A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker |
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| Authors: | Winfried Oswalda Walaiporn Tonpitaka Gisela Ohrta Gerald-F Gerlacha |
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| Institution: | 1. Department of Urology, College of Medicine, Chung-Ang University, Seoul, Republic of Korea;2. College of Medicine, Chung-Ang University, Seoul, Republic of Korea;3. College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea;4. Genitourinary Cancer Branch, Research Institute, National Cancer Center, Goyang, Republic of Korea;1. Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA;1. Department of Microbiology, Oregon State University, Corvallis, OR 97331, USA;2. Department of Integrative Biology, Oregon State University, Corvallis, OR 97331, USA;3. Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, 61029 Urbino (PU), Italy;1. State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, 430070, China;2. Hubei Key Laboratory of Animal Embryo and Molecular Breeding, Institute of Animal Husbandry and Veterinary Sciences, Hubei Academy of Agricultural Sciences, Wuhan 430070, China;3. The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China |
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| Abstract: | Research on the porcine respiratory tract pathogen Actinobacillus pleuropneumoniae requires the availability of improved genetic tools. Therefore, using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows rapid curing of an Escherichia coli-A. pleuropneumoniae shuttle vector as well as the introduction of unmarked mutations into the A. pleuropneumoniae chromosome. A cassette containing the Tn903 kanamycin resistance determinant (km(r)) and the sacB gene expressed from the A. pleuropneumoniae omlA promoter was introduced by homologous recombination into the ureC gene of A. pleuropneumoniae. The resultant stable plasmid cointegrates were kanamycin-resistant, sucrose-sensitive, and urease-positive. A simple counterselection on sucrose-containing agar plates without an additional transconjugation step allowed the efficient isolation of urease-negative A. pleuropneumoniae mutants that had lost the km(r)-sacB cassette. |
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| Keywords: | Actinobacillus pleuropneumoniae Counterselection sacB gene |
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