Mini-Tn7 vectors as genetic tools for gene cloning at a single copy number in an industrially important and phytopathogenic bacteria, Xanthomonas spp.

Authors

  • Thichakorn Jittawuttipoka,

    1. Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand
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  • Sarinya Buranajitpakorn,

    1. Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand
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  • Mayuree Fuangthong,

    1. Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok, Thailand
    2. Chulabhorn Graduate Institute, Lak Si, Bangkok, Thailand
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  • Herbert P. Schweizer,

    1. Department of Microbiology, Immunology and Pathology, Rocky Mountain Regional Center of Excellence for Biodefense and Infectious Diseases Research, Colorado State University, Fort Collins, CO, USA
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  • Paiboon Vattanaviboon,

    1. Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok, Thailand
    2. Chulabhorn Graduate Institute, Lak Si, Bangkok, Thailand
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  • Skorn Mongkolsuk

    1. Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand
    2. Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok, Thailand
    3. Chulabhorn Graduate Institute, Lak Si, Bangkok, Thailand
    4. Center of Emerging Bacterial Infection, Faculty of Science, Mahidol University, and Center for Environmental Health, Toxicology and Management of Chemicals, Bangkok, Thailand
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  • Editor: Reggie Lo

Correspondence: Skorn Mongkolsuk, Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand. Tel.: +66 2 574 0622, ext. 3816; fax: +66 2 574 2027; e-mail: skorn@cri.or.th

Abstract

Transposon mini-Tn7 vectors insert into the chromosome of several Gram-negative bacteria in a site-specific manner. Here, we showed the application of mini-Tn7 as single copy site-specific integration vector system for Xanthomonas campestris pv. campestris. The transposition of the mini-Tn7 into the bacterial genome was detected at a Tn7 attachment (attTn7) site located downstream of glmS1. Furthermore, using a newly constructed vector pBBR1FLP2 containing the flipase (FLP) recombinase for site-specific excision of the sequence between the FLP recombinase target (FRT) sites, and a sacB counter selection marker, an unmarked mini-Tn7 insertion mutant was created. Mini-Tn7 insertion did not affect bacterial virulence on the tested plant. The mini-Tn7 and FLP–FRT systems also work well in Xanthomonas oryzae pv. oryzae.

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