hlyE expression in Escherichia coli K-12. Mol Microbiol 66:685–698
    [Google Scholar]
  38. Mattison K., Oropeza R., Kenney L. J. 2002; The linker region plays an important role in the interdomain communication of the response regulator OmpR. J Biol Chem 277:32714–32721
    [Google Scholar]
  39. Mellies J. L., Elliott S. J., Sperandio V., Donnenberg M. S., Kaper J. B. 1999; The Per regulon of enteropathogenic Escherichia coli: identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler. Mol Microbiol 33:296–306
    [Google Scholar]
  40. Mellies J. L., Barron A. M., Carmona A. M. 2007a; Enteropathogenic and enterohemorrhagic Escherichia coli virulence gene regulation. Infect Immun 75:4199–4210
    [Google Scholar]
  41. Mellies J. L., Haack K. R., Galligan D. C. 2007b; SOS regulation of the type III secretion system of enteropathogenic Escherichia coli . J Bacteriol 189:2863–2872
    [Google Scholar]
  42. Miller J. H. 1972 Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  43. Mukerji M., Mahadevan S. 1997; Characterization of the negative elements involved in silencing the bgl operon of Escherichia coli: possible roles for DNA gyrase, H-NS, and CRP-cAMP in regulation. Mol Microbiol 24:617–627
    [Google Scholar]
  44. Müller C. M., Dobrindt U., Nagy G., Emèody L., Uhlin B. E., Hacker J. 2006; Role of histone-like proteins H-NS and StpA in expression of virulence determinants of uropathogenic Escherichia coli . J Bacteriol 188:5428–5438
    [Google Scholar]
  45. Navarre W. W., Porwollik S., Wang Y., McClelland M., Rosen H., Libby S. J., Fang F. C. 2006; Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella . Science 313:236–238
    [Google Scholar]
  46. Porter M. E., Mitchell P., Free A., Smith D. G., Gally D. L. 2005; The LEE1 promoters from both enteropathogenic and enterohemorrhagic Escherichia coli can be activated by PerC-like proteins from either organism. J Bacteriol 187:458–472
    [Google Scholar]
  47. Rumer L., Jores J., Kirsch P., Cavignac Y., Zehmke K., Wieler L. H. 2003; Dissemination of pheU- and pheV-located genomic islands among enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli and their possible role in the horizontal transfer of the locus of enterocyte effacement (LEE. Int J Med Microbiol 292:463–475
    [Google Scholar]
  48. Russell R. M., Sharp F. C., Rasko D. A., Sperandio V. 2007; QseA and GrlR/GrlA regulation of the locus of enterocyte effacement genes in enterohemorrhagic Escherichia coli . J Bacteriol 189:5387–5392
    [Google Scholar]
  49. Sánchez-SanMartín C., Bustamante V. H., Calva E., Puente J. L. 2001; Transcriptional regulation of the orf19 gene and the tir-cesT-eae operon of enteropathogenic Escherichia coli . J Bacteriol 183:2823–2833
    [Google Scholar]
  50. Sharp F. C., Sperandio V. 2007; QseA directly activates transcription of LEE1 in enterohemorrhagic Escherichia coli . Infect Immun 75:2432–2440
    [Google Scholar]
  51. Shin S., Castanie-Cornet M. P., Foster J. W., Crawford J. A., Brinkley C., Kaper J. B. 2001; An activator of glutamate decarboxylase genes regulates the expression of enteropathogenic Escherichia coli virulence genes through control of the plasmid-encoded regulator, Per. Mol Microbiol 41:1133–1150
    [Google Scholar]
  52. Simons R. W., Houman F., Kleckner N. 1987; Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53:85–96
    [Google Scholar]
  53. Sperandio V., Mellies J. L., Nguyen W., Shin S., Kaper J. B. 1999; Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli . Proc Natl Acad Sci U S A 96:15196–15201
    [Google Scholar]
  54. Sperandio V., Mellies J. L., Delahay R. M., Frankel G., Crawford J. A., Nguyen W., Kaper J. B. 2000; Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by Ler. Mol Microbiol 38:781–793
    [Google Scholar]
  55. Sperandio V., Torres A. G., Giron J. A., Kaper J. B. 2001; Quorum sensing is a global regulatory mechanism in enterohemorrhagic Escherichia coli O157 : H7. J Bacteriol 183:5187–5197
    [Google Scholar]
  56. Spurio R., Falconi M., Brandi A., Pon C. L., Gualerzi C. O. 1997; The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending. EMBO J 16:1795–1805
    [Google Scholar]
  57. Stella S., Spurio R., Falconi M., Pon C. L., Gualerzi C. O. 2005; Nature and mechanism of the in vivo oligomerization of nucleoid protein H-NS. EMBO J 24:2896–2905
    [Google Scholar]
  58. Tobe T., Beatson S. A., Taniguchi H., Abe H., Bailey C. M., Fivian A., Younis R., Matthews S., Marches O. other authors 2006; An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination. Proc Natl Acad Sci U S A 103:14941–14946
    [Google Scholar]
  59. Torres A. G., Lopez-Sanchez G. N., Milflores-Flores L., Patel S. D., Rojas-Lopez M., Martinez de la Pena C. F., Arenas-Hernandez M. M., Martinez-Laguna Y. 2007; Ler and H-NS, regulators controlling expression of the long polar fimbriae of Escherichia coli O157 : H7. J Bacteriol 189:5916–5928
    [Google Scholar]
  60. Typas A., Becker G., Hengge R. 2007; The molecular basis of selective promoter activation by the σ S subunit of RNA polymerase. Mol Microbiol 63:1296–1306
    [Google Scholar]
  61. Ueguchi C., Suzuki T., Yoshida T., Tanaka K., Mizuno T. 1996; Systematic mutational analysis revealing the functional domain organization of Escherichia coli nucleoid protein H-NS. J Mol Biol 263:149–162
    [Google Scholar]
  62. Ueguchi C., Seto C., Suzuki T., Mizuno T. 1997; Clarification of the dimerization domain and its functional significance for the Escherichia coli nucleoid protein H-NS. J Mol Biol 274:145–151
    [Google Scholar]
  63. Umanski T., Rosenshine I., Friedberg D. 2002; Thermoregulated expression of virulence genes in enteropathogenic Escherichia coli . Microbiology 148:2735–2744
    [Google Scholar]
  64. Walthers D., Tran V. K., Kenney L. J. 2003; Interdomain linkers of homologous response regulators determine their mechanism of action. J Bacteriol 185:317–324
    [Google Scholar]
  65. Williams R. M., Rimsky S., Buc H. 1996; Probing the structure, function, and interactions of the Escherichia coli H-NS and StpA proteins by using dominant negative derivatives. J Bacteriol 178:4335–4343
    [Google Scholar]
  66. Williamson H. S., Free A. 2005; A truncated H-NS-like protein from enteropathogenic Escherichia coli acts as an H-NS antagonist. Mol Microbiol 55:808–827
    [Google Scholar]
  67. Yu R. R., DiRita V. J. 2002; Regulation of gene expression in Vibrio cholerae by ToxT involves both antirepression and RNA polymerase stimulation. Mol Microbiol 43:119–134
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2008/023382-0
Loading
/content/journal/micro/10.1099/mic.0.2008/023382-0
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