{"abstracts":[{"sha1":"40194629b8bb55199b5a0ccbb767765c6b5a7562","content":"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 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 C. trahomatis tmeA negatively impacts expression of tmeB. We have adapted FRAEM technology by employing a gfp-bla cassette flanked by loxP sites. Conditional expression of Cre recombinase in tmeA Chlamydia containing a floxed cassette resulted in deletion of the marker and restoration of tmeB expression.\nIMPORTANCE C. trachomatis infections represent a significant burden to human health. The ability to genetically manipulate Chlamydia is overcoming historic, confounding barriers that have impeded rapid progress in understanding overall chlamydial pathogenesis. The current state of genetic manipulation in Chlamydia 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 Chlamydia molecular toolbox by facilitating deletion of genes within operons.","mimetype":"application/xml+jats"}],"refs":[],"contribs":[{"index":0,"raw_name":"G. Keb","role":"author","extra":{"seq":"first"}},{"index":1,"raw_name":"R. Hayman","role":"author"},{"index":2,"raw_name":"K.A. Fields","role":"author"}],"language":"en","publisher":"American Society for Microbiology","ext_ids":{"doi":"10.1128/jb.00479-18","pmid":"30224436"},"release_year":2018,"release_date":"2018-09-17","release_stage":"published","release_type":"article-journal","container_id":"fu4cl5wxynclfn3telu2mfbrna","webcaptures":[],"filesets":[],"files":[{"release_ids":["auihou5a2nbm5na2cxdl7bcqte"],"mimetype":"application/pdf","urls":[],"sha256":"ad108fea77f163efbfa48d8ce2f26b3fcd7d2a075de8ac7fa7e7cbdf97f76381","sha1":"7e141b1398e0bc66537da042d56e130c6b4fbe4d","md5":"ffd35fc48e3a151c998b276f5718647f","size":1267041,"extra":{"shadows":{"scimag_doi":"10.1128/JB.00479-18","scimag_id":"72025571"}},"revision":"e736a7ae-d397-4d55-9516-81b814f5afcc","ident":"fgzjcf4btndjjjqxfcyyq7huc4","state":"active"},{"release_ids":["auihou5a2nbm5na2cxdl7bcqte"],"mimetype":"application/pdf","urls":[{"url":"https://uknowledge.uky.edu/cgi/viewcontent.cgi?article=1121&context=microbio_facpub","rel":"web"},{"url":"https://web.archive.org/web/20200307172019/https://uknowledge.uky.edu/cgi/viewcontent.cgi?article=1121&context=microbio_facpub","rel":"webarchive"}],"sha256":"f182df8e3514a792fd6a5e208190d8e842488fc7c666d3bf5e07343278ebd058","sha1":"9c6ebb0438b514c0b4c167ef3a4fb7baa5d4055e","md5":"3827726fba9c9305307f4dec0d0472d0","size":1075003,"revision":"190aa424-ff00-4c0c-9bc7-bb276835114e","ident":"dyumcb4a7va45kgsio4wfavvmy","state":"active"},{"release_ids":["auihou5a2nbm5na2cxdl7bcqte"],"mimetype":"application/pdf","urls":[{"url":"https://web.archive.org/web/20190502105556/https://jb.asm.org/content/jb/200/24/e00479-18.full.pdf","rel":"webarchive"},{"url":"https://jb.asm.org/content/jb/200/24/e00479-18.full.pdf","rel":"web"}],"sha256":"6d200aa61bc3b46fb2e920801567be359cf74ba86c558c1bcfa610b1355bd34e","sha1":"b82042bea41fb20c312e8609463a932ea6040a2d","md5":"25c355725c6832e19108262effdeec44","size":974263,"revision":"8a51d8bc-a3a8-49b6-8653-25f7de426bdc","ident":"5x5hfmznhzchlfi3qrwrzeq2ni","state":"active"}],"container":{"wikidata_qid":"Q478419","issnp":"0021-9193","issne":"1098-5530","issnl":"0021-9193","publisher":"American Society for Microbiology","container_type":"journal","name":"Journal of Bacteriology","extra":{"abbrev":"J. Bacteriol.","country":"us","ia":{"sim":{"peer_reviewed":true,"pub_type":"Scholarly Journals","scholarly_peer_reviewed":true,"sim_pubid":"112","year_spans":[[1916,2015]]}},"kbart":{"hathitrust":{"year_spans":[[1916,1923],[1936,1949],[1955,1955],[1957,1957],[1974,1974]]},"lockss":{"year_spans":[[1916,2021]]}},"languages":["en"],"publisher_type":"society","sherpa_romeo":{"color":"green"},"urls":["https://jb.asm.org/"]},"revision":"3bb13a16-2002-4c43-983c-b5999a523559","ident":"fu4cl5wxynclfn3telu2mfbrna","state":"active"},"work_id":"cex6qy3duvarzj5gvjztpenl3a","title":"Floxed-cassette allelic exchange mutagenesis enables marker-less gene deletion in Chlamydia trachomatis and can reverse cassette-induced polar effects","state":"active","ident":"auihou5a2nbm5na2cxdl7bcqte","revision":"3fe55c47-c34d-49fb-8310-2370a964ac88","extra":{"crossref":{"alternative-id":["10.1128/JB.00479-18"],"subject":["Molecular Biology","Microbiology"],"type":"journal-article"},"pubmed":{"pub_types":["Journal Article"]}}}