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Data from: A platform supporting generation and isolation of random transposon mutants in Chlamydia trachomatis

Data files

Jan 10, 2025 version files 1.99 MB

Abstract

Chlamydia species represent a paradigm for understanding successful obligate intracellular parasitism. Despite limited genetic malleability, development of genetic tools has facilitated the elucidation of molecular mechanisms governing infectivity. Random mutagenesis approaches provide one of the most powerful strategies available to accomplish untargeted elucidation of gene function. Unfortunately, initial progress in transposon-mediated mutagenesis of Chlamydia has been challenging. To increase efficiency, we developed a plasmid-based system that couples conditional plasmid maintenance with a previously described strategy leveraging inducible expression of the Himar1-derived C9 transposase. Our pOri-Tn(Q) construct was maintained in C. trachomatis cultivated with antibiotics but was rapidly cured in the absence of antibiotic selection. pOri-Tn(Q) supported transposition events when transposase expression was induced during infection. Induction was accompanied by loss of the plasmid backbone when Penicillin G was used to select for only the transposable element. C9 induction during iterative passaging was used to increase overall insertion frequency and accumulate an expanded pool of transposon mutants. The approach supported isolation of individual mutant strains from the mixed pool, and whole-genome sequencing confirmed that the recovered strains harbored single insertions.