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E-coli recA gene improves gene targeted homologous recombination in Mycoplasma hyorhinis

文献类型： 外文期刊

作者： Ishag, Hassan Z. A. 1 ; Xiong, Qiyan 1 ; Liu, Maojun 1 ; Feng, Zhixin 1 ; Shao, Guoqing 1 ;

作者机构： 1.Jiangsu Acad Agr Sci, Inst Vet Med, Key Lab Vet Biol Engn & Technol, Minist Agr,Natl Res Ctr Engn & Technol Vet Biopro, Nanjing 210014, Jiangsu, Peoples R China

2.Univ Nyala, Coll Vet Sci, Nyala, Sudan

关键词： Mycoplasma hyorhinis;Suicide vector;recA gene;Hemolysin;Inactivation

期刊名称：JOURNAL OF MICROBIOLOGICAL METHODS （ 影响因子：2.363； 五年影响因子：2.24 ）

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收录情况： SCI

摘要： 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. (C) 2017 Elsevier B.V. All rights reserved.

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[1]Characterization of the role in adherence of Mycoplasma hyorhinis variable lipoproteins containing different repeat unit copy numbers. Xiong, Qiyan,Zhang, Bixiong,Wang, Jia,Ni, Bo,Ji, Yan,Wei, Yanna,Feng, Zhixin,Liu, Maojun,Shao, Guoqing,Zhang, Bixiong,Xiao, Shaobo,Shao, Guoqing.

[2]The functions of the variable lipoprotein family of Mycoplasma hyorhinis in adherence to host cells. Xiong, Qiyan,Wang, Jia,Ji, Yan,Ni, Bo,Zhang, Bixiong,Ma, Qinghong,Wei, Yanna,Feng, Zhixin,Liu, Maojun,Shao, Guoqing,Xiao, Shaobo.

[3]GFP as a marker for transient gene transfer and expression in Mycoplasma hyorhinis. Ishag, Hassan Z. A.,Liu, Maojun,Yang, Ruosong,Xiong, Qiyan,Feng, Zhixin,Shao, Guoqing,Ishag, Hassan Z. A.. 2016

[4]Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Mycoplasma hyorhinis. Du, Gai-Mei,Liu, Mao-Jun,Wu, Yu-Zi,Xiong, Qi-Yan,Bai, Fang-Fang,Feng, Zhi-Xin,Shao, Guo-Qing,Du, Gai-Mei. 2013

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