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The acetic acid resistance of acetic acid bacteria has been explained as a complex of several mechanisms. The\n                      <jats:italic toggle=\"yes\">aarC<\/jats:italic>\n                      gene, encoding succinyl-CoA:acetate CoA transferase, which catalyzes the committed step of acetate metabolism, plays a crucial role in acetic acid resistance in\n                      <jats:italic toggle=\"yes\">Acetobacter aceti<\/jats:italic>\n                      . Here, we constructed a mutant of\n                      <jats:italic toggle=\"yes\">Acetobacter pasteurianus<\/jats:italic>\n                      strain SKU1108, from which\n                      <jats:italic toggle=\"yes\">aarC<\/jats:italic>\n                      and its paralog\n                      <jats:italic toggle=\"yes\">aarC2<\/jats:italic>\n                      were deleted (named the \u2206\u2206 strain). The \u2206\u2206 strain failed to grow on a glucose- and glycerol-containing medium in the presence of acetic acid. However, when the \u2206\u2206 strain was cultivated with ethanol, it grew well and produced acetic acid at comparable levels to the parental strain. When ethanol was added to growing cell-based acetic acid resistance experiments, the \u2206\u2206 strain grew even in the presence of acetic acid, suggesting an ethanol-dependent but AarC-independent mechanism of acetic acid resistance. 2-Propanol and lactic acid were also oxidized by the \u2206\u2206 strain and improved acetic acid resistance, though not as much as ethanol. In addition to growing cell assays, we established a resting cell-based cell killing assay with acetic acid. A protonophore, carbonyl cyanide\n                      <jats:italic toggle=\"yes\">m<\/jats:italic>\n                      -chlorophenyl hydrazone, inhibited the ethanol-dependent acetic acid resistance of the \u2206\u2206 strain in the cell killing assay. Thus, overall, we find that\n                      <jats:italic toggle=\"yes\">A. pasteurianus<\/jats:italic>\n                      has AarC-independent but ethanol-dependent acetic acid resistance that is protonophore-sensitive.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Vinegar is produced by acetic acid fermentation by acetic acid bacteria such as\n                      <jats:italic toggle=\"yes\">Komagataeibacter<\/jats:italic>\n                      spp. and\n                      <jats:italic toggle=\"yes\">Acetobacter<\/jats:italic>\n                      spp. Resistance to acetic acid is an important feature of these microorganisms. At least two mechanisms have been proposed for acetic acid resistance: acetate metabolism and acetic acid efflux. The gene\n                      <jats:italic toggle=\"yes\">aarC<\/jats:italic>\n                      is crucial in the acetate metabolism of\n                      <jats:italic toggle=\"yes\">Acetobacter<\/jats:italic>\n                      sp. Here, a mutant derivative of\n                      <jats:italic toggle=\"yes\">Acetobacter pasteurianus<\/jats:italic>\n                      strain SKU1108, devoid of\n                      <jats:italic toggle=\"yes\">aarC,<\/jats:italic>\n                      failed to grow on acetate. In the absence of ethanol, the mutant was sensitive to acetic acid. However, in the presence of ethanol, it was resistant to acetic acid. These observations suggest a novel mechanism of AarC-independent but ethanol-dependent acetic acid resistance of\n                      <jats:italic toggle=\"yes\">A. pasteurianus<\/jats:italic>\n                      . We can now find acetic acid-resistant mechanisms independent of acetate metabolism in growing cell-based experiments, which may promote elucidation of mechanism(s) of acetic acid resistance that involve as-yet-unidentified molecules.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aem.02034-25","type":"journal-article","created":{"date-parts":[[2025,11,26]],"date-time":"2025-11-26T14:25:08Z","timestamp":1764167108000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Evidence for ethanol-dependent acetic acid resistance in\n                    <i>Acetobacter pasteurianus<\/i>\n                    strain SKU1108"],"prefix":"10.1128","volume":"91","author":[{"given":"Akari","family":"Narimatsu","sequence":"first","affiliation":[{"name":"Joint Degree Program of Kasetsart University and Yamaguchi University, Graduate School of Science and Technology for Innovation, Yamaguchi 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