Expression analysis of HAA1 and Haa1-regulated genes in the HAA1-OP strain. The expression data of the HAA1-OP strain were compared with those of the wild-type strain and are shown as relative expression levels. The values are the means of results from three independent experiments.

Growth phenotypes of the wild-type and HAA1-OP strains under conditions of acetic acid stress. Approximately 10⁵ cells and serial dilutions of 10⁻¹ to 10⁻⁴ (from left to right) of wild-type and HAA1-OP strains were spotted on YPD plates in the absence or presence of 0.7% (wt/vol) acetic acid (pH 4.2) or 3% (wt/vol) lactic acid (pH 2.9). Plates were incubated at 30°C for 1 day (YPD), 4 days (0.7% acetic acid), or 6 days (3% lactic acid).

Comparison of the levels of intracellular acetic acid and cell viability after exposure to acetic acid. (A) Intracellular acetic acid levels. Wild-type and HAA1-OP cells precultured in YPD medium (pH 6.8) were reinoculated into YPD medium (pH 4.2) in the absence (0%) or presence (0.2% to 1.0%) of acetic acid and incubated at 30°C for 60 min. Intracellular acetic acid was extracted and quantified using HPLC. The acetic acid concentration of each sample was compared with that of the wild-type sample grown in normal YPD medium (0% extracellular acetic acid, pH 6.8), and the results are shown as relative intracellular acetic acid levels. The values are the means of results from three independent experiments. (B) Viability of cells after exposure to acetic acid. Wild-type and HAA1-OP cells precultured in YPD medium (pH 6.8) were reinoculated into YPD medium containing 1.0% acetic acid (pH 4.2) and incubated for 60 min at 30°C. Cells were harvested and resuspended in YPD medium, and approximately 4 × 10⁵ cells and serial dilutions of 10⁻¹ to 10⁻⁴ (from left to right) were spotted onto YPD plates (pH 6.8).