1887

Microbiology Society logo

Volume 158, Issue 8

Heterologous expression of a plant RelA-SpoT homologue results in increased stress tolerance in by accumulation of the bacterial alarmone ppGpp Free

Abstract

The bacterial alarmone ppGpp is present only in bacteria and the chloroplasts of plants, but not in mammalian cells or eukaryotic micro-organisms such as yeasts and fungi. The importance of the ppGpp signalling system in eukaryotes has therefore been largely overlooked. Here, we demonstrated that heterologous expression of a homologue () isolated from the halophilic plant in the yeast results in accumulation of ppGpp, accompanied by enhancement of tolerance against various stress stimuli, such as osmotic stress, ethanol, hydrogen peroxide, high temperature and freezing. Unlike bacterial ppGpp accumulation, ppGpp was accumulated in the early growth phase but not in the late growth phase. Moreover, nutritional downshift resulted in a decrease in ppGpp level, suggesting that the observed Sj-RSH activity to synthesize ppGpp is not starvation-dependent, contrary to our expectations based on bacteria. Accumulated ppGpp was found to be present solely in the cytosolic fraction and not in the mitochondrial fraction, perhaps reflecting the ribosome-independent ppGpp synthesis in cells. Unlike bacterial inosine monophosphate (IMP) dehydrogenases, the IMP dehydrogenase of was insensitive to ppGpp. Microarray analysis showed that ppGpp accumulation gave rise to marked changes in gene expression, with both upregulation and downregulation, including changes in mitochondrial gene expression. The most prominent upregulation (38-fold) was detected in the hypothetical gene YBR072C–A of unknown function, followed by many other known stress-responsive genes. may provide new opportunities to uncover and analyse the ppGpp signalling system in eukaryotic cells.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.057638-0
2012-08-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/8/2213.html?itemId=/content/journal/micro/10.1099/mic.0.057638-0&mimeType=html&fmt=ahah

References

  1. Artsimovitch I., Patlan V., Sekine S., Vassylyeva M. N., Hosaka T., Ochi K., Yokoyama S., Vassylyev D. G. ( 2004). Structural basis for transcription regulation by alarmone ppGpp. Cell 117:299–310 [View Article][PubMed]
     [Google Scholar]
  2. Balzer G. J., McLean R. J. ( 2002). The stringent response genes relA and spoT are important for Escherichia coli biofilms under slow-growth conditions. Can J Microbiol 48:675–680 [View Article][PubMed]
     [Google Scholar]
  3. Baysse C., Cullinane M., Dénervaud V., Burrowes E., Dow J. M., Morrissey J. P., Tam L., Trevors J. T., O’Gara F. ( 2005). Modulation of quorum sensing in Pseudomonas aeruginosa through alteration of membrane properties. Microbiology 151:2529–2542 [View Article][PubMed]
     [Google Scholar]
  4. Bibb M. J. ( 2005). Regulation of secondary metabolism in streptomycetes. Curr Opin Microbiol 8:208–215 [View Article][PubMed]
     [Google Scholar]
  5. Braeken K., Moris M., Daniels R., Vanderleyden J., Michiels J. ( 2006). New horizons for (p)ppGpp in bacterial and plant physiology. Trends Microbiol 14:45–54 [View Article][PubMed]
     [Google Scholar]
  6. Cashel M., Rudd E. ( 1987). The stringent response. Escherichia coli and Salmonella: Cellular and Molecular Biology1410–1438 Neidhardt F. C. et al. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  7. Cashel M., Gentry D. R., Hernandez V. J., Vinella D. ( 1996). The stringent response. Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn.1458–1496 Neidhardt F. C. et al. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  8. Chatterji D., Ojha A. K. ( 2001). Revisiting the stringent response, ppGpp and starvation signaling. Curr Opin Microbiol 4:160–165 [View Article][PubMed]
     [Google Scholar]
  9. Chatterji D., Fujita N., Ishihama A. ( 1998). The mediator for stringent control, ppGpp, binds to the β-subunit of Escherichia coli RNA polymerase. Genes Cells 3:279–287 [View Article][PubMed]
     [Google Scholar]
  10. 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 A 100:10026–10031 [View Article][PubMed]
     [Google Scholar]
  11. 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 Immun 72:5638–5645 [View Article][PubMed]
     [Google Scholar]
  12. Gaynor E. C., Wells D. H., MacKichan J. K., Falkow S. ( 2005). The Campylobacter jejuni stringent response controls specific stress survival and virulence-associated phenotypes. Mol Microbiol 56:8–27 [View Article][PubMed]
     [Google Scholar]
  13. Givens R. M., Lin M. H., Taylor D. J., Mechold U., Berry J. O., Hernandez V. J. ( 2004). Inducible expression, enzymatic activity, and origin of higher plant homologues of bacterial RelA/SpoT stress proteins in Nicotiana tabacum . J Biol Chem 279:7495–7504 [View Article][PubMed]
     [Google Scholar]
  14. Godfrey H. P., Bugrysheva J. V., Cabello F. C. ( 2002). The role of the stringent response in the pathogenesis of bacterial infections. Trends Microbiol 10:349–351 [View Article][PubMed]
     [Google Scholar]
  15. 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 Bacteriol 185:4672–4682 [View Article][PubMed]
     [Google Scholar]
  16. Harris B. Z., Kaiser D., Singer M. ( 1998). The guanosine nucleotide (p)ppGpp initiates development and A-factor production in Myxococcus xanthus . Genes Dev 12:1022–1035 [View Article][PubMed]
     [Google Scholar]
  17. Hesketh A., Sun J., Bibb M. ( 2001). Induction of ppGpp synthesis in Streptomyces coelicolor A3(2) grown under conditions of nutritional sufficiency elicits actII-ORF4 transcription and actinorhodin biosynthesis. Mol Microbiol 39:136–144 [View Article][PubMed]
     [Google Scholar]
  18. Hogg T., Mechold U., Malke H., Cashel M., Hilgenfeld R. ( 2004). Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response [corrected]. Cell 117:57–68 [View Article][PubMed]
     [Google Scholar]
  19. Hoyt S., Jones G. H. ( 1999). relA is required for actinomycin production in Streptomyces antibioticus . J Bacteriol 181:3824–3829[PubMed]
     [Google Scholar]
  20. 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 Bacteriol 184:3923–3930 [View Article][PubMed]
     [Google Scholar]
  21. Inaoka T., Takahashi K., Ohnishi-Kameyama M., Yoshida M., Ochi K. ( 2003). Guanine nucleotides guanosine 5′-diphosphate 3′-diphosphate and GTP co-operatively regulate the production of an antibiotic bacilysin in Bacillus subtilis . J Biol Chem 278:2169–2176 [View Article][PubMed]
     [Google Scholar]
  22. Jenks M. H., Reines D. ( 2005). Dissection of the molecular basis of mycophenolate resistance in Saccharomyces cerevisiae . Yeast 22:1181–1190 [View Article][PubMed]
     [Google Scholar]
  23. Jishage M., Kvint K., Shingler V., Nyström T. ( 2002). Regulation of sigma factor competition by the alarmone ppGpp. Genes Dev 16:1260–1270 [View Article][PubMed]
     [Google Scholar]
  24. Kasai K., Usami S., Yamada T., Endo Y., Ochi K., Tozawa Y. ( 2002). A RelA-SpoT homolog (Cr-RSH) identified in Chlamydomonas reinhardtii generates stringent factor in vivo and localizes to chloroplasts in vitro . Nucleic Acids Res 30:4985–4992 [View Article][PubMed]
     [Google Scholar]
  25. 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 Res 32:5732–5741 [View Article][PubMed]
     [Google Scholar]
  26. Kasai K., Nishizawa T., Takahashi K., Hosaka T., Aoki H., Ochi K. ( 2006). Physiological analysis of the stringent response elicited in an extreme thermophilic bacterium, Thermus thermophilus . J Bacteriol 188:7111–7122 [View Article][PubMed]
     [Google Scholar]
  27. Köhler G. A., Gong X., Bentink S., Theiss S., Pagani G. M., Agabian N., Hedstrom L. ( 2005). The functional basis of mycophenolic acid resistance in Candida albicans IMP dehydrogenase. J Biol Chem 280:11295–11302 [View Article][PubMed]
     [Google Scholar]
  28. Korch S. B., Henderson T. A., Hill T. M. ( 2003). Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis. Mol Microbiol 50:1199–1213 [View Article][PubMed]
     [Google Scholar]
  29. Krásný L., Gourse R. L. ( 2004). An alternative strategy for bacterial ribosome synthesis: Bacillus subtilis rRNA transcription regulation. EMBO J 23:4473–4483 [View Article][PubMed]
     [Google Scholar]
  30. 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 Immun 72:1431–1440 [View Article][PubMed]
     [Google Scholar]
  31. Magnusson L. U., Farewell A., Nyström T. ( 2005). ppGpp: a global regulator in Escherichia coli . Trends Microbiol 13:236–242 [View Article][PubMed]
     [Google Scholar]
  32. Masuda S., Mizusawa K., Narisawa T., Tozawa Y., Ohta H., Takamiya K. ( 2008). The bacterial stringent response, conserved in chloroplasts, controls plant fertilization. Plant Cell Physiol 49:135–141 [View Article][PubMed]
     [Google Scholar]
  33. Meisinger C., Sommer T., Pfanner N. ( 2000). Purification of Saccharomcyes cerevisiae mitochondria devoid of microsomal and cytosolic contaminations. Anal Biochem 287:339–342 [View Article][PubMed]
     [Google Scholar]
  34. Moris M., Braeken K., Schoeters E., Verreth C., Beullens S., Vanderleyden J., Michiels J. ( 2005). Effective symbiosis between Rhizobium etli and Phaseolus vulgaris requires the alarmone ppGpp. J Bacteriol 187:5460–5469 [View Article][PubMed]
     [Google Scholar]
  35. Ochi K. ( 1987a). Metabolic initiation of differentiation and secondary metabolism by Streptomyces griseus: significance of the stringent response (ppGpp) and GTP content in relation to A factor. J Bacteriol 169:3608–3616[PubMed]
     [Google Scholar]
  36. Ochi K. ( 1987b). Changes in nucleotide pools during sporulation of Streptomyces griseus in submerged culture. J Gen Microbiol 133:2787–2795
     [Google Scholar]
  37. Ochi K. ( 2007). From microbial differentiation to ribosome engineering. Biosci Biotechnol Biochem 71:1373–1386 [View Article][PubMed]
     [Google Scholar]
  38. Ochi K., Kandala J. C., Freese E. ( 1981). Initiation of Bacillus subtilis sporulation by the stringent response to partial amino acid deprivation. J Biol Chem 256:6866–6875[PubMed]
     [Google Scholar]
  39. Okamoto S., Ochi K. ( 1998). An essential GTP-binding protein functions as a regulator for differentiation in Streptomyces coelicolor . Mol Microbiol 30:107–119 [View Article][PubMed]
     [Google Scholar]
  40. Pizarro-Cerdá J., Tedin K. ( 2004). The bacterial signal molecule, ppGpp, regulates Salmonella virulence gene expression. Mol Microbiol 52:1827–1844 [View Article][PubMed]
     [Google Scholar]
  41. Potrykus K., Cashel M. ( 2008). (p)ppGpp: still magical?. Annu Rev Microbiol 62:35–51 [View Article][PubMed]
     [Google Scholar]
  42. 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 [View Article][PubMed]
     [Google Scholar]
  43. Rodríguez-Vargas S., Sánchez-García A., Martínez-Rivas J. M., Prieto J. A., Randez-Gil F. ( 2007). Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt stress. Appl Environ Microbiol 73:110–116 [View Article][PubMed]
     [Google Scholar]
  44. Saito N., Xu J., Hosaka T., Okamoto S., Aoki H., Bibb M. J., Ochi K. ( 2006). EshA accentuates ppGpp accumulation and is conditionally required for antibiotic production in Streptomyces coelicolor A3(2). J Bacteriol 188:4952–4961 [View Article][PubMed]
     [Google Scholar]
  45. Sato M., Takahashi K., Ochiai Y., Hosaka T., Ochi K., Nabeta K. ( 2009). Bacterial alarmone, guanosine 5′-diphosphate 3′-diphosphate (ppGpp), predominantly binds the β′ subunit of plastid-encoded plastid RNA polymerase in chloroplasts. ChemBioChem 10:1227–1233 [View Article][PubMed]
     [Google Scholar]
  46. Shaw R. J., Wilson J. L., Smith K. T., Reines D. ( 2001). Regulation of an IMP dehydrogenase gene and its overexpression in drug-sensitive transcription elongation mutants of yeast. J Biol Chem 276:32905–32916 [View Article][PubMed]
     [Google Scholar]
  47. Sherman F. ( 1991). Getting started with yeast. Methods Enzymol 194:3–21 [View Article][PubMed]
     [Google Scholar]
  48. Song M., Kim H. J., Kim E. Y., Shin M., Lee H. C., Hong Y., Rhee J. H., Yoon H., Ryu S. & other authors ( 2004). ppGpp-dependent stationary phase induction of genes on Salmonella pathogenicity island 1. J Biol Chem 279:34183–34190 [View Article][PubMed]
     [Google Scholar]
  49. Sun J., Hesketh A., Bibb M. ( 2001). Functional analysis of relA and rshA, two relA/spoT homologues of Streptomyces coelicolor A3(2). J Bacteriol 183:3488–3498 [View Article][PubMed]
     [Google Scholar]
  50. Sun D., Lee G., Lee J. H., Kim H. Y., Rhee H. W., Park S. Y., Kim K. J., Kim Y., Kim B. Y. & other authors ( 2010). A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation responses. Nat Struct Mol Biol 17:1188–1194 [View Article][PubMed]
     [Google Scholar]
  51. Sy J., Ogawa Y., Lipmann F. ( 1973). Nonribosomal synthesis of guanosine 5′,3′-polyphosphates by the ribosomal wash of stringent Escherichia coli . Proc Natl Acad Sci U S A 70:2145–2148 [View Article][PubMed]
     [Google Scholar]
  52. Takahashi K., Kasai K., Ochi K. ( 2004). Identification of the bacterial alarmone guanosine 5′-diphosphate 3′-diphosphate (ppGpp) in plants. Proc Natl Acad Sci U S A 101:4320–4324 [View Article][PubMed]
     [Google Scholar]
  53. Tozawa Y., Nomura Y. ( 2011). Signalling by the global regulatory molecule ppGpp in bacteria and chloroplasts of land plants. Plant Biol (Stuttg) 13:699–709 [View Article][PubMed]
     [Google Scholar]
  54. Tozawa Y., Nozawa A., Kanno T., Narisawa T., Masuda S., Kasai K., Nanamiya H. ( 2007). Calcium-activated (p)ppGpp synthetase in chloroplasts of land plants. J Biol Chem 282:35536–35545 [View Article][PubMed]
     [Google Scholar]
  55. van Delden C., Comte R., Bally A. M. ( 2001). Stringent response activates quorum sensing and modulates cell density-dependent gene expression in Pseudomonas aeruginosa . J Bacteriol 183:5376–5384 [View Article][PubMed]
     [Google Scholar]
  56. van der Biezen E. A., Sun J., Coleman M. J., Bibb M. J., Jones J. D. ( 2000). Arabidopsis RelA/SpoT homologs implicate (p)ppGpp in plant signaling. Proc Natl Acad Sci U S A 97:3747–3752 [View Article][PubMed]
     [Google Scholar]
  57. Wagner R. ( 2002). Regulation of ribosomal RNA synthesis in E. coli: effects of the global regulator guanosine tetraphosphate (ppGpp). J Mol Microbiol Biotechnol 4:331–340[PubMed]
     [Google Scholar]
  58. Wang G., Tanaka Y., Ochi K. ( 2010). The G243D mutation (afsB mutation) in the principal sigma factor σHrdB alters intracellular ppGpp level and antibiotic production in Streptomyces coelicolor A3(2). Microbiology 156:2384–2392 [View Article][PubMed]
     [Google Scholar]
  59. Wells D. H., Long S. R. ( 2002). The Sinorhizobium meliloti stringent response affects multiple aspects of symbiosis. Mol Microbiol 43:1115–1127 [View Article][PubMed]
     [Google Scholar]
  60. Wells D. H., Long S. R. ( 2003). Mutations in rpoBC suppress the defects of a Sinorhizobium meliloti relA mutant. J Bacteriol 185:5602–5610 [View Article][PubMed]
     [Google Scholar]
  61. Xu J., Tozawa Y., Lai C., Hayashi H., Ochi K. ( 2002). A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2). Mol Genet Genomics 268:179–189 [View Article][PubMed]
     [Google Scholar]
  62. Yamada A., Tsutsumi K., Tanimoto S., Ozeki Y. ( 2003). Plant RelA/SpoT homolog confers salt tolerance in Escherichia coli and Saccharomyces cerevisiae . Plant Cell Physiol 44:3–9 [View Article][PubMed]
     [Google Scholar]
  63. Zhang H. B., Wang C., Zhang L. H. ( 2004). The quormone degradation system of Agrobacterium tumefaciens is regulated by starvation signal and stress alarmone (p)ppGpp. Mol Microbiol 52:1389–1401 [View Article][PubMed]
     [Google Scholar]
  64. Zhou Y. N., Jin D. J. ( 1998). The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like “stringent” RNA polymerases in Escherichia coli . Proc Natl Acad Sci U S A 95:2908–2913 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.057638-0
Loading
Heterologous expression of a plant RelA-SpoT homologue results in increased stress tolerance in Saccharomyces cerevisiae by accumulation of the bacterial alarmone ppGpp
Microbiology 158, 2213 (2012); https://doi.org/10.1099/mic.0.057638-0
/content/journal/micro/10.1099/mic.0.057638-0
/content/journal/micro/10.1099/mic.0.057638-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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