{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T16:48:31Z","timestamp":1790095711999,"version":"4.0.1"},"reference-count":40,"publisher":"Public Library of Science (PLoS)","issue":"11","license":[{"start":{"date-parts":[[2024,11,14]],"date-time":"2024-11-14T00:00:00Z","timestamp":1731542400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100015691","name":"Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01AI126785"],"award-info":[{"award-number":["R01AI126785"]}],"id":[{"id":"10.13039\/100015691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>The ability to efficiently target loci in the <jats:italic>Chlamydia trachomatis<\/jats:italic> genome for deletion remains a desirable goal in the field and new strategies need to be developed and refined. Here we describe the development and application of a lambda red recombineering system for <jats:italic>Chlamydia<\/jats:italic>. Using a non-replicative plasmid encoding key lambda Red components and targeting sequences, we demonstrate the efficient deletion of numerous gene targets in the model chlamydial strains <jats:italic>C<\/jats:italic>. <jats:italic>trachomatis<\/jats:italic> L2\/434 and <jats:italic>C<\/jats:italic>. <jats:italic>muridarum<\/jats:italic>. For initial development of the system, we targeted the <jats:italic>incA<\/jats:italic> gene of <jats:italic>C<\/jats:italic>. <jats:italic>trachomatis<\/jats:italic> for deletion. Deletion mutants containing a chloramphenicol resistance marker were recovered within 3 rounds of selection, or 2 rounds of passaging, in McCoy cells and the resulting clones (CT\u0394<jats:italic>incA<\/jats:italic>) were verified by PCR-based genotyping and whole genome sequencing. Phenotypic assessment of host cells infected with CT\u0394<jats:italic>incA<\/jats:italic> was performed by immunofluorescence microscopy and confirmed the lack of IncA expression and the uniform presence of nonfusogenic vacuoles (inclusions) across CT\u0394<jats:italic>incA<\/jats:italic>-infected monolayers. To explore the utility of this system, we deleted 5 additional candidate virulence factors in <jats:italic>C<\/jats:italic>. <jats:italic>trachomatis<\/jats:italic> and <jats:italic>C<\/jats:italic>. <jats:italic>muridarum<\/jats:italic>, including deletions of single and multiple genes. We expect lambda Red recombineering to offer a powerful new strategy for making gene deletion and\/or replacement mutants in <jats:italic>Chlamydia<\/jats:italic>.<\/jats:p>","DOI":"10.1371\/journal.pone.0311630","type":"journal-article","created":{"date-parts":[[2024,11,14]],"date-time":"2024-11-14T18:23:08Z","timestamp":1731608588000},"page":"e0311630","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":6,"title":["Development of a lambda Red based system for gene deletion in Chlamydia"],"prefix":"10.1371","volume":"19","author":[{"given":"Yibing","family":"Wang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert","family":"Suchland","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amy","family":"Hua","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven","family":"Carrell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Rockey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2967-3079","authenticated-orcid":true,"given":"Kevin","family":"Hybiske","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2024,11,14]]},"reference":[{"issue":"4","key":"pone.0311630.ref001","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1016\/j.joco.2016.08.011","article-title":"Trachoma: Past, present and future","volume":"28","author":"M Mohammadpour","year":"2016","journal-title":"J Curr Ophthalmol"},{"issue":"4","key":"pone.0311630.ref002","doi-asserted-by":"crossref","first-page":"222","DOI":"10.1097\/OLQ.0000000000001382","article-title":"Estimates of the Prevalence and Incidence of Chlamydia and Gonorrhea Among US Men and Women, 2018","volume":"48","author":"KM Kreisel","year":"2021","journal-title":"Sex Transm Dis"},{"issue":"2","key":"pone.0311630.ref003","first-page":"61","article-title":"Epidemiological synergy. 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Bacteriol"},{"issue":"6","key":"pone.0311630.ref016","doi-asserted-by":"crossref","first-page":"2544","DOI":"10.1128\/IAI.01324-09","article-title":"Genome sequencing of recent clinical Chlamydia trachomatis strains identifies loci associated with tissue tropism and regions of apparent recombination","volume":"78","author":"BM Jeffrey","year":"2010","journal-title":"Infect Immun"},{"issue":"11","key":"pone.0311630.ref017","doi-asserted-by":"crossref","first-page":"e0026522","DOI":"10.1128\/iai.00265-22","article-title":"Genomic Analysis of MSM Rectal Chlamydia trachomatis Isolates Identifies Predicted Tissue-Tropic Lineages Generated by Intraspecies Lateral Gene Transfer-Mediated Evolution","volume":"90","author":"RJ Suchland","year":"2022","journal-title":"Infect Immun"},{"issue":"8","key":"pone.0311630.ref018","doi-asserted-by":"crossref","first-page":"2063","DOI":"10.1128\/JB.180.8.2063-2071.1998","article-title":"Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli","volume":"180","author":"KC Murphy","year":"1998","journal-title":"J Bacteriol"},{"issue":"11","key":"pone.0311630.ref019","doi-asserted-by":"crossref","first-page":"5978","DOI":"10.1073\/pnas.100127597","article-title":"An efficient recombination system for chromosome engineering in Escherichia coli","volume":"97","author":"D Yu","year":"2000","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"12","key":"pone.0311630.ref020","doi-asserted-by":"crossref","first-page":"6640","DOI":"10.1073\/pnas.120163297","article-title":"One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products","volume":"97","author":"KA Datsenko","year":"2000","journal-title":"Proc Natl Acad Sci U S A"},{"key":"pone.0311630.ref021","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1186\/1471-2199-9-20","article-title":"Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa","volume":"9","author":"B Lesic","year":"2008","journal-title":"BMC Mol Biol"},{"key":"pone.0311630.ref022","doi-asserted-by":"crossref","unstructured":"Savage PJ, Leong JM, Murphy KC. Rapid allelic exchange in enterohemorrhagic Escherichia coli (EHEC) and other E. coli using lambda red recombination. 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Herod","year":"2022","journal-title":"Methods Mol Biol"},{"key":"pone.0311630.ref026","doi-asserted-by":"crossref","first-page":"2006.0008","DOI":"10.1038\/msb4100050","article-title":"Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection","volume":"2","author":"T Baba","year":"2006","journal-title":"Mol Syst Biol"},{"issue":"1","key":"pone.0311630.ref027","doi-asserted-by":"crossref","DOI":"10.1128\/ecosalplus.esp-0011-2015","article-title":"\u03bb Recombination and Recombineering","volume":"7","author":"KC Murphy","year":"2016","journal-title":"EcoSal Plus"},{"key":"pone.0311630.ref028","doi-asserted-by":"crossref","first-page":"2307","DOI":"10.3389\/fmicb.2018.02307","article-title":"A Multiplex Genome Editing Method for Escherichia coli Based on CRISPR-Cas12a","volume":"9","author":"X Ao","year":"2018","journal-title":"Front 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apparatus in Chlamydia trachomatis","volume":"24","author":"ML Barta","year":"2015","journal-title":"Protein Sci"},{"key":"pone.0311630.ref032","doi-asserted-by":"crossref","first-page":"e21465","DOI":"10.7554\/eLife.21465","article-title":"Chlamydia trachomatis-containing vacuole serves as deubiquitination platform to stabilize Mcl-1 and to interfere with host defense","volume":"6","author":"A Fischer","year":"2017","journal-title":"Elife"},{"issue":"1","key":"pone.0311630.ref033","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1080\/21505594.2015.1111509","article-title":"Chlamydial polymorphic membrane proteins: regulation, function and potential vaccine candidates","volume":"7","author":"S Vasilevsky","year":"2016","journal-title":"Virulence"},{"issue":"6","key":"pone.0311630.ref034","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1038\/nrmicro.2016.30","article-title":"Chlamydia cell biology and pathogenesis","volume":"14","author":"C Elwell","year":"2016","journal-title":"Nat Rev Microbiol"},{"issue":"17","key":"pone.0311630.ref035","doi-asserted-by":"crossref","first-page":"7189","DOI":"10.1073\/pnas.1102229108","article-title":"Generation of targeted Chlamydia trachomatis null mutants","volume":"108","author":"L Kari","year":"2011","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"4","key":"pone.0311630.ref036","doi-asserted-by":"crossref","first-page":"1263","DOI":"10.1073\/pnas.1117884109","article-title":"Virulence determinants in the obligate intracellular pathogen Chlamydia trachomatis revealed by forward genetic approaches","volume":"109","author":"BD Nguyen","year":"2012","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"5","key":"pone.0311630.ref037","doi-asserted-by":"crossref","first-page":"716","DOI":"10.1016\/j.chom.2015.03.014","article-title":"Integrating chemical mutagenesis and whole-genome sequencing as a platform for forward and reverse genetic analysis of 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