{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T16:37:05Z","timestamp":1768927025170,"version":"3.49.0"},"reference-count":41,"publisher":"American Society for Microbiology","issue":"3","license":[{"start":{"date-parts":[[2024,3,21]],"date-time":"2024-03-21T00:00:00Z","timestamp":1710979200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/doi.org\/10.1128\/ASMCopyrightv2"},{"start":{"date-parts":[[2024,3,21]],"date-time":"2024-03-21T00:00:00Z","timestamp":1710979200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100002347","name":"Bundesministerium f\u00fcr Bildung und Forschung","doi-asserted-by":"crossref","award":["01KI1824"],"award-info":[{"award-number":["01KI1824"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100002347","name":"Bundesministerium f\u00fcr Bildung und Forschung","doi-asserted-by":"crossref","award":["01KI2131"],"award-info":[{"award-number":["01KI2131"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Bacteriol"],"published-print":{"date-parts":[[2024,3,21]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:sec>\n            <jats:title\/>\n            <jats:p>\n              Cas9-based gene editing tools have revolutionized genetics, enabling the fast and precise manipulation of diverse bacterial species. However, widely applicable genetic tools for non-model gut bacteria are unavailable. Here, we present a two-plasmid Cas9-based system designed for gene deletion and knock-in complementation in three members of the\n              <jats:italic>Klebsiella oxytoca<\/jats:italic>\n              species complex (KoSC), which we applied to study the genetic factors underlying the role of these bacteria in competition against\n              <jats:italic>Klebsiella pneumoniae<\/jats:italic>\n              . Firstly, the system allowed efficient and precise full-length gene deletion via enhanced lambda Red expression. Furthermore, we tested the efficiency of two independent, functionally validated complementation strategies. Ultimately, the insertion of universal \u201cbookmark\u201d targets during gene deletion subsequently allows the most optimal genetic complementation in\n              <jats:italic>K. oxytoca<\/jats:italic>\n              ,\n              <jats:italic>Klebsiella michiganensis<\/jats:italic>\n              , and\n              <jats:italic>Klebsiella grimontii<\/jats:italic>\n              . This approach offers a significant advantage by enabling the use of a single high-efficiency \u201cbookmark\u201d for complementing other loci or strains, eliminating the need for site-specific design. We revealed that the carbohydrate permease CasA is critical in\n              <jats:italic>ex vivo<\/jats:italic>\n              assays for\n              <jats:italic>K. pneumoniae<\/jats:italic>\n              inhibition by\n              <jats:italic>K. oxytoca<\/jats:italic>\n              but is neither sufficient nor required for\n              <jats:italic>K. michiganensis<\/jats:italic>\n              and\n              <jats:italic>K. grimontii<\/jats:italic>\n              . Thus, the adaptation of state-of-the-art genetic tools to KoSC allows the identification of species-specific functions in microbial competition.\n            <\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>IMPORTANCE<\/jats:title>\n            <jats:p>\n              Cas9-based gene editing tools have revolutionized bacterial genetics, yet, their application to non-model gut bacteria is frequently hampered by various limitations. We utilized a two-plasmid Cas9-based system designed for gene deletion in\n              <jats:italic>Klebsiella pneumoniae<\/jats:italic>\n              and demonstrate after optimization its utility for gene editing in three members of the\n              <jats:italic>Klebsiella oxytoca<\/jats:italic>\n              species complex (KoSC) namely\n              <jats:italic>K. oxytoca<\/jats:italic>\n              ,\n              <jats:italic>Klebsiella michiganensis<\/jats:italic>\n              , and\n              <jats:italic>Klebsiella grimontii<\/jats:italic>\n              . We then adapted a recently developed protocol for functional complementation based on universal \u201cbookmark\u201d targets applicable to all tested species. In summary, species-specific adaptation of state-of-the-art genetic tools allows efficient gene deletion and complementation in type strains as well as natural isolates of KoSC members to study microbial interactions.\n            <\/jats:p>\n          <\/jats:sec>","DOI":"10.1128\/jb.00317-23","type":"journal-article","created":{"date-parts":[[2024,2,14]],"date-time":"2024-02-14T14:01:15Z","timestamp":1707919275000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":8,"title":["An adapted method for Cas9-mediated editing reveals the species-specific role of \u03b2-glucoside utilization driving competition between\n            <i>Klebsiella<\/i>\n            species"],"prefix":"10.1128","volume":"206","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5042-5478","authenticated-orcid":false,"given":"\u00c9va d. 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