PMC

Differences in membrane lipid composition in response to ethanol in S288c and YPS163. GC–MS analysis of the total membrane lipids in response to ethanol in S288c, YPS163, or the YPS163 (Y) elo1Δ mutant. The x-axis represents lipid chain length and level of saturation. Error bars represent standard error of biological triplicates. Asterisks denote significantly different comparisons between YPS163 and S288c (*, P < 0.05; **, P < 0.01; ***, P <0.001, paired t-test).

Acquired ethanol tolerance depends on Msn2 . (A) Acquired tolerance defect of an msn2Δ strain. Cells were pretreated with 5% (v/v) ethanol for 1 hr and then subjected to severe ethanol doses (x-axis) for 2 hr. Colony-forming units indicated percentage viability. Error bars represent standard deviation of biological triplicates. (B) Average log² expression change of 106 Msn2-dependent genes with significantly lower expression in S288c vs. YPS163, in S288c (S), M22 (M), YPS163 (Y), and YPS163 (Y) msn2Δ strains responding to ethanol.

Acquired ethanol tolerance in diverse yeast strains. (A) A representative acquired ethanol tolerance assay is shown. S288c (left) or YPS163 (right) was exposed to 5% ethanol or mock pretreatment for 60 min. Cells were exposed to one of seven indicated severe doses of ethanol for 2 hr and then plated onto a YPD plate to score viability. (B) Basal (red) and acquired (blue) percentage ethanol tolerated is shown for strains collected from diverse niches (clin, clinical; ferm, fermentations). The maximal dose survived was based on >50% spot density compared to the no-ethanol control. Data represent the average of biological duplicates. Strains and scores are found in File S1. (C) Cycloheximide blocks acquired ethanol tolerance in both YPS163 and M22. Error bars represent standard deviation of biological triplicates. Cycloheximide (CHX), if present, was added 20 min prior to either the mock or ethanol (5%) primary stress. (D) Results of a representative spot assay from the experiment shown in C. The ethanol doses were 12.5–17.5% (v/v) in 0.5% increments.

Genes necessary for acquired ethanol resistance. (A) Basal and acquired ethanol tolerance is shown for various mutant strains. The average and standard deviation of ethanol tolerance scores (see materials and methods) is shown for strains pretreated with 5% ethanol (blue) versus the mock-treated control (orange). Error bars represent standard deviation of biological triplicates. Asterisks denote significant differences in acquired ethanol resistance relative to the YPS163 wild-type strain (*, P < 0.05; ** = P < 0.01, t-test). (B) Basal ethanol sensitivity in mutants with defects in acquired ethanol resistance. Cells were grown at least eight generations to an OD₆₀₀ of 0.3–0.6, after which cells were normalized to an OD₆₀₀ of 0.15 and 10-fold serial dilutions were plated onto either a YPD (control) plate or YPD + 8% (v/v) ethanol (YPDE). Growth on the YPD plate was scored at 2 days, while growth on the YPDE plate was scored at 3 days. (C) A representative experiment showing strain basal tolerances to 2 hr exposure of indicated ethanol doses in S288c containing the indicated galactose-inducible plasmid constructs (see materials and methods). Data are as shown in . (D) As in C except viability was scored after a 2-hr exposure to 19% ethanol using flow cytometry to determine the proportion of propidium iodide negative (i.e., live) cells. Error bars represent standard deviation of biological triplicates. Asterisks denote significant differences in percentage viability relative to S288c carrying the vector only control (**, P < 0.01, t-test).

Variation in gene expression between S288c, M22, and YPS163. (A) Log2 ethanol-responsive expression changes of 2203 genes differentially expressed in either wild-strain vs. S288c (FDR is 0.05, paired t-test). Basal expression differences in M22 (M) or YPS163 (Y) vs. S288c (S) are shown on the left; time courses of the expression changes in response to ethanol are shown in the middle; and difference between ethanol response in each wild strain vs. S288c is shown on the right. Each row represents a gene and each column represents a strain or condition, with time-course samples indicated by triangles. Genes were organized by hierarchical clustering of the combined basal expression and time-course data. Differences in ethanol response across strains were subsequently added to the figure. Red indicates induced and green indicates repressed expression in response to ethanol. Blue indicates higher and yellow indicates lower expression in S288c relative to the wild strains. Complete GO categories enriched in each cluster are found in File S2. The top GO categories for each cluster listed below were chosen on the basis of lowest P-value for clearly nonoverlapping functional groups: (A) RNA localization, (B) translation, (C) regulation of translation, (D) cellular amine metabolism, (E) oxidative phosphorylation, (F) flocculation, (G) methionine biosynthesis, (H) oxidative phosphorylation, cell death, (I) protein folding, (J) vacuolar protein catabolism, trehalose metabolism, proteolysis, (K) catabolic process, (L) transposition, (M) transposition, (N) polyphosphate metabolism. (B) Venn diagram of differentially expressed genes across all possible pairwise strain comparisons. This comparison includes genes with significant differential expression in M22 vs. YPS163, which were omitted from the clustering analysis.