Generation of Chromosomal Deletions in Chlamydia trachomatis

Genetic manipulation of Chlamydia trachomatis can elucidate the determinants involved in pathogenesis but is difficult due to this bacterium's obligate intracellular lifestyle and comparatively low transformation rate. Deletion mutants can be generated in Chlamydia by allelic exchange via homologous recombination, but require conditionally replicating vectors that have to be maintained and propagated through several rounds of the intracellular developmental cycle. An additional challenge is that Chlamydia and E. coli cannot stably maintain each other's endogenous plasmids. As a solution, the ori (origin of replication) region from C. trachomatis endogenous plasmid pL2 was cloned into the E. coli pUC19 plasmid together with genes for fluorescent reporting and spectinomycin resistance to generate the pKW shuttle vector. pKW can be successfully propagated in both Chlamydia and E. coli, but is rapidly lost by chlamydial transformants upon removal of antibiotic selection pressure. In this chapter, we describe how to prepare the chlamydial stock and generate a pKW-based allelic exchange shuttle vector, with chromosomal sequences upstream and downstream of a target gene, and transform it into C. trachomatis. We provide step-by-step instructions for generating a targeted deletion mutant by growing the transformants under antibiotic selection to allow homologous recombination with the target gene and then removing the antibiotic to induce loss of the suicide vector.