{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T06:05:14Z","timestamp":1791266714411,"version":"4.1.0"},"reference-count":48,"publisher":"American Society for Microbiology","issue":"13","license":[{"start":{"date-parts":[[2012,7,1]],"date-time":"2012-07-01T00:00:00Z","timestamp":1341100800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2012,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            <jats:named-content content-type=\"genus-species\">Coxiella burnetii<\/jats:named-content>\n            is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness.\n            <jats:named-content content-type=\"genus-species\">C. burnetii<\/jats:named-content>\n            's former obligate intracellular nature significantly impeded the genetic characterization of putative virulence factors. However, recent host cell-free (axenic) growth of the organism has enabled development of shuttle vector, transposon, and inducible gene expression technologies, with targeted gene inactivation remaining an important challenge. In the present study, we describe two methods for generating targeted gene deletions in\n            <jats:named-content content-type=\"genus-species\">C. burnetii<\/jats:named-content>\n            that exploit pUC\/ColE1\n            <jats:italic>ori<\/jats:italic>\n            -based suicide plasmids encoding\n            <jats:italic>sacB<\/jats:italic>\n            for positive selection of mutants. As proof of concept,\n            <jats:named-content content-type=\"genus-species\">C. burnetii<\/jats:named-content>\n            dotA and\n            <jats:italic>dotB<\/jats:italic>\n            , encoding structural components of the type IVB secretion system (T4BSS), were selected for deletion. The first method exploited Cre-\n            <jats:italic>lox-<\/jats:italic>\n            mediated recombination. Two suicide plasmids carrying different antibiotic resistance markers and a\n            <jats:italic>loxP<\/jats:italic>\n            site were integrated into 5\u2032 and 3\u2032 flanking regions of\n            <jats:italic>dotA<\/jats:italic>\n            . Transformation of this strain with a third suicide plasmid encoding Cre recombinase resulted in the deletion of\n            <jats:italic>dotA<\/jats:italic>\n            under sucrose counterselection. The second method utilized a loop-in\/loop-out strategy to delete\n            <jats:italic>dotA<\/jats:italic>\n            and\n            <jats:italic>dotB.<\/jats:italic>\n            A single suicide plasmid was first integrated into 5\u2032 or 3\u2032 target gene flanking regions. Resolution of the plasmid cointegrant by a second crossover event under sucrose counterselection resulted in gene deletion that was confirmed by PCR and Southern blot. \u0394\n            <jats:italic>dotA<\/jats:italic>\n            and \u0394\n            <jats:italic>dotB<\/jats:italic>\n            mutants failed to secrete T4BSS substrates and to productively infect host cells. The repertoire of\n            <jats:named-content content-type=\"genus-species\">C. burnetii<\/jats:named-content>\n            genetic tools now allows ready fulfillment of molecular Koch's postulates for suspected virulence genes.\n          <\/jats:p>","DOI":"10.1128\/aem.00881-12","type":"journal-article","created":{"date-parts":[[2012,4,21]],"date-time":"2012-04-21T02:55:58Z","timestamp":1334976958000},"page":"4580-4589","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":105,"title":["Two Systems for Targeted Gene Deletion in Coxiella burnetii"],"prefix":"10.1128","volume":"78","author":[{"given":"Paul A.","family":"Beare","sequence":"first","affiliation":[{"name":"Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charles L.","family":"Larson","sequence":"additional","affiliation":[{"name":"Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stacey D.","family":"Gilk","sequence":"additional","affiliation":[{"name":"Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert A.","family":"Heinzen","sequence":"additional","affiliation":[{"name":"Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, 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