1887

Abstract

The sequencing of , encoding the transcriptional regulator of virulence genes, in 26 low-virulence field strains showed that eight strains exhibited the same single amino-acid substitution: PrfAK220T. These strains exhibited no expression of PrfA-regulated proteins and thus no virulence. This substitution inactivated PrfA, since expression of the PrfAK220T mutant gene in an EGDΔ strain did not restore the haemolytic and phosphatidylcholine phospholipase C activities, in contrast to the wild-type gene. The substitution of the lysine at position 220 occurred in the helix H. However, the data showed that the PrfAK220T protein is dimerized just as well as its wild-type counterpart, but does not bind to PrfA-boxes. PrfAK220T did not form a PrfA–DNA complex in electrophoretic mobility shift assays, but low concentrations of CI complexes (PrfAK220T–RNA polymerase–DNA complex) were formed by adding RNA polymerase, suggesting that PrfA interacted with RNA polymerase in solution in the absence of DNA. Formation of some transcriptionally active complexes was confirmed by runoff transcription assays and quantitative RT-PCR. Crystallographic analyses described the structure of native PrfA and highlighted the key role of allosteric changes in the activity of PrfA and especially the role of the Lys220 in the conformation of the helix–turn–helix (HTH) motif.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2006/002238-0
2007-04-01
2020-09-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/153/4/995.html?itemId=/content/journal/micro/10.1099/mic.0.2006/002238-0&mimeType=html&fmt=ahah

References

  1. Bockmann R., Dickneite C., Middendorf B., Goebel W., Sokolovic Z.. 1996; Specific binding of the Listeria monocytogenes transcriptional regulator PrfA to target sequences requires additional factor(s) and is influenced by iron. Mol Microbiol22:643–653[CrossRef]
    [Google Scholar]
  2. Bockmann R., Dickneite C., Goebel W., Bohne J.. 2000; PrfA mediates specific binding of RNA polymerase of Listeria monocytogenes to PrfA-dependent virulence gene promoters resulting in a transcriptionally active complex. Mol Microbiol36:487–497[CrossRef]
    [Google Scholar]
  3. Brunger A. T., Adams P. D., Clore G. M., Gros P., Grosse-Kunsteleve R. W., Jiang J. S., Kuszewski J., Nilges M., other authors DeLano W. L.. 1998; Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr54:905–921
    [Google Scholar]
  4. Chico-Calero I., Suarez M., Gonzalez-Zorn B., Scortti M., Slaghuis J., Goebel W., Vazquez-Boland J. A.. 2002; Hpt, a bacterial homolog of the microsomal glucose-6-phosphate translocase, mediates rapid intracellular proliferation in Listeria. Proc Natl Acad Sci U S A99:431–436[CrossRef]
    [Google Scholar]
  5. Collaborative Computational Project 1994; The CCP4 Suite: programs for protein crystallography. Acta Crystallogr4:760–763
    [Google Scholar]
  6. Cowtan K.. 1994; ‘dm’: An automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography31:34–38
    [Google Scholar]
  7. Dickneite C., Bockmann R., Spory A., Goebel W., Sokolovic Z.. 1998; Differential interaction of the transcription factor PrfA and the PrfA-activating factor (paf) of Listeria monocytogenes with target sequences. Mol Microbiol27:915–928[CrossRef]
    [Google Scholar]
  8. Domann E., Wehland J., Rohde M., Pistor S., Hartl M., Goebel W., Leimeisterwachter M., Wuenscher M., Chakraborty T.. 1992; A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. EMBO J11:1981–1986
    [Google Scholar]
  9. Dramsi S., Biswas I., Maguin E., Braun L., Mastroeni P., Cossart P.. 1995; Entry of Listeria monocytogenes into hepatocytes requires expression of InlB, a surface protein of the internalin multigene family. Mol Microbiol16:251–261[CrossRef]
    [Google Scholar]
  10. Eiting M., Hagelueken G., Schubert W.-D., Heinz D. W.. 2004; PrfA, transcriptional regulator in Listeria monocytogenes . Deposition 2004-11-24http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=2beo
  11. Eiting M., Hageluken G., Schubert W. D., Heinz D. W.. 2005; The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes , increases DNA-binding affinity by stabilizing the HTH motif. Mol Microbiol56:433–446[CrossRef]
    [Google Scholar]
  12. Gaillard J. L., Berche P., Frehel C., Gouin E., Cossart P.. 1991; Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from Gram-positive cocci. Cell65:1127–1141[CrossRef]
    [Google Scholar]
  13. Geoffroy C., Raveneau J., Beretti J. L., Lecroisey A., Vazquezboland J. A., Alouf J. E., Berche P.. 1991; Purification and characterization of an extracellular 29-kilodalton phospholipase-C from Listeria monocytogenes. Infect Immun59:2382–2388
    [Google Scholar]
  14. Glaser P., Frangeul L., Buchrieser C., Rusniok C., Amend A., Baquero F., Berche P., Bloecker H., Brandt P.. other authors 2001; Comparative genomics of Listeria species. Science294:849–852
    [Google Scholar]
  15. Glatron M. F., Rapoport G.. 1972; Biosynthesis of the parasporal inclusion of Bacillus thuringiensis : half-life of its corresponding messenger RNA. Biochimie54:1291–1301[CrossRef]
    [Google Scholar]
  16. Goebel W., Kathariou S., Kuhn M., Sokolovic Z., Kreft S., Kohler S., Funke T., Chakraborty T., Leimeister-Wachter M.. 1988; Hemolysin from Listeria – biochemistry, genetics and function in pathogenesis. Infection16 (Suppl. 2:S149–S156[CrossRef]
    [Google Scholar]
  17. Harman J. G.. 2001; Allosteric regulation of the cAMP receptor protein. Biochim Biophys Acta1547:1–17[CrossRef]
    [Google Scholar]
  18. Herler M., Bubert A., Goetz M., Vega Y., Vazquez-Boland J. A., Goebel W.. 2001; Positive selection of mutations leading to loss or reduction of transcriptional activity of PrfA, the central regulator of Listeria monocytogenes virulence. J Bacteriol183:5562–5570[CrossRef]
    [Google Scholar]
  19. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard. 1991; Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A47:110–119[CrossRef]
    [Google Scholar]
  20. Lampidis R., Gross R., Sokolovic Z., Goebel W., Kreft J.. 1994; The virulence regulator protein of Listeria ivanovii is highly homologous to PrfA from Listeria monocytogenes and both belong to the Crp-Fnr family of transcription regulators. Mol Microbiol13:141–151[CrossRef]
    [Google Scholar]
  21. Laskowski R. A., Rullmannn J. A., MacArthur M. W., Kaptein R., Thornton J. M.. 1996; aqua and procheck-nmr: programs for checking the quality of protein structures solved by NMR. J Biomol NMR8:477–486
    [Google Scholar]
  22. Lereclus D., Arantes O.. 1992; spbA locus ensures the segregational stability of pTH1030, a novel type of gram-positive replicon. Mol Microbiol6:35–46[CrossRef]
    [Google Scholar]
  23. Matthews B. W.. 1968; Solvent content of protein crystals. J Mol Biol33:491–497[CrossRef]
    [Google Scholar]
  24. Mengaud J., Dramsi S., Gouin E., Vazquezboland J. A., Milon G., Cossart P.. 1991; Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated. Mol Microbiol5:2273–2283[CrossRef]
    [Google Scholar]
  25. Renzoni A., Klarsfeld A., Dramsi S., Cossart P.. 1997; Evidence that PrfA, the pleiotropic activator of virulence genes in Listeria monocytogenes , can be present but inactive. Infect Immun65:1515–1518
    [Google Scholar]
  26. Ripio M. T., Dominguezbernal G., Suarez M., Brehm K., Berche P., Vazquezboland J. A.. 1996; Transcriptional activation of virulence genes in wild-type strains of Listeria monocytogenes in response to a change in the extracellular medium composition. Res Microbiol147:371–384[CrossRef]
    [Google Scholar]
  27. Ripio M. T., Dominguezbernal G., Lara M., Suarez M., Vazquezboland J. A.. 1997; A gly145ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes. J Bacteriol179:1533–1540
    [Google Scholar]
  28. Roche S. M., Velge P., Bottreau E., Durier C., Pardon P., Marquet-van der Mee N.. 2001; Assessment of the virulence of Listeria monocytogenes : agreement between a plaque-forming assay with HT-29 cells and infection of immunocompetent mice. Int J Food Microbiol68:33–44[CrossRef]
    [Google Scholar]
  29. Roche S. M., Gracieux P., Albert I., Gouali M., Jacquet C., Martin P. M., Velge P.. 2003; Experimental validation of low virulence in field strains of Listeria monocytogenes. Infect Immun71:3429–3436[CrossRef]
    [Google Scholar]
  30. Roche S. M., Gracieux P., Milohanic E., Albert I., Virlogeux-Payant I., Temoin S., Grepinet O., Kerouantan A., Jacquet C.. other authors 2005; Investigation of specific substitutions in virulence genes characterizing phenotypic groups of low-virulence field strains of Listeria monocytogenes. Appl Environ Microbiol71:6039–6048[CrossRef]
    [Google Scholar]
  31. Rocourt J., Cossart P.. 1997; Listeria monocytogenes . In Food Microbiology, Fundamentals and Frontiers pp337–352 Edited by Doyle M. P., Beuchat L. R., Montville T. J.. Washington, DC: American Society for Microbiology Press;
    [Google Scholar]
  32. Schultz S. C., Shields G. C., Steitz T. A.. 1991; Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees. Science253:1001–1007[CrossRef]
    [Google Scholar]
  33. Sheehan B., Klarsfeld A., Msadek T., Cossart P.. 1995; Differential activation of virulence gene expression by PrfA, the Listeria monocytogenes virulence regulator. J Bacteriol177:6469–6476
    [Google Scholar]
  34. Sheehan B., Klarsfeld A., Ebright R., Cossart P.. 1996; A single substitution in the putative helix-turn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence. Mol Microbiol20:785–797[CrossRef]
    [Google Scholar]
  35. Smith G. A., Portnoy D. A.. 1993; The role of two phospholipases in the pathogenicity of Listeria monocytogenes. Infect Agents Dis2:183–185
    [Google Scholar]
  36. Stritzker J., Schoen C., Goebel W.. 2005; Enhanced synthesis of internalin A in aro mutants of Listeria monocytogenes indicates posttranscriptional control of the inlAB mRNA. J Bacteriol187:2836–2845[CrossRef]
    [Google Scholar]
  37. Thirumuruhan R., Rajashankar K., Fedorov A. A., Dodatko T., Chance M. R., Cossart P., Almo S. C.. 2003; Crystal structure of PrfA, the transcriptional regulator in Listeria monocytogenes . Deposition 2003-02-25http://www.rcsb.org/pdb/explore/explore.do?structureId=1OMI
  38. Tompkin R. B.. 2002; Control of Listeria monocytogenes in the food-processing environment. J Food Prot65:709–725
    [Google Scholar]
  39. Vazquez Boland J. A., Kocks C., Dramsi S., Ohayon H., Geoffroy C., Mengaud J., Cossart P.. 1992; Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread. Infect Immun60:219–230
    [Google Scholar]
  40. Vazquez-Boland J. A., Dominguez-Bernal G., Gonzalez-Zorn B., Kreft J., Goebel W.. 2001; Pathogenicity islands and virulence evolution in Listeria. Microbes Infect3:571–584[CrossRef]
    [Google Scholar]
  41. Vega Y., Rauch M., Banfield M. J., Ermolaeva S., Scortti M., Goebel W., Vazquez-Boland J. A.. 2004; New Listeria monocytogenes prfA * mutants, transcriptional properties of PrfA* proteins and structure–function of the virulence regulator PrfA. Mol Microbiol52:1553–1565[CrossRef]
    [Google Scholar]
  42. Williams J. R., Thayyullathil C., Freitag N. E.. 2000; Sequence variations within PrfA DNA binding sites and effects on Listeria monocytogenes virulence gene expression. J Bacteriol182:837–841[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2006/002238-0
Loading
/content/journal/micro/10.1099/mic.0.2006/002238-0
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