Fig 1.
Relationship between yeast strain fitness in high copper medium and CUP1 copy number.
The fitness value shown on the x-axis is the mean of two independent experiments. Significant observations are recorded as 0, 1 or 2 if there was evidence (P < 0.05) that the log2 fold change differed relative to the control condition in independent fitness experiments (n = 3 for each of 2 independent experiments). The y-axis shows the mean absolute CUP1 copy number of each strain. Error bars show standard deviation (n = 3). Strains pictured with a larger point size (3019, 3032 and 3029) were the parental strains used in subsequent bulk segregant QTL experiments.
Fig 2.
Single nucleotide variant (SNV) frequency in spores generated from diploids following crosses between A) AWRI 3471 and AWRI 3470, and B) AWRI 3471 and AWRI 3807. SNV frequency is shown for each parent, the diploid generated from each cross (AWRI 3001 and AWRI 3811) and spore pools where each spore in the pool was classified as either copper tolerant (Cu-tol) or copper sensitive (Cu-sens).
Fig 3.
Heritability of SSU1 dependent copper tolerance and sensitivity assessed in defined medium containing either 0.25 or 10 mg/L copper.
(A) Growth of yeast AWRI 3471 (●) and AWRI 3807 (■), haploid derivatives of AWRI 796 and AWRI 1487 respectively. (B) Growth of AWRI 3811, a diploid derived from a cross between 3471 x 3807. (C) Growth of AWRI 3901 and AWRI 3902, two derivatives of the AWRI 3811 diploid each containing a deletion of SSU1 at chromosome XVI and VIII::XVI respectively. Filled lines; standard defined medium, dashed lines; defined medium containing 10 mg/L copper. Points show mean of three replicates with error bars indicating standard deviation.
Fig 4.
Effect of copper on fermentation, gene expression and protein production in AWRI 3471 and AWRI 4052.
(A) Growth as indicated by absorbance at 600 nm and (B) Sugar consumption of SSU1 wild type (AWRI 3471, blue) and ssu1(pr)Δ::ECM34(pr) (AWRI 4052, red) yeast strains during growth in defined medium with and without copper at 10 mg/L. Filled lines; standard defined medium, dashed lines; defined medium containing 10 mg/L copper. Arrow in (A) and (B) show sampling points used for RNAseq analysis. Points show mean of three replicates with error bars indicating standard deviation. (C) Relative transcript abundance grouped by Gene Ontology summary category for the contrast AWRI 4052 HCu–AWRI 3471 HCu, expressed as Log2 Fold Change (Log2FC) with red showing increased and blue decreased expression. Colour intensity highlights the P value score obtained from differential expression analysis undertaken with DEseq2. SSU1 was omitted from over-representation analysis and therefore does not appear in enriched ontology summaries. (D) Relative protein abundance shown as volcano plots with colours indicating increased (red) and decreased (blue) relative fold change for four contrasts i) AWRI 4052 –AWRI 3471, ii) AWRI 4052 HCu–AWRI 3471 HCu. Colours indicating increased (orange) and decreased (light blue) are used in plots iii) AWRI 4052 HCu–AWRI 4052, iv) AWRI 3471 HCu–AWRI 3471. Vertical dotted lines indicate Log2FC = 1 and horizontal dotted lines indicate an adjusted P value = 0.005. n = 3 in all cases. SSU1 is shown despite having P value > 0.005 in panel ii).
Fig 5.
Diagrammatic representation of the sulfate assimilation pathway.
Gene names in red and blue indicate up-regulated and down-regulated genes, respectively, comparing SSU1 over-expressing (AWRI 4052) yeast with control yeast (AWRI 3471) growing in medium containing 10 mg/L copper. SSU1 over-expression is indicated by a red helix. Gene names in black indicate no change in expression. Green circles represent copper ions. The structure of Cup1p is adapted from the crystal structure determined by Calderone et al [48]. Cysteine residues in the structure are coloured yellow. APS; Adenosine-5’-phosphosulfate, PAPS; phosphoadenosine phosphosulfate. The image was created with BioRender.com.
Fig 6.
Effect of copper concentration and MET3-MET14-MET16 over-expression on growth and, SO2 and H2S production in AWRI 3471 and AWRI 4052.
(A) Growth of AWRI 3471 in medium containing 0.25 mg/L and 10 mg/L copper. (B) Growth of AWRI 4052 in medium containing 0.25 mg/L and 10 mg/L copper. (C) The concentration of SO2 produced by AWRI 3471 and AWRI 4052 in medium containing 0.25 mg/L and 10 mg/L copper. (D) The concentration of H2S produced by AWRI 3471 and AWRI 4052 in medium containing 0.25 mg/L copper. Blue lines and bars; standard defined medium, red lines and bars; defined medium containing 10 mg/L copper, filled lines; plasmid carrying KANMX marker only, dashed lines; plasmid carrying KANMX, MET3, MET14 and MET16. Points and bars show mean of three replicates with error bars indicating standard deviation.
Fig 7.
The combined effect of copper concentration (0.25, 4 and 6 mg/L) and SO4 concentration (20 and 195 mg/L) on the growth and fermentation kinetics of the yeast strain AWRI 4052.
Points show the mean of three replicates with error bars indicating standard deviation.
Table 1.
Yeast strains used in this work.