Creation of a transposon mutant library in live vaccine strain Francisella tularensis
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Tularemia is a disease of small animals and humans caused by the bacterium Francisella tularensis. The most virulent subspecies to humans is F. tularensis subspecies tularensis. Due to its highly-infectious nature, this pathogen was classified as a category A agent by the Center for Disease Control and Prevention. The Live Vaccine Strain (LVS) is derived from F. tularensis subspecies holarctica. LVS is avirulent in humans, but still replicates in macrophages and causes a lethal infection in mice. Therefore, identifying LVS genes that are required for intra-macrophage survival should also aid in efforts to attenuate the category A strain. The transposon Tn5 is a mobile genetic element that inserts into the target DNA with very little site-specificity, resulting in virtually-random transposition. To generate a transposon system that functions in Francisella species, a Tn5 transposome was constructed containing the gene encoding green fluorescent protein (GFP) and the gene encoding hygromycin resistance under the control of Francisella promoters. Transformation into LVS should result in GFP-expressing transformants resistant to hygromycin. As transposition is a rare event, experiments are underway to optimize transformation into LVS. The optimized conditions should enable the isolation of a large set of transposon mutants which can then be screened for failure to replicate in cultured macrophages. The transposon is engineered to facilitate the identification of its chromosomal location. Identifying the disrupted genes is a first step in understanding the mechanisms by which F. tularensis bacteria escape killing by the host.