PROJECT SUMMARY/ABSTRACT For bacterial pathogens, the first studies that begin to define the microorganism’s functional genetic nature on a genome-wide scale, frequently represent a landmark and highly impactful achievement. The major human pathogen, Chlamydia trachomatis, is a bacterium for which such studies have not been performed, in large part due to Chlamydia’s obligate intracellular nature and historical intractability to modern genetic perturbations. With the development of two major genetic tools for Chlamydia by our group—transposon mutagenesis and conditional temperature-sensitive mutants—functional genomic investigations in Chlamydia are now possible. Our long term goal is to globally define and functionally characterize the genetic correlates to C. trachomatis infection and pathogenesis. Using the new approaches described above, we propose to: (i) build defined mutant libraries of transposon and temperature-sensitive conditional mutants in model Chlamydia strains, (ii) screen these libraries for hallmark in vitro and in vivo growth defects using a clinically relevant murine genital tract model, and (iii) determine the Chlamydia genes associated with host adaptation by exploiting interspecies lateral gene transfer for Chlamydia and the inability of human parental strains to infect mice. These efforts are essential for gaining a functional appreciation of the role chlamydial genes play in the organism’s growth and development, and also Chlamydia’s evolutionary adaptation to successfully maintain infection and pathogenesis in complex mammalian hosts. We anticipate that Chlamydia will serve as a model obligate intracellular bacterium, and, therefore, the knowledge obtained from this work will broadly extend to other pathogens that have similarly evolved an obligate intracellular niche in their mammalian hosts. Identification of these loci is critical for our understanding the mechanism of Chlamydia adaptation to mammalian hosts, and is an important step towards elucidating the major virulence correlates of C. trachomatis in humans.