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The direct electron donors to nitrogenase are reduced ferredoxin (Fd) and flavodoxin (Fld). The enzymes that reduce Fd or Fld for N\n                      <jats:sub>2<\/jats:sub>\n                      fixation mainly include pyruvate-flavodoxin (ferredoxin) oxidoreductase (PFOR), ferredoxin-NADP\n                      <jats:sup>+<\/jats:sup>\n                      reductase (FNR),\n                      <jats:italic toggle=\"yes\">R<\/jats:italic>\n                      hodobacter\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      itrogen\n                      <jats:italic toggle=\"yes\">f<\/jats:italic>\n                      ixation protein complex (Rnf), and bifurcating electron transfer flavoprotein complex (Fix).\n                      <jats:italic toggle=\"yes\">Paenibacillus polymyxa<\/jats:italic>\n                      is a model for studies of nitrogen fixation, but the pathways of electron transport to nitrogenase are unknown. In this study, we characterized the electron transport pathway to nitrogenase in\n                      <jats:italic toggle=\"yes\">P. polymyxa<\/jats:italic>\n                      . Deletion analysis showed that among 15 genes encoding Fds, Fld, and Fd-\/Fld-reducing enzymes, only\n                      <jats:italic toggle=\"yes\">cbiW<\/jats:italic>\n                      gene and\n                      <jats:italic toggle=\"yes\">pfoAB<\/jats:italic>\n                      operon decreased nitrogenase activity (31% and 37% relative to wild type, respectively). Triple deletion of\n                      <jats:italic toggle=\"yes\">cbiW<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">pfoAB<\/jats:italic>\n                      led to a reduction of nitrogenase activity by 61%. Elemental analysis and ultraviolet-visible absorption spectroscopy revealed that the purified Fd protein encoded by\n                      <jats:italic toggle=\"yes\">cbiW<\/jats:italic>\n                      is a [2Fe-2S] Fd. The purified Fd protein was able to substitute for\n                      <jats:italic toggle=\"yes\">Klebsiella<\/jats:italic>\n                      NifF (Fld) to transfer electrons from pyruvate to\n                      <jats:italic toggle=\"yes\">Klebsiella<\/jats:italic>\n                      nitrogenase\n                      <jats:italic toggle=\"yes\">in vitro.<\/jats:italic>\n                      The\n                      <jats:italic toggle=\"yes\">pfoAB<\/jats:italic>\n                      operon encodes PFOR, which is composed of two subunits.\n                      <jats:italic toggle=\"yes\">Klebsiella oxytoca<\/jats:italic>\n                      NifJ (PFOR) was able to support nitrogen fixation in the \u0394\n                      <jats:italic toggle=\"yes\">pfoAB<\/jats:italic>\n                      mutant. These findings suggest that [2Fe-2S] Fd encoded by\n                      <jats:italic toggle=\"yes\">cbiW<\/jats:italic>\n                      and PFOR encoded by the\n                      <jats:italic toggle=\"yes\">pfoAB<\/jats:italic>\n                      operon constitute an electron transport pathway to\n                      <jats:italic toggle=\"yes\">Paenibacillus<\/jats:italic>\n                      nitrogenase.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Gram-positive and facultatively anaerobic\n                      <jats:italic toggle=\"yes\">Paenibacillus polymyxa<\/jats:italic>\n                      is a model diazotroph whose\n                      <jats:italic toggle=\"yes\">nif<\/jats:italic>\n                      (nitrogen fixation) genes encoding nitrogenase have been used for engineering yeast and plants. However, the pathway of electron transfer to\n                      <jats:italic toggle=\"yes\">Paenibacillus<\/jats:italic>\n                      nitrogenase is unknown. In this study, we demonstrate that Fd encoded by\n                      <jats:italic toggle=\"yes\">cbiW<\/jats:italic>\n                      and PFOR encoded by the\n                      <jats:italic toggle=\"yes\">pfoAB<\/jats:italic>\n                      operon constitute an electron transport pathway to\n                      <jats:italic toggle=\"yes\">Paenibacillus<\/jats:italic>\n                      nitrogenase. Fd encoded by\n                      <jats:italic toggle=\"yes\">cbiW<\/jats:italic>\n                      is a [2Fe-2S] Fd, and PFOR encoded by the\n                      <jats:italic toggle=\"yes\">pfoAB<\/jats:italic>\n                      operon is a heterodimer. In contrast, in many gram-negative diazotrophs, [4Fe-4S] Fds are widely used as electron donors to nitrogenase, and PFORs (NifJs) are homodimers. These results suggest that there may be some differences in the mechanisms of electron transfer to nitrogenase between gram-negative and gram-positive bacteria. The identified electron transport components in\n                      <jats:italic toggle=\"yes\">P. polymyxa<\/jats:italic>\n                      will have great potential for use in engineering eukaryotes.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aem.01681-26","type":"journal-article","created":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T13:01:58Z","timestamp":1792674118000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Electron transfer to nitrogenase in\n                    <i>Paenibacillus polymyxa<\/i>"],"prefix":"10.1128","author":[{"given":"Tianyi","family":"Hao","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04v3ywz14","id-type":"ROR","asserted-by":"publisher"}],"name":"College of Biological Sciences, China Agricultural University","place":["Beijing, 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