{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T09:06:59Z","timestamp":1790845619104,"version":"4.1.0"},"reference-count":36,"publisher":"American Society for Microbiology","issue":"20","license":[{"start":{"date-parts":[[2009,10,15]],"date-time":"2009-10-15T00:00:00Z","timestamp":1255564800000},"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":[[2009,10,15]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Burkholderia pseudomallei<\/jats:italic>\n                    is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for\n                    <jats:italic>B. pseudomallei<\/jats:italic>\n                    based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a\n                    <jats:italic>lacZ<\/jats:italic>\n                    \u03b1 gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed\n                    <jats:italic>gusA<\/jats:italic>\n                    indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or\n                    <jats:italic>sacB-<\/jats:italic>\n                    based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an\n                    <jats:italic>araC<\/jats:italic>\n                    -\n                    <jats:italic>\n                      P\n                      <jats:sub>BAD<\/jats:sub>\n                      -I\n                    <\/jats:italic>\n                    -\n                    <jats:italic>sceI<\/jats:italic>\n                    expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved \u0394\n                    <jats:italic>asd Escherichia coli<\/jats:italic>\n                    mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI- and\n                    <jats:italic>sacB<\/jats:italic>\n                    -based methods afforded by pEXKm5 in conjunction with\n                    <jats:italic>E. coli<\/jats:italic>\n                    RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory\n                    <jats:italic>B. pseudomallei<\/jats:italic>\n                    strains. Finally,\n                    <jats:italic>sacB<\/jats:italic>\n                    -based counterselection was employed to isolate a defined chromosomal\n                    <jats:italic>fabD<\/jats:italic>\n                    (Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme.\n                  <\/jats:p>","DOI":"10.1128\/aem.01669-09","type":"journal-article","created":{"date-parts":[[2009,8,21]],"date-time":"2009-08-21T21:50:29Z","timestamp":1250891429000},"page":"6496-6503","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":181,"title":["Versatile Dual-Technology System for Markerless Allele Replacement in\n                    <i>Burkholderia pseudomallei<\/i>"],"prefix":"10.1128","volume":"75","author":[{"given":"Carolina M.","family":"Lo\u0301pez","sequence":"first","affiliation":[{"name":"Department of Microbiology, Immunology and Pathology, Rocky Mountain Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, Colorado State University, Fort Collins, Colorado 80523-2025"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Drew A.","family":"Rholl","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology and Pathology, Rocky Mountain Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, Colorado State University, Fort Collins, Colorado 80523-2025"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lily A.","family":"Trunck","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology and Pathology, Rocky Mountain Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, Colorado State University, Fort Collins, Colorado 80523-2025"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Herbert P.","family":"Schweizer","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology and Pathology, Rocky Mountain Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, Colorado State University, Fort Collins, Colorado 80523-2025"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.00531-08"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1099\/00207713-48-1-317"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.18.2.383-416.2005"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.02430-07"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0035-9203(08)70002-6"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.179.2.538-540.1997"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.179.7.2116-2125.1997"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1462-2920.2008.01576.x"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.gene.2008.10.011"},{"key":"e_1_3_2_11_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0403302101"},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.74.3.1949-1953.2006"},{"key":"e_1_3_2_13_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00440-06"},{"key":"e_1_3_2_14_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.181.17.5498-5504.1999"},{"key":"e_1_3_2_15_2","first-page":"13","volume":"9","year":"1987","unstructured":"Liss, L. 1987. 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