Stress survival depends on stress type–specific and general parameters
The obvious requirement to be able to adapt to a changed environment is survival when the change happens. It has already been well established that survival of individual yeast cells to severe stress conditions can be increased by preexposure of the population to a less severe stress. Such a pretreatment protects yeast cells not only from lethal levels of the same stress but also of other, apparently unrelated, stresses, a phenomenon called acquired stress (cross-) tolerance or resistance (McAlister and Finkelstein, 1980
). This phenomenon can be interpreted as the existence of a GSR in yeast. We chose to study yeast survival after high doses of various types of stress (defined as a condition causing at least 40% loss of viability within a 10-min treatment) to determine the deletants' intrinsic stress tolerance (). Next we determined the protective effect of various mild stress conditions (defined as a stress causing a significantly increased severe stress survival upon exposure to severe dose of a related stress without in itself causing loss of viability in a 24-h period) on survival of high levels of the same stresses. We determined the tolerance to severe oxidative, weak acid, and heat stress treatments in carefully controlled batch cultures that had been preexposed for 2 h to control or to mild heat, cold, acid, and oxidative stress conditions (). We chose a pretreatment time of 2 h since all mild stress–severe stress combinations showed maximal survival after this period (although in several cases the maximum had been reached sooner). We observed that preexposure to any of the mild stress conditions led to an increased survival under three of the severe stress conditions, the exception being the heat shock after the cold stress.
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, (more ...)
Measurement of the extent of the increase in survival generated by the four pretreatments revealed that the three high-level stresses did not increase survival proportionally, which would be expected if one general stress response underlies the acquisition of tolerance; one general mechanism activated to various extents could increase, for example, heat resistance more strongly than oxidative stress resistance but should then always do so. This was not the case, although there were clear similarities in the induction of stress survival among acid-, oxidative-, and high temperature–induced tolerances (Supplemental Figure S1). Cold stress seems to induce tolerance by a deviating mechanism. Therefore, although induction of stress cross-tolerance occurred with all conditions tested, the underlying mechanisms generating the tolerance are likely to represent a combination of stress-specific and general processes.
A general stress response increases survival after stress pretreatment
To identify the processes required for stress survival and for acquisition of stress cross-tolerance, we performed a genome-wide analysis using a pooled yeast knockout collection. We cultivated this pooled collection in batch fermentors to early exponential growth. We sampled three times to determine the growth rates of the individual mutants. The culture was divided in two, and one-half was exposed to a mild heat stress (3 h at 38°C, time approximating one generation time for most of the deletion collection in these conditions, leading to induction of maximal severe stress survival of the total pooled collection), whereas the other half was maintained at the normal growth temperature of 30°C. Subsequently, aliquots of both cultures were exposed to severe stress treatments (10-min exposure to 10 mM H2O2, 327 mM acetic acid at pH 3.0, or 48°C; ). The treated cells were analyzed for survival (). We determined the fraction of the population represented by individual deletant strains using Tag3 microarrays. Thus we determined growth rates, stress survival, and acquired stress tolerance for 4066 single-gene deletants (Supplemental Data File 1). The pretreatment increased survival of the mutant population of all three severe stresses to similar levels (), whereas control resistance differed by orders of magnitude.
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 (more ...)
We performed pairwise correlation analysis of the survival of all mutants toward oxidative, acid, and heat stress (), both with and without the pretreatment, to understand whether the tolerance toward the three stresses depended on shared or on unrelated sets of genes. There was a mild but significant correlation in mutant survival after exposure to the three different severe stresses (r = 0.32–0.39, p < 0.0001). Mechanistic overlap can be estimated from the r2 value, and therefore the comparison of mutant survival from one stress to another suggests that the mechanistic overlap would be between 10 and 15% with the stresses we used. To understand the similarity of the mechanisms of initial survival and induced survival, we analyzed the correlation of mutant survival upon each severe stress before and after pretreatment at 38°C. Here r ranging from 0.33 for acid stress to a fully uncorrelated −0.04 for heat stress suggests that the mechanisms responsible for stress survival in cells growing in relatively optimal conditions are different from those causing the (increased) stress resistance after a mild stress pretreatment. Finally, tolerance toward the three different severe stresses of all mutants became more strongly correlated with each other after the 38°C pretreatment (r = 0.43–0.49, or estimated mechanistic overlap between 18 and 24%) than before (p values for increase <0.001 in all three cases). This suggests that the response that led to the increased stress survival of all three stresses depends at least in part on common mechanisms. Together, these observations imply that the acclimatory response leading to increased stress survival has a significant general aspect and that this is unrelated to the mechanisms responsible for the inherent tolerance to the individual stresses in unstressed exponential-phase batch cultures.
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 (more ...)
Slow growth increases stress survival
The correlations of mutant survival of one stress versus survival of another suggest that only 10–25% of the results can be explained by mechanistic overlap, whereas the mechanisms of mutant survival of a single stress before and after the 38°C pretreatment underlies overlap even less. However, in the course of the experiment we also determined the growth rates of the individual mutants. Remarkably, there was a mild to strong correlation of mutant stress survival with growth rate, revealing a significant inverse relation (, white bars at 30°C). This means that slow-growing mutants have increased changes of surviving strong oxidative, acid, or heat stress. On average, the correlations with growth rate were stronger than those of survival of one stress with survival of another (). Thus, based on the correlation, differences of growth rate in the mutants could explain up to half of the differences in severe stress survival (for heat stress r = −0.70, and therefore r2 = 0.5). In addition, the survival of severe stress after the 38°C pretreatment showed an inverse correlation with the growth rate at 38°C, although not as strong (, gray bars at 38°C), suggesting again that if mutants grow more slowly at 38°C, they will be more tolerant toward severe stress. Finally, the increase in stress survival for all mutants upon mild heat pretreatment showed a clear correlation with their decrease in growth rate after the switch to 38°C (, black bars). Thus mutants that had a stronger growth rate response upon temperature shift also had the strongest stress tolerance acquisition. This strongly suggests that mutant growth rate is one of the important determinants of stress tolerance.
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. (more ...)
To determine whether this relation between growth rate and stress survival is a general trait of yeast cultures and not just of mutants, we performed chemostat cultures of wild-type yeast at different dilution rates and compared survival after high-dose stress treatment (). For all three severe stresses tested, we observed an inverse relation between growth rate and stress survival, showing that in wild type, a decrease in growth rate by itself already leads to an increased stress resistance. Thus slow growth, whether induced by mutation during exponential growth on glucose or induced by nutrient-limited growth in chemostats, led to an increased tolerance for all three severe stresses tested. The effect was strongest for heat stress survival, corroborating the finding that the strongest correlation observed in the mutant analysis was that between growth rate and heat-stress survival. This suggests that the GSR may well be closely related to processes involved in the modulation of growth rate.
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 (more ...)
One would expect that a greater investment in stress resistance activities would be costly for a cell in terms of resources and energy. Analyzing the steady-state carbon fluxes, we found that the yeast cultures were fully respiratory at dilution rates of 0.1, 0.21, and 0.25 h−1, whereas they had a mixed respirofermentative metabolism at the highest growth rate of 0.35 h−1 (Supplemental Table S1). Of interest, assuming similar biomass compositions and a constant respiratory efficiency, it can be deduced that the cultures grow more efficiently at lower dilution rates, with a higher biomass yield on ATP () This suggests that scarcity of glucose leads not only to a lower growth rate, but also to a more-than-proportional saving of energy. This would liberate energy to be invested in other processes, such as those involved in stress survival.
Functional requirements for growth rate–coupled stress survival
Alteration of growth rate caused either by a mutation or by nutrient limitation in a chemostat affected high-dose stress survival. We asked which cellular functions affected both growth rate and stress sensitivity. This question can be answered by looking at significantly affected mutants and determining the enrichment for particular functional groups, but we can also directly assess whether a priori–defined functional groups are deviating from the rest of the population as a group
. Such an approach is robust, as its reliability does not depend on error-prone determinations for a single mutant values (see Supplemental Figure S2) and no cutoffs for the significance of individual mutant traits have to be made. In addition, the distribution of stress survival was far from normal and usually showed strong tailing in one direction. Multiple sampling from such distributions inherently corrects for this (Kim and Volsky, 2005
), such that simpler statistics can be used (Boorsma et al., 2005
). We therefore determined the group-average growth rate or stress survival percentages for all Gene Ontology (GO) groups with six or more mutants measured in our screen. Next we calculated the distribution of group averages for 2000 drawings of random groups of the same number of mutants. We then determined whether the group average of a GO group was significantly different from the expected value for a group of the same size (two-tailed p of 0.05 after correction for multiple testing; Supplemental Data File 2). Mutant groups that lead to slow growth at 30°C are, as expected, generally involved in ribosome biogenesis and translation, as well as general transcription (Supplemental Table S2). At 38°C, we additionally observed groups involved in vesicular transport within the secretory pathway, as well as GTPase activities, largely involved in vesicle fusion. These activities are relevant for cellular remodeling in response to the temperature shift.
Mathematical uncoupling of mutant growth rate from gene-specific effects on stress survival
Because growth rate and stress survival were so strongly coupled, it was difficult to point out the functional groups that couple growth to survival. Knowing that growth rate directly affects stress survival, we corrected for this contribution to severe stress survival in each mutant using linear regression (see Materials and Methods). To identify those functional groups that are important for the coupling of growth rate and stress survival, we looked for those groups for which an observed survival phenotype was in fact mainly caused by a growth phenotype and therefore lose their significant phenotype upon growth rate correction (Supplemental Table S3). These groups were usually composed of slow growers, which are intrinsically stress resistant, and the growth rate correction led to decreased resistance. In general, the groups functioned in general (RNA polymerase II mediated) transcription and translation and in DNA and chromosome organization. A number of groups were involved in biogenesis and maintenance of mitochondria. Mutants in these groups were slow growers, notwithstanding the presence of high glucose concentrations, and correction for growth rate often revealed significant sensitivity, mostly for severe oxidative and acid stress. It seems therefore that the general regulation of ribosome biogenesis, RNA polymerase II–based transcription, and mitochondrial functions is the most prominent component that coordinates the direct coupling between growth rate and stress survival. This suggests that the coupling has integrated at a very basic level of cellular growth control.
Functional requirements for survival of specific stresses
After correction of mutant survival for growth rate, the phenotypes of mutants before and after the 38°C treatment became more similar (; p < 0.0001 in all cases). This revealed that the contribution of stress-specific survival mechanisms has become more prominent upon growth rate correction. However, the correlation between any two severe stresses, either before or after the 38°C treatment, decreased
after this correction, which confirmed that the general aspect of the acclimatization—growth rate—which had now been now corrected for, was a major determinant of the cross-tolerance. The groups that remain affected in stress survival after the growth rate correction allow us to analyze the specific functions required for the survival of the three severe stresses individually (Supplemental Table S4). Indeed, in the mutant pools growing at 30°C, we no longer found groups that were significantly sensitive to multiple stresses, suggesting that growth rate was the most prominent general aspect of stress survival. After the 38°C pretreatment, we did find general stress sensitivity of mutant groups involved in vesicle-mediated transport and cytoskeleton organization. These groups are likely important for cellular remodeling upon environmental change (Hillenmeyer et al., 2008
; Mousley et al., 2008
), and in our experiments they appear to be generally important for the response to a nonlethal stress, leading to increased survival. In general, we conclude that there is a difference in the groups of genes responsible for survival of severe stress and those involved in growth
in the presence of and phenotypic adaptation to chemical or physical stress.
Correlation coefficients of severe stress survival of all deletants after growth rate correction.
Stress tolerance acquisition
The study was designed to identify processes that play a role in the acquisition of stress (cross-) tolerance. We identified the reduction in growth rate to be an important general factor in the induction of stress survival. We know, however, that most stresses lead to the down-regulation of transcription of mRNA involved in translation and ribosome biogenesis but also the up-regulation of stress-related genes by Msn2p and Msn4p (Gasch et al., 2000
; Causton et al., 2001
). No indication of a role for these genes in the acquisition of stress tolerance could be found in our screen, and we decided to test the involvement of the Msn2/4p–regulated genes directly, using a double knockout for these transcription factors. The msn2 msn4
double-deletion mutant had much longer lag phases upon inoculation in fresh medium, but the maximal growth rate was not significantly different from that of the wild-type strain (0.38 ± 0.01 for wild-type and 0.36 ± 0.05 for the double-deletion strain). Remarkably, although higher numbers of nonviable cells were present during both normal exponential growth and during the 38°C treatment, both stress survival and stress cross-tolerance acquisition were largely unaffected (). These results were confirmed in the BY4741 background (unpublished data). Only the acquisition of acid-induced heat tolerance was much lower than in the isogenic wild-type strain. These data strongly suggest that Msn2p and Msn4p are largely dispensable for the acquisition of stress tolerance
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 (more ...)
Deficiency of stress tolerance acquisition relied on roughly three major cellular functions ( and Supplemental Table S5). Deletants of genes in several groups involved in vesicular protein transport and the actin cytoskeleton were defective in severe stress tolerance acquisition, as well as those in several groups involved in transcription, mRNA modification, and translation, in addition to import and export from the nucleus. Of interest, deletion of genes in quite a number of groups involved in chromatin remodeling, notably with the Swr1 and Rpd3 histone deacetylase complexes, resulted in a stress tolerance acquisition deficiency toward acid and heat stress but not oxidative stress. These groups were previously shown to be relevant for the transient up- and down-regulation of transcription in response to environmental change (Alejandro-Osorio et al., 2009
; Ruiz-Roig et al., 2010
). Although the functions that were required for tolerance acquisition toward three severe stresses were related, the exact aspects of these functions differed among the individual stresses.
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, (more ...)