As obligate intracellular bacteria, <jats:italic>Chlamydia</jats:italic> 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 insertion of a selection cassette encoding antibiotic resistance and 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 <jats:italic>C. trahomatis</jats:italic> <jats:italic>tmeA</jats:italic> negatively impacts expression of <jats:italic>tmeB</jats:italic>. We have adapted FRAEM technology by employing a <jats:italic>gfp-bla</jats:italic> cassette flanked by <jats:italic>loxP</jats:italic> sites. Conditional expression of Cre recombinase in <jats:italic>tmeA Chlamydia</jats:italic> containing a floxed cassette resulted in deletion of the marker and restoration of <jats:italic>tmeB</jats:italic> expression.
<jats:bold>IMPORTANCE</jats:bold> <jats:italic>C. trachomatis</jats:italic> infections represent a significant burden to human health. The ability to genetically manipulate <jats:italic>Chlamydia</jats:italic> is overcoming historic, confounding barriers that have impeded rapid progress in understanding overall chlamydial pathogenesis. The current state of genetic manipulation in <jats:italic>Chlamydia</jats:italic> requires further development, including mechanisms to generate marker-less gene disruption. We leveraged a step-wise Cre-lox approach to excise selection marker genes from a deleted gene locus. We found this process to be efficient and removal of extraneous elements resulted in reversal of a negative polar effect on a down-stream gene. This technique facilitates a more direct assessment of gene function and adds to the <jats:italic>Chlamydia</jats:italic> molecular toolbox by facilitating deletion of genes within operons.
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