Development of a replicable oriC plasmid for Mycoplasma gallisepticum and Mycoplasma imitans, and gene disruption through homologous recombination in M. gallisepticum
Development of a replicable oriC plasmid for Mycoplasma gallisepticum and Mycoplasma imitans, and gene disruption through homologous recombination in M. gallisepticum
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DOI：
10.1099/mic.0.2008/019208-0
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发表时间：
2008-09-01
期刊：
影响因子：
2.8
通讯作者：
Markham, P. F.
中科院分区：
文献类型：
--
作者：
Lee, S. -W;Browning, G. F.;Markham, P. F.
鸡毒支原体菌株R（低）的基因组已被完全测序，但随后的遗传研究受到缺乏可复制载体系统的限制。本研究构建了M.鸡毒支原体和仿支原体使用soj基因上游的oriC区域。将M. gallisepticum（pGTLori）和M. imitans（pMlori）在两个物种中复制，但肺炎支原体不能支持pGTLori的复制。克隆了M.发现鸡毒支原体是质粒复制所需的最小区域。鸡毒支原体菌株S6，定义为支原体的最短oriC区域。目的基因vlhA 1.1的破坏。使用这些oriC质粒尝试了鸡败血症S6。在pPLoriC7中制备的构建体整合到M.在pMlori中制备的质粒破坏了vlhA1.2基因，其与vlhA1.1基因具有97%的DNA序列同一性。在体外传代过程中，抗菌剂选择压力不影响染色体整合率。因此，这些oriC质粒将是有用的遗传研究，包括失活或选定的基因的表达，在M。鸡毒M. imitans，并将导致更好地了解其分子生物学。据我们所知，它们是第一个可复制的质粒，用于支原体的进化系统群。
The genome of Mycoplasma gallisepticum strain R(low) has been sequenced completely, but subsequent genetic studies have been limited by the lack of a replicable vector system. In this study, replicable plasmids were constructed for M. gallisepticum and Mycoplasma imitans using the oriC region upstream from the soj gene. The oriC plasmids of M. gallisepticum (pGTLori) and M. imitans (pMlori) replicated in both species, but Mycoplasma pneumoniae could not support replication of pGTLori. A 180 bp section of the oriC region of M. gallisepticum was found to be the minimal region required for plasmid replication in M. gallisepticum strain S6, the shortest oriC region defined for mycoplasmas. Targeted gene disruption of vlhA 1.1 of M. gallisepticum S6 was attempted using these oriC plasmids. Constructs made in pPLoriC7 integrated into the M. gallisepticum genomic oriC region, not into the targeted gene, whereas those made in pMlori disrupted the vlhA1.2 gene, which has 97% DNA sequence identity with the vlhA1.1 gene. During in vitro passages, antimicrobial selection pressure did not influence the rate of chromosomal integration. These oriC plasmids will thus be useful for genetic studies, including inactivation or expression of selected genes, in M. gallisepticum and M. imitans, and will lead to a better understanding of their molecular biology. They are, to our knowledge, the first replicable plasmids developed for the Pneumoniae phylogenetic group of mycoplasmas.