1887

Abstract

Guanosine penta- and tetraphosphate [(p)ppGpp] are two unusual nucleotides implied in the bacterial stringent response. In many pathogenic bacteria, mutants unable to synthesize these molecules lose their virulence. In Gram-positive bacteria such as , the synthesis and degradation of (p)ppGpp mainly depend on the activity of a bifunctional enzyme, encoded by the gene. By analysing Δ and Δ (which encodes a protein harbouring a ppGpp synthetase activity) deletion mutants, we showed that RelA is by far the main system leading to (p)ppGpp production under our experimental conditions, and during the development of a stringent response induced by mupirocin. We also constructed a mutant (Δ) in which a small part of the gene (about 0.7 kbp) encoding the carboxy-terminal domain of the RelA protein was deleted. Both mutants were more resistant than the wild-type strain to 0.3 % bile salts, 25 % ethanol and acid (pH 2.3) challenges. Interestingly, the Δ mutant grew better than the two other strains in the presence of 1 mM HO, but did not display increased tolerance when subjected to lethal doses of HO (45 mM). By contrast, the Δ mutant was highly sensitive to 45 mM HO and displayed reduced growth in a medium containing 1 M NaCl. The two mutants also displayed contrasting virulence phenotypes towards larvae of the Greater Wax Moth infection model . Indeed, although the Δ mutant did not display any phenotype, the Δ mutant was more virulent than the wild-type strain. This virulent phenotype should stem from its increased ability to proliferate under oxidative environments.

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2009-10-01
2020-10-22
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References

  1. Abranches J., Martinez A. R., Kajfasz J. K., Chavez V., Garsin D. A., Lemos J. A.. 2009; The molecular alarmone (p)ppGpp mediates stress responses, vancomycin tolerance and virulence in Enterococcus faecalis. J Bacteriol191:2248–2256
    [Google Scholar]
  2. Arnaud M., Chastanet A., Débarbouillé M.. 2004; New vector for efficient allelic replacement in naturally nontransformable low-GC-content, Gram-positive bacteria. Appl Environ Microbiol70:6887–6891
    [Google Scholar]
  3. Benachour A., Muller C., Dabrowski-Coton M., Le Breton Y., Giard J. C., Rincé A., Auffray Y., Hartke A.. 2005; The Enterococcus faecalis SigV protein is an extracytoplasmic function sigma factor contributing to survival following heat, acid, and ethanol treatments. J Bacteriol187:1022–1035
    [Google Scholar]
  4. Bennett H. J., Pearce D. M., Glenn S., Taylor C. M., Kuhn M., Sonenshein A. L., Andrew P. W., Roberts I. S.. 2007; Characterization of relA and codY mutants of Listeria monocytogenes: identification of the CodY regulon and its role in virulence. Mol Microbiol63:1453–1467
    [Google Scholar]
  5. Bergin D., Reeves E. P., Renwick J., Wientjes F. B., Kavanagh K.. 2005; Superoxide production in Galleria mellonella hemocytes: identification of proteins homologous to the NADPH oxidase complex of human neutrophils. Infect Immun73:4161–4170
    [Google Scholar]
  6. Cashel M., Gentry D. R., Hernandez V. J., Vinella D.. 1996; The stringent response. In Escherichia coli and Salmonella: Cellular and Molecular Biology , 2nd edn. pp1458–1496 Edited by Neidhardt F. C., Curtiss R. I., Ingraham J. L., Lin E. C. C., Low K. B., Magasanik B., Reznikoff W. S., Riley M., Schaechter M., Umbarger H. E. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  7. Chopin A., Chopin M. C., Moillo-Batt A., Langella P.. 1984; Two plasmid-determined restriction and modification systems in Streptococcus lactis. Plasmid11:260–263
    [Google Scholar]
  8. Dahl J. L., Kraus C. N., Boshoff H. I., Doan B., Foley K., Avarbock D., Kaplan G., Mizrahi V., Rubin H., Barry C. E..III: 2003; The role of RelMtb-mediated adaptation to stationary phase in long-term persistence of Mycobacterium tuberculosis in mice. Proc Natl Acad Sci U S A100:10026–10031
    [Google Scholar]
  9. Erickson D. L., Lines J. L., Pesci E. C., Venturi V., Storey D. G.. 2004; Pseudomonas aeruginosa relA contributes to virulence in Drosophila melanogaster. Infect Immun72:5638–5645
    [Google Scholar]
  10. Flahaut S., Boutibonnes P., Auffray Y.. 1997; Enterococci in human environment. Can J Microbiol43:699–708
    [Google Scholar]
  11. Gasson M. J.. 1983; Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol154:1–9
    [Google Scholar]
  12. Giard J. C., Laplace J. M., Rincé A., Pichereau V., Benachour A., Leboeuf C., Flahaut S., Auffray Y., Hartke A.. 2001; The stress proteome of Enterococcus faecalis. Electrophoresis22:2947–2954
    [Google Scholar]
  13. Giard J. C., Riboulet E., Verneuil N., Sanguinetti M., Auffray Y., Hartke A.. 2006; Characterization of Ers, a PrfA-like regulator of Enterococcus faecalis. FEMS Immunol Med Microbiol46:410–418
    [Google Scholar]
  14. Giraffa G.. 2003; Functionality of enterococci in dairy products. Int J Food Microbiol88:215–222
    [Google Scholar]
  15. Greenway D. L., England R. R.. 1999; The intrinsic resistance of Escherichia coli to various antimicrobial agents requires ppGpp and σ S. Lett Appl Microbiol29:323–326
    [Google Scholar]
  16. Haralalka S., Nandi S., Bhadra R. K.. 2003; Mutation in the relA gene of Vibrio cholerae affects in vitro and in vivo expression of virulence factors. J Bacteriol185:4672–4682
    [Google Scholar]
  17. Hugas M., Garriga M., Aymerich M. T.. 2003; Functionality of enterococci in meat products. Int J Food Microbiol88:223–233
    [Google Scholar]
  18. Inaoka T., Ochi K.. 2002; RelA protein is involved in induction of genetic competence in certain Bacillus subtilis strains by moderating the level of intracellular GTP. J Bacteriol184:3923–3930
    [Google Scholar]
  19. Jander G., Rahme L. G., Ausubel F. M.. 2000; Positive correlation between virulence of Pseudomonas aeruginosa mutants in mice and insects. J Bacteriol182:3843–3845
    [Google Scholar]
  20. Jin W., Kim H. K., Kim J. Y., Kang S. G., Lee S. H., Lee K. J.. 2004a; Cephamycin C production is regulated by relA and rsh genes in Streptomyces clavuligerus ATCC 27064. J Biotechnol114:81–87
    [Google Scholar]
  21. Jin W., Ryu Y. G., Kang S. G., Kim S. K., Saito N., Ochi K., Lee S. H., Lee K. J.. 2004b; Two relA/ spoT homologous genes are involved in the morphological and physiological differentiation of Streptomyces clavuligerus. Microbiology150:1485–1493
    [Google Scholar]
  22. Kasai K., Kanno T., Endo Y., Wakasa K., Tozawa Y.. 2004; Guanosine tetra- and pentaphosphate synthase activity in chloroplasts of a higher plant: association with 70S ribosomes and inhibition by tetracycline. Nucleic Acids Res32:5732–5741
    [Google Scholar]
  23. Le Breton Y., Boël G., Benachour A., Prévost H., Auffray Y., Rincé A.. 2003; Molecular characterization of Enterococcus faecalis two-component signal transduction pathways related to environmental stresses. Environ Microbiol5:329–337
    [Google Scholar]
  24. Lemos J. A., Brown T. A. Jr, Burne R. A.. 2004; Effects of RelA on key virulence properties of planktonic and biofilm populations of Streptococcus mutans. Infect Immun72:1431–1440
    [Google Scholar]
  25. Lemos J. A., Lin V. K., Nascimento M. M., Abranches J., Burne R. A.. 2007; Three gene products govern (p)ppGpp production by Streptococcus mutans. Mol Microbiol65:1568–1581
    [Google Scholar]
  26. Mechold U., Cashel M., Steiner K., Gentry D., Malke H.. 1996; Functional analysis of a relA/spoT gene homolog from Streptococcus equisimilis. J Bacteriol178:1401–1411
    [Google Scholar]
  27. Mechold U., Murphy H., Brown L., Cashel M.. 2002; Intramolecular regulation of the opposing (p)ppGpp catalytic activities of Rel Seq , the Rel/Spo enzyme from Streptococcus equisimilis. J Bacteriol184:2878–2888
    [Google Scholar]
  28. Mercenier A., Pavan S., Pot B.. 2003; Probiotics as biotherapeutic agents: present knowledge and future prospects. Curr Pharm Des9:175–191
    [Google Scholar]
  29. Mittenhuber G.. 2001; Comparative genomics and evolution of genes encoding bacterial (p)ppGpp synthetases/hydrolases (the Rel, RelA and SpoT proteins. J Mol Microbiol Biotechnol3:585–603
    [Google Scholar]
  30. Miyata S., Casey M., Frank D. W., Ausubel F. M., Drenkard E.. 2003; Use of the Galleria mellonella caterpillar as a model host to study the role of the type III secretion system in Pseudomonas aeruginosa pathogenesis. Infect Immun71:2404–2413
    [Google Scholar]
  31. Mostertz J., Scharf C., Hecker M., Homuth G.. 2004; Transcriptome and proteome analysis of Bacillus subtilis gene expression in response to superoxide and peroxide stress. Microbiology150:497–512
    [Google Scholar]
  32. Muller C., Le Breton Y., Morin T., Benachour A., Auffray Y., Rincé A.. 2006; The response regulator CroR modulates expression of the secreted stress induced SalB protein in Enterococcus faecalis. J Bacteriol188:2636–2645
    [Google Scholar]
  33. Muller C., Sanguinetti M., Riboulet E., Hébert L., Posteraro B., Fadda G., Auffray Y., Rincé A.. 2008; Characterization of two signal transduction systems involved in intracellular macrophage survival and environmental stress response in Enterococcus faecalis. J Mol Microbiol Biotechnol14:59–66
    [Google Scholar]
  34. Murray K. D., Bremer H.. 1996; Control of spoT-dependent ppGpp synthesis and degradation in Escherichia coli. J Mol Biol259:41–57
    [Google Scholar]
  35. Nanamiya H., Kasai K., Nozawa A., Yun C. S., Narisawa T., Murakami K., Natori Y., Kawamura F., Tozawa Y.. 2008; Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis. Mol Microbiol67:291–304
    [Google Scholar]
  36. Noskin G. A., Till M., Patterson B. K., Clarke J. T., Warren J. R.. 1991; High level gentamycin resistance in Enterococcus faecalis bacteremia. J Infect Dis164:1212–1215
    [Google Scholar]
  37. Okada Y., Makino S., Tobe T., Okada N., Yamazaki S.. 2002; Cloning of rel from Listeria monocytogenes as an osmotolerance involvement gene. Appl Environ Microbiol68:1541–1547
    [Google Scholar]
  38. Park S. Y., Kim K. M., Lee J. H., Seo S. J., Lee I. H.. 2007; Extracellular gelatinase of Enterococcus faecalis destroys a defense system in insect hemolymph and human serum. Infect Immun75:1861–1869
    [Google Scholar]
  39. Paulsen I. T., Banerjei L., Myers G. S., Nelson K. E., Seshadri R., Read T. D., Fouts D. E., Eisen J. A., Gill S. R.. other authors 2003; Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis. Science299:2071–2074
    [Google Scholar]
  40. Pichereau V., Bourot S., Flahaut S., Blanco C., Auffray Y., Bernard T.. 1999; The osmoprotectant glycine betaine inhibits salt-induced cross-tolerance towards lethal treatment in Enterococcus faecalis. Microbiology145:427–435
    [Google Scholar]
  41. Pizarro-Cerda J., Tedin K.. 2004; The bacterial signal molecule, ppGpp, regulates Salmonella virulence gene expression. Mol Microbiol52:1827–1844
    [Google Scholar]
  42. Rallu F., Gruss A., Ehrlich S. D., Maguin E.. 2000; Acid- and multistress-resistant mutant of Lactococcus lactis: identification of intracellular stress signals. Mol Microbiol35:517–528
    [Google Scholar]
  43. Sahm D. F., Kissinger J., Gilmore M. S., Murray P. R., Mulder R., Solliday J., Clarke B.. 1989; In vitro susceptibility studies of vancomycin-resistant Enterococcus faecalis. Antimicrob Agents Chemother33:1588–1591
    [Google Scholar]
  44. Schaberg D. R., Culver D. H., Gaynes R. P.. 1991; Major trends in microbial etiology of nosocomial infection. Am J Med91:72S–75S
    [Google Scholar]
  45. Seed K. D., Dennis J. J.. 2008; Development of Galleria mellonella as an alternative infection model for the Burkholderia cepacia complex. Infect Immun76:1267–1275
    [Google Scholar]
  46. Sherman J. M.. 1937; The streptococci. Bacteriol Rev1:3–97
    [Google Scholar]
  47. Silva A. J., Benitez J. A.. 2006; A Vibrio cholerae relaxed ( relA) mutant expresses major virulence factors, exhibits biofilm formation and motility, and colonizes the suckling mouse intestine. J Bacteriol188:794–800
    [Google Scholar]
  48. Sun J., Hesketh A., Bibb M.. 2001; Functional analysis of relA and rshA, two relA/spoT homologues of Streptomyces coelicolor A3(2. J Bacteriol183:3488–3498
    [Google Scholar]
  49. Taylor C. M., Beresford M., Epton H. A., Sigee D. C., Shama G., Andrew P. W., Roberts I. S.. 2002; Listeria monocytogenes relA and hpt mutants are impaired in surface-attached growth and virulence. J Bacteriol184:621–628
    [Google Scholar]
  50. Verneuil N., Sanguinetti M., Le Breton Y., Posteraro B., Fadda G., Auffray Y., Hartke A., Giard J. C.. 2004; Effects of the Enterococcus faecalis hypR gene encoding a new transcriptional regulator on oxidative stress response and intracellular survival within macrophages. Infect Immun72:4424–4431
    [Google Scholar]
  51. Verneuil N., Rincé A., Sanguinetti M., Posteraro B., Fadda G., Auffray Y., Hartke A., Giard J. C.. 2005; Contribution of a PerR-like regulator to the oxidative-stress response and virulence of Enterococcus faecalis. Microbiology151:3997–4004
    [Google Scholar]
  52. Wendrich T. M., Marahiel M. A.. 1997; Cloning and characterization of a relA/spoT homologue from Bacillus subtilis. Mol Microbiol26:65–79
    [Google Scholar]
  53. Wendrich T. M., Blaha G., Wilson D. N., Marahiel M. A., Nierhaus K. H.. 2002; Dissection of the mechanism for the stringent factor RelA. Mol Cell10:779–788
    [Google Scholar]
  54. Yang X., Ishiguro E. E.. 2003; Temperature-sensitive growth and decreased thermotolerance associated with relA mutations in Escherichia coli. J Bacteriol185:5765–5771
    [Google Scholar]
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