{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T16:48:34Z","timestamp":1790095714111,"version":"4.0.1"},"reference-count":33,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2021,4,13]],"date-time":"2021-04-13T00:00:00Z","timestamp":1618272000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"crossref","award":["202755"],"award-info":[{"award-number":["202755"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["wellcomeopenresearch.org"],"crossmark-restriction":false},"short-container-title":["Wellcome Open Res"],"abstract":"<ns4:p>\n                    <ns4:bold>Background<\/ns4:bold>\n                  <\/ns4:p>\n                  <ns4:p>\n                    Genetic systems have been developed for\n                    <ns4:italic>Chlamydia<\/ns4:italic>\n                    but the extremely low transformation frequency remains a significant bottleneck.\u00a0 Our goal is to develop a self-replicating transposon delivery vector for\n                    <ns4:italic>C. trachomatis<\/ns4:italic>\n                    which can be expanded prior to transposase induction.\n                  <\/ns4:p>\n                  <ns4:p\/>\n                  <ns4:p>\n                    <ns4:bold>Methods<\/ns4:bold>\n                  <\/ns4:p>\n                  <ns4:p>\n                    We made\n                    <ns4:italic>E. coli<\/ns4:italic>\n                    \/\n                    <ns4:italic>C. trachomatis<\/ns4:italic>\n                    shuttle vectors bearing the\n                    <ns4:italic>Himar1<\/ns4:italic>\n                    C9\u00a0 transposase under control of the\n                    <ns4:italic>tet<\/ns4:italic>\n                    promoter and a novel rearrangement of the\n                    <ns4:italic>Himar1<\/ns4:italic>\n                    transposon with the \u03b2-lactamase gene.\u00a0 Activity of the transposase was monitored by immunoblot and by DNA sequencing.\n                  <\/ns4:p>\n                  <ns4:p\/>\n                  <ns4:p>\n                    <ns4:bold>Results<\/ns4:bold>\n                  <\/ns4:p>\n                  <ns4:p>\n                    We constructed pSW2-mCh-C9, a\n                    <ns4:italic>C. trachomatis<\/ns4:italic>\n                    plasmid designed to act as a self-replicating vector carrying both the\n                    <ns4:italic>Himar1<\/ns4:italic>\n                    C9\u00a0 transposase under\n                    <ns4:italic>tet<\/ns4:italic>\n                    promoter control and its transposon.\u00a0 However, we were unable to recover this plasmid in\n                    <ns4:italic>C. trachomatis<\/ns4:italic>\n                    following multiple attempts at transformation.\n                  <\/ns4:p>\n                  <ns4:p>\n                    Therefore, we assembled two new deletion plasmids pSW2-mCh-C9-\u0394Tpon carrying only the\n                    <ns4:italic>Himar1<\/ns4:italic>\n                    C9\u00a0 transposase (under\n                    <ns4:italic>tet<\/ns4:italic>\n                    promoter control) and a sister vector (same sequence backbone) pSW2-mCh-C9-\u0394Tpase carrying its cognate transposon.\u00a0 We demonstrated that the biological components that make up both pSW2-mCh-C9-\u0394Tpon and pSW2-mCh-C9-\u0394Tpase are active in\n                    <ns4:italic>E. coli.\u00a0<\/ns4:italic>\n                    Both these plasmids could be independently recovered in\n                    <ns4:italic>C. trachomatis.<\/ns4:italic>\n                  <\/ns4:p>\n                  <ns4:p>\n                    We attempted to perform lateral gene transfer by transformation and mixed infection with\n                    <ns4:italic>C. trachomatis<\/ns4:italic>\n                    strains bearing\n                    <ns4:italic\/>\n                    pSW2-mCh-C9-\u0394Tpon and pSW2-RSGFP-Tpon\n                    <ns4:italic\/>\n                    (a green fluorescent version of\n                    <ns4:italic\/>\n                    pSW2-mCh-C9-\u0394Tpase).\u00a0 Despite success in achieving mixed infections, it was not possible to recover progeny bearing both versions of these plasmids.\n                  <\/ns4:p>\n                  <ns4:p\/>\n                  <ns4:p>\n                    <ns4:bold>Conclusions<\/ns4:bold>\n                  <\/ns4:p>\n                  <ns4:p>\n                    We have designed a self-replicating plasmid vector pSW2-mCh-C9 for\n                    <ns4:italic>C. trachomatis<\/ns4:italic>\n                    carrying the\n                    <ns4:italic>Himar1<\/ns4:italic>\n                    C9\u00a0 transposase under\n                    <ns4:italic>tet<\/ns4:italic>\n                    promoter control.\u00a0 Whilst this can be transformed into\n                    <ns4:italic>E. coli<\/ns4:italic>\n                    it cannot be recovered in\n                    <ns4:italic>C. trachomatis.<\/ns4:italic>\n                    \u00a0Based on selected deletions and phenotypic analyses we conclude that low level expression from the\n                    <ns4:italic>tet<\/ns4:italic>\n                    inducible promoter is responsible for premature transposition and hence plasmid loss early on in the transformation process.\n                  <\/ns4:p>","DOI":"10.12688\/wellcomeopenres.16665.1","type":"journal-article","created":{"date-parts":[[2021,4,13]],"date-time":"2021-04-13T08:22:14Z","timestamp":1618302134000},"page":"82","update-policy":"https:\/\/doi.org\/10.12688\/wellcomeopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":7,"title":["Progress towards an inducible, replication-proficient transposon delivery vector for Chlamydia trachomatis"],"prefix":"10.12688","volume":"6","author":[{"given":"Rachel J.","family":"Skilton","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Colette","family":"O'Neill","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas R.","family":"Thomson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David J.","family":"Lampe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4938-1620","authenticated-orcid":false,"given":"Ian N.","family":"Clarke","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2021,4,13]]},"reference":[{"key":"ref-1","doi-asserted-by":"publisher","first-page":"673-91","DOI":"10.1111\/j.1365-3156.2010.02521.x","article-title":"Epidemiology and control of trachoma: systematic review.","volume":"15","author":"V Hu","year":"2010","journal-title":"Trop Med Int Health."},{"key":"ref-2","doi-asserted-by":"publisher","first-page":"e0143304","DOI":"10.1371\/journal.pone.0143304","article-title":"Global Estimates of the Prevalence and Incidence of Four Curable Sexually Transmitted Infections in 2012 Based on Systematic Review and Global Reporting.","volume":"10","author":"L Newman","year":"2015","journal-title":"PLoS One."},{"key":"ref-3","doi-asserted-by":"publisher","first-page":"109-115","DOI":"10.1093\/oxfordjournals.bmb.a071800","article-title":"Chlamydial classification, development and 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status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/wellcomeopenres.18376.r43469, Isabelle Derr\u00e9, Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA, 16 Apr 2021, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-82\/v1#referee-response-43469","order":0,"name":"referee-response-43469","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.18376.r43470, Ted Hackstadt, Zoe Dimond, Host-Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, NIAID, National Institutes of Health\u00a0(NIH), Hamilton, MT, USA, 04 May 2021, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-82\/v1#referee-response-43470","order":1,"name":"referee-response-43470","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was funded by Wellcome Trust grant number 202755\/Z\/16\/Z entitled \u2018Saturation transposon mutagenesis of Chlamydia trachomatis\u2019. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":2,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Data associated with the article are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).","order":0,"name":"copyright-info","label":"Copyright"}]}}