Summary: Francisella tularensis is an intracellular pathogen capable of causing severe infections with high mortality. This organism has been under-studied and its pathogenesis is not completely understood. Our long-term goal is to define the molecular mechanisms responsible for this organism's intracellular survival and virulence. This long-term goal is hampered by the paucity of tools available for molecular genetic studies. In particular, antibiotic resistance has been widely used to select for genetically-modified strains, raising concerns of creating highly pathogenic Francisellae that are resistant to antibiotics. The work described in this application focuses on novel strategies to select genetically-modified F. tularensis without the use of antibiotics. Our specific aims include: Aim 1: Develop counterselectable marker systems in F. tularensis. We will exploit selection systems widely used in yeast and other bacteria that rely on non-antibiotic drug resistance and auxotrophy at the pyrF and trpC loci, permitting reciprocal positive and negative selection for these genes. Our preliminary data indicate the pyrF and trpC genes in F. tularensis are legitimate targets for a positive and negative selection system. pyrF? and trpC? mutants generated in this aim will serve as host strains for plasmid transformation and gene deletion experiments. Aim 2: Adapt the TargeTron system to F. tularensis using fluorescence selection. Group II introns, commercialized as TargeTron, are efficient tools to disrupt genes in a variety of bacterial species. We will adapt to F. tularensis and improve this system by incorporating a marker for fluorescence screening. These experiments will develop an efficient gene disruption system and a rapid, high-throughput fluorescence selection strategy to identify transformed Francisellae. Aim 3: Develop immunoaffinity selection system in F. tularensis. Antibodies are highly specific and are widely used to detect antigens in complex mixtures. In this aim we will develop selection methods using neoantigen expression on F. tularensis and affinity purification of bacteria using antibodies. This approach is expected to be rapid and, under the appropriate promoter, reversible, permitting novel promoter screens as well as routine selection. Successful completion of these aims will create several independent and complementary methods for selecting genetically-altered F. tularensis for use in studies of virulence and pathogenesis. These tools will also be applicable to studies of other biodefense and non-biodefense pathogens. [unreadable] [unreadable] [unreadable]