{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,10]],"date-time":"2026-10-10T00:24:07Z","timestamp":1791591847626,"version":"4.3.4"},"reference-count":53,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2025,9,10]],"date-time":"2025-09-10T00:00:00Z","timestamp":1757462400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,9,10]],"date-time":"2025-09-10T00:00:00Z","timestamp":1757462400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100002387","name":"National Cancer Institute, Cairo University","doi-asserted-by":"publisher","award":["R01CA154426"],"award-info":[{"award-number":["R01CA154426"]}],"id":[{"id":"10.13039\/501100002387","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000289","name":"Cancer Research UK","doi-asserted-by":"publisher","award":["C51981-A29063"],"award-info":[{"award-number":["C51981-A29063"]}],"id":[{"id":"10.13039\/501100000289","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mBio"],"published-print":{"date-parts":[[2025,9,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                      is an oncobacterium associated with colorectal cancer (CRC) development. Despite that\n                      <jats:italic toggle=\"yes\">F. animalis<\/jats:italic>\n                      (\n                      <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                      ) dominates the CRC niche in humans, studies on the pathogenesis of\n                      <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                      have been limited by the paucity of genetically tractable\n                      <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                      strains. Here, we constructed a conjugative DNA transfer system for genetic manipulation of\n                      <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                      clinical isolates. A mobilizable shuttle plasmid can be transferred by conjugation from\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      to various\n                      <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                      strains, including\n                      <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                      7_1, which has been considered genetically intractable using electroporation methods. The conjugation efficiency of\n                      <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                      7_1 was further improved by ethanol treatment. Next, we manipulated the\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      donor strain to express\n                      <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                      methyltransferases, which can protect plasmids from degradation by restriction systems in\n                      <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                      recipient strains. With this donor strain, we could isolate transconjugants of\n                      <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                      harboring chromosomally integrated plasmid. As a test case, we disrupted\n                      <jats:italic toggle=\"yes\">megL<\/jats:italic>\n                      which is involved in cysteine metabolism and hydrogen sulfide (H\n                      <jats:sub>2<\/jats:sub>\n                      S) production. The plasmid integrated into the\n                      <jats:italic toggle=\"yes\">megL<\/jats:italic>\n                      locus was stably maintained without antibiotic selective pressure. The\n                      <jats:italic toggle=\"yes\">megL<\/jats:italic>\n                      disruption in\n                      <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                      7_1 significantly decreased the level of H\n                      <jats:sub>2<\/jats:sub>\n                      S production\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      in the presence of cysteine and resulted in lower cecal H\n                      <jats:sub>2<\/jats:sub>\n                      S level in a mouse colonization model. The same conjugation-based system was successfully applied to another\n                      <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                      strain CTI-1, although this strain showed 10-fold lower conjugation efficiency compared to\n                      <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                      7_1. Altogether, this work suggests that conjugation is an efficient method for genetic manipulation of\n                      <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                      strains, allowing us to mechanistically study their pathogenicity in CRC at the molecular level.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Among the\n                        <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                        species associated with colorectal cancer (CRC),\n                        <jats:italic toggle=\"yes\">F. animalis<\/jats:italic>\n                        (\n                        <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                        ) is the most clinically relevant species showing distinct genetic features. However, due to its genetic intractability, little is known experimentally about the molecular factors of\n                        <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                        contributing to CRC development. Here, we showed that foreign DNA can be transferred from\n                        <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                        to\n                        <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                        via conjugation. The conjugation system was developed by expressing\n                        <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                        -derived methyltransferases in the\n                        <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                        donor strain to transfer methylated plasmid to\n                        <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                        . Finally, the\n                        <jats:italic toggle=\"yes\">megL<\/jats:italic>\n                        gene, encoding an enzyme responsible for hydrogen sulfide (H\n                        <jats:sub>2<\/jats:sub>\n                        S) production, was successfully disrupted in\n                        <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                        strains. The effects of the\n                        <jats:italic toggle=\"yes\">megL<\/jats:italic>\n                        disruption in\n                        <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                        on H\n                        <jats:sub>2<\/jats:sub>\n                        S production were verified both\n                        <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                        and\n                        <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                        . This conjugation-based approach would be applied to not only\n                        <jats:italic toggle=\"yes\">Fa<\/jats:italic>\n                        but also a broader range of\n                        <jats:italic toggle=\"yes\">Fusobacterium<\/jats:italic>\n                        species, expanding our understanding of their virulence traits.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.01714-25","type":"journal-article","created":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T13:00:20Z","timestamp":1753880420000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":4,"title":["Conjugation-based genetic manipulation of\n                    <i>Fusobacterium animalis<\/i>"],"prefix":"10.1128","volume":"16","author":[{"given":"Duhyun","family":"Ko","sequence":"first","affiliation":[{"name":"Department of Immunology & Infectious Diseases, Harvard T. 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