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Artificial chromosomes for antibiotic-producing actinomycetes

Nature Biotechnology volume 18, pages 343–345 (2000)Cite this article

Abstract

Bacteria belonging to the order Actinomycetales produce most microbial metabolites thus far described, several of which have found applications in medicine and agriculture. However, most strains were discovered by their ability to produce a given molecule and are, therefore, poorly characterized physiologically and genetically. Thus, methodologies for genetic manipulation of actinomycetes are not available and efficient tools have been developed for just a few strains. This constitutes a serious limitation to applying molecular genetics approaches to strain development and structural manipulation of microbial metabolites. To overcome this hurdle, we have developed bacterial artificial chromosomes (BAC)1,2 that can be shuttled among Escherichia coli, where they replicate autonomously, and a suitable Streptomyces host, where they integrate site-specifically into the chromosome. The existence of gene clusters3 and of genetically amenable host strains, such as Streptomyces coelicolor or Streptomyces lividans4, makes this a sensible approach. We report here that 100 kb segments of actinomycete DNA can be cloned into these vectors and introduced into genetically accessible S. lividans, where they are stably maintained in integrated form in its chromosome.

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Figure 1: Escherichia coli–Streptomyces artificial chromosome (ESAC) vectors and ESAC clones.
Figure 2: Genomic segment and resulting artificial chromosome (ESAC) clone.
Figure 3: Southern hybridization of S. lividans transformants.

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References

  1. Shizuya, H. et al. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. USA 89, 8794–8797 (1992).

    Article  CAS  Google Scholar 

  2. Ioannou, P.A. et al. A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nat. Genet. 6, 84–89 (1994).

    Article  CAS  Google Scholar 

  3. Martìn, J.F. & Liras, P. Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annu. Rev. Microbiol. 43, 173–206 (1989).

    Article  Google Scholar 

  4. Hopwood, D.A. In: Proceedings from the Genetics and Molecular Biology and Industrial Microorganisms (GMBIM) conference (eds.Baltz, R.H., Hegeman, G.D. & Skatrud, P.L.) 1–6 (Society of Industrial Microbiology, Fairfax, VA; 1997).

    Google Scholar 

  5. Malpartida, F. & Hopwood, D.A. Molecular cloning of the whole biosynthetic pathway of a Streptomyces antibiotic and its expression in a heterologous host. Nature 309, 462–464 (1984).

    Article  CAS  Google Scholar 

  6. Kao, C.M., Katz, L. & Khosla, C. Engineered biosynthesis of a complete macrolactone in a heterologous host. Science 265, 509–512 (1994).

    Article  CAS  Google Scholar 

  7. Schwecke, T. et al. The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin. Proc. Natl. Acad. Sci. USA 92, 7839–7843 (1995).

    Article  CAS  Google Scholar 

  8. August P.R. et al. Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chem. Biol. 5, 69–79 (1998).

    Article  CAS  Google Scholar 

  9. Kuhstoss, S. & Rao, R.N. Analysis of the integration function of the streptomycete bacteriophage phi C31. J. Mol. Biol. 222, 897–908 (1991).

    Article  CAS  Google Scholar 

  10. Hopwood, D.A. et al. Genetic manipulation of Streptomyces: a laboratory manual (The John Innes Foundation, Norwich, UK; 1985).

    Google Scholar 

  11. Ioannou, P.A. & de Jong, P.J. in Current protocols in human genetics (eds Dracopoli, N.C. et al.) 5.15.1–5.15.24 (Wiley, New York; 1996).

    Google Scholar 

  12. Kieser, H.M., Kieser, T. & Hopwood, D.A. A combined genetic and physical map of the chromosome of Streptomyces coelicolor A3(2). J. Bacteriol. 174, 5496–5507 (1992).

    Article  CAS  Google Scholar 

  13. Redenbach, M. et al. A set of ordered cosmids and a detailed genetic and physical map of the 8Mb Streptomyces coelicolor A3(2) chromosome. Mol. Microbiol. 21, 77–96 (1996).

    Article  CAS  Google Scholar 

  14. Leblond, P., Redenbach, M. & Cullum, J. Physical map of the Streptomyces lividans 66 genome and comparison with that of the related strain Streptomyces coelicolor A3(2). J. Bacteriol. 175, 3422–3429 (1993).

    Article  CAS  Google Scholar 

  15. Xu, Y., Jiang, L., Murray, B.E. & Weinstock, G.M. Enterococcus faecalis antigens in human infections. Infect. Immun. 65, 4207–4215 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Brosch, R. et al. Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics. Infect. Immun. 66, 2221–2229 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Rondon, M.R., Raffel, S.J., Goodman, R.M. & Handelsman, J. Towards functional genomics in bacteria: analysis of expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus. Proc. Natl. Acad. Sci. USA 96, 6451–6455 (1999).

    Article  CAS  Google Scholar 

  18. Hamilton, C.M., Frary, A., Lewis, C. & Tanskley, S.D. Stable transfer of intact high molecular weight DNA into plant chromosomes. Proc. Natl. Acad. Sci. USA 93, 9975–9979 (1996).

    Article  CAS  Google Scholar 

  19. Liu, Y.-G. et al. Complementation of plant mutations with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning. Proc. Natl. Acad. Sci. USA 96, 6535–6540 (1999).

    Article  CAS  Google Scholar 

  20. Birren, B. & Lai, E. Pulsed field gel electrophoresis: a practical guide (Academic Press, New York, NY; 1993).

    Google Scholar 

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Acknowledgements

We thank Luca Dolce and Rosa Alduina for performing some experiments on the ESAC library. We are grateful to Gianni Dehò and Charles Thompson for critical reading of the manuscript, and to David Hopwood and Pieter de Jong, and Sidney Kushner for plasmids and strains. This work was partially supported by the Italian CNR, PF Biotecnologie.

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Author notes

  1. Francesco Giusino

    Present address: Wyeth-Lederle SpA, Catania, Italy

  2. Carmela Cappellano

    Present address: Rhône-Poulenc Rorer, Vitry sur Seine, France

  3. Margherita Sosio and Francesco Giusino: The first two authors contributed equally to this research.

Authors and Affiliations

  1. Biosearch Italia SpA, Gerenzano, 21040, Italy

    Margherita Sosio, Elena Bossi & Stefano Donadio

  2. Department of Cellular and Developmental Biology, Università di Palermo, Palermo, 90128, Italy

    Francesco Giusino & Anna Maria Puglia

Authors
  1. Margherita Sosio
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  2. Francesco Giusino
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  4. Elena Bossi
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Correspondence to Stefano Donadio.

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Sosio, M., Giusino, F., Cappellano, C. et al. Artificial chromosomes for antibiotic-producing actinomycetes. Nat Biotechnol 18, 343–345 (2000). https://doi.org/10.1038/73810

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