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

As obligate intracellular bacteria, spp. have evolved numerous, likely intricate, mechanisms to create and maintain a privileged intracellular niche. Recent progress in elucidating and characterizing these processes has been bolstered by the development of techniques enabling basic genetic tractability. Florescence-reported allelic exchange mutagenesis (FRAEM) couples chromosomal gene deletion with the insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Similar to other bacteria, many chlamydial genes exist within polycistronic operons, raising the possibility of polar effects mediated by insertion cassettes. Indeed, FRAEM-mediated deletion of negatively impacts the expression of We have adapted FRAEM technology by employing a cassette flanked by sites. Conditional expression of Cre recombinase in containing a floxed cassette resulted in deletion of the marker and restoration of expression. infections represent a significant burden to human health. The ability to genetically manipulate spp. is overcoming historic confounding barriers that have impeded rapid progress in understanding overall chlamydial pathogenesis. The current state of genetic manipulation in 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 molecular toolbox by facilitating the deletion of genes within operons.