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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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Single-cell Cd resistance in wild-type cells is non-inheritable. Exponential-phase wild-type (BY4743) or gsh1Δ cells were originally plated onto YPD supplemented with Cd(NO3)2 concentrations that gave either 100% viability (minus-Cd control), ∼50% viability (wild type, 150 μM Cd; gsh1Δ, 80 μM) or ∼10% viability (wild type, 200 μM Cd; gsh1Δ, 100 μM); viability being defined as colony-forming ability. Single-colony isolates from these plates (which in the case of the Cd-supplemented plates were Cd-resistant colonies) were subsequently inoculated directly in to liquid YPD medium in the absence of Cd(NO3)2 and, after 24 h exponential growth, % resistance to Cd was retested by plating aliquots of each isolate onto YPD agar supplemented or not with Cd(NO3)2. Data are shown for eight wild-type and eight gsh1Δ isolates, each obtained from plates that originally yielded the % viabilities indicated, and each retested for % resistance at the Cd concentrations which normally give ∼50% (□) or ∼10% viability (▪) of the relevant strains (see above for relevant concentrations). Percentage viabilities were calculated with reference to growth on minus-Cd control plates, and data are averaged from three replicate determinations ± SEM. Asterisks denote isolates exhibiting Cd resistance that is significantly higher (P < 0.05, according to Student's t-test) at both tested Cd concentrations than for control cultures not previously exposed to Cd.
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fig02: Single-cell Cd resistance in wild-type cells is non-inheritable. Exponential-phase wild-type (BY4743) or gsh1Δ cells were originally plated onto YPD supplemented with Cd(NO3)2 concentrations that gave either 100% viability (minus-Cd control), ∼50% viability (wild type, 150 μM Cd; gsh1Δ, 80 μM) or ∼10% viability (wild type, 200 μM Cd; gsh1Δ, 100 μM); viability being defined as colony-forming ability. Single-colony isolates from these plates (which in the case of the Cd-supplemented plates were Cd-resistant colonies) were subsequently inoculated directly in to liquid YPD medium in the absence of Cd(NO3)2 and, after 24 h exponential growth, % resistance to Cd was retested by plating aliquots of each isolate onto YPD agar supplemented or not with Cd(NO3)2. Data are shown for eight wild-type and eight gsh1Δ isolates, each obtained from plates that originally yielded the % viabilities indicated, and each retested for % resistance at the Cd concentrations which normally give ∼50% (□) or ∼10% viability (▪) of the relevant strains (see above for relevant concentrations). Percentage viabilities were calculated with reference to growth on minus-Cd control plates, and data are averaged from three replicate determinations ± SEM. Asterisks denote isolates exhibiting Cd resistance that is significantly higher (P < 0.05, according to Student's t-test) at both tested Cd concentrations than for control cultures not previously exposed to Cd.

Mentions: To confirm that the single-cell phenotypes were not inheritable (i.e. not genotypic), a number of ‘resistant’ colonies isolated after an initial exposure to the stressor on agar were subcultured to YPD broth in the absence of stressor. These were grown for 24 h in the broth before resistance was retested (non-inheritable stress resistance is normally lost within 24 h of out-growth) (Bishop et al., 2007). Cadmium was selected for these tests because it yielded the strongest phenotype in gsh1Δ cells and because of its genotoxicity (Jin et al., 2003). This should enhance detection of any genotypic variation. The initial isolation of ‘resistant’ wild-type and gsh1Δ cells (via colony formation on Cd) was at Cd concentrations (see Fig. 2 legend) that gave either 100% viability (minus-Cd control), ∼50% viability or ∼10% viability. Resistant isolates subcultured from these plates (see above) were retested for resistance at each Cd concentration. Wild-type isolates that had not previously been exposed to Cd (‘control’ isolates) exhibited the anticipated ∼50% and ∼10% colony formation when tested at the relevant Cd concentrations (Fig. 2). With one exception out of six colonies tested, the same was true of cultures derived from Cd-resistant wild-type colonies (‘50’ and ‘10’ isolates in Fig. 2). This indicated that these cells did not retain their resistances during the intervening growth period in the absence of Cd. Therefore, the variable Cd resistances of wild-type cells was primarily due to phenotypic rather than genotypic (inheritable) heterogeneity. In contrast, occasional Cd resistance in gsh1Δ cultures appeared to be primarily genotypic rather than phenotypic: five out of six resistant isolates exhibited inheritable Cd resistance (Fig. 2) (note that the higher Cd concentration gave closer to 0% than the normal 10% viability in some gsh1Δ retests, reflecting the difficulty of reproducing equivalent dosages in successive experiments where the kill gradient is very steep; Fig. 1A). The results indicated that much of the residual detected variability in gsh1Δ cells was probably of genotypic origin. Therefore, the relative gradients shown in Fig. 1A actually underestimate the true impact of GSH on phenotypic (non-genotypic) heterogeneity. It is concluded that elimination of GSH synthesis eliminates most of the non-genotypic heterogeneity in Cd resistance, i.e. there is no GSH-independent mechanism that makes a substantial contribution to this heterogeneity phenotype.


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

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

Single-cell Cd resistance in wild-type cells is non-inheritable. Exponential-phase wild-type (BY4743) or gsh1Δ cells were originally plated onto YPD supplemented with Cd(NO3)2 concentrations that gave either 100% viability (minus-Cd control), ∼50% viability (wild type, 150 μM Cd; gsh1Δ, 80 μM) or ∼10% viability (wild type, 200 μM Cd; gsh1Δ, 100 μM); viability being defined as colony-forming ability. Single-colony isolates from these plates (which in the case of the Cd-supplemented plates were Cd-resistant colonies) were subsequently inoculated directly in to liquid YPD medium in the absence of Cd(NO3)2 and, after 24 h exponential growth, % resistance to Cd was retested by plating aliquots of each isolate onto YPD agar supplemented or not with Cd(NO3)2. Data are shown for eight wild-type and eight gsh1Δ isolates, each obtained from plates that originally yielded the % viabilities indicated, and each retested for % resistance at the Cd concentrations which normally give ∼50% (□) or ∼10% viability (▪) of the relevant strains (see above for relevant concentrations). Percentage viabilities were calculated with reference to growth on minus-Cd control plates, and data are averaged from three replicate determinations ± SEM. Asterisks denote isolates exhibiting Cd resistance that is significantly higher (P < 0.05, according to Student's t-test) at both tested Cd concentrations than for control cultures not previously exposed to Cd.
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Related In: Results  -  Collection

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fig02: Single-cell Cd resistance in wild-type cells is non-inheritable. Exponential-phase wild-type (BY4743) or gsh1Δ cells were originally plated onto YPD supplemented with Cd(NO3)2 concentrations that gave either 100% viability (minus-Cd control), ∼50% viability (wild type, 150 μM Cd; gsh1Δ, 80 μM) or ∼10% viability (wild type, 200 μM Cd; gsh1Δ, 100 μM); viability being defined as colony-forming ability. Single-colony isolates from these plates (which in the case of the Cd-supplemented plates were Cd-resistant colonies) were subsequently inoculated directly in to liquid YPD medium in the absence of Cd(NO3)2 and, after 24 h exponential growth, % resistance to Cd was retested by plating aliquots of each isolate onto YPD agar supplemented or not with Cd(NO3)2. Data are shown for eight wild-type and eight gsh1Δ isolates, each obtained from plates that originally yielded the % viabilities indicated, and each retested for % resistance at the Cd concentrations which normally give ∼50% (□) or ∼10% viability (▪) of the relevant strains (see above for relevant concentrations). Percentage viabilities were calculated with reference to growth on minus-Cd control plates, and data are averaged from three replicate determinations ± SEM. Asterisks denote isolates exhibiting Cd resistance that is significantly higher (P < 0.05, according to Student's t-test) at both tested Cd concentrations than for control cultures not previously exposed to Cd.
Mentions: To confirm that the single-cell phenotypes were not inheritable (i.e. not genotypic), a number of ‘resistant’ colonies isolated after an initial exposure to the stressor on agar were subcultured to YPD broth in the absence of stressor. These were grown for 24 h in the broth before resistance was retested (non-inheritable stress resistance is normally lost within 24 h of out-growth) (Bishop et al., 2007). Cadmium was selected for these tests because it yielded the strongest phenotype in gsh1Δ cells and because of its genotoxicity (Jin et al., 2003). This should enhance detection of any genotypic variation. The initial isolation of ‘resistant’ wild-type and gsh1Δ cells (via colony formation on Cd) was at Cd concentrations (see Fig. 2 legend) that gave either 100% viability (minus-Cd control), ∼50% viability or ∼10% viability. Resistant isolates subcultured from these plates (see above) were retested for resistance at each Cd concentration. Wild-type isolates that had not previously been exposed to Cd (‘control’ isolates) exhibited the anticipated ∼50% and ∼10% colony formation when tested at the relevant Cd concentrations (Fig. 2). With one exception out of six colonies tested, the same was true of cultures derived from Cd-resistant wild-type colonies (‘50’ and ‘10’ isolates in Fig. 2). This indicated that these cells did not retain their resistances during the intervening growth period in the absence of Cd. Therefore, the variable Cd resistances of wild-type cells was primarily due to phenotypic rather than genotypic (inheritable) heterogeneity. In contrast, occasional Cd resistance in gsh1Δ cultures appeared to be primarily genotypic rather than phenotypic: five out of six resistant isolates exhibited inheritable Cd resistance (Fig. 2) (note that the higher Cd concentration gave closer to 0% than the normal 10% viability in some gsh1Δ retests, reflecting the difficulty of reproducing equivalent dosages in successive experiments where the kill gradient is very steep; Fig. 1A). The results indicated that much of the residual detected variability in gsh1Δ cells was probably of genotypic origin. Therefore, the relative gradients shown in Fig. 1A actually underestimate the true impact of GSH on phenotypic (non-genotypic) heterogeneity. It is concluded that elimination of GSH synthesis eliminates most of the non-genotypic heterogeneity in Cd resistance, i.e. there is no GSH-independent mechanism that makes a substantial contribution to this heterogeneity phenotype.

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