Skip to main content
SpringerLink
Log in

Deletion of ku homologs increases gene targeting frequency in Streptomyces avermitilis

  • Genetics and Molecular Biology of Industrial Organisms
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Streptomyces avermitilis is an industrially important soil bacterium known for production of avermectins, which are antiparasitic agents useful in animal health care, agriculture, and treatment of human infections. ku genes play a key role in the non-homologous end-joining pathway for repair of DNA double strand breaks. We identified homologs of eukaryotic ku70 and ku80 genes, termed ku1 and ku2, in S. avermitilis. Mutants with deletion of ku1, ku2, and both genes were constructed and their phenotypic changes were characterized. Deletion of ku genes had no apparent adverse effects on growth, spore formation, or avermectin production. The ku mutants, in comparison to wild-type strain, were slightly more sensitive to the DNA-damaging agent ethyl methanesulfonate, but not to UV exposure or to bleomycin. Gene targeting frequencies by homologous recombination were higher in the ku mutants than in wild-type strain. We conclude that ku-deleted strains will be useful hosts for efficient gene targeting and will facilitate functional analysis of genes in S. avermitilis and other industrially important bacterial strains.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

DSBs:

Double strand breaks

HR:

Homologous recombination

NHEJ:

Non-homologous end-joining

HPLC:

High-performance liquid chromatography

EMS:

Ethyl methanesulfonate

MMS:

Methyl methanesulfonate

References

  1. Aravind L, Koonin EV (2001) Prokaryotic double-strand break repair system Ku, novel domains in the Ku protein and prediction of a prokaryotic homologs of the eukaryotic DNA-end-binding protein. Genome Res 11:1365–1374

    Article  PubMed  CAS  Google Scholar 

  2. Bierman M, Logan R, O’Brien K, Seno ET, Rao RN, Schoner BE (1992) Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116:43–49

    Article  PubMed  CAS  Google Scholar 

  3. Burg RW, Miller BM, Baker EE, Birnbaum J, Currie SA, Hartman R, Kong YL, Monaqhan RL, Olson G, Putter I, Tunac JB, Wallick H, Stapley EO, Oiwa R, Omura S (1979) Avermectins, new family of potent anthelmintic agents: producing organism and fermentation. Antimicrob Agents Chemother 15:361–367

    PubMed  CAS  Google Scholar 

  4. Chen Z, Wen J, Song Y, Wen Y, Li JL (2007) Enhancement and selective production of avermectin B by recombinants of Streptomyces avermitilis via intraspecific protoplast fusion. Chin Sci Bull 52:616–622

    Article  CAS  Google Scholar 

  5. da Silva Ferreira ME, Kress MR, Savoldi M, Goldman MH, Härtl A, Heinekamp T, Brakhage AA, Goldman GH (2006) The akuB KU80 mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot Cell 5:207–211

    Article  PubMed  Google Scholar 

  6. Demain AL (1999) Pharmaceutically active secondary metabolites of microorganisms. Appl Microbiol Biotechnol 52:455–463

    Article  PubMed  CAS  Google Scholar 

  7. Gong C, Bongiorno P, Martins A, Stephanou NC, Zhu H, Shuman S, Glickman MS (2005) Mechanism of nonhomologous end-joining in mycobacteria: a low-fidelity repair system driven by Ku, ligase D and ligase C. Nat Struct Mol Biol 12:304–312

    Article  PubMed  CAS  Google Scholar 

  8. Haarmann T, Lorenz N, Tudzynski P (2008) Use of a nonhomologous end joining deficient strain (Deltaku70) of the ergot fungus Claviceps purpurea for identification of a nonribosomal peptide synthetase gene involved in ergotamine biosynthesis. Fungal Genet Biol 45:35–44

    Article  PubMed  CAS  Google Scholar 

  9. Ikeda H, Kotaki H, Tanaka H, Omura S (1988) Involvement of glucose catabolism in avermectin production by Streptomyces avermitilis. Antimicrob Agents Chemother 32:282–284

    PubMed  CAS  Google Scholar 

  10. Inbar O, Kupiec M (1999) Homology search and choice of homologous partner during mitotic recombination. Mol Cell Biol 19:4134–4142

    PubMed  CAS  Google Scholar 

  11. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. The John Innes Foundation, Norwich

    Google Scholar 

  12. Kobayashi H, Simmons LA, Yuan DS, Broughton WJ, Walker GC (2008) Multiple Ku orthologues mediate DNA non-homologous end-joining in the free-living form and during chronic infection of Sinorhizobium meliloti. Mol Microbiol 67:350–363

    Article  PubMed  CAS  Google Scholar 

  13. Krappmann S (2007) Gene targeting in filamentous fungi: the benefits of impaired repair. Fungal Biol Rev 21:25–29

    Article  Google Scholar 

  14. Krappmann S, Sasse C, Braus GH (2006) Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous end-joining-deficient genetic background. Eukaryot Cell 5:212–215

    Article  PubMed  CAS  Google Scholar 

  15. Li M, Chen Z, Zhang X, Song Y, Wen Y, Li JL (2010) Enhancement of avermectin and ivermectin production by overexpression of the maltose ATP-binding cassette transporter in Streptomyces avermitilis. Bioresour Technol 101:9228–9235

    Article  PubMed  CAS  Google Scholar 

  16. MacNeil DJ, Klapko LM (1987) Transformation of Streptomyces avermitilis by plasmid DNA. J Ind Microbiol 2:209–218

    Article  CAS  Google Scholar 

  17. Meyer V, Arentshorst M, El-Ghezal A, Drews AC, Kooistra R, van den Hondel CA, Ram AF (2007) Highly efficient gene targeting in the Aspergillus niger kusA mutant. J Biotechnol 128:770–775

    Article  PubMed  CAS  Google Scholar 

  18. Moeller R, Stackebrandt E, Reitz G, Berger T, Rettberg P, Doherty AJ, Horneck G, Nicholson WL (2007) Role of DNA repair by nonhomologous-end joining in Bacillus subtilis spore resistance to extreme dryness, mono- and polychromatic UV, and ionizing radiation. J Bacteriol 189:3306–3311

    Google Scholar 

  19. Nayak T, Szewczyk E, Oakley CE, Osmani A, Ukil L, Murray SL, Hynes MJ, Osmani SA, Oakley BR (2006) A versatile and efficient gene-targeting system for Aspergillus nidulans. Genetics 172:1557–1566

    Article  PubMed  CAS  Google Scholar 

  20. Ninomiya Y, Suzuki K, Ishii C, Inoue H (2004) Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci U S A 101:12248–12253

    Article  PubMed  CAS  Google Scholar 

  21. Omura S, Ikeda H, Ishikawa J, Hanamoto A, Takahashi C, Shinose M, Takahashi Y, Horikawa H, Nakazawa H, Osonoe T, Kikuchi H, Shiba T, Sakaki Y, Hattori M (2001) Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites. Proc Natl Acad Sci U S A 98:12215–12220

    Article  PubMed  CAS  Google Scholar 

  22. Osipovich O, Durum SK, Muegge K (1997) Defining the minimal domain of Ku80 for interaction with Ku70. J Biol Chem 272:27259–27265

    Article  PubMed  CAS  Google Scholar 

  23. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  24. Takahashi T, Masuda T, Koyama Y (2006) Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae. Mol Gen Genomics 275:460–470

    Article  CAS  Google Scholar 

  25. Takanhashi T, Masuda T, Koyama Y (2006) Identification and analysis of Ku70 and Ku80 homologs in the koji molds Aspergillus sojae and Aspergillus oryzae. Biosci Biotechnol Biochem 70:135–143

    Article  Google Scholar 

  26. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  PubMed  CAS  Google Scholar 

  27. Van Dyck E, Stasiak AZ, Stasiak A, West SC (1999) Binding of double-strand breaks in DNA by human Rad52 protein. Nature 398:728–731

    Article  PubMed  Google Scholar 

  28. Villalba F, Collemare J, Landraud P (2008) Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for non-homologous end joining. Fungal Genet Biol 45:68–75

    Article  PubMed  CAS  Google Scholar 

  29. Weller GR, Kysela B, Roy R, Tonkin LM, Scanlan E, Della M, Devine SK, Day JP, Wilkinson A, d’Adda di Fagagna F, Devine KM, Bowater RP, Jeggo PA, Jackson SP, Doherty AJ (2002) Identification of a DNA nonhomologous end-Joining complex in bacteria. Science 297:1686–1689

    Google Scholar 

  30. William SD, Sunghan Y (1998) Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids. Nucleic Acids Res 26:71551–71559

    Google Scholar 

  31. Zhang GT, Lukas H, Andre S (2009) Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina. J Biotechnol 139:146–151

    Article  Google Scholar 

  32. Zhao JL, Wen Y, Chen Z, Song Y, Li JL (2007) An adpA homologue in Streptomyces avermitilis is involved in regulation of morphogenesis and melanogenesis. Chin Sci Bull 52:623–630

    Article  CAS  Google Scholar 

  33. Zhao XJ, Wang YX, Wang SW, Chen Z, Wen Y, Song Y (2009) Construction of a doramectin producing mutant from an avermectin-overproducing industrial strain of Streptomyces avermitilis. Can J Microbiol 55:1355–1363

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the National Basic Research Program of China (Grant No. 2009CB118905).

Author information

Authors and Affiliations

  1. State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University, Beijing, 100193, People’s Republic of China

    Xiaojuan Zhang, Wei Chen, Yang Zhang, Libin Jiang, Zhi Chen, Ying Wen & Jilun Li

Authors
  1. Xiaojuan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wei Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Libin Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhi Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ying Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jilun Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ying Wen.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 108 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, X., Chen, W., Zhang, Y. et al. Deletion of ku homologs increases gene targeting frequency in Streptomyces avermitilis . J Ind Microbiol Biotechnol 39, 917–925 (2012). https://doi.org/10.1007/s10295-012-1097-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-012-1097-x

Keywords

Search

Navigation