title: E. coli recA gene improves gene targeted homologous recombination in Mycoplasma hyorhinis.
abstract: Mycoplasma hyorhinis is an opportunistic pathogen of pigs. Recently, it has been shown to transform cell cultures, increasing the attention of the researchers. Studies on the pathogenesis require specific genetic tool that is not yet available for the pathogen. To address this limitation, we constructed two suicide plasmids pGEMT-tetM/LR and pGEMT-recA-tetM/LR having a tetracycline resistance marker flanked by two hemolysin gene arms. The latter plasmid encodes an E. coli recA, a gene involved in DNA recombination, repair and maintenance of DNA. Using inactivation of the hemolysin gene, which results in a detectable and measurable phenotype, we found that each plasmid can disrupt the hemolysin gene of M. hyorhinis through a double cross-over homologous recombination. However, inclusion of the E. coli recA gene in the construct resulted in 9-fold increase in the frequency of hemolysin gene mutants among the screened tetracycline resistance colonies. The resultant hemolysin mutant strain lacks the ability to lyse mouse bed blood cells (RBC) when tested in vitro (p<0.001). The host-plasmid system described in this study, has applications for the genetic manipulation of this pathogen and potentially other mycoplasmas.
authors.affiliation: Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Research Center for Engineering and Technology of Veterinary Bio-products, Nanjing 210014, China; College of Veterinary Sciences, University of Nyala, Nyala, Sudan.
authors.forename: Hassan Z A
authors.initials: HZA
authors.lastname: Ishag
authors.affiliation: Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Research Center for Engineering and Technology of Veterinary Bio-products, Nanjing 210014, China. Electronic address: qiyanxiongnj@163.com.
authors.forename: Qiyan
authors.initials: Q
authors.lastname: Xiong
authors.affiliation: Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Research Center for Engineering and Technology of Veterinary Bio-products, Nanjing 210014, China.
authors.forename: Maojun
authors.initials: M
authors.lastname: Liu
authors.affiliation: Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Research Center for Engineering and Technology of Veterinary Bio-products, Nanjing 210014, China.
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Feng
authors.affiliation: Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Research Center for Engineering and Technology of Veterinary Bio-products, Nanjing 210014, China.
authors.forename: Guoqing
authors.initials: G
authors.lastname: Shao
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006460:Hemolysin Proteins / Q000235:genetics; D059765:Homologous Recombination; D051379:Mice; D009154:Mutation; D009174:Mycoplasma / Q000235:genetics; D009175:Mycoplasma Infections / Q000175:diagnosis; D045790:Mycoplasma hyorhinis / Q000235:genetics* / Q000472:pathogenicity; D010641:Phenotype; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011938:Rec A Recombinases / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D011995:Recombination, Genetic; D059767:Recombinational DNA Repair; D013552:Swine; D013753:Tetracycline Resistance / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; D011938:Rec A Recombinases
keywords: Hemolysin; Inactivation; Mycoplasma hyorhinis; Suicide vector; recA gene