{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T23:28:57Z","timestamp":1790724537471,"version":"4.1.0"},"reference-count":39,"publisher":"American Society for Microbiology","issue":"4","license":[{"start":{"date-parts":[[2026,4,7]],"date-time":"2026-04-07T00:00:00Z","timestamp":1775520000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,4,7]],"date-time":"2026-04-07T00:00:00Z","timestamp":1775520000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"name":"Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (OPS MHESI), Thailand Science Research and Innovation","award":["RGNS 65-025"],"award-info":[{"award-number":["RGNS 65-025"]}]},{"DOI":"10.13039\/501100004776","name":"Faculty of Medicine, Chulalongkorn University","doi-asserted-by":"publisher","award":["RA-MF-24\/66 and RA-MF-05\/68"],"award-info":[{"award-number":["RA-MF-24\/66 and RA-MF-05\/68"]}],"id":[{"id":"10.13039\/501100004776","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,4,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Burkholderia cenocepacia<\/jats:italic>\n                      , a member of the\n                      <jats:italic toggle=\"yes\">Burkholderia cepacia<\/jats:italic>\n                      complex (Bcc) poses a significant threat to immunocompromised individuals, particularly those with cystic fibrosis. Progress in understanding its pathogenesis has been hindered by the availability of efficient genetic tools. We selected the\n                      <jats:italic toggle=\"yes\">purM<\/jats:italic>\n                      gene for functional analysis because it is essential for growth under purine-limited conditions, intracellular survival, and virulence in the closely related species,\n                      <jats:italic toggle=\"yes\">Burkholderia pseudomallei<\/jats:italic>\n                      . We adapted the non-replicative, trimethoprim-resistance (TMP\n                      <jats:sup>r<\/jats:sup>\n                      ) vector pEDL1005 for markerless gene deletion in\n                      <jats:italic toggle=\"yes\">B. cenocepacia<\/jats:italic>\n                      using an optimized\n                      <jats:italic toggle=\"yes\">sacB<\/jats:italic>\n                      -based counterselection strategy, enabling in-frame deletion of\n                      <jats:italic toggle=\"yes\">purM<\/jats:italic>\n                      , which encodes phosphoribosylaminoimidazole synthetase, in both the reference strain K56-2 and the clinical isolate SCBC075. Notably, efficient\n                      <jats:italic toggle=\"yes\">sacB<\/jats:italic>\n                      -based counterselection required strain-specific sucrose concentrations (20% for K56-2 and 25% for SCBC075). To restore\n                      <jats:italic toggle=\"yes\">purM<\/jats:italic>\n                      function, we employed a mini-Tn7-based complementation system (pUC18T-mini-Tn7T-TMP) for site-specific chromosomal insertion downstream of one of the four\n                      <jats:italic toggle=\"yes\">glmS<\/jats:italic>\n                      homologs identified in this bacterium. A PCR-based method verified insertion 18\u201320 bp downstream of each target locus. Functional analysis revealed that\n                      <jats:italic toggle=\"yes\">\u0394purM<\/jats:italic>\n                      mutants were strictly adenine auxotrophic, confirming the essential role of\n                      <jats:italic toggle=\"yes\">purM<\/jats:italic>\n                      in\n                      <jats:italic toggle=\"yes\">de novo<\/jats:italic>\n                      purine biosynthesis, consistent with findings in\n                      <jats:italic toggle=\"yes\">B. pseudomallei<\/jats:italic>\n                      . However, unlike in\n                      <jats:italic toggle=\"yes\">B. pseudomallei<\/jats:italic>\n                      , the \u0394\n                      <jats:italic toggle=\"yes\">purM<\/jats:italic>\n                      mutant of SCBC075 showed no significant attenuation in intracellular survival within RAW264.7 macrophages. These results validate pEDL1005 and the mini-Tn7 system as effective tools for unmarked gene deletion and chromosomal complementation in\n                      <jats:italic toggle=\"yes\">B. cenocepacia<\/jats:italic>\n                      , expanding the molecular toolkit for this pathogen and enabling future studies of gene function, metabolism, and pathogenesis.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Genetic manipulation of\n                        <jats:italic toggle=\"yes\">Burkholderia cenocepacia<\/jats:italic>\n                        , a challenging opportunistic pathogen, is essential for elucidating its pathogenesis. However, the available genetic tools remain limited. This study addresses this gap by adapting a pair of trimethoprim-selectable systems: the pEDL1005 vector for markerless gene deletion using strain-specific\n                        <jats:italic toggle=\"yes\">sacB<\/jats:italic>\n                        -based counterselection, and a mini-Tn7-based strategy for site-specific chromosomal complementation. A key advancement is the development of a PCR-based method to verify precise transposon insertion downstream of\n                        <jats:italic toggle=\"yes\">glmS<\/jats:italic>\n                        . Using these systems, we functionally characterized the\n                        <jats:italic toggle=\"yes\">purM<\/jats:italic>\n                        gene, demonstrating that its deletion causes a purine-specific growth defect in minimal media. Together, these tools provide a robust platform for functional studies and enable future research into gene function and pathogenesis in this clinically relevant pathogen.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.02492-25","type":"journal-article","created":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T14:07:29Z","timestamp":1772633249000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Functional analysis of\n                    <i>purM<\/i>\n                    in\n                    <i>Burkholderia cenocepacia<\/i>\n                    using a trimethoprim-selectable allelic exchange and mini-Tn7 complementation approach"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-7106-5243","authenticated-orcid":false,"given":"May 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