Rasko David A, Esteban Carlos D, Sperandio Vanessa
University of Texas Southwestern Medical Center at Dallas, Department of Microbiology, 6000 Harry Hines Blvd, Room NA6.138, Dallas, TX 75235-9048, USA.
Plasmid. 2007 Sep;58(2):159-66. doi: 10.1016/j.plasmid.2007.03.002. Epub 2007 Apr 24.
Francisella tularensis is a category A bioterror pathogen which in some cases can cause a severe and fatal human infection. Very few virulence factors are known in this species due to the difficulty in working with it as well as the lack of tools for genetic manipulation. This work describes the construction of a shuttle vector that can replicate in Escherichia coli and F. tularensis as well as two distinct promoter trap constructs based on the shuttle vector backbone. Replication in F. tularensis is based on the promiscuous origin of replication from the Staphylococcus aureus plasmid pC194. We demonstrate the novel plasmids can coexist with established F. tularensis vectors based on the pFNL10 plasmid, the current workhorse of F. tularensis genetics. Our promoter trap can identify promoters that are activated during intracellular growth and survival. These new vectors provide additional tools for the genetic manipulation of F. tularensis.
土拉弗朗西斯菌是一种A类生物恐怖病原体，在某些情况下可导致严重且致命的人类感染。由于处理该菌存在困难以及缺乏基因操作工具，目前已知的该菌毒力因子非常少。这项工作描述了一种穿梭载体的构建，该载体可在大肠杆菌和土拉弗朗西斯菌中复制，以及基于该穿梭载体骨架的两种不同的启动子捕获构建体。土拉弗朗西斯菌中的复制基于金黄色葡萄球菌质粒pC194的混杂复制起点。我们证明了这些新型质粒可以与基于pFNL10质粒（土拉弗朗西斯菌遗传学目前的常用载体）的已建立的土拉弗朗西斯菌载体共存。我们的启动子捕获可以识别在细胞内生长和存活期间被激活的启动子。这些新载体为土拉弗朗西斯菌的基因操作提供了额外的工具。
