{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T12:59:27Z","timestamp":1792673967371,"version":"4.5.0"},"reference-count":24,"publisher":"National Academy of Sciences","issue":"17","content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2011,4,26]]},"abstract":"<jats:p>\n                    <jats:italic>Chlamydia trachomatis<\/jats:italic>\n                    is an obligate intracellular bacterial pathogen that infects hundreds of millions of individuals globally, causing blinding trachoma and sexually transmitted disease. More effective chlamydial control measures are needed, but progress toward this end has been severely hampered by the lack of a tenable chlamydial genetic system. Here, we describe a reverse-genetic approach to create isogenic\n                    <jats:italic>C. trachomatis<\/jats:italic>\n                    mutants.\n                    <jats:italic>C. trachomatis<\/jats:italic>\n                    was subjected to low-level ethyl methanesulfonate mutagenesis to generate chlamydiae that contained less then one mutation per genome. Mutagenized organisms were expanded in small subpopulations that were screened for mutations by digesting denatured and reannealed PCR amplicons of the target gene with the mismatch specific endonuclease CEL I. Subpopulations with mutations were then sequenced for the target region and plaque-cloned if the desired mutation was detected. We demonstrate the utility of this approach by isolating a tryptophan synthase gene (\n                    <jats:italic>trpB<\/jats:italic>\n                    ) null mutant that was otherwise isogenic to its parental clone as shown by de novo genome sequencing. The mutant was incapable of avoiding the anti-microbial effect of IFN-\u03b3\u2013induced tryptophan starvation. The ability to genetically manipulate chlamydiae is a major advancement that will enhance our understanding of chlamydial pathogenesis and accelerate the development of new anti-chlamydial therapeutic control measures. Additionally, this strategy could be applied to other medically important bacterial pathogens with no or difficult genetic systems.\n                  <\/jats:p>","DOI":"10.1073\/pnas.1102229108","type":"journal-article","created":{"date-parts":[[2011,4,12]],"date-time":"2011-04-12T06:00:50Z","timestamp":1302588050000},"page":"7189-7193","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":136,"title":["Generation of targeted\n                    <i>Chlamydia trachomatis<\/i>\n                    null mutants"],"prefix":"10.1073","volume":"108","author":[{"given":"Laszlo","family":"Kari","sequence":"first","affiliation":[{"name":"Laboratory of Intracellular Parasites, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Morgan M.","family":"Goheen","sequence":"additional","affiliation":[{"name":"Laboratory of Intracellular Parasites, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Linnell B.","family":"Randall","sequence":"additional","affiliation":[{"name":"Laboratory of Intracellular Parasites, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lacey D.","family":"Taylor","sequence":"additional","affiliation":[{"name":"Laboratory of Intracellular Parasites, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John H.","family":"Carlson","sequence":"additional","affiliation":[{"name":"Laboratory of Intracellular Parasites, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William M.","family":"Whitmire","sequence":"additional","affiliation":[{"name":"Laboratory of Intracellular Parasites, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dezso","family":"Virok","sequence":"additional","affiliation":[{"name":"Institute of Clinical Microbiology, University of Szeged, H-6725 Szeged, Hungary;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Krithika","family":"Rajaram","sequence":"additional","affiliation":[{"name":"Department of Biology, Indiana University, Bloomington, IN 47405;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valeria","family":"Endresz","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology and Immunobiology, University of Szeged, H-6720 Szeged, Hungary; and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Grant","family":"McClarty","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada R3T 2N2"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David E.","family":"Nelson","sequence":"additional","affiliation":[{"name":"Department of Biology, Indiana University, Bloomington, IN 47405;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Harlan D.","family":"Caldwell","sequence":"additional","affiliation":[{"name":"Laboratory of Intracellular Parasites, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840;"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2011,4,11]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"crossref","first-page":"428","DOI":"10.1056\/NEJM197802232980805","article-title":"Chlamydial infections (first of three parts)","volume":"298","author":"Schachter J","year":"1978","unstructured":"J Schachter, Chlamydial infections (first of three parts). N Engl J Med 298, 428\u2013435 (1978).","journal-title":"N Engl J Med"},{"key":"e_1_3_4_2_2","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1056\/NEJM197803092981005","article-title":"Chlamydial infections (third of three parts)","volume":"298","author":"Schachter J","year":"1978","unstructured":"J Schachter, Chlamydial infections (third of three parts). N Engl J Med 298, 540\u2013549 (1978).","journal-title":"N Engl J Med"},{"key":"e_1_3_4_3_2","doi-asserted-by":"crossref","first-page":"490","DOI":"10.1056\/NEJM197803022980905","article-title":"Chlamydial infections (second of three parts)","volume":"298","author":"Schachter J","year":"1978","unstructured":"J Schachter, Chlamydial infections (second of three parts). N Engl J Med 298, 490\u2013495 (1978).","journal-title":"N Engl J Med"},{"key":"e_1_3_4_4_2","doi-asserted-by":"crossref","first-page":"S10","DOI":"10.1007\/BF01640709","article-title":"The relation of basic biology to pathogenic potential in the genus Chlamydia","volume":"10","author":"Moulder JW","year":"1982","unstructured":"JW Moulder, The relation of basic biology to pathogenic potential in the genus Chlamydia. Infection 10, S10\u2013S18 (1982).","journal-title":"Infection"},{"key":"e_1_3_4_5_2","doi-asserted-by":"crossref","first-page":"4295","DOI":"10.1128\/JB.186.13.4295-4306.2004","article-title":"Recombination in the genome of Chlamydia trachomatis involving the polymorphic membrane protein C gene relative to ompA and evidence for horizontal gene transfer","volume":"186","author":"Gomes JP","year":"2004","unstructured":"JP Gomes, WJ Bruno, MJ Borrego, D Dean, Recombination in the genome of Chlamydia trachomatis involving the polymorphic membrane protein C gene relative to ompA and evidence for horizontal gene transfer. J Bacteriol 186, 4295\u20134306 (2004).","journal-title":"J Bacteriol"},{"key":"e_1_3_4_6_2","doi-asserted-by":"crossref","first-page":"991","DOI":"10.1128\/JB.00845-06","article-title":"Lateral gene transfer in vitro in the intracellular pathogen Chlamydia trachomatis","volume":"189","author":"DeMars R","year":"2007","unstructured":"R DeMars, J Weinfurter, E Guex, J Lin, Y Potucek, Lateral gene transfer in vitro in the intracellular pathogen Chlamydia trachomatis. J Bacteriol 189, 991\u20131003 (2007).","journal-title":"J Bacteriol"},{"key":"e_1_3_4_7_2","doi-asserted-by":"crossref","first-page":"4604","DOI":"10.1128\/AAC.00477-09","article-title":"Horizontal transfer of tetracycline resistance among Chlamydia spp. in vitro","volume":"53","author":"Suchland RJ","year":"2009","unstructured":"RJ Suchland, KM Sandoz, BM Jeffrey, WE Stamm, DD Rockey, Horizontal transfer of tetracycline resistance among Chlamydia spp. in vitro. 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Nucleic Acids Res 26, 4597\u20134602 (1998).","journal-title":"Nucleic Acids Res"},{"key":"e_1_3_4_12_2","volume-title":"Drosophila: A Laboratory Handbook","author":"Ashburner M","year":"1990","unstructured":"M Ashburner Drosophila: A Laboratory Handbook (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990)."},{"key":"e_1_3_4_13_2","doi-asserted-by":"crossref","first-page":"1605","DOI":"10.1128\/JB.01592-07","article-title":"Interstrain gene transfer in Chlamydia trachomatis in vitro: Mechanism and significance","volume":"190","author":"DeMars R","year":"2008","unstructured":"R DeMars, J Weinfurter, Interstrain gene transfer in Chlamydia trachomatis in vitro: Mechanism and significance. J Bacteriol 190, 1605\u20131614 (2008).","journal-title":"J Bacteriol"},{"key":"e_1_3_4_14_2","doi-asserted-by":"crossref","first-page":"2316","DOI":"10.1128\/AAC.47.7.2316-2318.2003","article-title":"Detection of nucleotide variability in rpoB in both rifampin-sensitive and rifampin-resistant strains of Chlamydia trachomatis","volume":"47","author":"Dreses-Werringloer U","year":"2003","unstructured":"U Dreses-Werringloer, I Padubrin, L K\u00f6hler, AP Hudson, Detection of nucleotide variability in rpoB in both rifampin-sensitive and rifampin-resistant strains of Chlamydia trachomatis. 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J Antimicrob Chemother 61, 91\u201394 (2008).","journal-title":"J Antimicrob Chemother"},{"key":"e_1_3_4_17_2","doi-asserted-by":"crossref","first-page":"1120","DOI":"10.1128\/AAC.49.3.1120-1126.2005","article-title":"Rifampin-resistant RNA polymerase mutants of Chlamydia trachomatis remain susceptible to the ansamycin rifalazil","volume":"49","author":"Suchland RJ","year":"2005","unstructured":"RJ Suchland, A Bourillon, E Denamur, WE Stamm, DM Rothstein, Rifampin-resistant RNA polymerase mutants of Chlamydia trachomatis remain susceptible to the ansamycin rifalazil. 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Infect Immun 73, 6407\u20136418 (2005).","journal-title":"Infect Immun"},{"key":"e_1_3_4_20_2","doi-asserted-by":"crossref","first-page":"1757","DOI":"10.1172\/JCI17993","article-title":"Polymorphisms in Chlamydia trachomatis tryptophan synthase genes differentiate between genital and ocular isolates","volume":"111","author":"Caldwell HD","year":"2003","unstructured":"HD Caldwell, et al., Polymorphisms in Chlamydia trachomatis tryptophan synthase genes differentiate between genital and ocular isolates. J Clin Invest 111, 1757\u20131769 (2003).","journal-title":"J Clin Invest"},{"key":"e_1_3_4_21_2","doi-asserted-by":"crossref","first-page":"26893","DOI":"10.1074\/jbc.M203937200","article-title":"Molecular basis defining human Chlamydia trachomatis tissue tropism. A possible role for tryptophan synthase","volume":"277","author":"Fehlner-Gardiner C","year":"2002","unstructured":"C Fehlner-Gardiner, et al., Molecular basis defining human Chlamydia trachomatis tissue tropism. A possible role for tryptophan synthase. J Biol Chem 277, 26893\u201326903 (2002).","journal-title":"J Biol Chem"},{"key":"e_1_3_4_22_2","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1086\/525285","article-title":"Pathogenic diversity among Chlamydia trachomatis ocular strains in nonhuman primates is affected by subtle genomic variations","volume":"197","author":"Kari L","year":"2008","unstructured":"L Kari, et al., Pathogenic diversity among Chlamydia trachomatis ocular strains in nonhuman primates is affected by subtle genomic variations. J Infect Dis 197, 449\u2013456 (2008).","journal-title":"J Infect Dis"},{"key":"e_1_3_4_23_2","doi-asserted-by":"crossref","first-page":"2273","DOI":"10.1128\/IAI.00102-08","article-title":"The Chlamydia trachomatis plasmid is a transcriptional regulator of chromosomal genes and a virulence factor","volume":"76","author":"Carlson JH","year":"2008","unstructured":"JH Carlson, et al., The Chlamydia trachomatis plasmid is a transcriptional regulator of chromosomal genes and a virulence factor. Infect Immun 76, 2273\u20132283 (2008).","journal-title":"Infect Immun"},{"key":"e_1_3_4_24_2","doi-asserted-by":"crossref","first-page":"3660","DOI":"10.1128\/IAI.00386-10","article-title":"Frameshift mutations in a single novel virulence factor alter the in vivo pathogenicity of Chlamydia trachomatis for the female murine genital tract","volume":"78","author":"Sturdevant GL","year":"2010","unstructured":"GL Sturdevant, et al., Frameshift mutations in a single novel virulence factor alter the in vivo pathogenicity of Chlamydia trachomatis for the female murine genital tract. Infect Immun 78, 3660\u20133668 (2010).","journal-title":"Infect Immun"}],"container-title":["Proceedings of the National Academy of Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pnas.org\/doi\/pdf\/10.1073\/pnas.1102229108","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,6,7]],"date-time":"2022-06-07T03:39:59Z","timestamp":1654573199000},"score":1,"resource":{"primary":{"URL":"https:\/\/pnas.org\/doi\/full\/10.1073\/pnas.1102229108"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2011,4,11]]},"references-count":24,"journal-issue":{"issue":"17","published-print":{"date-parts":[[2011,4,26]]}},"alternative-id":["10.1073\/pnas.1102229108"],"URL":"https:\/\/doi.org\/10.1073\/pnas.1102229108","relation":{},"ISSN":["0027-8424","1091-6490"],"issn-type":[{"value":"0027-8424","type":"print"},{"value":"1091-6490","type":"electronic"}],"subject":[],"published":{"date-parts":[[2011,4,11]]},"assertion":[{"value":"2011-04-11","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}