Genome-wide analysis of yeast stress survival and tolerance acquisition to analyze the central trade-off between growth rate and cellular robustness

Abstract

All organisms have evolved to cope with changes in environmental conditions, ensuring the optimal combination of proliferation and survival. In yeast, exposure to a mild stress leads to an increased tolerance for other stresses. This suggests that yeast uses information from the environment to prepare for future threats. We used the yeast knockout collection to systematically investigate the genes and functions involved in severe stress survival and in the acquisition of stress (cross-) tolerance. Besides genes and functions relevant for survival of heat, acid, and oxidative stress, we found an inverse correlation between mutant growth rate and stress survival. Using chemostat cultures, we confirmed that growth rate governs stress tolerance, with higher growth efficiency at low growth rates liberating the energy for these investments. Cellular functions required for stress tolerance acquisition, independent of the reduction in growth rate, were involved in vesicular transport, the Rpd3 histone deacetylase complex, and the mitotic cell cycle. Stress resistance and acquired stress tolerance in Saccharomyces cerevisiae are governed by a combination of stress-specific and general processes. The reduction of growth rate, irrespective of the cause of this reduction, leads to redistribution of resources toward stress tolerance functions, thus preparing the cells for impending change.

FIGURE 1:

Severe stress survival before and after mild-stress treatment. Cultures of BY4741 were pregrown in batch fermentors at 30°C to an OD₆₀₀ of ∼0.1. Independent cultures were exposed to a nonlethal mild stress treatment of 39°C, 10°C, 0.15 mM H₂O₂, or 1.2 mM sorbic acid at pH 5.0 for 2 h, whereas a control culture continued growth at 30°C. From each fermentor, triplicate samples were taken and exposed to severe stress treatments of 327 mM acetic acid at pH 3.0 (white bars), 10 mM H₂O₂ (gray bars), or 48°C (black bars), during 10 min. These samples, as well as triplicate nontreated control samples, were diluted and plated. CFUs were determined after 3 d at 30°C. Bars represent averages and standard deviations of biological quadruplicates; symbols indicate significance of the difference with the untreated samples (**p < 0.001; *p < 0.05; in a one-tailed t test assuming unequal variance).

FIGURE 2:

Genome-wide analysis of the impact of single-gene deletions on stress survival and acquired stress tolerance. (A) Design of the genome-wide analysis of stress survival and acquisition of stress tolerance. (B) Survival of 10-min treatments of high levels of oxidative, acid, or heat stress of pooled deletion collection samples (± SD) of five independent replicates. (C–E) Distribution of deletant stress survival percentages within 4067 deletants analyzed, with (black bars) and without (gray bars) a 3-h pretreatment at 38°C. Stress survival was measured after 10 min of (C) oxidative stress (10 mM H₂O₂), (D) acid stress (327 mM acetic acid), or (E) heat stress (48°C).

FIGURE 3:

Mild stress pretreatment causes an increase in the correlated stress survival of the deletant collection. (A) Stress survival of all individual deletants under high acid stress (327 mM acetic acid) vs. their stress survival of high oxidative stress (10 mM H₂O₂). The correlation between the survival was higher after 3 h of growth at 38°C (black symbols) than without this pretreatment (gray symbols), as indicated by their r (p < 0.0001). (B) Pearson correlation coefficients (p for all correlations <0.0001) of the comparison of survival percentages of all mutants upon a high-dose stress pulse of one stress compared with survival upon a pulse of another stress, either with or without a 3-h acclimatory period at 38°C, or comparison of the survival of one single pulse stress after the acclimation period with survival of the same pulse stress before the acclimation period. ^aPretreatment of 3 h at 38°C. ^bSevere stress treatment of 10 min with 10 mM H₂O₂ (H₂O₂), 327 mM acetic acid at pH 3.0 (HAc), or 48°C (48°C).

FIGURE 4:

Inverse correlation of stress survival with growth rate. (A) Survival percentages of individual mutants after 10 min at 48°C plotted against their specific growth rate just before this treatment reveal an inverse correlation between the two parameters. (B) Correlation coefficients of pairwise comparison of severe stress survival and growth rate of the mutant collection. Consistent inverse correlation of survival upon severe stress is observed with the growth rate at the moment of exposure, so of survival percentages without pretreatment with growth rate at 30°C (white bars), of survival upon preexposure to 38°C with the growth rate at 38°C (gray bars), and of tolerance acquisition with the change in growth rate (μ38°C/μ30°C) upon a shift from 30 to 38°C (black bars). Survival percentages were calculated from the normalized signal intensities of all probes for one ORF before and after severe stress treatment, correcting for the viable cell counts of the total pooled deletion collection (see Materials and Methods).

FIGURE 5:

Growth rate affects severe stress tolerance. Culture samples from glucose-limited chemostats grown at different dilution rates were exposed to 10-min treatments of the different stressors. (A) Survival after exposure to 20 mM H₂O₂, 327 mM acetic acid at pH 3.0, 48°C; survival percentages were determined. (B) Biomass yield on ATP at the different dilution rates. Bars, average and SE of duplicate experiments.

FIGURE 6:

Msn2p and Msn4p are not required for stress tolerance acquisition. Stress survival after 10-min exposure to 10 mM H₂O₂ (white bars), 327 mM acetic acid (gray bars), or 48°C (black bars) of (A) wild type (W303-1a) and (B) the isogenic msn2 msn4 double-deletion strain in control conditions or after a 3-h treatment with 38°C or 1.2 mM sorbic acid. Bars, average and SD of biological triplicates.

FIGURE 7:

Functional groups involved in tolerance acquisition to different stresses are related but not identical. Mutant groups were selected if tolerance acquisition for at least one severe stress was significantly reduced (Bonferroni-corrected p <0.05, indicated with an asterisk) after correcting for growth rate–associated stress tolerance acquisition. A cutoff line designates Z values of 2, indicating noncorrected significance. White bars, tolerance acquisition for 10 mM H₂O₂; gray bars, tolerance acquisition for 327 mM acetic acid at pH 3.0; black bars, tolerance acquisition for 48°C.