Development of oriC-plasmids for use in Mycoplasma hyorhinis

文献类型： 外文期刊

第一作者： Ishag, Hassan Z. A.

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

作者机构：

期刊名称：SCIENTIFIC REPORTS （ 影响因子：4.379； 五年影响因子：5.133 ）

ISSN： 2045-2322

年卷期： 2017 年 7 卷

页码：

收录情况： SCI

摘要： Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pig pathogen, belonging to the class Mollicutes. It causes polyserositis, arthritis and cancers in vitro, increasing attention of the researchers. Currently, there is no available genetic tool to manipulate its genome. This study describes a development of oriC-plasmids harboring either large (pGEMT-LoriC) or minimum (pGEMT-MoriC) origin of replication (oriC) of M. hyorhinis along with tetracycline resistance marker. These plasmids were successfully transformed into M. hyorhinis with average transformation frequency of 1.5 x 10(-4) and 2.0 x 10(-5) transformants/CFU for pGEMT-LoriC and pGEMT-MoriC respectively, and were integrated at the chromosomal oriC as well as remained freely replicating. We also constructed a Mini-oriC-HT1 targeting plasmid by inclusion of hlyC arms and was used to inactivate hlyC at average frequency of 50%. The efficiency of hlyC inactivation was further improved (by 90%) when Mini-oriC-HT2 that contains E. coli recA was used. In both cases, hemolysin mutant bacteria diminished the ability to lyse mouse RBCs compared to wildtype (P < 0.001). OriC-plasmids described in this study may, therefore open the way for functional genomics in M. hyorhinis. Furthermore, this is a first study demonstrated the gene associated with a hemolytic phenotype in mycoplasmas.

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