Floxed-Cassette Allelic Exchange Mutagenesis Enables Markerless Gene Deletion in Chlamydia trachomatis and Can Reverse Cassette-Induced Polar Effects

Abstract

C. trachomatis infections represent a significant burden to human health. The ability to genetically manipulate Chlamydia spp. is overcoming historic confounding barriers that have impeded rapid progress in understanding overall chlamydial pathogenesis. The current state of genetic manipulation in Chlamydia spp. requires further development, including mechanisms to generate markerless gene disruption. We leveraged a stepwise Cre-lox approach to excise selection marker genes from a deleted gene locus. We found this process to be efficient, and the removal of extraneous elements resulted in the reversal of a negative polar effect on a downstream gene. This technique facilitates a more direct assessment of gene function and adds to the Chlamydia molecular toolbox by facilitating the deletion of genes within operons.