Floxed-cassette allelic exchange mutagenesis enables markerless gene deletion in Chlamydia trachomatis and can reverse cassette-induced polar effects

As obligate intracellular bacteria, Chlamydia 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 Chlamydia trachomatis tmeA negatively impacts the expression of tmeB. We have adapted FRAEM technology by employing a gfp-bla cassette flanked by loxP sites. Conditional expression of Cre recombinase in Chlamydia tmeA containing a floxed cassette resulted in deletion of the marker and restoration of tmeB expression.