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Glutathione and Gts1p drive beneficial variability in the cadmium resistances of individual yeast cells.

Smith MC, Sumner ER, Avery SV - Mol. Microbiol. (2007)

Bottom Line: Gts1p stabilizes these oscillations and was found to be required for heterogeneous Cd and hydrogen-peroxide resistance, through the same pathway as Gsh1p.Expression of GTS1 from a constitutive tet-regulated promoter suppressed oscillations and heterogeneity in GSH content, and resulted in decreased variation in stress resistance.The results establish a novel molecular mechanism for single-cell heterogeneity, and demonstrate experimentally fitness advantages that depend on deterministic variation in gene expression within cell populations.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Institute of Genetics, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
Phenotypic heterogeneity among individual cells within isogenic populations is widely documented, but its consequences are not well understood. Here, cell-to-cell variation in the stress resistance of Saccharomyces cerevisiae, particularly to cadmium, was revealed to depend on the antioxidant glutathione. Heterogeneity was decreased strikingly in gsh1 mutants. Furthermore, cells sorted according to differing reduced-glutathione (GSH) contents exhibited differing stress resistances. The vacuolar GSH-conjugate pathway of detoxification was implicated in heterogeneous Cd resistance. Metabolic oscillations (ultradian rhythms) in yeast are known to modulate single-cell redox and GSH status. Gts1p stabilizes these oscillations and was found to be required for heterogeneous Cd and hydrogen-peroxide resistance, through the same pathway as Gsh1p. Expression of GTS1 from a constitutive tet-regulated promoter suppressed oscillations and heterogeneity in GSH content, and resulted in decreased variation in stress resistance. This enabled manipulation of the degree of gene expression noise in cultures. It was shown that cells expressing Gts1p heterogeneously had a competitive advantage over more-homogeneous cell populations (with the same mean Gts1p expression), under continuous and fluctuating stress conditions. The results establish a novel molecular mechanism for single-cell heterogeneity, and demonstrate experimentally fitness advantages that depend on deterministic variation in gene expression within cell populations.

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Influence of Gsh1p and Ycf1p on heterogeneous resistance to Cd and H2O2. Exponential-phase cultures of S. cerevisiae BY4743 (○) or isogenic deletion strains gsh1Δ (•) or ycf1Δ (□) were plated onto YPD supplemented with either Cd(NO3)2 (A) or H2O2 (B). Viability (colony formation) was determined after incubation for up to 8 days at 30°C, and converted to percentages by reference to growth on unsupplemented control medium. The points represent means from three replicate determinations ± SEM. Typical results from one of at least three independent experiments are shown.
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fig01: Influence of Gsh1p and Ycf1p on heterogeneous resistance to Cd and H2O2. Exponential-phase cultures of S. cerevisiae BY4743 (○) or isogenic deletion strains gsh1Δ (•) or ycf1Δ (□) were plated onto YPD supplemented with either Cd(NO3)2 (A) or H2O2 (B). Viability (colony formation) was determined after incubation for up to 8 days at 30°C, and converted to percentages by reference to growth on unsupplemented control medium. The points represent means from three replicate determinations ± SEM. Typical results from one of at least three independent experiments are shown.

Mentions: It was hypothesized that any variation in the cellular GSH contents of individual yeast cells could cause heterogeneity in GSH-dependent phenotypes. To test this, heterogeneity in a gsh1Δ-deletion strain (defective for the rate-limiting step of glutathione synthesis) was compared with that of wild-type cells. Heterogeneity was compared according to the relative gradients of dose–response curves, as described previously (Sumner et al., 2003; Bishop et al., 2007). Dose–response plots for the gsh1Δ mutant were shifted left relative to the wild type (Fig. 1). This indicated a culture-averaged sensitisation of gsh1Δ cells to Cd and H2O2, consistent with previous results (Wu and Moye-Rowley, 1994; Grant et al., 1996). Moreover, loss of Gsh1p was also associated with decreased cell-to-cell variability. Thus, loss of viability of mutant cells occurred over a narrower range of stressor doses (producing a steeper dose–response curve) than wild-type cells. An approximate 15-fold decline in viability of gsh1Δ cells (from 35% to 2.4%) resulted from only an ∼11% increase in Cd concentration (from 90 to 100 μM), whereas a similar loss of viability in wild-type cells required a > 70% increase in Cd concentration (Fig. 1A). No viable gsh1Δ cells could be detected at > 100 μM Cd. This degree of homogeneity resulting from a single gene knockout [equating to a heterogeneity ratio (HR) value ∼0.41; see Experimental procedures] was unprecedented in our experience, and was highly reproducible (further plots for wild-type and gsh1Δ cells are shown in Fig. 4B and D). GSH also contributed slightly to heterogeneous H2O2 resistance (Fig. 1B), but this effect (i.e. the difference in kill gradient for gsh1Δ versus wild-type cells) was considerably less marked than for Cd.


Glutathione and Gts1p drive beneficial variability in the cadmium resistances of individual yeast cells.

Smith MC, Sumner ER, Avery SV - Mol. Microbiol. (2007)

Influence of Gsh1p and Ycf1p on heterogeneous resistance to Cd and H2O2. Exponential-phase cultures of S. cerevisiae BY4743 (○) or isogenic deletion strains gsh1Δ (•) or ycf1Δ (□) were plated onto YPD supplemented with either Cd(NO3)2 (A) or H2O2 (B). Viability (colony formation) was determined after incubation for up to 8 days at 30°C, and converted to percentages by reference to growth on unsupplemented control medium. The points represent means from three replicate determinations ± SEM. Typical results from one of at least three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2167119&req=5

fig01: Influence of Gsh1p and Ycf1p on heterogeneous resistance to Cd and H2O2. Exponential-phase cultures of S. cerevisiae BY4743 (○) or isogenic deletion strains gsh1Δ (•) or ycf1Δ (□) were plated onto YPD supplemented with either Cd(NO3)2 (A) or H2O2 (B). Viability (colony formation) was determined after incubation for up to 8 days at 30°C, and converted to percentages by reference to growth on unsupplemented control medium. The points represent means from three replicate determinations ± SEM. Typical results from one of at least three independent experiments are shown.
Mentions: It was hypothesized that any variation in the cellular GSH contents of individual yeast cells could cause heterogeneity in GSH-dependent phenotypes. To test this, heterogeneity in a gsh1Δ-deletion strain (defective for the rate-limiting step of glutathione synthesis) was compared with that of wild-type cells. Heterogeneity was compared according to the relative gradients of dose–response curves, as described previously (Sumner et al., 2003; Bishop et al., 2007). Dose–response plots for the gsh1Δ mutant were shifted left relative to the wild type (Fig. 1). This indicated a culture-averaged sensitisation of gsh1Δ cells to Cd and H2O2, consistent with previous results (Wu and Moye-Rowley, 1994; Grant et al., 1996). Moreover, loss of Gsh1p was also associated with decreased cell-to-cell variability. Thus, loss of viability of mutant cells occurred over a narrower range of stressor doses (producing a steeper dose–response curve) than wild-type cells. An approximate 15-fold decline in viability of gsh1Δ cells (from 35% to 2.4%) resulted from only an ∼11% increase in Cd concentration (from 90 to 100 μM), whereas a similar loss of viability in wild-type cells required a > 70% increase in Cd concentration (Fig. 1A). No viable gsh1Δ cells could be detected at > 100 μM Cd. This degree of homogeneity resulting from a single gene knockout [equating to a heterogeneity ratio (HR) value ∼0.41; see Experimental procedures] was unprecedented in our experience, and was highly reproducible (further plots for wild-type and gsh1Δ cells are shown in Fig. 4B and D). GSH also contributed slightly to heterogeneous H2O2 resistance (Fig. 1B), but this effect (i.e. the difference in kill gradient for gsh1Δ versus wild-type cells) was considerably less marked than for Cd.

Bottom Line: Gts1p stabilizes these oscillations and was found to be required for heterogeneous Cd and hydrogen-peroxide resistance, through the same pathway as Gsh1p.Expression of GTS1 from a constitutive tet-regulated promoter suppressed oscillations and heterogeneity in GSH content, and resulted in decreased variation in stress resistance.The results establish a novel molecular mechanism for single-cell heterogeneity, and demonstrate experimentally fitness advantages that depend on deterministic variation in gene expression within cell populations.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Institute of Genetics, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
Phenotypic heterogeneity among individual cells within isogenic populations is widely documented, but its consequences are not well understood. Here, cell-to-cell variation in the stress resistance of Saccharomyces cerevisiae, particularly to cadmium, was revealed to depend on the antioxidant glutathione. Heterogeneity was decreased strikingly in gsh1 mutants. Furthermore, cells sorted according to differing reduced-glutathione (GSH) contents exhibited differing stress resistances. The vacuolar GSH-conjugate pathway of detoxification was implicated in heterogeneous Cd resistance. Metabolic oscillations (ultradian rhythms) in yeast are known to modulate single-cell redox and GSH status. Gts1p stabilizes these oscillations and was found to be required for heterogeneous Cd and hydrogen-peroxide resistance, through the same pathway as Gsh1p. Expression of GTS1 from a constitutive tet-regulated promoter suppressed oscillations and heterogeneity in GSH content, and resulted in decreased variation in stress resistance. This enabled manipulation of the degree of gene expression noise in cultures. It was shown that cells expressing Gts1p heterogeneously had a competitive advantage over more-homogeneous cell populations (with the same mean Gts1p expression), under continuous and fluctuating stress conditions. The results establish a novel molecular mechanism for single-cell heterogeneity, and demonstrate experimentally fitness advantages that depend on deterministic variation in gene expression within cell populations.

Show MeSH
Related in: MedlinePlus