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L. iners<\/jats:italic>\n                      , the most prevalent vaginal microbe globally, is the most fastidious of the vaginal lactobacilli, has the smallest genome, and produces less lactic acid (only the L-isoform).\n                      <jats:italic toggle=\"yes\">L. iners<\/jats:italic>\n                      is also less protective against bacterial vaginosis and uniquely encodes a cholesterol-dependent cytolysin, inerolysin, suggesting it may be a pathobiont. Despite its central role in the health of over one billion females,\n                      <jats:italic toggle=\"yes\">L. iners<\/jats:italic>\n                      biology remains poorly understood, in part, due to a lack of genetic editing tools. Here, we present findings that\n                      <jats:italic toggle=\"yes\">L. iners<\/jats:italic>\n                      is naturally competent and can be transformed easily by exogenous DNA. Natural competence was leveraged to disrupt the\n                      <jats:italic toggle=\"yes\">iny<\/jats:italic>\n                      gene encoding inerolysin, and\n                      <jats:italic toggle=\"yes\">comGA<\/jats:italic>\n                      , encoding the ATPase component of the competence pilus. Both gene disruptions were accomplished using PCR-assembled DNA fragments comprising a drug resistance gene cassette (\n                      <jats:italic toggle=\"yes\">tetM<\/jats:italic>\n                      or\n                      <jats:italic toggle=\"yes\">ermB<\/jats:italic>\n                      ) flanked by ~2 kb regions of homology to the\n                      <jats:italic toggle=\"yes\">L. iners<\/jats:italic>\n                      chromosome. We further demonstrate that\n                      <jats:italic toggle=\"yes\">comGA<\/jats:italic>\n                      is essential for\n                      <jats:italic toggle=\"yes\">L. iners<\/jats:italic>\n                      transformation. The ability to rapidly perform targeted deletions in\n                      <jats:italic toggle=\"yes\">L. iners<\/jats:italic>\n                      with\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      generated DNA templates provides a straightforward and much-needed method to probe the genetics and physiology of these important vaginal bacteria.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      This study describes, to our knowledge, the first method for genetically manipulating\n                      <jats:italic toggle=\"yes\">L. iners<\/jats:italic>\n                      , the most prevalent bacteria of the human vaginal microbiota. This work paves the way for the rapid development of genetic tools to explore the physiology of\n                      <jats:italic toggle=\"yes\">L. iners<\/jats:italic>\n                      in the context of the vaginal microbiome and potentially alter its properties as a probiotic.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aem.00441-26","type":"journal-article","created":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T13:00:51Z","timestamp":1786366851000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Utilizing natural competence to genetically manipulate\n                    <i>Lactobacillus iners<\/i>"],"prefix":"10.1128","volume":"92","author":[{"given":"Kevin Y.","family":"Cao","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03dbr7087","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Genetics, University of Toronto","place":["Toronto, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniella","family":"Serrador","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03dbr7087","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Genetics, University of Toronto","place":["Toronto, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jhenielle R.","family":"Campbell","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03dbr7087","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Genetics, University of Toronto","place":["Toronto, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1586-4093","authenticated-orcid":true,"given":"Rupert","family":"Kaul","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03dbr7087","id-type":"ROR","asserted-by":"publisher"}],"name":"Departments of Medicine and Immunology, University of Toronto","place":["Toronto, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9293-9220","authenticated-orcid":true,"given":"William Wiley","family":"Navarre","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03dbr7087","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Genetics, University of Toronto","place":["Toronto, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-022-01083-2"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1007\/s11908-023-00798-5"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1000086107"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tim.2016.11.007"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/aem.02123-22"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1126\/scitranslmed.3003605"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00694-10"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1186\/2049-2618-1-12"},{"key":"e_1_3_3_10_2","unstructured":"Rampersaud R. 2014. 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