Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae.
Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae.
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DOI：
10.1186/1297-9716-44-63
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发表时间：
2013-07-29
影响因子：
4.4
通讯作者：
BRaDP1T consortium
中科院分区：
文献类型：
--
作者：
Maglennon GA;Cook BS;Matthews D;Deeney AS;Bossé JT;Langford PR;Maskell DJ;Tucker AW;Wren BW;Rycroft AN;BRaDP1T consortium
肺炎支原体是一种流行的猪呼吸道病原体，是造成养猪生产者经济损失的主要原因。控制可通过结合使用抗微生物药物、疫苗接种和管理措施来实现，但目前的疫苗只能提供部分控制，需要改进预防战略。在了解肺炎支原体发病机制和开发新疫苗方面取得进展的一个主要障碍是缺乏基因操纵该生物体的工具。我们描述的发展和优化的第一个成功的质粒为基础的系统为猪肺炎支原体的遗传操作。我们的人工质粒包含肺炎支原体的复制起源（oriC）和tetM，赋予对四环素的抗性。利用这些质粒，我们成功地通过电穿孔转化了猪肺炎支原体菌株232，产生了四环素耐药菌。染色体外质粒的持续存在和质粒DNA的连续传代表明，这些人工质粒在肺炎支原体中具有自我复制的能力。除了证明肺炎支原体对基因操作的适应性和优化成功转化所需的条件外，我们还使用该系统确定了肺炎支原体的最小功能oriC。在此过程中，我们开发了一种具有小oriC的质粒，该质粒可以在多次传代中稳定维持，这可能有助于产生靶向基因破坏。总之，我们已经生成了一组对猪肺炎支原体发病机制的研究有价值的质粒，并为研究这一重要的猪病原体迈出了重要的一步。
Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss to pig producers. Control is achieved by a combination of antimicrobials, vaccination and management practices, but current vaccines offer only partial control and there is a need for improved preventative strategies. A major barrier to advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lack of tools to genetically manipulate the organism. We describe the development and optimisation of the first successful plasmid-based system for the genetic manipulation of M. hyopneumoniae. Our artificial plasmids contain the origin of replication (oriC) of M. hyopneumoniae along with tetM, conferring resistance to tetracycline. With these plasmids, we have successfully transformed M. hyopneumoniae strain 232 by electroporation, generating tetracycline resistant organisms. The persistence of extrachromosomal plasmid and maintenance of plasmid DNA over serial passages shows that these artificial plasmids are capable of self-replication in M. hyopneumoniae. In addition to demonstrating the amenability of M. hyopneumoniae to genetic manipulation and in optimising the conditions necessary for successful transformation, we have used this system to determine the minimum functional oriC of M. hyopneumoniae. In doing so, we have developed a plasmid with a small oriC that is stably maintained over multiple passages that may be useful in generating targeted gene disruptions. In conclusion, we have generated a set of plasmids that will be valuable in studies of M. hyopneumoniae pathogenesis and provide a major step forward in the study of this important swine pathogen.
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DOI：
10.1093/dnares/dsr045
发表时间：
2012-04
期刊：
DNA research : an international journal for rapid publication of reports on genes and genomes
影响因子：
--
作者：
Weber Sde S;Sant'Anna FH;Schrank IS
通讯作者：
Schrank IS
影响因子：
4.9
作者：
Algire, Mikkel A.;Lartigue, Carole;Merryman, Chuck
通讯作者：
Merryman, Chuck
影响因子：
3.2
作者：
Yang, Falong;Tang, Cheng;Yue, Hua
通讯作者：
Yue, Hua
影响因子：
2.6
作者：
Whetzel, PL;Hnatow, LL;Dohms, JE
通讯作者：
Dohms, JE
影响因子：
3.3
作者：
Maes D;Segales J;Meyns T;Sibila M;Pieters M;Haesebrouck F
通讯作者：
Haesebrouck F