1887

Abstract

In , production of guanosine tetraphosphate/guanosine pentaphosphate [(p)ppGpp], the effector molecule of the stringent response, is controlled by the bifunctional synthetase/hydrolase RelA and the monofunctional synthetase RelQ. Previously, the (p)ppGpp profiles of strains lacking , or both genes indicated that RelA is the primary enzyme responsible for (p)ppGpp synthesis under stress conditions, while the contributions of RelQ to the stringent response and cell homeostasis remained elusive. Here, survival within the mouse-derived macrophage cell line J774A.1 and killing of supported initial evidence that virulence was attenuated in the (p)ppGpp ΔΔ strain but not in the Δ or Δ strains. We performed, for the first time to our knowledge, global transcriptome analysis in a documented (p)ppGpp Gram-positive bacterium and provided the first insights into the role of a Gram-positive monofunctional (p)ppGpp synthetase in transcriptional regulation. Transcription profiling after mupirocin treatment confirmed that RelA is the major enzyme responsible for the (p)ppGpp-mediated transcriptional repression of genes associated with macromolecular biosynthesis, but also revealed that RelQ is required for full and timely stringent response induction. The delayed transcriptional response of Δ could not be correlated with reduced or slower production of (p)ppGpp, in part because RelA-dependent (p)ppGpp accumulation occurred very rapidly. Comparisons of the transcriptional responses of Δ or ΔΔ strains with the parent strain under starvation conditions revealed upregulation of operons involved in energy metabolism in the (p)ppGpp strain. Thus, while Δ and ΔΔ cannot use (p)ppGpp to sense and respond to stresses, fitness of ΔΔ may be further impaired due to an unbalanced metabolism.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.060236-0
2012-08-01
2019-11-14
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/8/1994.html?itemId=/content/journal/micro/10.1099/mic.0.060236-0&mimeType=html&fmt=ahah

References

  1. Abranches J., Candella M. M., Wen Z. T., Baker H. V., Burne R. A.. ( 2006;). Different roles of EIIABMan and EIIGlc in regulation of energy metabolism, biofilm development, and competence in Streptococcus mutans. . J Bacteriol 188:, 3748–3756. [CrossRef][PubMed]
    [Google Scholar]
  2. Abranches J., Martinez A. R., Kajfasz J. K., Chávez V., Garsin D. A., Lemos J. A.. ( 2009;). The molecular alarmone (p)ppGpp mediates stress responses, vancomycin tolerance, and virulence in Enterococcus faecalis. . J Bacteriol 191:, 2248–2256. [CrossRef][PubMed]
    [Google Scholar]
  3. Arias C. A., Murray B. E.. ( 2012;). The rise of the Enterococcus: beyond vancomycin resistance. . Nat Rev Microbiol 10:, 266–278. [CrossRef][PubMed]
    [Google Scholar]
  4. Battesti A., Bouveret E.. ( 2009;). Bacteria possessing two RelA/SpoT-like proteins have evolved a specific stringent response involving the acyl carrier protein–SpoT interaction. . J Bacteriol 191:, 616–624. [CrossRef][PubMed]
    [Google Scholar]
  5. 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 Microbiol 63:, 1453–1467. [CrossRef][PubMed]
    [Google Scholar]
  6. Blumenthal R. M., Lemaux P. G., Neidhardt F. C., Dennis P. P.. ( 1976;). The effects of the relA gene on the synthesis of aminoacyl-tRNA synthetases and other transcription and translation proteins in Escherichia coli A. . Mol Gen Genet 149:, 291–296. [CrossRef][PubMed]
    [Google Scholar]
  7. Boucher H. W., Talbot G. H., Bradley J. S., Edwards J. E., Gilbert D., Rice L. B., Scheld M., Spellberg B., Bartlett J.. ( 2009;). Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. . Clin Infect Dis 48:, 1–12. [CrossRef][PubMed]
    [Google Scholar]
  8. Bourgogne A., Garsin D. A., Qin X., Singh K. V., Sillanpaa J., Yerrapragada S., Ding Y., Dugan-Rocha S., Buhay C.. & other authors ( 2008;). Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF. . Genome Biol 9:, R110. [CrossRef][PubMed]
    [Google Scholar]
  9. Brockmann-Gretza O., Kalinowski J.. ( 2006;). Global gene expression during stringent response in Corynebacterium glutamicum in presence and absence of the rel gene encoding (p)ppGpp synthase. . BMC Genomics 7:, 230. [CrossRef][PubMed]
    [Google Scholar]
  10. 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., vol. 1, pp. 1458–1496. Edited by Neidhardt F. C., Curtiss R. C. III, Ingraham J. L., Lin E. C. C., Low K. B., Magasanik B., Reznikoff W. S., Riley M., Schaechter M., Umbarger A. E... Washington, DC:: American Society for Microbiology;.
    [Google Scholar]
  11. Choudhury T., Singh K. V., Sillanpää J., Nallapareddy S. R., Murray B. E.. ( 2011;). Importance of two Enterococcus faecium loci encoding Gls-like proteins for in vitro bile salts stress response and virulence. . J Infect Dis 203:, 1147–1154. [CrossRef][PubMed]
    [Google Scholar]
  12. Crosse A. M., Greenway D. L., England R. R.. ( 2000;). Accumulation of ppGpp and ppGp in Staphylococcus aureus 8325-4 following nutrient starvation. . Lett Appl Microbiol 31:, 332–337. [CrossRef][PubMed]
    [Google Scholar]
  13. de Oliveira N. E., Abranches J., Gaca A. O., Laport M. S., Damaso C. R., Bastos M. C., Lemos J. A., Giambiagi-deMarval M.. ( 2011;). clpB, a class III heat-shock gene regulated by CtsR, is involved in thermotolerance and virulence of Enterococcus faecalis. . Microbiology 157:, 656–665. [CrossRef][PubMed]
    [Google Scholar]
  14. den Hengst C. D., van Hijum S. A., Geurts J. M., Nauta A., Kok J., Kuipers O. P.. ( 2005;). The Lactococcus lactis CodY regulon: identification of a conserved cis-regulatory element. . J Biol Chem 280:, 34332–34342. [CrossRef][PubMed]
    [Google Scholar]
  15. Durfee T., Hansen A. M., Zhi H., Blattner F. R., Jin D. J.. ( 2008;). Transcription profiling of the stringent response in Escherichia coli. . J Bacteriol 190:, 1084–1096. [CrossRef][PubMed]
    [Google Scholar]
  16. Eymann C., Homuth G., Scharf C., Hecker M.. ( 2002;). Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis. . J Bacteriol 184:, 2500–2520. [CrossRef][PubMed]
    [Google Scholar]
  17. Geiger T., Goerke C., Fritz M., Schäfer T., Ohlsen K., Liebeke M., Lalk M., Wolz C.. ( 2010;). Role of the (p)ppGpp synthase RSH, a RelA/SpoT homolog, in stringent response and virulence of Staphylococcus aureus. . Infect Immun 78:, 1873–1883. [CrossRef][PubMed]
    [Google Scholar]
  18. Green N. J., Grundy F. J., Henkin T. M.. ( 2010;). The T box mechanism: tRNA as a regulatory molecule. . FEBS Lett 584:, 318–324. [CrossRef][PubMed]
    [Google Scholar]
  19. Hartke A., Giard J. C., Laplace J. M., Auffray Y.. ( 1998;). Survival of Enterococcus faecalis in an oligotrophic microcosm: changes in morphology, development of general stress resistance, and analysis of protein synthesis. . Appl Environ Microbiol 64:, 4238–4245.[PubMed]
    [Google Scholar]
  20. Kajfasz J. K., Mendoza J. E., Gaca A. O., Miller J. H., Koselny K. A., Giambiagi-Demarval M., Wellington M., Abranches J., Lemos J. A.. ( 2012;). The Spx regulator modulates stress responses and virulence in Enterococcus faecalis. . Infect Immun. (In press) [CrossRef][PubMed]
    [Google Scholar]
  21. Kazmierczak K. M., Wayne K. J., Rechtsteiner A., Winkler M. E.. ( 2009;). Roles of relSpn in stringent response, global regulation and virulence of serotype 2 Streptococcus pneumoniae D39. . Mol Microbiol 72:, 590–611. [CrossRef][PubMed]
    [Google Scholar]
  22. Kohanski M. A., Dwyer D. J., Hayete B., Lawrence C. A., Collins J. J.. ( 2007;). A common mechanism of cellular death induced by bactericidal antibiotics. . Cell 130:, 797–810. [CrossRef][PubMed]
    [Google Scholar]
  23. Lebreton F., Riboulet-Bisson E., Serror P., Sanguinetti M., Posteraro B., Torelli R., Hartke A., Auffray Y., Giard J. C.. ( 2009;). ace, Which encodes an adhesin in Enterococcus faecalis, is regulated by Ers and is involved in virulence. . Infect Immun 77:, 2832–2839. [CrossRef][PubMed]
    [Google Scholar]
  24. 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 Microbiol 65:, 1568–1581. [CrossRef][PubMed]
    [Google Scholar]
  25. Lemos J. A., Nascimento M. M., Lin V. K., Abranches J., Burne R. A.. ( 2008;). Global regulation by (p)ppGpp and CodY in Streptococcus mutans. . J Bacteriol 190:, 5291–5299. [CrossRef][PubMed]
    [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 Bacteriol 178:, 1401–1411.[PubMed]
    [Google Scholar]
  27. Mechold U., Murphy H., Brown L., Cashel M.. ( 2002;). Intramolecular regulation of the opposing (p)ppGpp catalytic activities of RelSeq, the Rel/Spo enzyme from Streptococcus equisimilis. . J Bacteriol 184:, 2878–2888. [CrossRef][PubMed]
    [Google Scholar]
  28. Murray B. E.. ( 1990;). The life and times of the Enterococcus. . Clin Microbiol Rev 3:, 46–65.[PubMed]
    [Google Scholar]
  29. 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 Microbiol 67:, 291–304. [CrossRef][PubMed]
    [Google Scholar]
  30. Nascimento M. M., Lemos J. A., Abranches J., Lin V. K., Burne R. A.. ( 2008;). Role of RelA of Streptococcus mutans in global control of gene expression. . J Bacteriol 190:, 28–36. [CrossRef][PubMed]
    [Google Scholar]
  31. Nguyen D., Joshi-Datar A., Lepine F., Bauerle E., Olakanmi O., Beer K., McKay G., Siehnel R., Schafhauser J.. & other authors ( 2011;). Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. . Science 334:, 982–986. [CrossRef][PubMed]
    [Google Scholar]
  32. Nishino T., Gallant J., Shalit P., Palmer L., Wehr T.. ( 1979;). Regulatory nucleotides involved in the Rel function of Bacillus subtilis. . J Bacteriol 140:, 671–679.[PubMed]
    [Google Scholar]
  33. 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. . Science 299:, 2071–2074. [CrossRef][PubMed]
    [Google Scholar]
  34. Potrykus K., Cashel M.. ( 2008;). (p)ppGpp: still magical?. Annu Rev Microbiol 62:, 35–51. [CrossRef][PubMed]
    [Google Scholar]
  35. Ratnayake-Lecamwasam M., Serror P., Wong K. W., Sonenshein A. L.. ( 2001;). Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels. . Genes Dev 15:, 1093–1103. [CrossRef][PubMed]
    [Google Scholar]
  36. Reiss S., Pané-Farré J., Fuchs S., François P., Liebeke M., Schrenzel J., Lindequist U., Lalk M., Wolz C.. & other authors ( 2012;). Global analysis of the Staphylococcus aureus response to mupirocin. . Antimicrob Agents Chemother 56:, 787–804. [CrossRef][PubMed]
    [Google Scholar]
  37. Sajish M., Kalayil S., Verma S. K., Nandicoori V. K., Prakash B.. ( 2009;). The significance of EXDD and RXKD motif conservation in Rel proteins. . J Biol Chem 284:, 9115–9123. [CrossRef][PubMed]
    [Google Scholar]
  38. Shi L., Reid L. H., Jones W. D., Shippy R., Warrington J. A., Baker S. C., Collins P. J., de Longueville F., Kawasaki E. S.. & other authors ( 2006;). The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements. . Nat Biotechnol 24:, 1151–1161. [CrossRef][PubMed]
    [Google Scholar]
  39. Shivers R. P., Dineen S. S., Sonenshein A. L.. ( 2006;). Positive regulation of Bacillus subtilis ackA by CodY and CcpA: establishing a potential hierarchy in carbon flow. . Mol Microbiol 62:, 811–822. [CrossRef][PubMed]
    [Google Scholar]
  40. Sonenshein A. L.. ( 2005;). CodY, a global regulator of stationary phase and virulence in Gram-positive bacteria. . Curr Opin Microbiol 8:, 203–207. [CrossRef][PubMed]
    [Google Scholar]
  41. Sonenshein A. L.. ( 2007;). Control of key metabolic intersections in Bacillus subtilis. . Nat Rev Microbiol 5:, 917–927. [CrossRef][PubMed]
    [Google Scholar]
  42. Terleckyj B., Willett N. P., Shockman G. D.. ( 1975;). Growth of several cariogenic strains of oral streptococci in a chemically defined medium. . Infect Immun 11:, 649–655.[PubMed]
    [Google Scholar]
  43. Traxler M. F., Summers S. M., Nguyen H. T., Zacharia V. M., Hightower G. A., Smith J. T., Conway T.. ( 2008;). The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli. . Mol Microbiol 68:, 1128–1148. [CrossRef][PubMed]
    [Google Scholar]
  44. 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. . Microbiology 151:, 3997–4004. [CrossRef][PubMed]
    [Google Scholar]
  45. Yan X., Zhao C., Budin-Verneuil A., Hartke A., Rincé A., Gilmore M. S., Auffray Y., Pichereau V.. ( 2009;). The (p)ppGpp synthetase RelA contributes to stress adaptation and virulence in Enterococcus faecalis V583. . Microbiology 155:, 3226–3237. [CrossRef][PubMed]
    [Google Scholar]
  46. Zhao C., Hartke A., La Sorda M., Posteraro B., Laplace J. M., Auffray Y., Sanguinetti M.. ( 2010;). Role of methionine sulfoxide reductases A and B of Enterococcus faecalis in oxidative stress and virulence. . Infect Immun 78:, 3889–3897. [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.060236-0
Loading
/content/journal/micro/10.1099/mic.0.060236-0
Loading

Data & Media loading...

Supplements

Tables S1 - S4 legends 

PDF

Table S1 

EXCEL

Table S2 

EXCEL

Table S3 

EXCEL

Table S4 

EXCEL
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