Skip to main content
SpringerLink
Log in

Site-specific recombination system based on actinophage TG1 integrase for gene integration into bacterial genomes

  • Applied Genetics and Molecular Biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Phage integrases are enzymes that catalyze unidirectional site-specific recombination between the attachment sites of phage and host bacteria, attP and attB, respectively. We recently developed an in vivo intra-molecular site-specific recombination system based on actinophage TG1 serine-type integrase that efficiently acts between attP and attB on a single plasmid DNA in heterologous Escherichia coli cells. Here, we developed an in vivo inter-molecular site-specific recombination system that efficiently acted between the att site on exogenous non-replicative plasmid DNA and the corresponding att site on endogenous plasmid or genomic DNA in E. coli cells, and the recombination efficiencies increased by a factor of ~101–3 in cells expressing TG1 integrase over those without. Moreover, integration of attB-containing incoming plasmid DNA into attP-inserted E. coli genome was more efficient than that of the reverse substrate configuration. Together with our previous result that purified TG1 integrase functions efficiently without auxiliary host factors in vitro, these in vivo results indicate that TG1 integrase may be able to introduce attB-containing circular DNAs efficiently into attP-inserted genomes of many bacterial species in a site-specific and unidirectional manner. This system thus may be beneficial to genome engineering for a wide variety of bacterial species.

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

Similar content being viewed by others

References

  • Bibb LA, Hancox MI, Hatfull GF (2005) Integration and excision by the large serine recombinase ϕRv1 integrase. Mol Microbiol 55:1896–1910

    Article  CAS  Google Scholar 

  • Biswas T, Aihara H, Radman-Livaja M, Filman D, Landy A, Ellenberger T (2005) A structural basis for allosteric control of DNA recombination by λ integrase. Nature 435:1059–1066

    Article  CAS  Google Scholar 

  • Campbell A (1962) Episomes. Adv Genet 11:101–145

    Article  Google Scholar 

  • Campbell A (1992) Chromosomal insertion site for phages and plasmids. J Bacteriol 174:7495–7499

    CAS  Google Scholar 

  • Foor F, Roberts GP, Morin N, Snyder L, Hwang M, Gibbons PH, Paradiso MJ, Stotish RL, Ruby CL, Wolanski B, Streicher SL (1985) Isolation and characterization of the Streptomyces cattleya temperate phage TG1. Gene 39:11–16

    Article  CAS  Google Scholar 

  • Ghosh P, Kim AI, Hatfull GF (2003) The orientation of mycobacteriophage Bxb1 integration is solely dependent on the central dinucleotide of attP and attB. Mol Cell 12:1101–1111

    Article  CAS  Google Scholar 

  • Goryshin IY, Jendrisak J, Hoffman LM, Meis R, Reznikoff WS (2000) Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes. Nat Biotechnol 18:97–100

    Article  CAS  Google Scholar 

  • Gregory MA, Till R, Smith MCM (2003) Integration site for Streptomyces phage ϕBT1 and development of site-specific integrating vectors. J Bacteriol 185:5320–5323

    Article  CAS  Google Scholar 

  • Groth AC, Calos MP (2004) Phage integrases: biology and applications. J Mol Biol 335:667–678

    Article  CAS  Google Scholar 

  • Groth AC, Olivares EC, Thyagarajan B, Calos MP (2000) A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci USA 97:5995–6000

    Article  CAS  Google Scholar 

  • Ikeda H, Ishikawa J, Hanamoto A, Shinose M, Kikuchi H, Shiba T, Sakaki Y, Hattori M, Omura S (2003) Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat Biotechnol 21:526–531

    Article  Google Scholar 

  • Landy A (1989) Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Annu Rev Biochem 58:913–949

    Article  CAS  Google Scholar 

  • Li W, Kamtekar S, Xiong Y, Sarkis GJ, Grindley NDF, Steitz TA (2005) Structure of a synaptic γδ resolvase tetramer covalently linked to two cleaved DNAs. Science 309:1210–1215

    Article  CAS  Google Scholar 

  • Matsuura M, Noguchi T, Yamaguchi D, Aida T, Asayama M, Takahashi H, Shirai M (1996) The sre gene (ORF469) encodes a site-specific recombinase responsible for integration of the R4 phage genome. J Bacteriol 178:3374–3376

    CAS  Google Scholar 

  • Morita K, Yamamoto T, Fusada N, Komatsu M, Ikeda H, Hirano N, Takahashi H (2009a) In vitro characterization of the site-specific recombination system based on actinophage TG1 integrase. Mol Genet Genomics 282:607–616

    Article  CAS  Google Scholar 

  • Morita K, Yamamoto T, Fusada N, Komatsu M, Ikeda H, Hirano N, Takahashi H (2009b) The site-specific recombination system of actinophage TG1. FEMS Microbiol Lett 297:234–240

    Article  CAS  Google Scholar 

  • Olivares EC, Hollis RP, Calos MP (2001) Phage R4 integrase mediates site-specific integration in human cells. Gene 278:167–176

    Article  CAS  Google Scholar 

  • Rashel M, Uchiyama J, Ujihara T, Takemura I, Hoshiba H, Matsuzaki S (2008) A novel site-specific recombination system derived from bacteriophage ϕMR11. Biochem Biophys Res Commun 368:192–198

    Article  CAS  Google Scholar 

  • Russell JP, Chang DW, Tretiakova A, Padidam M (2006) Phage Bxb1 integrase mediates highly efficient site-specific recombination in mammalian cells. Biotechniques 40:460–464

    Article  CAS  Google Scholar 

  • Smith MCM, Thorpe HM (2002) Diversity in the serine recombinases. Mol Microbiol 44:299–307

    Article  CAS  Google Scholar 

  • Stoll SM, Ginsburg DS, Calos MP (2002) Phage TP901-1 site-specific integrase functions in human cells. J Bacteriol 184:3657–3663

    Article  CAS  Google Scholar 

  • Thorpe HM, Smith MCM (1998) In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci USA 95:5505–5510

    Article  CAS  Google Scholar 

  • Thyagarajan B, Olivares EC, Hollis RP, Ginsburg DS, Calos MP (2001) Site-specific genomic integration in mammalian cells mediated by phage ϕC31 integrase. Mol Cell Biol 21:3926–3934

    Article  CAS  Google Scholar 

  • Xu Z, Lee NCO, Dafhnis-Calas F, Malla S, Smith MCM, Brown WRA (2008) Site-specific recombination in Schizosaccharomyces pombe and systematic assembly of a 400 kb transgene array in mammalian cells using the integrase of Streptomyces phage ϕBT1. Nucleic Acids Res 36:e9

    Article  Google Scholar 

Download references

Acknowledgments

We thank S. Nonoda for technical support. This work is supported in part by a Grant-in-Aid for Young Scientists (B) No. 10017551, and a grant to promote advanced scientific research, and Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Research Grant of College of Engineering, Nihon University for 2010.

Author information

Authors and Affiliations

  1. Department of Chemical Biology & Applied Chemistry, College of Engineering, Nihon University, 1 Nakagawara, Tokusada, Tamura-machi, Koriyama, Fukushima, 963-8642, Japan

    Nobutaka Hirano, Tetsurou Muroi, Yoshihiko Kihara, Ryuichi Kobayashi & Mitsuru Haruki

  2. Department of Applied Biological Science, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa, 252-8510, Japan

    Hideo Takahashi

Authors
  1. Nobutaka Hirano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tetsurou Muroi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yoshihiko Kihara
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ryuichi Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hideo Takahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mitsuru Haruki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nobutaka Hirano.

Additional information

Authors Nobutaka Hirano and Tetsurou Muroi contributed equally to this work and should be regarded as joint first authors.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 31 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hirano, N., Muroi, T., Kihara, Y. et al. Site-specific recombination system based on actinophage TG1 integrase for gene integration into bacterial genomes. Appl Microbiol Biotechnol 89, 1877–1884 (2011). https://doi.org/10.1007/s00253-010-3003-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-010-3003-7

Keywords

Search

Navigation