Advertisement

Molecular Genetics and Genomics

, Volume 270, Issue 1, pp 66–77 | Cite as

Shigella flexneri 2a strain 2457T expresses three members of the H-NS-like protein family: characterization of the Sfh protein

  • C. Beloin
  • P. Deighan
  • M. Doyle
  • C. J. Dorman
Original Paper

Abstract

Shigella flexneri 2a is known to express the H-NS nucleoid-structuring protein and the paralogous protein StpA. Using bioinformatic analysis we have now discovered a third member of the H-NS protein family, Sfh (S higella f lexneri H-NS-like protein), in strain 2457T. This protein is encoded by the sfh gene, which is located on a high-molecular-mass plasmid that is closely related to the self-transmissible plasmid R27. When expressed in Escherichia coli, the Sfh protein can complement an hns null mutation, restoring wild-type Bgl, porin protein, and mucoidy phenotypes, and wild-type expression of the fliC and proU genes. While a knockout mutation in the sfh gene alone had no effect on the expression of virulence genes in S. flexneri, an additive effect on virulence gene derepression was seen when the sfh lesion was combined with a mutation in hns. Over-expression of the sfh gene repressed expression of the VirB virulence regulatory protein and transcription of a VirB-dependent structural gene promoter. The purified Sfh protein bound specifically to DNA sequences containing the promoters of the virF and virB virulence regulatory genes. These findings show that Sfh has the ability to influence genetic events beyond the genetic element that encodes it, including the expression of the S. flexneri virulence genes. They raise the possibility of a triangular relationship among three closely related proteins with broad consequences for genetic events in the bacterium that harbours them.

Keywords

Sfh protein Plasmid R27 H-NS protein StpA protein  Shigella flexneri

Notes

Acknowledgements

We thank Philippe Bertin for the E. coli strains BE1414 and YK3421, Maria Mavris for S. flexneri strains, and Pablo Garcia Bravo and Antonio Cerqueira for help with protein purification. This work was supported by European Training and Mobility of Researchers Award ERBFMRXCT98-0164, Wellcome Trust Award 064284/Z01/Z and Enterprise Ireland Award SC/2002/22. P. Deighan was supported by a Wellcome Trust Prize Fellowship. The research described in this paper was performed in compliance with current laws governing genetic experimentation in the Republic of Ireland.

References

  1. Adler B, Sasakawa C, Tobe T, Makino S, Komatsu K, Yoshikawa M (1989) A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence-associated antigens of Shigella flexneri. Mol Microbiol 3:627–635PubMedGoogle Scholar
  2. Altschul W, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefPubMedGoogle Scholar
  3. Atlung T, Ingmer H (1997) H-NS: a modulator of environmentally regulated gene expression. Mol Microbiol 24:7–17PubMedGoogle Scholar
  4. Beloin C, Dorman CJ (2003) An extended role for the nucleoid structuring protein H-NS in the virulence gene regulatory cascade of Shigella flexneri. Mol Microbiol 47:825–838CrossRefPubMedGoogle Scholar
  5. Beloin C, McKenna S, Dorman CJ (2002) Molecular dissection of VirB, a key regulator of the virulence cascade of Shigella flexneri. J Biol Chem 277:15333–15344Google Scholar
  6. Bertin P, Terao E, Lee EH, Lejeune P, Colson C, Danchin A, Collatz E (1994) The H-NS protein is involved in the biogenesis of flagella in Escherichia coli. J Bacteriol 176:5537–5540PubMedGoogle Scholar
  7. 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 proteins is evolutionarily conserved in Gram-negative bacteria. Mol Microbiol 31:319–330CrossRefPubMedGoogle Scholar
  8. Bracco L, Kotlarz D, Kolb A, Diekmann S, Buc H (1989) Synthetic curved DNA sequences can act as transcriptional activators in Escherichia coli. EMBO J 8:4289–4296Google Scholar
  9. Bräu B, Pilz U, Piepersberg W (1984) Genes for gentamycin-(3)-acetyltransferase III and IV: I. Nucleotide sequence of the AAC(3)-IV gene and possible involvement of an IS140 element in its expression. Mol Gen Genet 183:179–187Google Scholar
  10. Buchrieser C, Glaser P, Rusniok C, Nedjari H, d'Hauteville H, Kunst F, Sansonetti P, Parsot C (2000) The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri. Mol Microbiol 38:760–771CrossRefPubMedGoogle Scholar
  11. Casadaban MJ (1976) Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol 104:541–555PubMedGoogle Scholar
  12. Chaveroche MK, Ghigo JM, d'Enfert C (2000) A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res 28E97Google Scholar
  13. Csonka LN, Epstein W (1996) Osmoregulation. In: Curtiss, R III, Ingraham JL, Lin ECC, Brooks Low K, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella: cellular and molecular biology. ASM Press, Washington D.C., pp1210–1223Google Scholar
  14. Cusick ME, Belfort M (1998) Domain structure and RNA annealing activity of the Escherichia coli regulatory protein StpA. Mol Microbiol 28:847–857CrossRefPubMedGoogle Scholar
  15. Dame RT, Wyman C, Goosen N (2000) H-NS mediated compaction of DNA visualized by atomic force microscopy. Nucleic Acids Res 28:3504–3510CrossRefPubMedGoogle Scholar
  16. Datsenko KA, Wanner BL. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645CrossRefPubMedGoogle Scholar
  17. Deighan P, Free A, Dorman CJ (2000) A role for the Escherichia coli H-NS-like protein StpA in OmpF porin expression through modulation of micF RNA stability. Mol Microbiol 38:126–139CrossRefPubMedGoogle Scholar
  18. Deighan P, Beloin C, Dorman CJ (2003) Three-way interactions among the Sfh, StpA, and H-NS nucleoid-structuring proteins of Shigella flexneri 2a strain 2457T. Mol Microbiol 48:1401–1416.CrossRefPubMedGoogle Scholar
  19. Dersch P, Schmidt K, Bremer E (1993) Synthesis of the Escherichia coli K-12 nucleoid-associated DNA-binding protein H-NS is subjected to growth-phase control and autoregulation. Mol Microbiol 8:875–889PubMedGoogle Scholar
  20. Dorman CJ, Hinton JCD, Free A (1999) Domain organization and oligomerization among H-NS-like nucleoid-associated proteins in bacteria. Trends Microbiol 7:124–128CrossRefPubMedGoogle Scholar
  21. Falconi M, Colonna B, Prosseda G, Micheli G, Gualerzi CO (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 17:7033–7043CrossRefPubMedGoogle Scholar
  22. Falconi M, Prosseda G, Giangrossi M, Beghetto E, Colonna B (2001) Involvement of FIS in the H-NS-mediated regulation of virF gene of Shigella and enteroinvasive Escherichia coli. Mol Microbiol 42:439–452CrossRefPubMedGoogle Scholar
  23. Formal SB, Gustave JD, LaBrec EH, Schneider H (1958) Experimental shigella infections: characteristics of a fatal infection produced in guinea pigs. J Bacteriol 75:604–610Google Scholar
  24. Free A, Williams RM, Dorman CJ (1998) The StpA protein functions as a molecular adapter to mediate repression of the bgl operon by truncated H-NS in Escherichia coli. J Bacteriol 180:994–997PubMedGoogle Scholar
  25. Free A, Porter ME, Deighan P, Dorman CJ (2001) Requirement for the molecular adapter function of StpA at the Escherichia coli bgl promoter depends upon the level of truncated H-NS protein. Mol Microbiol 42:903–918CrossRefPubMedGoogle Scholar
  26. Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130PubMedGoogle Scholar
  27. Higgins CF, Dorman CJ, Stirling DA, Waddell L, Booth IR, May G, Bremer E (1988) A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli. Cell 52:569–584PubMedGoogle Scholar
  28. Hommais F, Krin E, Laurent-Winter C, Soutourina O, Malpertuy A, Le Caer JP, Danchin A, Bertin P (2001) Large-scale monitoring of pleiotropic regulation of gene expression by the prokaryotic nucleoid-associated protein, H-NS. Mol Microbiol 40:20–36CrossRefPubMedGoogle Scholar
  29. Hromockyj AE, Maurelli AT (1992) Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1 tyr). Mol Microbiol 6:2113–2124PubMedGoogle Scholar
  30. Jin Q, et al (2002) Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157. Nucleic Acids Res 30:4432–4441CrossRefPubMedGoogle Scholar
  31. Johansson J, Uhlin BE (1999) Differential protease-mediated turnover of H-NS and StpA revealed by a mutation altering protein stability and stationary phase survival of Escherichia coli. Proc Natl Acad Sci USA 96:10776–10781CrossRefPubMedGoogle Scholar
  32. Johansson J, Eriksson S, Sondén B, Wai SN, Uhlin BE (2001) Heteromeric interactions among nucleoid-associated bacterial proteins: localization of StpA-stabilizing regions in H-NS of Escherichia coli. J Bacteriol 183:2343–2347CrossRefPubMedGoogle Scholar
  33. Lucht JM, Dersch P, Kempf B, Bremer E (1994) Interactions of the nucleoid-associated DNA-binding protein H-NS with the regulatory region of the osmotically controlled proU operon in Escherichia coli. J Biol Chem 269:6578–6586PubMedGoogle Scholar
  34. Maher D, Taylor DE (1993) Host range and transfer efficiency of incompatibility group HI plasmids. Can J Microbiol 39:581–587PubMedGoogle Scholar
  35. Maurelli AT, Sansonetti PJ (1988) Identification of a chromosomal gene controlling temperature-regulated expression of virulence genes in Shigella flexneri. Proc Natl Acad Sci USA 85:2820–2824PubMedGoogle Scholar
  36. Maurelli AT, Blackmon B, Curtiss R III (1984) Temperature-dependent expression of virulence genes in Shigella flexneri. Infect Immun 43:196–201Google Scholar
  37. Maurelli AT, Baudry B, d'Hauteville H, Hale TL, Sansonetti PJ (1985) Cloning of plasmid DNA sequences involved in invasion of HeLa cells by Shigella flexneri. Infect Immun 49:164–171PubMedGoogle Scholar
  38. Miller JH (1992) A Short course in bacterial genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
  39. Nakayama S-I, Watanabe H (1995) Involvement of cpxA, a sensor of a two-component regulatory system, in the pH-dependent regulation of expression of Shigella sonnei virF gene. J Bacteriol 177:5062–5069PubMedGoogle Scholar
  40. Nakayama S-I, Watanabe H (1998) Identification of cpxR as a positive regulator essential for expression of the Shigella sonnei virF gene. J Bacteriol 180:3522–3528PubMedGoogle Scholar
  41. Owen-Hughes TA, Pavitt GD, Santos DS, Sidebotham JM, Hulton CSJ, Hinton JCD, Higgins CF (1992) The chromatin-associated protein H-NS interacts with curved DNA to influence DNA topology and gene expression. Cell 71:255–265PubMedGoogle Scholar
  42. Page RDM (1996) TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Appl Biosci 12:357–358Google Scholar
  43. Parkhill J, et al (2001) Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature 413:848–852CrossRefPubMedGoogle Scholar
  44. Porter ME (1998) The Regulation of virulence gene expression in Shigella flexneri. PhD thesis, University of DublinGoogle Scholar
  45. Porter ME, Dorman CJ (1994) A role for H-NS in the thermo-osmotic regulation of virulence gene expression in Shigella flexneri. J Bacteriol 176:4187–4191PubMedGoogle Scholar
  46. Prosseda G, Fradiani PA, Di Lorenzo M, Falconi M, Micheli G, Casalino M, Nicoletti M, Colonna B (1998) A role for H-NS in the regulation of the virF gene of Shigella and enteroinvasive Escherichia coli. Res Microbiol 149:15–25CrossRefPubMedGoogle Scholar
  47. Sasakawa C, Kamata K, Sakai T, Makino S, Yamada M, Okada N, Yoshikawa M (1988) Virulence-associated genetic region comprising 31 kilobases of the 230-kilobase plasmid in Shigella flexneri 2a. J Bacteriol 170:2480–2484PubMedGoogle Scholar
  48. Schröder O, Wagner R (2002) The bacterial regulatory protein H-NS – a versatile modulator of nucleic acid structures. J Biol Chem 383:945–960Google Scholar
  49. Sherburne CK, Lawley TD, Gilmoir MW, Blattner FR. Burland V, Grotbeck E, Rose DJ, Taylor DE (2000) The complete DNA sequence and analysis of R27, a large IncHI plasmid from Salmonella typhi that is temperature sensitive for transfer. Nucleic Acids Res 28:2177–2186CrossRefPubMedGoogle Scholar
  50. Smyth CP, Lundbäck T, Rezoni D, Siligardi G, Beavil R, Layton M, Sidebotham JM, Hinton JCD, Driscoll PC, Higgins CF, Ladbury JE (2000) Oligomerization of the chromatin-structuring protein H-NS. Mol Microbiol 36:962–972CrossRefPubMedGoogle Scholar
  51. Sondén B, Uhlin BE (1996) Coordinated and differential expression of histone-like proteins in Escherichia coli: regulation and function of the H-NS analog StpA. EMBO J 15:4970–4980PubMedGoogle Scholar
  52. Sonnenfield JM, Burns CM, Higgins CF, Hinton JC (2001) The nucleoid-associated protein StpA binds curved DNA, has a greater DNA-binding affinity than H-NS and is present in significant levels in hns mutants. Biochimie 83:243–249CrossRefPubMedGoogle Scholar
  53. Soutourina O, Kolb A, Krin E, Laurent-Winter C, Rimsky S, Danchin A, Bertin P (1999) Multiple control of flagella 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 181:7500–7508PubMedGoogle Scholar
  54. Spurio R, Falconi M, Brandi A, Pon CL, Gualerzi CO (1997) The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending. EMBO J 16:1795–1805CrossRefPubMedGoogle Scholar
  55. Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130PubMedGoogle Scholar
  56. Taylor DE, Levine JG (1980) Studies of temperature-sensitive transfer and maintenance of H incompatibility group plasmids. J Gen Microbiol 116:475–484PubMedGoogle Scholar
  57. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighing, position-specific gap penalties and weigh matrix choice. Nucleic Acids Res 22:4673–4680PubMedGoogle Scholar
  58. Tobe T, Nagai S, Okada N, Adler B, Yoshikawa M, Sasakawa C (1991) Temperature-regulated expression of invasion genes in Shigella flexneri is controlled through the transcriptional activation of the virB gene on the large virulence plasmid. Mol Microbiol 5:887–893PubMedGoogle Scholar
  59. Tobe T, Yoshikawa M, Mizuno T, Sasakawa C (1993) Transcriptional control of the invasion regulatory gene virB of Shigella flexneri: activation by VirF and repression by H-NS. J Bacteriol 175:6142–6149PubMedGoogle Scholar
  60. Tupper AE, Owen-Hughes TA, Ussery DW, Santos DS, Ferguson FJP, Sidebotham JM, Hinton JCD, Higgins CF (1994) The chromatin-associated protein H-NS alters DNA topology in vitro. EMBO J 13:258–268PubMedGoogle 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 263:149–162CrossRefPubMedGoogle 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 274:145–151CrossRefPubMedGoogle Scholar
  63. Venkatesan MM, Goldberg MB, Rose DJ, Grotbeck EJ, Burland V, Blattner FR (2001) Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri. Infect Immun 69:3271–3285CrossRefPubMedGoogle Scholar
  64. Wei J, et al (2003) Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T. Infect Immun 71:2775–2786.CrossRefPubMedGoogle Scholar
  65. Williams RM, Rimsky S (1997) Molecular aspects of the E. coli nucleoid protein, H-NS: a central controller of gene regulatory networks. FEMS Microbiol Lett 156:175–185CrossRefPubMedGoogle Scholar
  66. Williams RM, 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 178:4335–4343PubMedGoogle Scholar
  67. Yamada H, Muramatsu S, Mizuno T (1990) An Escherichia coli protein that preferentially binds to sharply curved DNA. J Biochem (Tokyo) 108:420–425Google Scholar
  68. Zhang A, Belfort M (1992) Nucleotide sequence of a newly identified Escherichia coli gene, stpA, encoding an H-NS-like protein. Nucleic Acids Res 20:6735PubMedGoogle Scholar
  69. Zhang A, Rimsky S, Reaban ME, Buc H, Belfort M (1996) Escherichia coli protein analogs StpA and H-NS: regulatory networks, similar and disparate effects on nucleic acid dynamics. EMBO J 15:1340–1349PubMedGoogle Scholar
  70. Zhang Y, Buchholz F, Muyrers JP, Stewart AF (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20:123–128CrossRefPubMedGoogle Scholar
  71. Zuber F, Kotlars D, Rimsky S, Buc H (1994) Modulated expression of promoters containing upstream curved DNA sequences by the Escherichia coli nucleoid protein H-NS. Mol Microbiol 12:231–240PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • C. Beloin
    • 1
    • 2
  • P. Deighan
    • 1
  • M. Doyle
    • 1
  • C. J. Dorman
    • 1
  1. 1.Department of Microbiology, Moyne Institute of Preventive MedicineUniversity of Dublin, Trinity CollegeDublin 2Ireland
  2. 2.Groupe de Génétique des BiofilmsInstitut PasteurParisFrance

Personalised recommendations