Burkholderia pseudomallei is the causative agent of melioidosis and a category B select agent for bioterrorism. Strains of B. pseudomallei display high levels of genomic diversity. Much of this diversity is localized to regions of difference larger than 10-kb in size. Characterizing the contributions of these regions of difference to pathogenic phenotypes requires the ability to both delete as well as capture and insert these large regions into B. pseudomallei to generate isogenic strains. However the capture and insertion of large genomic fragment is technically challenging. We propose to develop a broad-host- range, select agent compliant, large fragment capture system for use in B. pseudomallei. The capture system will be based on the site specific recombination that occurs between two directed origins of transfer during conjugation. The oriT directed capture system will additionally be functionalized with the broad-host-range mini-Tn7 single copy integration system. Utilizing our developed capture system and techniques for constructing large deletions, two B. pseudomallei systems with pathogenic phenotypes linked to large regions of difference will be characterized. The first is based on the virulent clinical B. pseudomallei isolate 708a which carries a >130-kb natural deletion of several virulence factors including the malleobactin primary siderophore system. The second system is based on two B. pseudomallei gene clusters, Yersinia-like fimbriae (YLF) and Burkholderia thailandensis flagella and chemotaxis (BTFC). The clusters are mutually exclusive and are both geographically and clinically biased, with YLF being the dominant group in Thai and clinical isolates. PUBLIC HEALTH RELEVANCE: Characterization of natural gene loss, acquisition and strain to strain variability are likely to be important factors in understanding B. pseudomallei pathogenicity and phenotypic diversity. Additionally, the oriT directed fragment capture system will be broadly applicable in other bacteria and will bridge an important technical gap for genetic characterization of many Gram-negative pathogens.