Chen John, Ram Geeta, Yoong Pauline, Penadés José R, Shopsin Bo, Novick Richard P
Skirball Institute Program in Molecular Pathogenesis, Departments of Microbiology and Medicine, New York University Medical Center, New York, NY 10016, USA.
Skirball Institute Program in Molecular Pathogenesis, Departments of Microbiology and Medicine, New York University Medical Center, New York, NY 10016, USA.
Plasmid. 2015 May;79:8-14. doi: 10.1016/j.plasmid.2015.02.002. Epub 2015 Feb 7.
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.
金黄色葡萄球菌是最成功的细菌病原体之一，其武器库中拥有大量毒力因子。因此，对金黄色葡萄球菌染色体DNA进行基因操作是研究宿主中毒力和生存相关基因的重要工具。先前报道的用于金黄色葡萄球菌的等位基因交换载体是穿梭载体，可在大肠杆菌中繁殖，从而能够进行标准的基因操作。目前使用的大多数载体携带最初为枯草芽孢杆菌开发的温度敏感复制子（pE194ts）。在这里我们表明，在金黄色葡萄球菌中，pE194ts载体在标准非允许温度（42°C）下的热敏感性不完全，质粒复制受到损害但未被消除。我们报道了基于rpsL的反选择载体，其具有改进的温度敏感复制子（pT181 repC3），在非允许温度和标准生长温度（37°C）下，该复制子在金黄色葡萄球菌中的复制完全被阻断。我们还描述了一组可在标准生长温度下消除的温度敏感载体。这些载体为快速产生等位基因替换突变和消除表达质粒提供了高效工具，并扩展了可用于研究金黄色葡萄球菌的基因工具集。
