{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T15:40:11Z","timestamp":1792510811414,"version":"4.5.0"},"reference-count":50,"publisher":"American Society for Microbiology","issue":"10","license":[{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32370583, 32370023, 31970036, 31900401, 32401044"],"award-info":[{"award-number":["32370583, 32370023, 31970036, 31900401, 32401044"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100005145","name":"Basic Research Program of Jiangsu Province","doi-asserted-by":"publisher","award":["BK20241961, BK20231350"],"award-info":[{"award-number":["BK20241961, BK20231350"]}],"id":[{"id":"10.13039\/501100005145","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Natural Science Foundation of Xuzhou Municipality","award":["KC23301"],"award-info":[{"award-number":["KC23301"]}]},{"name":"China Manned Space Program","award":["KJZ-YY-NSM0518"],"award-info":[{"award-number":["KJZ-YY-NSM0518"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,10,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      The anaerobic decolorization of azo dyes by\n                      <jats:italic toggle=\"yes\">Shewanella<\/jats:italic>\n                      species relies on an intricate electron transfer network, yet the full complement of genes involved remains incompletely defined. In this study, we used mini-Tn5 transposon mutagenesis to identify genes required for the decolorization of the azo dye acid yellow 36 in\n                      <jats:italic toggle=\"yes\">Shewanella putrefaciens<\/jats:italic>\n                      CN32. Two genes were uncovered:\n                      <jats:italic toggle=\"yes\">menD<\/jats:italic>\n                      , encoding a key enzyme in menaquinone biosynthesis, and\n                      <jats:italic toggle=\"yes\">lutB<\/jats:italic>\n                      , involved in lactate metabolism. Deletion of\n                      <jats:italic toggle=\"yes\">menD<\/jats:italic>\n                      caused a nearly complete loss of decolorization capacity without affecting growth. Supplementation with vitamin K\n                      <jats:sub>2<\/jats:sub>\n                      or spent supernatant from the wild-type strain restored decolorization, demonstrating that menaquinone is essential. We systematically identified the menaquinone biosynthesis gene cluster of\n                      <jats:italic toggle=\"yes\">S. putrefaciens<\/jats:italic>\n                      CN32, including\n                      <jats:italic toggle=\"yes\">menA<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">menB<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">menC<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">menE<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">menF<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">menG<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">menH<\/jats:italic>\n                      , and\n                      <jats:italic toggle=\"yes\">paaI<\/jats:italic>\n                      . Deleting most of these genes severely impaired decolorization, whereas deletions of\n                      <jats:italic toggle=\"yes\">menH<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">paaI<\/jats:italic>\n                      , or an adjacent LysR-family regulator gene had no significant effect. Furthermore, acid yellow 36 induced biofilm formation in a dose-dependent manner, and this induction was abolished in most menaquinone synthesis mutants. Acid yellow 36 did not substantially upregulate transcription of the\n                      <jats:italic toggle=\"yes\">men<\/jats:italic>\n                      gene cluster, suggesting that the dye promotes biofilm formation through a direct mechanism rather than by enhancing menaquinone synthesis. Collectively, our results establish menaquinone as a critical component for both azo dye decolorization and biofilm formation in\n                      <jats:italic toggle=\"yes\">S. putrefaciens<\/jats:italic>\n                      CN32, and they point to the existence of a CymA-independent electron transfer branch in this strain.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Azo dyes are environmental pollutants that can be effectively removed by\n                        <jats:italic toggle=\"yes\">Shewanella<\/jats:italic>\n                        bacteria through anaerobic respiration. Understanding the genetic basis of azo dye decolorization is essential for improving bioremediation strategies. This study identifies menaquinone, a respiratory chain cofactor, as indispensable for the decolorization of acid yellow 36 in\n                        <jats:italic toggle=\"yes\">Shewanella putrefaciens<\/jats:italic>\n                        CN32. By systematically mapping the menaquinone biosynthesis gene cluster, we show that an intact synthesis pathway is required for both dye reduction and biofilm formation, a lifestyle that enhances bacterial survival and metabolic activity in wastewater treatment systems. Importantly, the severe decolorization defect of menaquinone-deficient mutants contrasts with the relatively mild effect of deleting cymA, the canonical electron hub, suggesting the existence of a CymA-independent electron transfer route from menaquinone to azo dyes. These findings expand the current model of extracellular electron transfer in\n                        <jats:italic toggle=\"yes\">Shewanella<\/jats:italic>\n                        and provide new genetic targets for engineering more efficient azo dye-degrading strains.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.01996-26","type":"journal-article","created":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T16:53:24Z","timestamp":1788368004000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Menaquinone synthesis genes are critical for the decolorization of acid yellow 36 in\n                    <i>Shewanella putrefaciens<\/i>\n                    CN32"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5104-831X","authenticated-orcid":false,"given":"Jiawen","family":"Liu","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/051hvcm98","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, School of Life Sciences, Jiangsu Normal University","place":["Xuzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Yang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/051hvcm98","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, School of Life Sciences, Jiangsu Normal University","place":["Xuzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aofei","family":"Sun","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/051hvcm98","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, School of Life Sciences, 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