1887

Abstract

Deficient antibiotic production in an mutant, BH5, of A3(2) was recently shown to be due to a mutation (G243D) in region 1.2 of the primary sigma factor . Here we show that intracellular ppGpp levels during growth, as well as after amino acid depletion, in the mutant BH5 are lower than those of the parent strain. The introduction of certain rifampicin resistance () mutations, which bypassed the requirement of ppGpp for transcription of pathway-specific regulatory genes, II-ORF4 and , for actinorhodin and undecylprodigiosin, respectively, completely restored antibiotic production by BH5. Antibiotic production was restored also by introduction of a new class of thiostrepton-resistance () mutations, which provoked aberrant accumulation of intracellular ppGpp. Abolition of ppGpp synthesis in the mutant Tsp33 again abolished antibiotic production. These results indicate that intracellular ppGpp level is finely tuned for successful triggering of antibiotic production in the wild-type strain, and that this fine tuning was absent from the mutant BH5, resulting in a failure to initiate antibiotic production in this strain.

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2010-08-01
2019-12-08
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References

  1. Aigle, B., Wietzorrek, A., Takano, E. & Bibb, M. J. ( 2000; ). A single amino acid substitution in region 1.2 of the principal σ factor of Streptomyces coelicolor A3(2) results in pleiotropic loss of antibiotic production. Mol Microbiol 37, 995–1004.[CrossRef]
    [Google Scholar]
  2. 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.[CrossRef]
    [Google Scholar]
  3. Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M., Challis, G. L., Thomson, N. R., James, K. D., Harris, D. E., Quail, M. A., Kieser, H. & other authors ( 2002; ). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141–147.[CrossRef]
    [Google Scholar]
  4. Bibb, M. J. ( 2005; ). Regulation of secondary metabolism in streptomycetes. Curr Opin Microbiol 8, 208–215.[CrossRef]
    [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.[CrossRef]
    [Google Scholar]
  6. Brown, K. L., Wood, S. & Buttner, M. J. ( 1992; ). Isolation and characterization of the major vegetative RNA polymerase of Streptomyces coelicolor A3(2); renaturation of a sigma subunit using GroEL. Mol Microbiol 6, 1133–1139.[CrossRef]
    [Google Scholar]
  7. Buttner, M. J. & Lewis, C. G. ( 1992; ). Construction and characterization of Streptomyces coelicolor A3(2) mutants that are multiply deficient in the non-essential hrd-encoded RNA polymerase sigma factors. J Bacteriol 174, 5165–5167.
    [Google Scholar]
  8. Buttner, M. J., Chater, K. F. & Bibb, M. J. ( 1990; ). Cloning, disruption, and transcriptional analysis of three RNA polymerase sigma factor genes of Streptomyces coelicolor A3(2). J Bacteriol 172, 3367–3378.
    [Google Scholar]
  9. 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, pp. 1458–1496. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
  10. Chakraburtty, R. & Bibb, M. ( 1997; ). The ppGpp synthetase gene (relA) of Streptomyces coelicolor A3(2) plays a conditional role in antibiotic production and morphological differentiation. J Bacteriol 179, 5854–5861.
    [Google Scholar]
  11. 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.[CrossRef]
    [Google Scholar]
  12. Fujii, T., Gramajo, H. C., Takano, E. & Bibb, M. J. ( 1996; ). redD and actII-ORF4, pathway-specific regulatory genes for antibiotic production in Streptomyces coelicolor A3(2), are transcribed in vitro by an RNA polymerase holoenzyme containing σhrdD . J Bacteriol 178, 3402–3405.
    [Google Scholar]
  13. Gramajo, H. C., Takano, E. & Bibb, M. J. ( 1993; ). Stationary-phase production of the antibiotic actinorhodin in Streptomyces coelicolor A3(2) is transcriptionally regulated. Mol Microbiol 7, 837–845.[CrossRef]
    [Google Scholar]
  14. Hara, O., Horinouchi, S., Uozumi, T. & Beppu, T. ( 1983; ). Genetic analysis of A-factor synthesis in Streptomyces coelicolor A3(2) and Streptomyces griseus. J Gen Microbiol 129, 2939–2944.
    [Google Scholar]
  15. 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.[CrossRef]
    [Google Scholar]
  16. Hesketh, A., Chen, W. J., Ryding, J., Chang, S. & Bibb, M. J. ( 2007; ). The global role of ppGpp synthesis in morphological differentiation and antibiotic production in Streptomyces coelicolor A3(2). Genome Biol 8, R161 [CrossRef]
    [Google Scholar]
  17. Hosaka, T., Ohnishi-Kameyama, M., Muramatsu, H., Murakami, K., Tsurumi, Y., Kodani, S., Yoshida, M., Fujie, A. & Ochi, K. ( 2009; ). Antibacterial discovery in actinomycetes strains with mutations in RNA polymerase or ribosomal protein S12. Nat Biotechnol 27, 462–464.[CrossRef]
    [Google Scholar]
  18. Hu, H., Zhang, Q. & Ochi, K. ( 2002; ). Activation of antibiotic biosynthesis by specific mutations in the rpoB gene (encoding the RNA polymerase β subunit) of Streptomyces lividans. J Bacteriol 184, 3984–3991.[CrossRef]
    [Google Scholar]
  19. Huang, J., Lih, C. J., Pan, K. H. & Cohen, S. N. ( 2001; ). Global analysis of growth phase responsive gene expression and regulation of antibiotic biosynthetic pathways in Streptomyces coelicolor using DNA microarrays. Genes Dev 15, 3183–3192.[CrossRef]
    [Google Scholar]
  20. 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.[CrossRef]
    [Google Scholar]
  21. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. ( 2000; ). Practical Streptomyces Genetics. Norwich, UK: John Innes Foundation.
  22. Lai, C., Xu, J., Tozawa, Y., Okamoto-Hosoya, Y., Yao, X. & Ochi, K. ( 2002; ). Genetic and physiological characterization of rpoB mutations that activate antibiotic production in Streptomyces lividans. Microbiology 148, 3365–3378.
    [Google Scholar]
  23. Nishimura, K., Hosaka, T., Tokuyama, S., Okamoto, S. & Ochi, K. ( 2007; ). Mutations in rsmG, encoding a 16S rRNA methyltransferase, result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3(2). J Bacteriol 189, 3876–3883.[CrossRef]
    [Google Scholar]
  24. Ochi, K. ( 1987; ). 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.
    [Google Scholar]
  25. Ochi, K. ( 1990a; ). Streptomyces relC mutants with an altered ribosomal protein ST-L11 and genetic analysis of a Streptomyces griseus relC mutant. J Bacteriol 172, 4008–4016.
    [Google Scholar]
  26. Ochi, K. ( 1990b; ). A relaxed (rel) mutant of Streptomyces coelicolor A3(2) with a missing ribosomal protein lacks the ability to accumulate ppGpp, A-factor and prodigiosin. J Gen Microbiol 136, 2405–2412.[CrossRef]
    [Google Scholar]
  27. Ochi, K. ( 2007; ). From microbial differentiation to ribosome engineering. Biosci Biotechnol Biochem 71, 1373–1386.[CrossRef]
    [Google Scholar]
  28. Ochi, K., Zhang, D., Kawamoto, S. & Hesketh, A. ( 1997; ). Molecular and functional analysis of the ribosomal L11 and S12 protein genes (rplK and rpsL) of Streptomyces coelicolor A3(2). Mol Gen Genet 256, 488–498.
    [Google Scholar]
  29. Okamoto-Hosoya, Y., Hosaka, T. & Ochi, K. ( 2003; ). An aberrant protein synthesis activity is linked with antibiotic overproduction in rpsL mutants of Streptomyces coelicolor A3(2). Microbiology 149, 3299–3309.[CrossRef]
    [Google Scholar]
  30. 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.[CrossRef]
    [Google Scholar]
  31. Shima, J., Hesketh, A., Okamoto, S., Kawamoto, S. & Ochi, K. ( 1996; ). Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2). J Bacteriol 178, 7276–7284.
    [Google Scholar]
  32. 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.[CrossRef]
    [Google Scholar]
  33. Takano, E., Gramajo, H. C., Strauch, E., Andres, N., White, J. & Bibb, M. J. ( 1992; ). Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2). Mol Microbiol 6, 2797–2804.[CrossRef]
    [Google Scholar]
  34. Tala, A., Wang, G., Zemanova, M., Okamoto, S., Ochi, K. & Alifano, P. ( 2009; ). Activation of dormant bacterial genes by Nonomuraea sp. strain ATCC 39727 mutant-type RNA polymerase. J Bacteriol 191, 805–814.[CrossRef]
    [Google Scholar]
  35. Wang, G., Hosaka, T. & Ochi, K. ( 2008; ). Dramatic activation of antibiotic production in Streptomyces coelicolor by cumulative drug resistance mutations. Appl Environ Microbiol 74, 2834–2840.[CrossRef]
    [Google Scholar]
  36. 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 Cell 10, 779–788.[CrossRef]
    [Google Scholar]
  37. 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.[CrossRef]
    [Google Scholar]
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vol. , part 8, pp. 2384–2392

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Act production by strains A700 (wild-type) and BH5 ( ) in the presence of a limited amount of phosphate source. The reverse sides of the plates are shown. Strains were incubated for 7 days on a chemically defined medium (Ochi, 1990), which contained an excess (0.2%) or limited (0.01%) amount of yeast extract instead of 50 mM phosphate as a sole phosphate source. The blue colour represents the antibiotic Act.

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