Advanced Genetic Tools for Studying Chlamydia

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PROJECT SUMMARY/ABSTRACT Chlamydiae are obligate intracellular bacterial pathogens that cause disease in human and animal populations Chlamydia trachomatis is the major cause of both bacterial sexually transmitted disease and infectious blindness in the world. Despite great strides over the past decade, tools for genetic study and manipulation of Chlamydia spp. remain severely limited. This proposal will develop new and powerful genetic tools including tightly negatively regulated promoter expression systems, a Tn7-based single chromosomal insertion transgene system, and application of the latter to build new, smaller shuttle plasmids for transformation. Specific Aim 1 – Development of a tightly negatively regulated inducible promoter for Chlamydia. This aim will provide one of the key missing tools in the repertoire of genetic tools for Chlamydia: a tightly negatively regulated, inducible, and titratable promoter for performing expression / overexpression studies. In bacterial physiology studies, it is important to be able to express and overexpress cloned genes in a mutant or wild type genetic background. One also must be able to tightly repress the cloned gene to avoid undesirable phenotypes associated with leaky expression of the cloned gene. The two subaims will design and test inducible promoter systems derived from two well-established Escherichia coli systems: the lac operon and the arabinose operon. Both systems are tightly repressed in the absence of inducer and rapidly upregulated when inducer is present. Our rigorous validation plan will use three reporter genes to measure promoter activity in the presence and absence of inducer. The lac operon promoters will allow us to develop a range of promoters of varying induction strength and induction ratios to provide flexibility for future genetic studies. Specific Aim 2 – Development of a chromosomal Tn7 transgene insertion system for single copy gene complementation studies in Chlamydia. Shuttle plasmids used in Chlamydia complementation studies are based on the native Chlamydia plasmid, which is present at 7.6 copies per genome. The major shortcoming of expressing a cloned gene from a multi-copy plasmid is that even low copies of the plasmid may lead to non-physiological levels of gene expression and aberrant phenotypes that complicate interpretation of the complementation results. The Tn7 transgene system provides high frequency, site- specific, single copy chromosomal insertion of any gene with the additional advantage of removing the need for antibiotic selection for transgene maintenance. We will adapt the Tn7 transgene system to Chlamydia. Our proposal includes a rigorous strategy to test the transgene system and plans to optimize the system by selection for transposase mutants that more efficiently recognize the chlamydial Tn7 attachment site. We will apply the Tn7 transgene system to build and validate new, smaller shuttle plasmids, which should improve transformation efficiencies. We will make all tools developed in this proposal available to the research community. Thus, successful completion of these aims will enhance genetic studies in Chlamydia.