Differential Stringent Responses of Streptomyces coelicolor M600 to Starvation of Specific Nutrients

  • Ryu, Yong-Gu (School of Biological Sciences, Seoul National University) ;
  • Kim, Eun-Sook (School of Biological Sciences, Seoul National University) ;
  • Kim, Dae-Wi (School of Biological Sciences, Seoul National University) ;
  • Kim, Sung-Keun (School of Biological Sciences, Seoul National University) ;
  • Lee, Kye-Joon (School of Biological Sciences, Seoul National University)
  • Published : 2007.02.28

Abstract

This study focused on the involvement of the unusual nucleotide (p)ppGpp, a stringent factor, during the morphological and physiological differentiation of Streptomyces coelicolor. Two genes, relA and rshA, were disrupted to demonstrate the roles of the stringent factor in the differentiation. The intracellular concentration of (p)ppGpp in the wild-type (M600) and disrupted mutants was measured in relation to the intentional starvation of a specific nutrient, such as carbon, nitrogen, and phosphate or the in situ depletion of nutrients in a batch culture. As a result, it was found that the morphological characteristic of the ${\Delta}relA$ mutant was a bld phenotype forming condensed mycelia, whereas the ${\Delta}rshA$ mutant grew fast-forming spores and straightforward mycelia. In both mutants, the production of actinorhodin (Act) was completely abolished, yet the undecylprodigiosin (Red) production was increased. Intracellular (p)ppGpp was detected in the ${\Delta}relA$ mutant in the case of limited phosphate, yet not with limited carbon or nitrogen sources. In contrast, (p)ppGpp was produced in the ${\Delta}rshA$ mutant under limited carbon and nitrogen conditions. Therefore, (p)ppGpp in S. coelicolor was found to be selectively regulated by either the RelA or RshA protein, which was differentially expressed in response to the specific nutrient limitation. These results were also supported by the in situ ppGpp production during a batch culture. Furthermore, it is suggested that RelA and RshA are bifunctional proteins that possess the ability to both synthesize and hydrolyze (p)ppGpp.

Keywords

References

  1. Bascaran, V., L. Sanchez, C. Hardisson, and A. F. Brana. 1991. Stringent response and initiation of secondary metabolism in Streptomyces clavuligerus. J. Gen. Microbiol. 137: 1625- 1634 https://doi.org/10.1099/00221287-137-7-1625
  2. Bystrykh, L. V., M. A. Fernandez-Moreno, J. K. Herrema, F. Malpartida, D. A. Hopwood, and L. Dijkhuizen. 1996. Production of actinorhodin-related blue pigments by Streptomyces coelicolor A3(2). J. Bacteriol. 178: 2238- 2244 https://doi.org/10.1128/jb.178.8.2238-2244.1996
  3. Cashel, M., D. R. Gentry, V. J. Hernandez, and D. Viella. 1996. In Neidhardt, F. C. et al. (eds.). Escherichia coli and Salmonella: Cellular and Molecular Biology, pp. 1458- 1496. Washington, D.C. American Society for Microbiology
  4. Chater, K. F. and M. J. Bibb. 1997. Regulation of bacterial antibiotic production, pp. 57-105. In Kleinkauf, H. and Dohren, H. V. (eds.), Biotechnology. Vol. 7: Products of Secondary Metabolism. VCH Press, Weinheim, Germany
  5. Chakraburtty, R., J. White, E. Takano, and M. J. Bibb. 1996. Cloning, characterization and disruption of a (p)ppGpp synthetase gene (relA) of Streptomyces coelicolor A3(2). Mol. Microbiol. 19: 357-368 https://doi.org/10.1046/j.1365-2958.1996.390919.x
  6. Fawcett, J. K. and J. E. Scott. 1960. A rapid and precise method for the determination of urea. J. Clin. Path. 13: 156- 159 https://doi.org/10.1136/jcp.13.2.156
  7. Gentry, D. R. and M. Cashel. 1996. Mutational analysis of the Escherichia coli spoT gene identifies distinct but overlapping regions involved in ppGpp synthesis and degradation. Mol. Microbiol. 19: 1373-1384 https://doi.org/10.1111/j.1365-2958.1996.tb02480.x
  8. Gust, B., T. Kieser, and K. F. Chater. 2002. REDIRECT technology: PCR-targeting system in Streptomyces coelicolor
  9. Haseltine, W. A. and R. Block. 1973. Synthesis of guanosine tetra- and pentaphosphate requires the presence of a codonspecific, uncharged transfer ribonucleic acid in the acceptor site of ribosomes. Proc. Natl. Acad. Sci. USA 70: 1564-1568
  10. Hesketh, A., J. Sun, and M. Bibb. 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 https://doi.org/10.1046/j.1365-2958.2001.02221.x
  11. Hobbs, G., C. M. Frazer, D. C. J. Garner, J. A. Cullum, and S. G. Oliver. 1989. Dispersed growth of Streptomyces in liquid culture. Appl. Microbiol. Biotechnol. 31: 272-277
  12. Hoyt, S. and G. H. Jones. 1999. relA is required for actinomycin production in Streptomyces antibioticus. J. Bacteriol. 181: 3824-3829
  13. Jin, W., H. K. Kim, J. Y. Kim, S. G. Kang, S. H. Lee, and K. J. Lee. 2004. Cephamycin C production is regulated by relA and rsh genes in Streptomyces clavuligerus ATCC 27064. J. Biotechnol. 114: 81-87 https://doi.org/10.1016/j.jbiotec.2004.06.010
  14. Jaishy, B. P., S. K. Lim, I. D. Yoo, and J. C. Yoo. 2006. Cloning and characterization of a gene cluster for the production of polyketide macrolide dihydrochalcomycin in Streptomyces sp. KCTC 0041BP. J. Microbiol. Biotechnol. 16: 764-770
  15. Jin, W., Y. G. Ryu, S. G. Kang, S. K. Kim, N. Saito, K. Ochi, S. H. Lee, and K. J. Lee. 2004. Two relA/spoT homologous genes are involved in the morphological and physiological differentiation of Streptomyces clavuligerus. Microbiology 150: 1485-1493 https://doi.org/10.1099/mic.0.26811-0
  16. Jones, G. H. and M. J. Bibb. 1996. Guanosine pentaphosphate synthetase from Streptomyces antibioticus is also a polynucleotide phosphorylase. J. Bacteriol. 178: 4281-4288 https://doi.org/10.1128/jb.178.14.4281-4288.1996
  17. Kang, S. G., W. Jin, M. J. Bibb, and K. J. Lee. 1998. Actinorhodin and undecylprodigiosin production in wild type and relA mutant strains of Streptomyces coelicolor A3(2) grown in continuous culture. FEMS Microbiol. Lett. 168: 221-226 https://doi.org/10.1111/j.1574-6968.1998.tb13277.x
  18. Keston, A. S. and B. Katchen. 1956. Incorporation of glycine-2-$C^{14}$ into homologous antibody by rabbit tissue slices. J. Immunol. 76: 253-258
  19. Kieser, T., M. J. Bibb, M. J. Buttner, K. F. Chater, and D. A. Hopwood. 2000. Practical Streptomyces Genetics. John Innes Foundation, Norwich, U.K
  20. Kim, S. Y. and J. Y. Cho. 2005. A modified PCR-directed replacement method using $\lambda$-red recombination function in Escherichia coli. J. Microbiol. Biotechnol. 15: 1346-1352
  21. MacNeil, D. J. 1989. Characterization of a unique methylspecific restriction system in Streptomyces avermitilis. J. Bacteriol. 170: 5607-5612
  22. Metzger, S., E. Sarubbi, G. Glaser, and M. Cashel. 1989. Protein sequences encoded by the relA and the spoT genes of Escherichia coli are interrelated. J. Biol. Chem. 264: 9122- 9125
  23. Metzger, S., G. Schreiber, E. Aizenman, M. Cashel, and G. Glaser. 1989. Characterization of the relA1 mutation and a comparison of relA1 with new relA null alleles in Escherichia coli. J. Biol. Chem. 264: 21146-21152
  24. Mittenhuber, G. 2001. Comparative genomics of prokaryotic GTP-binding proteins (the Era, Obg, EngA, ThdF (TrmE), YchF, and YihA families) and their relationship to eukaryotic GTB-binding (the DRG, ARF, RAB, RAN, RAS, and RHO families). J. Mol. Microbiol. Biotechnol. 3: 21-35
  25. Murray, K. D. and H. Bremer, 1996. Control of spoT-dependent ppGpp synthesis and degradation in Escherichia coli. J. Mol. Biol. 259: 41-57 https://doi.org/10.1006/jmbi.1996.0300
  26. Ochi, K. 1986. Occurrence of the stringent response in Streptomyces sp. and its significance for the initiation of morphological and physiological differentiation. J. Gen. Microbiol. 132: 2621-2631
  27. Ochi, K. 1987. A rel mutation abolishes the enzyme induction needed for actinomycin synthesis by Streptomyces antibioticus. Agric. Biol. Chem. 51: 829-835 https://doi.org/10.1271/bbb1961.51.829
  28. Park, H. S., S. H. Kang, H. J. Park, and E. S. Kim. 2005. Doxorubicin productivity improvement by recombination Streptomyces peucetius with high-copy regulatory genes cultured in the optimized media composition. J. Microbiol. Biotechnol. 15: 66-71
  29. Pospiech, A. and B. Neumann. 1995. A versatile quick-prep of genomic DNA from Gram-positive bacteria. Trends Genet. 11: 213-218 https://doi.org/10.1016/0168-9525(95)90476-X
  30. Plummer, T. H. Jr., A. W. Phelan, and A. L. Tarentino. 1987. An Introduction to Practical Biochemistry, 3rd Edition. McGraw-Hill, London
  31. Ryu, Y. G., W. Jin, J. Y. Kim, J. Y. Kim, S. H. Lee, and K. J. Lee. 2004. Stringent factor regulates antibiotics production and morphological differentiation of Streptomcyes clavuligerus. J. Microbiol. Biotechnol. 14: 1170-1175
  32. Strauch, E., E. Takano, H. A. Baylis, and M. J. Bibb. 1991. The stringent response in Streptomyces coelicolor A3(2). Mol. Microbiol. 5: 289-298 https://doi.org/10.1111/j.1365-2958.1991.tb02109.x
  33. Sun, J. H., A. Hesketh, and M. J. Bibb. 2001. Functional analysis of relA and rshA, two relA/spot homologues of Streptomyces coelicolor A3(2). J. Bacteriol. 3488-3498
  34. Wendrich, T. M., C. L. Beckering, and M. A. Marahiel. 2000. Characterization of the relA/spoT gene from Bacillus stearothermophilus FEMS Microbiol. Lett. 190: 195-201 https://doi.org/10.1111/j.1574-6968.2000.tb09286.x
  35. Williams, R. P., J. A. Green, and D. A. Rappoport. 1956. Studies on pigmentation of Serratia marcescens. 1. Spectral and paper chromatographic properties of prodigiosin. J. Bacteriol. 71: 115-120
  36. Yang, Y.-Y., X.-Q. Zhao, Y.-Y. Jin, J.-H. Huh, J.-H. Cheng, D. Singh, H.-J. Kwon, and J.-W. Suh. 2006. Novel function of cytokinin: A signaling molecule for promotion of antibiotic production in Streptomycetes. J. Microbiol. Biotechnol. 16: 896-900