Evidence for ethanol-dependent acetic acid resistance in Acetobacter pasteurianus strain SKU1108

Acetic acid bacteria such as Acetobacter spp. produce high concentrations of acetic acid by oxidizing ethanol while reducing molecular oxygen to water on the cell surface. The acetic acid resistance of acetic acid bacteria has been explained as a complex of several mechanisms. The aarC gene, encoding succinyl-CoA:acetate CoA transferase, which catalyzes the committed step of acetate metabolism, plays a crucial role in acetic acid resistance in Acetobacter aceti. Here, we constructed a mutant of Acetobacter pasteurianus strain SKU1108, from which aarC and its paralog aarC2 were deleted (named the ∆∆ strain). The ∆∆ strain failed to grow on a glucose- and glycerol-containing medium in the presence of acetic acid. However, when the ∆∆ 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 ∆∆ 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 ∆∆ 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 m-chlorophenyl hydrazone, inhibited the ethanol-dependent acetic acid resistance of the ∆∆ strain in the cell killing assay. Thus, overall, we find that A. pasteurianus has AarC-independent but ethanol-dependent acetic acid resistance that is protonophore-sensitive.

Vinegar is produced by acetic acid fermentation by acetic acid bacteria such as Komagataeibacter spp. and Acetobacter 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 aarC is crucial in the acetate metabolism of Acetobacter sp. Here, a mutant derivative of Acetobacter pasteurianus strain SKU1108, devoid of aarC, 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 A. pasteurianus. 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.