1887

Abstract

The complete natamycin (NTM) biosynthetic gene cluster of was cloned and confirmed by the disruption of pathway-specific activator genes. Comparative cluster analysis with its counterpart in revealed different cluster architecture between these two clusters. Compared with the highly conserved coding sequences, sequence variations appear to occur frequently in the intergenic regions. The evolutionary change of nucleotide sequence in the intergenic regions has given rise to different transcriptional organizations in the two clusters and resulted in altered gene regulation. These results provide insight into the evolution of antibiotic biosynthetic gene clusters. In addition, we cloned a pleitropic regulator gene, , in . Using the genetic system that we developed for this strain, was deleted from the genome of . The Δ mutant showed a conditionally sparse aerial mycelium formation phenotype and defects in sporulation; it also lost the ability to produce NTM and a diffusible yellow pigment normally produced by . RT-PCR analysis revealed that transcription of was constitutive in YEME liquid medium. By using rapid amplification of 5′ complementary DNA ends, two transcription start sites were identified upstream of the coding region. Quantitative transcriptional analysis showed that the expression level of the NTM regulatory gene decreased 20-fold in the Δ mutant strain, while the transcription of the other activator gene was not significantly affected. Electrophoretic mobility shift assay (EMSA) showed that AdpA binds to its own promoter but fails to bind to the promoter region of , indicating that the control of by AdpA is exerted in an indirect way. This work not only provides a platform and a new potential target for increasing the titre of NTM by genetic manipulation, but also advances the understanding of the regulation of NTM biosynthesis.

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2011-05-01
2019-10-18
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References

  1. Ahlert J. , Shepard E. , Lomovskaya N. , Zazopoulos E. , Staffa A. , Bachmann B. O. , Huang K. , Fonstein L. , Czisny A. et al. ( 2002; ). The calicheamicin gene cluster and its iterative type I enediyne PKS. . Science 297:, 1173–1176. [CrossRef] [PubMed]
    [Google Scholar]
  2. Antón N. , Mendes M. V. , Martín J. F. , Aparicio J. F. . ( 2004; ). Identification of PimR as a positive regulator of pimaricin biosynthesis in Streptomyces natalensis . . J Bacteriol 186:, 2567–2575. [CrossRef] [PubMed]
    [Google Scholar]
  3. Antón N. , Santos-Aberturas J. , Mendes M. V. , Guerra S. M. , Martín J. F. , Aparicio J. F. . ( 2007; ). PimM, a PAS domain positive regulator of pimaricin biosynthesis in Streptomyces natalensis. . Microbiology 153:, 3174–3183. [CrossRef] [PubMed]
    [Google Scholar]
  4. Aparicio J. F. , Fouces R. , Mendes M. V. , Olivera N. , Martín J. F. . ( 2000; ). A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosynthesis of the 26-membered polyene macrolide pimaricin in Streptomyces natalensis . . Chem Biol 7:, 895–905. [CrossRef] [PubMed]
    [Google Scholar]
  5. Aparicio J. F. , Caffrey P. , Gil J. A. , Zotchev S. B. . ( 2003; ). Polyene antibiotic biosynthesis gene clusters. . Appl Microbiol Biotechnol 61:, 179–188.[PubMed] [CrossRef]
    [Google Scholar]
  6. Chater K. F. , Chandra G. . ( 2008; ). The use of the rare UUA codon to define “expression space” for genes involved in secondary metabolism, development and environmental adaptation in Streptomyces . . J Microbiol 46:, 1–11. [CrossRef] [PubMed]
    [Google Scholar]
  7. Darling A. C. , Mau B. , Blattner F. R. , Perna N. T. . ( 2004; ). Mauve: multiple alignment of conserved genomic sequence with rearrangements. . Genome Res 14:, 1394–1403. [CrossRef] [PubMed]
    [Google Scholar]
  8. Du Y. L. , Chen S. F. , Cheng L. Y. , Shen X. L. , Tian Y. , Li Y. Q. . ( 2009; ). Identification of a novel Streptomyces chattanoogensis L10 and enhancing its natamycin production by overexpressing positive regulator ScnRII. . J Microbiol 47:, 506–513. [CrossRef] [PubMed]
    [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. [CrossRef] [PubMed]
    [Google Scholar]
  10. Hara H. , Ohnishi Y. , Horinouchi S. . ( 2009; ). DNA microarray analysis of global gene regulation by A-factor in Streptomyces griseus . . Microbiology 155:, 2197–2210. [CrossRef] [PubMed]
    [Google Scholar]
  11. Kato J. Y. , Ohnishi Y. , Horinouchi S. . ( 2005; ). Autorepression of AdpA of the AraC/XylS family, a key transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus . . J Mol Biol 350:, 12–26. [CrossRef] [PubMed]
    [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. Lee K. M. , Lee C. K. , Choi S. U. , Park H. R. , Kitani S. , Nihira T. , Hwang Y. I. . ( 2005; ). Cloning and in vivo functional analysis by disruption of a gene encoding the γ-butyrolactone autoregulator receptor from Streptomyces natalensis . . Arch Microbiol 184:, 249–257. [CrossRef] [PubMed]
    [Google Scholar]
  14. Lee K. M. , Lee C. K. , Choi S. U. , Park H. R. , Hwang Y. I. . ( 2008; ). Functional analysis of a BarX homologue (SngA) as a pleiotropic regulator in Streptomyces natalensis . . Arch Microbiol 189:, 569–577. [CrossRef] [PubMed]
    [Google Scholar]
  15. Mendes M. V. , Tunca S. , Antón N. , Recio E. , Sola-Landa A. , Aparicio J. F. , Martín J. F. . ( 2007; ). The two-component phoR-phoP system of Streptomyces natalensis: inactivation or deletion of phoP reduces the negative phosphate regulation of pimaricin biosynthesis. . Metab Eng 9:, 217–227. [CrossRef] [PubMed]
    [Google Scholar]
  16. Ohnishi Y. , Kameyama S. , Onaka H. , Horinouchi S. . ( 1999; ). The A-factor regulatory cascade leading to streptomycin biosynthesis in Streptomyces griseus: identification of a target gene of the A-factor receptor. . Mol Microbiol 34:, 102–111. [CrossRef] [PubMed]
    [Google Scholar]
  17. Ohnishi Y. , Yamazaki H. , Kato J. Y. , Tomono A. , Horinouchi S. . ( 2005; ). AdpA, a central transcriptional regulator in the A-factor regulatory cascade that leads to morphological development and secondary metabolism in Streptomyces griseus . . Biosci Biotechnol Biochem 69:, 431–439. [CrossRef] [PubMed]
    [Google Scholar]
  18. Recio E. , Colinas A. , Rumbero A. , Aparicio J. F. , Martín J. F. . ( 2004; ). PI factor, a novel type quorum-sensing inducer elicits pimaricin production in Streptomyces natalensis . . J Biol Chem 279:, 41586–41593. [CrossRef] [PubMed]
    [Google Scholar]
  19. Sambrook J. , Fritsch E. F. , Maniatis T. . ( 1989; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory;.
    [Google Scholar]
  20. Santos-Aberturas J. , Vicente C. M. , Guerra S. M. , Payero T. D. , Martin J. F. , Aparicio J. F. . ( 2011; ). Molecular control of polyene macrolide biosynthesis: direct binding of the regulator PimM to eight promoters of pimaricin genes. Identification of binding boxes. . J Biol Chem 286:, 9150–9161. [CrossRef] [PubMed]
    [Google Scholar]
  21. Seco E. M. , Pérez-Zúñiga F. J. , Rolón M. S. , Malpartida F. . ( 2004; ). Starter unit choice determines the production of two tetraene macrolides, rimocidin and CE-108, in Streptomyces diastaticus var. 108. . Chem Biol 11:, 357–366. [CrossRef] [PubMed]
    [Google Scholar]
  22. Shirling E. B. , Gottlieb D. . ( 1966; ). Methods for characterization of Streptomyces species. . Int J Syst Bacteriol 16:, 313–340. [CrossRef]
    [Google Scholar]
  23. Sun Y. , Zhou X. , Liu J. , Bao K. , Zhang G. , Tu G. , Kieser T. , Deng Z. . ( 2002; ). Streptomyces nanchangensis’, a producer of the insecticidal polyether antibiotic nanchangmycin and the antiparasitic macrolide meilingmycin, contains multiple polyketide gene clusters. . Microbiology 148:, 361–371.[PubMed]
    [Google Scholar]
  24. Takano E. , Tao M. , Long F. , Bibb M. J. , Wang L. , Li W. , Buttner M. J. , Bibb M. J. , Deng Z. X. , Chater K. F. . ( 2003; ). A rare leucine codon in adpA is implicated in the morphological defect of bldA mutants of Streptomyces coelicolor . . Mol Microbiol 50:, 475–486. [CrossRef] [PubMed]
    [Google Scholar]
  25. Vicente C. M. , Santos-Aberturas J. , Guerra S. M. , Payero T. D. , Martín J. F. , Aparicio J. F. . ( 2009; ). PimT, an amino acid exporter controls polyene production via secretion of the quorum sensing pimaricin-inducer PI-factor in Streptomyces natalensis . . Microb Cell Fact 8:, 33. [CrossRef] [PubMed]
    [Google Scholar]
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