Uracil

The following fungal strains were used: Candida albicans ATCC 10,231, Candida glabrata ATCC 90,030, Candida krusei ATCC 6258, Candida parapsilosis ATCC 22,019, Saccharomyces cerevisiae ATCC 9763, Candida albicans clinical isolates^{40 (link),41 (link)}: B3, B4, Gu4, Gu5, F2, F5, Candida glabrata clinical isolates^{34 (link)}: CZD 373, CZD 377, CZD 513, Gd 310, Saccharomyces cerevisiae strains^{35 (link),36 (link),42 (link)}: AD-MDR1-GFP, AD1-8u⁻, AD-CDR1-GFP, AD-CDR2-GFP. Fungal strains were routinely grown over 18 h at 30 °C in YPG liquid medium (1% m/V yeast extract, 1% m/V peptone, 2% m/V glucose) in a shaking incubator (INFORS HT Bottmingen, Switzerland). For growth on solid media, 1.5% m/V agar was added to the YPG medium. For antimicrobial activity assays RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) medium buffered to pH 7.0 was used for all strains except for Saccharomyces cerevisiae mutants AD-MDR1-GFP, AD1-8u⁻, AD-CDR1-GFP, AD-CDR2-GFP. Saccharomyces cerevisiae mutants were grown in 0.67% m/V yeast nitrogen base medium without amino acids, folic acid, p-aminobenzoic acid, with ammonium sulphate (MP Biomedicals, Irvine, CA, USA) supplemented with 2% m/V glucose, 0.192% m/V yeast synthetic drop-out medium supplement without uracil (Sigma-Aldrich, St. Louis, MO, USA) and 0.0076% m/V uracil (Sigma-Aldrich, St. Louis, MO, USA).

H. volcanii strain H26 was grown in selective CA medium (Allers et al., 2004 (link)) (0.5 g/L Bacto™ Casamino acids; pH 7.2 adjusted with KOH) modified with an expanded trace element solution (referred to as CAB) (Duggin et al., 2015 (link)). Cells were grown at 45°C in liquid medium while rotating (volumes up to 5 ml) or shaking (volumes > 5 ml).
S. acidocaldarius strain MW001 was grown in basal Brock medium (pH 3.5) (Brock et al., 1972 (link)) supplemented with 0.1% (w/v) NZ-amine (Sigma) and 0.2% (w/v) dextrin. Cells were grown at 75°C in liquid medium while shaking.
Since both strains are auxotroph for uracil (H26, ΔpyrE2 and MW001, ΔpyrEF), growth media were supplemented with uracil (Sigma) at a defined concentration (50 μg/ml for H26; 10 μg/ml for MW001). Further details on H26 and MW001 are listed in Supplementary Table 1.

Defined minimal medium contained 6.7 g/L of yeast nitrogen base without amino acids (catalog no. Y0626; Sigma-Aldrich, MO, USA), 20 g/L of glucose, 380 mg/L leucine (catalog no. 172130250; Acros Organics, CA, USA), 76 mg/L uracil (catalog no. 157301000; Acros Organics, CA, USA), and various concentrations of [EMIM][OAc] (>95% purity) (IoLiTec, AL, USA). Synthetic complete medium without leucine and uracil (SC-Leu-Ura) was prepared with 6.7 g/L of yeast nitrogen base without amino acids; 1.46 g/L of yeast synthetic dropout medium supplement without uracil, leucine, and tryptophan (catalog no. Y1771; Sigma-Aldrich, MO, USA); 76 mg/L tryptophan (catalog no. 172110250; Acros Organics, CA, USA), 20 g/L glucose, and various concentrations of [EMIM][OAc]. SC without leucine (SC-Leu) was prepared by adding 76 mg/L uracil to SC-Leu-Ura medium.

E. coli DH5ɑ was used for propagating all plasmids and grown at 37 °C in Luria Broth (LB) medium containing the appropriate antibiotics for plasmid selection (ampicillin 100 μg/mL, chloramphenicol 34 μg/mL, or kanamycin 50 μg/mL). S. cerevisiae strain BY4741^{39 (link)} (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used for all yeast experiments. For succinic acid experiments, fully complemented yeast strains were created by restoring the missing auxotrophic markers on a single-copy plasmid^{37 (link)}. Yeast extract peptone dextrose (YPD) was used for culturing cells in preparation for transformation: 1% (w/v) Bacto Yeast Extract (Merck), 2% (w/v) Bacto Peptone (Merck), 2% glucose (VWR). Fluorescent reporter assay experiments were performed in synthetic complete (SC) medium: 2% (w/v) glucose (VWR), 0.67% (w/v) Yeast Nitrogen Base without amino acids (Sigma), 0.14% (w/v) Yeast Synthetic Drop-out Medium Supplements without histidine, leucine, tryptophan, and uracil (Sigma), 20 mg/L uracil (Sigma), 100 mg/L leucine (Sigma), 20 mg/L histidine (Sigma), and 20 mg/mL tryptophan (Sigma). Succinic acid production experiments were performed in synthetic minimal (SD) medium: 2% (w/v) glucose (VWR), and 0.67% (w/v) Yeast Nitrogen Base without amino acids (Sigma).

S. cerevisiae strains used in this study are listed in Table 1. Yeast cells were grown aerobically at 26 °C in an orbital shaker (at 140 rpm), with a ratio of flask volume:medium volume of 5:1. The growth medium used was synthetic complete (SC) medium, containing drop-out, 2% (w/v) glucose (ThermoFisher Scientific, Waltham, MA, USA), and 0.67% (w/v) yeast nitrogen base without amino acids (BD BioSciences, San Jose, CA, USA), supplemented with appropriate amino acids or nucleotides [0.008% (w/v) histidine (Sigma Aldrich, St. Louis, MO, USA), 0.038% (w/v) methionine (Sigma Aldrich, St. Louis, MO, USA), 0.04% (w/v) leucine (Sigma Aldrich, St. Louis, MO, USA), and 0.008% (w/v) uracil (Sigma Aldrich, St. Louis, MO, USA)]. Deletion of AFT1 in ncr1Δ cells was performed using a deletion fragment containing HIS3 and the flanking regions of AFT1. Deletion of YCK3 in wild-type cells was performed using a deletion fragment containing KanMX4 and the flanking regions of YCK3. Yeast cells were transformed using the lithium acetate/single-stranded carrier DNA/PEG method as described [96 (link)]. Cells were selected by growing in a medium lacking histidine or containing geneticin (300 µg mL⁻¹), and gene deletion was confirmed by standard PCR procedures.