1887

Abstract

The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin contains two putative regulatory genes, i.e. and . The predicted gene product of shows a putative helix–turn–helix DNA-binding motif and shares sequence similarity with StrR, a well-studied pathway-specific transcriptional activator of streptomycin biosynthesis. Here functional proof is provided, by genetic and biochemical approaches, for the role of NovG as a positive regulator of novobiocin biosynthesis. The entire novobiocin cluster of the producer organism was expressed in the heterologous host M512, and additional strains were produced which lacked the gene within the heterologously expressed cluster. These Δ strains produced only 2 % of the novobiocin formed by the M512 strains carrying the intact novobiocin cluster. The production could be restored by introducing an intact copy of into the mutant. The presence of on a multicopy plasmid in the strain containing the intact cluster led to almost threefold overproduction of the antibiotic, suggesting that novobiocin biosynthesis is limited by the availability of NovG protein. Furthermore, purified N-terminal His-tagged NovG showed specific DNA-binding activity for the and the intergenic regions of the novobiocin and clorobiocin biosynthetic gene clusters, respectively. By comparing the DNA sequences of the fragments binding NovG, conserved inverted repeats were identified in both fragments, similar to those identified as the binding sites for StrR. The consensus sequence for the StrR and the putative NovG binding sites was GTTCRACTG(N)CRGTYGAAC. Therefore, NovG and StrR apparently belong to the same family of DNA-binding regulatory proteins.

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2005-06-01
2020-04-04
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References

  1. Antón N., Mendes M. V., Aparicio J. F, Martín J. F.. 2004; Identification of PimR as a positive regulator of pimaricin biosynthesis in Streptomyces natalensis. J Bacteriol186:2567–2575[CrossRef]
    [Google Scholar]
  2. Arias P., Malpartida F, Fernández-Moreno M. A.. 1999; Characterization of the pathway-specific positive transcriptional regulator for actinorhodin biosynthesis in Streptomyces coelicolor A3(2) as a DNA-binding protein. J Bacteriol181:6958–6968
    [Google Scholar]
  3. Bentley S. D., Chater K. F., 40 other authors Cerdeño-Tárraga A. M.. 2002; Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature417:141–147[CrossRef]
    [Google Scholar]
  4. Chater K. F., Bibb M. J. 1997; Regulation of bacterial antibiotic production. In Biotechnologyvol. 7Products of Secondary Metabolism pp57–105 Edited by Kleinkauf H., von Döhren H.. Weinheim: VCH;
    [Google Scholar]
  5. Chater K. F., Horinouchi S. 2003; Signalling early developmental events in two highly diverged Streptomyces species. Mol Microbiol48:9–15[CrossRef]
    [Google Scholar]
  6. Chen H., Walsh C. T. 2001; Coumarin formation in novobiocin biosynthesis: β-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI. Chem Biol8:301–312[CrossRef]
    [Google Scholar]
  7. Chiu H.-T., Hubbard B. K., Shah A. N., Eide J., Fredenburg R. A., Walsh C. T., Khosla C. 2001; Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster. Proc Natl Acad Sci U S A98:8548–8553[CrossRef]
    [Google Scholar]
  8. Datsenko K. A., Wanner B. L. 2000; One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A97:6640–6645[CrossRef]
    [Google Scholar]
  9. Eustáquio A. S., Gust B., Luft T., Li S.-M., Chater K. F., Heide L. 2003a; Clorobiocin biosynthesis in Streptomyces. Identification of the halogenase and generation of structural analogs. Chem Biol10:279–288[CrossRef]
    [Google Scholar]
  10. Eustáquio A. S., Luft T., Wang Z.-X., Gust B., Chater K. F., Li S.-M., Heide L. 2003b; Novobiocin biosynthesis: inactivation of the putative regulatory gene novE and heterologous expression of genes involved in aminocoumarin ring formation. Arch Microbiol180:25–32[CrossRef]
    [Google Scholar]
  11. Eustáquio A. S., Gust B., Li S.-M., Pelzer S., Wohlleben W., Chater K. F., Heide L. 2004; Production of 8′-halogenated and 8′-unsubstituted novobiocin derivatives in genetically engineered Streptomyces coelicolor strains. Chem Biol11:1561–1572[CrossRef]
    [Google Scholar]
  12. Eustáquio A. S., Gust B., Galm U., Li S.-M., Chater K. F., Heide L. 2005; Heterologous expression of novobiocin and clorobiocin biosynthetic gene clusters. Appl Environ Microbiol71: in press).
    [Google Scholar]
  13. Floriano B., Bibb M. 1996; afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production inStreptomyces coelicolor A3(2). Mol Microbiol21:385–396[CrossRef]
    [Google Scholar]
  14. Galm U., Dessoy M. A., Schmidt J., Wessjohann L. A., Heide L. 2004; In vitro and in vivo production of new aminocoumarins by a combined biochemical, genetic and synthetic approach. Chem Biol11:173–183[CrossRef]
    [Google Scholar]
  15. 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 Microbiol7:837–845[CrossRef]
    [Google Scholar]
  16. Gust B., Challis G. L., Fowler K., Kieser T., Chater K. F. 2003; PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci U S A100:1541–1546[CrossRef]
    [Google Scholar]
  17. Hojati Z., Milne C., Harvey B.. 9 other authors 2002; Structure, biosynthetic origin, and engineered biosynthesis of calcium-dependent antibiotics from Streptomyces coelicolor. Chem Biol9:1175–1187[CrossRef]
    [Google Scholar]
  18. Hussain H. A., Ritchie D. A. 1991; High frequency transformation of Streptomyces niveus protoplasts by plasmid DNA. J Appl Bacteriol71:422–427[CrossRef]
    [Google Scholar]
  19. Kieser T., Bibb M. J., Buttner M. J., Chater K. F., Hopwood D. A. 2000; Practical Streptomyces Genetics, 2nd edn. Norwich: John Innes Foundation;
    [Google Scholar]
  20. Kominek L. A. 1972; Biosynthesis of novobiocin by Streptomyces niveus. Antimicrob Agents Chemother1:123–134[CrossRef]
    [Google Scholar]
  21. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227:680–685[CrossRef]
    [Google Scholar]
  22. Leskiw B. K., Bibb M. J., Chater K. F. 1991; The use of a rare codon specifically during development?. Mol Microbiol5:2861–2867[CrossRef]
    [Google Scholar]
  23. Li S.-M., Heide L. 2004; Functional analysis of biosynthetic genes of aminocoumarins and production of hybrid antibiotics. Curr Med Chem Anti-Infective Agents3:279–295[CrossRef]
    [Google Scholar]
  24. Lombó F., Braña A. F., Méndez C., Salas J. A. 1999; The mithramycin gene cluster of Streptomyces argillaceus contains a positive regulatory gene and two repeated DNA sequences that are located at both ends of the cluster. J Bacteriol181:642–647
    [Google Scholar]
  25. MacNeil D. J., Gewain K. M., Ruby C. L., Dezeny G., Gibbons P. H., MacNeil T. 1992; Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene111:61–68[CrossRef]
    [Google Scholar]
  26. Maxwell A. 1993; The interaction between coumarin drugs and DNA gyrase. Mol Microbiol9:681–686[CrossRef]
    [Google Scholar]
  27. Otten S. L., Ferguson J., Hutchinson C. R. 1995; Regulation of daunorubicin production in Streptomyces peucetius by the dnrR2 locus. J Bacteriol177:1216–1224
    [Google Scholar]
  28. Otten S. L., Olano C., Hutchinson C. R. 2000; The dnrO gene encodes a DNA-binding protein that regulates daunorubicin production in Streptomyces peucetius by controlling expression of the dnrN pseudo response regulator gene. Microbiology146:1457–1468
    [Google Scholar]
  29. Pabo C. O., Sauer R. T. 1992; Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem61:1053–1095[CrossRef]
    [Google Scholar]
  30. Pelzer S., Heckmann D., Recktenwald J., Huber P., Jung G., Wohlleben W, Süßmuth R.. 1999; Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis mediterranei DSM5908. Antimicrob Agents Chemother43:1565–1573
    [Google Scholar]
  31. Pérez-Llarena F. J., Liras P., Rodríguez-García A., Martín J. F. 1997; A regulatory gene (ccaR) required for cephamycin and clavulanic acid production inStreptomyces clavuligerus: amplification results in overproduction of both β-lactam compounds. J Bacteriol179:2053–2059
    [Google Scholar]
  32. Peschke U., Schmidt H., Zhang H. Z., Piepersberg W. 1995; Molecular characterization of the lincomycin-production gene cluster of Streptomyces lincolnensis 78-11. Mol Microbiol16:1137–1156[CrossRef]
    [Google Scholar]
  33. Pojer F., Li S.-M., Heide L. 2002; Molecular cloning and sequence analysis of the clorobiocin biosynthetic gene cluster: new insights into the biosynthesis of aminocoumarin antibiotics. Microbiology148:3901–3911
    [Google Scholar]
  34. Retzlaff L., Distler J. 1995; The regulator of streptomycin gene expression, StrR, of Streptomyces griseus is a DNA binding activator protein with multiple recognition sites. Mol Microbiol18:151–162[CrossRef]
    [Google Scholar]
  35. Rost B. 1996; PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol266:525–539
    [Google Scholar]
  36. Sambrook J., Russell D. W. 2001; Molecular Cloning: a Laboratory Manual, 3rd edn. New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  37. Sekurova O. N., Brautaset T., Sletta H., Borgos S. E., Jakobsen M. O., Ellingsen T. E., Strom A. R., Valla S., Zotchev S. B. 2004; In vivo analysis of the regulatory genes in the nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455 reveals their differential control over antibiotic biosynthesis. J Bacteriol186:1345–1354[CrossRef]
    [Google Scholar]
  38. Sheldon P. J., Busarow S. B., Hutchinson C. R. 2002; Mapping the DNA-binding domain and target sequences of the Streptomyces peucetius daunorubicin biosynthesis regulatory protein, DnrI. Mol Microbiol44:449–460[CrossRef]
    [Google Scholar]
  39. Sosio M., Stinchi S., Beltrametti F., Lazzarini A., Donadio S. 2003; The gene cluster for the biosynthesis of the glycopeptide antibiotic A40926 by Nonomuraea species. Chem Biol10:541–549[CrossRef]
    [Google Scholar]
  40. Sosio M., Kloosterman H., Bianchi A., de Vreugd P., Dijkhuizen L., Donadio S. 2004; Organization of the teicoplanin gene cluster in Actinoplanes teichomyceticus. Microbiology150:95–102[CrossRef]
    [Google Scholar]
  41. Steffensky M., Wang Z.-X., Li S.-M., Heide L, Mühlenweg A.. 2000; Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11891. Antimicrob Agents Chemother44:1214–1222[CrossRef]
    [Google Scholar]
  42. Stutzman-Engwall K. J., Otten S. L., Hutchinson C. R. 1992; Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius. J Bacteriol174:144–154
    [Google Scholar]
  43. Thamm S., Distler J. 1997; Properties of C-terminal truncated derivatives of the activator, StrR, of the streptomycin biosynthesis in Streptomyces griseus. FEMS Microbiol Lett149:265–272[CrossRef]
    [Google Scholar]
  44. Thiara A. S., Cundliffe E. 1988; Cloning and characterization of a DNA gyrase B gene from Streptomyces sphaeroides that confers resistance to novobiocin. EMBO J7:2255–2259
    [Google Scholar]
  45. Thiara A. S., Cundliffe E. 1989; Interplay of novobiocin-resistant and -sensitive DNA gyrase activities in self-protection of the novobiocin producer, Streptomyces sphaeroides. Gene81:65–72[CrossRef]
    [Google Scholar]
  46. Thorpe H. M., Wilson S. E., Smith M. C. 2000; Control of directionality in the site-specific recombination system of the Streptomyces phage πC31. Mol Microbiol38:232–241[CrossRef]
    [Google Scholar]
  47. Vara J., Lewandowska-Skarbek M., Wang Y. G., Donadio S., Hutchinson C. R. 1989; Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea(Streptomyces erythreus). J Bacteriol171:5872–5881
    [Google Scholar]
  48. Wang Z.-X., Li S.-M., Heide L. 2000; Identification of the coumermycin A1 biosynthetic gene cluster ofStreptomyces rishiriensis DSM 40489. Antimicrob Agents Chemother44:3040–3048[CrossRef]
    [Google Scholar]
  49. Wietzorrek A., Bibb M. 1997; A novel family of proteins that regulates antibiotic production in streptomycetes appears to contain an OmpR-like DNA-binding fold. Mol Microbiol25:1181–1184[CrossRef]
    [Google Scholar]
  50. Wilson D. J., Xue Y., Reynolds K. A., Sherman D. H. 2001; Characterization and analysis of the PikD regulatory factor in the pikromycin biosynthetic pathway of Streptomyces venezuelae. J Bacteriol183:3468–3475[CrossRef]
    [Google Scholar]
  51. Xu H., Heide L., Li S. M. 2004; New aminocoumarin antibiotics formed by a combined mutational and chemoenzymatic approach utilizing the carbamoyltransferase NovN. Chem Biol11:655–662[CrossRef]
    [Google Scholar]
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