{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T19:44:34Z","timestamp":1768506274592,"version":"3.49.0"},"posted":{"date-parts":[[2023,3,14]]},"group-title":"Microbiology","reference-count":64,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,3,14]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  <jats:italic>Chlamydia trachomatis<\/jats:italic>\n                  is an obligate intracellular bacterium that is responsible for the most prevalent bacterial sexually transmitted infections. Changes in DNA topology in this pathogen have been linked to its pathogenicity-associated developmental cycle. Here, evidence is provided that the balanced activity of DNA topoisomerases (Topos) contributes to\n                  <jats:italic>Chlamydia<\/jats:italic>\n                  developmental processes. Utilizing catalytically inactivated Cas12 (dCas12) based-clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology, we demonstrate targeted knockdown of chromosomal\n                  <jats:italic>topA<\/jats:italic>\n                  transcription in\n                  <jats:italic>C. trachomatis<\/jats:italic>\n                  without detected toxicity of dCas12. Repression of\n                  <jats:italic>topA<\/jats:italic>\n                  impaired the growth of\n                  <jats:italic>C. trachomatis<\/jats:italic>\n                  mostly through disruption of its differentiation from a replicative form to an infectious form. Consistent with this, expression of late developmental genes of\n                  <jats:italic>C. trachomatis<\/jats:italic>\n                  was downregulated while early genes maintained their expression. Importantly, the growth defect associated with\n                  <jats:italic>topA<\/jats:italic>\n                  knockdown was rescued by overexpressing\n                  <jats:italic>topA<\/jats:italic>\n                  at an appropriate degree and time, directly linking the growth patterns to the levels of\n                  <jats:italic>topA<\/jats:italic>\n                  expression. Interestingly,\n                  <jats:italic>topA<\/jats:italic>\n                  knockdown had pleiotropic effects on DNA gyrase expression, indicating a potential compensatory mechanism for survival to offset TopA deficiency.\n                  <jats:italic>C. trachomatis<\/jats:italic>\n                  with\n                  <jats:italic>topA<\/jats:italic>\n                  knocked down displayed hypersensitivity to moxifloxacin that targets DNA gyrase in comparison with the wild type. These data underscore the requirement of integrated topoisomerase actions to support the essential development and transcriptional processes of\n                  <jats:italic>C. trachomatis<\/jats:italic>\n                  .\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>\n                    We used genetic and chemical tools to demonstrate the relationship of topoisomerase activities and their obligatory role for the chlamydial developmental cycle. Successfully targeting the essential gene\n                    <jats:italic>topA<\/jats:italic>\n                    with a CRISPRi approach, using dCas12, in\n                    <jats:italic>C. trachomatis<\/jats:italic>\n                    indicates that this method will facilitate the characterization of the essential genome. These findings have an important impact on our understanding of the mechanism(s) by which well-balanced topoisomerase activities enable\n                    <jats:italic>C. trachomatis<\/jats:italic>\n                    to adapt to unfavorable growth conditions imposed by antibiotics.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2023.03.14.532001","type":"posted-content","created":{"date-parts":[[2023,3,15]],"date-time":"2023-03-15T00:10:16Z","timestamp":1678839016000},"source":"Crossref","is-referenced-by-count":0,"title":["Targeted repression of DNA topoisomerase I by CRISPRi reveals a critical function for it in the\n                  <i>Chlamydia trachomatis<\/i>\n                  developmental cycle"],"prefix":"10.64898","author":[{"given":"Li","family":"Shen","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leiqiong","family":"Gao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Abigail R.","family":"Swoboda","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Scot P.","family":"Ouellette","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2023031714350427000_2023.03.14.532001v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/nrm831"},{"key":"2023031714350427000_2023.03.14.532001v1.2","doi-asserted-by":"publisher","DOI":"10.1007\/s12551-016-0238-2"},{"key":"2023031714350427000_2023.03.14.532001v1.3","first-page":"69","article-title":"Mechanistic aspects of DNA topoisomerases","volume":"8","year":"1986","journal-title":"Adv Protein Chem"},{"key":"2023031714350427000_2023.03.14.532001v1.4","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-061809-100002"},{"key":"2023031714350427000_2023.03.14.532001v1.5","first-page":"105","article-title":"Regulation of the genes for E","volume":"34","year":"1983","journal-title":"coli DNA gyrase: homeostatic control of DNA supercoiling. 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