Le Pihive E, Blaha D, Chenavas S, Thibault F, Vidal D, Valade E
Laboratoire de bactériologie/UMR-MD1, Département de biologie des agents transmissibles, Centre de Recherches du Service de Santé des Armées Emile Pardé, La Tronche Cedex, France.
Plasmid. 2009 Nov;62(3):147-57. doi: 10.1016/j.plasmid.2009.07.001. Epub 2009 Jul 15.
Francisella tularensis is the causative agent of tularemia, a zoonotic disease often transmitted to humans by infected animals. The lack of useful specific genetic tools has long hampered the study of F. tularensis subspecies. We identified and characterized two new plasmids, pF242 and pF243, isolated from Francisella philomiragia strains ATCC 25016 and ATCC 25017, respectively. Sequence analysis revealed that pF242 and pF243 are closely related to pC194 and pFNL10 plasmids, respectively. Two generations of pF242- and pF243-based shuttle vectors, harboring several antibiotic resistance markers, were developed. We used the first generation to compare transformation efficiencies in two virulent F. tularensis subspecies. We found that electroporation was more efficient than cryotransformation: almost all vectors tested were successfully introduced by electroporation into Francisella strains with a high level of efficiency. The second generation of shuttle vectors, containing a multiple cloning site and/or gfp gene downstream of Francisella groES promotor, was used for GFP production in F. tularensis. The development of new shuttle vectors offers new perspectives in the genetic manipulation of F. tularensis, helping to elucidate the mechanisms underlying its virulence.
土拉弗朗西斯菌是兔热病的病原体，兔热病是一种人畜共患病，通常由受感染的动物传播给人类。长期以来，缺乏有用的特定遗传工具一直阻碍着对土拉弗朗西斯菌亚种的研究。我们分别从土拉弗朗西斯菌菌株ATCC 25016和ATCC 25017中鉴定并表征了两个新质粒pF242和pF243。序列分析表明，pF242和pF243分别与pC194和pFNL10质粒密切相关。开发了两代基于pF242和pF243的穿梭载体，它们携带几种抗生素抗性标记。我们使用第一代载体比较了两种有毒力的土拉弗朗西斯菌亚种中的转化效率。我们发现电穿孔比冷冻转化更有效：几乎所有测试的载体都通过电穿孔以高效率成功导入弗朗西斯菌菌株。第二代穿梭载体在弗朗西斯菌groES启动子下游含有多克隆位点和/或gfp基因，用于在土拉弗朗西斯菌中产生绿色荧光蛋白（GFP）。新穿梭载体的开发为土拉弗朗西斯菌的基因操作提供了新的视角，有助于阐明其毒力的潜在机制。
