A Conditional Knockout Mutant Approach to Study Essential Genes in Chlamydia in Cell Culture and In Vivo

Identification and characterization of essential Chlamydia-specific factors are vital for the generation of effective therapeutics and vaccines. Despite delays in the development of genetic tools for Chlamydia, significant advancements in Chlamydia genetic manipulation have been made over the last decade and a half, now allowing for the targeted interruption or deletion of chromosomal genes. However, because of the obligate intracellular nature of the bacteria and its unique biphasic developmental cycle, it remains challenging to inactivate essential genes and study the function of their encoded proteins. Here, we describe a method to generate conditional knockout Chlamydia mutants by introducing, prior to gene inactivation, a complementation plasmid containing the gene of interest under the control of an anhydrotetracycline (aTc)-inducible promoter. The chromosomal copy of the gene can then be selectively deleted to produce a conditional knockout mutant in which expression of the gene is controlled with aTc. We detail how this conditional knockout mutant can be used to determine if the targeted gene is essential for Chlamydia infection in cell culture and in vivo.