Floxed-cassette allelic exchange mutagenesis enables marker-less gene deletion in Chlamydia trachomatis and can reverse cassette-induced polar effects release_auihou5a2nbm5na2cxdl7bcqte

by G. Keb, R. Hayman, K.A. Fields

Published in Journal of Bacteriology by American Society for Microbiology.



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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Type  article-journal
Stage   published
Date   2018-09-17
Language   en ?
DOI  10.1128/jb.00479-18
PubMed  30224436
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ISSN-L:  0021-9193
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