An rpsL-based allelic exchange vector for Staphylococcus aureus.
An rpsL-based allelic exchange vector for Staphylococcus aureus.
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DOI：
10.1016/j.plasmid.2015.02.002
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发表时间：
2015-05
期刊：
影响因子：
2.6
通讯作者：
Novick RP
中科院分区：
文献类型：
--
作者：
Chen J;Ram G;Yoong P;Penadés JR;Shopsin B;Novick RP
金黄色葡萄球菌是最成功的细菌病原体之一，其武库中含有大量的毒力因子。因此，S.金黄色葡萄球菌染色体DNA是研究与宿主毒力和存活有关的基因的重要工具。先前报道的S.金黄色葡萄球菌是可以在大肠杆菌中繁殖的穿梭载体，因此可以进行标准的遗传操作。目前使用的大多数载体携带最初开发用于枯草芽孢杆菌的温度敏感性复制子（pE194ts）。在这里，我们表明，在S。在金黄色葡萄球菌中，pE194ts载体的热敏性在标准非允许温度（42 °C）下是不完全的，并且质粒的复制受损但未消除。我们报告了基于rpsL的反选择载体，其具有改进的温度敏感复制子（pT181 repC3），该复制子在S.金黄色葡萄球菌在非允许和标准生长温度（37 °C）。我们还描述了一组温度敏感的载体，可以在标准生长温度下固化。这些载体为快速产生等位基因置换突变和治愈表达质粒提供了高效的工具，并扩展了可用于S.金黄色。
Staphylococcus aureus is one of the most successful bacterial pathogens, harboring a vast repertoire of virulence factors in its arsenal. As such, the genetic manipulation of S. aureus chromosomal DNA is an important tool for the study of genes involved in virulence and survival in the host. Previously reported allelic exchange vectors for S. aureus are shuttle vectors that can be propagated in Escherichia coli, so that standard genetic manipulations can be carried out. Most of the vectors currently in use carry the temperature-sensitive replicon (pE194ts) that was originally developed for use in Bacillus subtilis. Here we show that in S. aureus, the thermosensitivity of a pE194ts vector is incomplete at standard non-permissive temperatures (42 °C), and replication of the plasmid is impaired but not abolished. We report rpsL-based counterselection vectors, with an improved temperature-sensitive replicon (pT181 repC3) that is completely blocked for replication in S. aureus at non-permissive and standard growth temperature (37 °C). We also describe a set of temperature-sensitive vectors that can be cured at standard growth temperature. These vectors provide highly effective tools for rapidly generating allelic replacement mutations and curing expression plasmids, and expand the genetic tool set available for the study of S. aureus.