1887

Abstract

The aminocoumarin antibiotic novobiocin is a gyrase inhibitor formed by a strain. The biosynthetic gene cluster of novobiocin spans 23.4 kb and contains 20 coding sequences, among them the two regulatory genes and . We investigated the location of transcriptional promoters within this cluster by insertion of transcriptional terminator cassettes and RT-PCR analysis of the resulting mutants. The cluster was found to contain eight DNA regions with promoter activity. The regulatory protein NovG binds to a previously identified binding site within the promoter region located upstream of , but apparently not to any of the other seven promoters. Quantitative real-time PCR was used to compare the number of transcripts in a strain carrying an intact novobiocin cluster with strains carrying mutated clusters. Both in-frame deletion of the regulatory gene and insertion of a terminator cassette into the biosynthetic gene led to a strong reduction of the number of transcripts of the genes located between and . This suggested that these 16 biosynthetic genes form a single operon. Three internal promoters are located within this operon but appear to be of minor importance, if any, under our experimental conditions. Transcription of was found to depend on the presence of NovE, suggesting that the two regulatory genes, and , act in a cascade-like mechanism. The resistance gene , encoding an aminocoumarin-resistant gyrase B subunit, may initially be co-transcribed with the genes from to . However, when the gyrase inhibitor novobiocin accumulates in the cultures, is transcribed from its own promoter. Previous work has suggested that this promoter is controlled by the superhelical density of chromosomal DNA.

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2009-12-01
2024-03-29
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References

  1. Blondelet-Rouault M. H., Weiser J., Lebrihi A., Branny P., Pernodet J. L. 1997; Antibiotic resistance gene cassettes derived from the Ω interposon for use in E. coli and Streptomyces . Gene 190:315–317
    [Google Scholar]
  2. Chandra G., Chater K. F. 2008; Evolutionary flux of potentially bldA-dependent Streptomyces genes containing the rare leucine codon TTA. Antonie Van Leeuwenhoek 94:111–126
    [Google Scholar]
  3. Dangel V., Eustáquio A. S., Gust B., Heide L. 2008; novE and novG act as positive regulators of novobiocin biosynthesis. Arch Microbiol 190:509–519
    [Google Scholar]
  4. Duetz W. A., Rüedi L., Hermann R., O'Connor K., Büchs J., Witholt B. 2000; Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates. Appl Environ Microbiol 66:2641–2646
    [Google Scholar]
  5. Eustáquio A. S., Luft T., Wang Z. X., Gust B., Chater K. F., Li S. M., Heide L. 2003; Novobiocin biosynthesis: inactivation of the putative regulatory gene novE and heterologous expression of genes involved in aminocoumarin ring formation. Arch Microbiol 180:25–32
    [Google Scholar]
  6. Eustáquio A. S., Gust B., Galm U., Li S. M., Chater K. F., Heide L. 2005a; Heterologous expression of novobiocin and clorobiocin biosynthetic gene clusters. Appl Environ Microbiol 71:2452–2459
    [Google Scholar]
  7. Eustáquio A. S., Li S. M., Heide L. 2005b; NovG, a DNA-binding protein acting as a positive regulator of novobiocin biosynthesis. Microbiology 151:1949–1961
    [Google Scholar]
  8. Fischbach M. A., Walsh C. T. 2006; Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: logic, machinery, and mechanisms. Chem Rev 106:3468–3496
    [Google Scholar]
  9. 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 A 100:1541–1546
    [Google Scholar]
  10. Gust B., Chandra G., Jakimowicz D., Yuqing T., Bruton C. J., Chater K. F. 2004; λ Red-mediated genetic manipulation of antibiotic-producing Streptomyces . Adv Appl Microbiol 54:107–128
    [Google Scholar]
  11. Heide L., Gust B., Anderle C., Li S. M. 2008; Combinatorial biosynthesis, metabolic engineering and mutasynthesis for the generation of new aminocoumarin antibiotics. Curr Top Med Chem 8:667–679
    [Google Scholar]
  12. Kieser T., Bibb M. J., Buttner M. J., Chater K. F., Hopwood D. A. 2000 Practical Streptomyces Genetics Norwich, UK: John Innes Foundation;
    [Google Scholar]
  13. Kominek L. A. 1972; Biosynthesis of novobiocin by Streptomyces niveus . Antimicrob Agents Chemother 1:123–134
    [Google Scholar]
  14. Laing E., Mersinias V., Smith C. P., Hubbard S. J. 2006; Analysis of gene expression in operons of Streptomyces coelicolor . Genome Biol 7:R46
    [Google Scholar]
  15. Li S. M., Heide L. 2004; Functional analysis of biosynthetic genes of aminocoumarins and production of hybrid antibiotics. Curr Med Chem Anti-Infect Agents 3:279–295
    [Google Scholar]
  16. Li S. M., Heide L. 2005; New aminocoumarin antibiotics from genetically engineered Streptomyces strains. Curr Med Chem 12:419–427
    [Google Scholar]
  17. Li S. M., Heide L. 2006; The biosynthetic gene clusters of aminocoumarin antibiotics. Planta Med 72:1093–1099
    [Google Scholar]
  18. 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. Gene 111:61–68
    [Google Scholar]
  19. Maxwell A., Lawson D. M. 2003; The ATP-binding site of type II topoisomerases as a target for antibacterial drugs. Curr Top Med Chem 3:283–303
    [Google Scholar]
  20. Pojer F., Kahlich R., Kammerer B., Li S. M., Heide L. 2003a; CloR, a bifunctional non-heme iron oxygenase involved in clorobiocin biosynthesis. J Biol Chem 278:30661–30668
    [Google Scholar]
  21. Pojer F., Wemakor E., Kammerer B., Chen H., Walsh C. T., Li S. M., Heide L. 2003b; CloQ, a prenyltransferase involved in clorobiocin biosynthesis. Proc Natl Acad Sci U S A 100:2316–2321
    [Google Scholar]
  22. Prentki P., Krisch H. M. 1984; In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303–313
    [Google Scholar]
  23. Raynal A., Karray F., Tuphile K., Darbon-Rongere E., Pernodet J. L. 2006; Excisable cassettes: new tools for functional analysis of Streptomyces genomes. Appl Environ Microbiol 72:4839–4844
    [Google Scholar]
  24. Sambrook J., Russell D. W. 2001 Molecular Cloning: a Laboratory Manual , 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  25. Shawky R. M., Puk O., Wietzorrek A., Pelzer S., Takano E., Wohlleben W., Stegmann E. 2007; The border sequence of the balhimycin biosynthesis gene cluster from Amycolatopsis balhimycina contains bbr, encoding a StrR-like pathway-specific regulator. J Mol Microbiol Biotechnol 13:76–88
    [Google Scholar]
  26. Siebenberg S., Bapat P. M., Lantz A. E., Gust B., Heide L. 2009; Reducing the variability of antibiotic production in Streptomyces by cultivation in 24-square deepwell plates. J Biosci Bioeng in press
  27. Thiara A. S., Cundliffe E. 1989; Interplay of novobiocin-resistant and -sensitive DNA gyrase activities in self-protection of the novobiocin producer, Streptomyces sphaeroides . Gene 81:65–72
    [Google Scholar]
  28. Tomono A., Tsai Y., Yamazaki H., Ohnishi Y., Horinouchi S. 2005; Transcriptional control by A-factor of strR, the pathway-specific transcriptional activator for streptomycin biosynthesis in Streptomyces griseus . J Bacteriol 187:5595–5604
    [Google Scholar]
  29. Westpheling J., Brawner M. 1989; Two transcribing activities are involved in expression of the Streptomyces galactose operon. J Bacteriol 171:1355–1361
    [Google Scholar]
  30. Wilkinson B., Micklefield J. 2007; Mining and engineering natural-product biosynthetic pathways. Nat Chem Biol 3:379–386
    [Google Scholar]
  31. Zerikly M., Challis G. L. 2009; Strategies for the discovery of new natural products by genome mining. ChemBioChem 10:625–633
    [Google Scholar]
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