{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,6,4]],"date-time":"2025-06-04T10:31:06Z","timestamp":1749033066230},"reference-count":56,"publisher":"American Society for Microbiology","issue":"3","license":[{"start":{"date-parts":[[2015,2,1]],"date-time":"2015-02-01T00:00:00Z","timestamp":1422748800000},"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":[[2015,2]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            Targeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirochete\n            <jats:named-content content-type=\"genus-species\">Borrelia burgdorferi<\/jats:named-content>\n            . A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers than\n            <jats:named-content content-type=\"genus-species\">B. burgdorferi<\/jats:named-content>\n            plasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.\n            <jats:named-content content-type=\"genus-species\">B. burgdorferi<\/jats:named-content>\n            has over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochete\n            <jats:italic>in vivo<\/jats:italic>\n            but relatively unstable during\n            <jats:italic>in vitro<\/jats:italic>\n            cultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number and\n            <jats:italic>in vivo<\/jats:italic>\n            stability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into the\n            <jats:italic>bbe02<\/jats:italic>\n            locus, a site on lp25 that was previously shown to be nonessential during both\n            <jats:italic>in vitro<\/jats:italic>\n            and\n            <jats:italic>in vivo<\/jats:italic>\n            growth. We demonstrate the functional utility of this strategy by restoring infectivity to an\n            <jats:italic>ospC<\/jats:italic>\n            mutant through complementation at this site on lp25 and stable maintenance of the\n            <jats:italic>ospC<\/jats:italic>\n            gene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation in\n            <jats:named-content content-type=\"genus-species\">B. burgdorferi<\/jats:named-content>\n            .\n          <\/jats:p>","DOI":"10.1128\/aem.03657-14","type":"journal-article","created":{"date-parts":[[2014,12,2]],"date-time":"2014-12-02T04:19:53Z","timestamp":1417493993000},"page":"1038-1046","update-policy":"http:\/\/dx.doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":12,"title":["Use of an Endogenous Plasmid Locus for Stable in\n            <i>trans<\/i>\n            Complementation in Borrelia burgdorferi"],"prefix":"10.1128","volume":"81","author":[{"given":"Irene N.","family":"Kasumba","sequence":"first","affiliation":[{"name":"Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aaron","family":"Bestor","sequence":"additional","affiliation":[{"name":"Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kit","family":"Tilly","sequence":"additional","affiliation":[{"name":"Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patricia A.","family":"Rosa","sequence":"additional","affiliation":[{"name":"Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.7043737"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.182.9.2445-2452.2000"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1159\/000073406"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.185.22.6723-6727.2003"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.178.20.5946-5953.1996"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1099\/00221287-143-2-519"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.176.10.3072-3075.1994"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.080068797"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.183.22.6558-6564.2001"},{"key":"e_1_3_2_11_2","first-page":"291","volume-title":"Plasmid biology","author":"Stewart PE","year":"2004","unstructured":"Stewart PE, Rosa PA, Tilly K. 2004. 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