The completion of the genome sequences for many NIAID category A, B and C priority pathogens, which are also listed as select agent bacteria, now greatly facilitates the design and execution of genetic experiments aimed at understanding the biology and pathogenesis of these bacteria. However, these endeavors are greatly complicated by the fact that most genetic tools available today rely on the use of antibiotic selection markers, many of which are prohibited for use in these bacteria by NIH Guidelines for Recombinant DNA Research because they potentially compromise the therapeutic use of the respective drugs. We hypothesize that non-antibiotic resistance markers belonging to the biocide, herbicide and heavy metal families can be engineered to enhance the repertoire of genetic tools available for bacteria on the select agent list. To test this hypothesis, two specific aims will be pursued. In aim 1, we will modify the fabL, bar and ILV, and kilAtelAB genes, which encode triclosan (biocide), bialaphos and chlorimuron ethyl (herbicide), and tellurite (heavy metal) resistance, respectively. Expression cassettes will be constructed by placing the respective genes under the control of modified Escherichia coli lac operon promoters. Yeast Flp recombinase target (FRT) sites will provide an option for in vivo excision and therefore recycling of the selectable markers. In aim 2, the applicability of the newly constructed markers for priority pathogen research will be tested by using the examples of Burkholderia mallei, B. pseudomallei, Francisella tulerensis and Yersinia pestis. This will be done by construction and testing of multi-copy replicative and single-copy gene integration vectors with the option of Flp recombinase-mediated marker excision. In the end, we expect to have developed a panel of versatile genetic tools containing different non-antibiotic resistance markers for use in the select agents B. mallei, B. pseudomallei, F. tularensis and Y. pestis, as well as other bacterial pathogens for which one faces similar restrictions regarding use of antibiotic selection markers for genetic manipulation. [unreadable] [unreadable] [unreadable]