Description of two new plasmids isolated from Francisella philomiragia strains and construction of shuttle vectors for the study of Francisella tularensis.
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.
Le Pihive
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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.
Blaha
D
D
Chenavas
S
S
Thibault
F
F
Vidal
D
D
Valade
E
E
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005604:Francisella tularensis / Q000187:drug effects / Q000235:genetics* / Q000472:pathogenicity; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial
D000900:Anti-Bacterial Agents; D049452:Green Fluorescent Proteins