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other·microbiology, genetics, oenology, evolutionary biology, biochemistry·PMC10081759

SO2 and copper tolerance exhibit an evolutionary trade-off in Saccharomyces cerevisiae

PLOS Genetics · 12 authors, 3 centres

AI SUMMARY

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POPULATIONSaccharomyces cerevisiae wine yeast strains (94 strains for copy number analysis; specific strains AWRI 796, AWRI 1537, AWRI 1487 and their derivatives for mechanistic studies)

INTERVENTIONCopper exposure at varying concentrations (0.25–10 mg/L); SSU1 overexpression via ECM34 promoter replacement; MET3/MET14/MET16 overexpression; sulfate-limited medium

COMPARISONCopper-tolerant vs copper-sensitive strains with high or low CUP1 copy number; SSU1 wild-type vs SSU1-overexpressing isogenic strains; sulfate-replete vs sulfate-limited conditions

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TL;DR

This study reveals an evolutionary trade-off between copper and sulfur dioxide (SO₂) tolerance in wine yeast (Saccharomyces cerevisiae). While high copy number of the CUP1 gene is known to confer copper tolerance, the authors found that CUP1 copy number alone is a poor predictor of copper tolerance in wine yeasts. Instead, overexpression of the SSU1 gene (which confers SO₂ tolerance) induces sulfur limitation under copper stress, making even high-CUP1-copy strains copper-sensitive, with important implications for commercial yeast strain development and winemaking practices.

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**Background:** Copper and sulfur dioxide (SO₂) are widely used in the wine industry—copper as a fungicide and for aroma management, SO₂ as an antimicrobial and antioxidant. Saccharomyces cerevisiae has evolved tolerance mechanisms for both: copper tolerance is primarily mediated by copy number variation of the metallothionein gene CUP1, while SO₂ tolerance is conferred by overexpression of the efflux pump SSU1, often via a reciprocal translocation between chromosomes VIII and XVI that places SSU1 under control of the ECM34 promoter. Previous work had noted a negative association between SO₂ and copper tolerance in wine yeasts, but the mechanistic basis was unknown.

**Methods:** The authors first determined CUP1 copy number in 94 wine yeast strains using qPCR, normalised against a single-copy molecular barcode inserted at the HO locus. They compared copy number to competitive fitness in medium containing 10 mg/L copper. Bulk segregant QTL analysis was performed on haploid progeny from crosses between a copper-tolerant, high-CUP1-copy strain (AWRI 796, ~25.6 copies) and two copper-sensitive parents—one with low CUP1 copy (AWRI 1537, ~2.5 copies) and one with high CUP1 copy (AWRI 1487, ~31.7 copies). Reciprocal hemizygosity analysis was used to validate SSU1's role. Transcriptomic (RNA-seq) and label-free quantitative proteomic analyses were conducted on isogenic strains differing only in SSU1 promoter (wild-type vs ECM34 promoter replacement) grown in 0.25 and 10 mg/L copper. Fermentation trials assessed growth and sugar utilisation under varying copper and sulfate concentrations. Overexpression of MET3, MET14, and MET16 (upstream of H₂S production in the sulfate assimilation pathway) was tested for effects on copper sensitivity.

**Key Results:** CUP1 copy number varied dramatically among 94 wine yeast strains (2 to 55 copies per cell) but showed no correlation with copper tolerance—many high-copy strains were copper-sensitive. QTL analysis of the cross between high-CUP1-copy tolerant and high-CUP1-copy sensitive parents identified loci on chromosomes VIII and XVI corresponding to the SSU1-ECM34 translocation region. Reciprocal deletion of SSU1 from the translocated chromosome (but not the wild-type chromosome) restored copper tolerance in the sensitive hybrid. Promoter replacement (ssu1(pr)Δ::ECM34(pr)) in the tolerant background was sufficient to confer copper sensitivity, causing a mean biomass decrease of 2.1 g/L DCW [95%CI: 1.8, 2.4] and a mean increase in residual sugar of 78 g/L [95%CI: 67, 86] at day 17. Transcriptomic analysis showed that SSU1 overexpression did not suppress CUP1 transcription; CUP1 transcript abundance was equivalent between genotypes. Instead, SSU1-overexpressing cells upregulated genes for sulfate uptake (SUL1, SOA1), sulfonate catabolism (JLP1), and sulfur-containing amino acid uptake (MUP1, MUP3, YCT1, OPT1, AGP3), indicating sulfur limitation. Proteomic analysis confirmed equivalent or increased Cup1p production in SSU1-overexpressing cells (6.5-fold relative increase, Padj=0.02), ruling out metallothionein limitation. Ssu1p abundance increased 107-fold (Padj=3.23e⁻¹³) with the ECM34 promoter. Overexpression of MET3/14/16 increased SO₂ production (mean increase 12.7 mg/L [95%CI: 9.7, 15.7] in AWRI 3471; 12.0 mg/L [95%CI: 0.2, 23.8] in AWRI 4052) and H₂S production (20.3 mg/L [95%CI: 15.2, 25.5] in AWRI 3471; 4.7 mg/L [95%CI: 1.5, 7.7] in AWRI 4052) but did not improve copper tolerance. In sulfate-limited medium (20 mg/L SO₄), the SSU1-overexpressing strain showed dramatically increased copper sensitivity—as little as 2 mg/L copper suppressed growth, and 10 mg/L almost completely abolished sugar utilisation. Fermentation time increased by 5.5 days [95%CI: 3.3, 7.6] at 4 mg/L copper under sulfate limitation, and fermentations at 6 mg/L copper were incomplete after 20 days.

**Clinical Implications:** While this is not a clinical study, the findings have direct practical implications for the wine industry and yeast biotechnology. The demonstration that SO₂ tolerance (via SSU1 overexpression) creates a metabolic trade-off with copper tolerance—even in strains with high CUP1 copy number—has important implications for commercial yeast strain selection and breeding. The authors suggest that less aggressive SSU1 promoter variants (natural variation in ECM34 promoter strength exists) should be considered during strain development to reduce metabolic burden. The findings also suggest that agricultural or oenological application of SO₂ may inadvertently select for continued CUP1 amplification, providing an evolutionary driver for the extreme CUP1 copy numbers observed in some yeasts.

PICO

PPOPULATION

Saccharomyces cerevisiae wine yeast strains (94 strains for copy number analysis; specific strains AWRI 796, AWRI 1537, AWRI 1487 and their derivatives for mechanistic studies)

IINTERVENTION

Copper exposure at varying concentrations (0.25–10 mg/L); SSU1 overexpression via ECM34 promoter replacement; MET3/MET14/MET16 overexpression; sulfate-limited medium

OOUTCOME

Copper tolerance (growth, biomass, fermentation kinetics); CUP1 copy number; SSU1 expression; transcriptomic and proteomic profiles; SO₂ and H₂S production

STUDY TYPE

other

SPECIALTY

microbiology

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100% · A

ORIGINAL PAPER

PMC10081759

SO2 and copper tolerance exhibit an evolutionary trade-off in Saccharomyces cerevisiae

PLOS Genetics

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ABSTRACT

Abstract Copper tolerance and SO 2 tolerance are two well-studied phenotypic traits of Saccharomyces cerevisiae . The genetic bases of these traits are the allelic expansion at the CUP1 locus and reciprocal translocation at the SSU1 locus, respectively. Previous work identified a negative association between SO 2 and copper tolerance in S . cerevisiae wine yeasts. Here we probe the relationship between SO 2 and copper tolerance and show that an increase in CUP1 copy number does not always impart copper tolerance in S . cerevisiae wine yeast. Bulk-segregant QTL analysis was used to identify variance at SSU1 as a causative factor in copper sensitivity, which was verified by reciprocal hemizygosity analysis in a strain carrying 20 copies of CUP1 . Transcriptional and proteomic analysis demonstrated that SSU1 over-expression did not suppress CUP1 transcription or constrain protein production and provided evidence that SSU1 over-expression induced sulfur limitation during exposure to copper. Finally, an SSU1 over-expressing strain exhibited increased sensitivity to moderately elevated copper concentrations in sulfur-limited medium, demonstrating that SSU1 over-expression burdens the sulfate assimilation pathway. Over-expression of MET 3/14/16 , genes upstream of H 2 S production in the sulfate assimilation pathway increased the production of SO 2 and H 2 S but did not improve copper sensitivity in an SSU1 over-expressing background. We conclude that copper and SO 2 tolerance are conditional traits in S . cerevisiae and provide evidence of the metabolic basis for their mutual exclusivity. These findings suggest an evolutionary driver for the extreme amplification of CUP1 observed in some yeasts.

CITATION EXPORT

Cristobal A Onetto, Dariusz R Kutyna, Radka Kolouchova et al.. SO2 and copper tolerance exhibit an evolutionary trade-off in Saccharomyces cerevisiae. PLOS Genetics. (2023). https://doi.org/10.1371/journal.pgen.1010692

AUTHORS

12 · 3 CENTRES

Cristobal A Onetto, Dariusz R Kutyna, Radka Kolouchova, Jane McCarthy, Anthony R Borneman, Simon A Schmidt, Justin C Fay, Geraldine Butler, Justin C Fay, Geraldine Butler, Justin C Fay, Geraldine Butler

1The Australian Wine Research Institute, Glen Osmond, South Australia, Australia
2School of Agriculture, Food and Wine, Faculty of Sciences, University of Adelaide, Adelaide, South Australia, Australia
3University of Rochester, UNITED STATES