Potassium acetate

Curing of the [GAR⁺] phenotype (Figure 2B) was achieved by transformation of lactic acid induced cells with PDJ281 (Hygromycin resistant, Supplementary file 1D), restreaking multiple transformants to single colonies on YPD plates containing 400 ug/mL Hygromycin (Gold Biotechnology, Olivette, MO) a total of five times, then restreaking in the same manner on YPD plates twice before confirming each clone had lost the plasmid by testing on YPD+Hygromycin. These clones were grown in liquid YPD cultures before testing on GLY + GlcN media.
Spontaneous and lactic acid-induced [GAR⁺] diploids (W303 background, Figure 2—figure supplement 1) were isolated from single colonies grown on GLY + GlcN or GLY + GlcN + lactic acid plates and grown in Pre-SPO liquid media (YPD with 6% glucose) for 3 days at room temperature. Cells were then pelleted and washed twice in SPO media (1% Potassium Acetate (Sigma-Aldrich), 320 mg CSM-Met powder (Sunrise Science, San Diego, CA), 20 mg Methionine (Sigma-Aldrich) per liter), and then diluted ten-fold to obtain 3 mL cultures in SPO media. These cultures were incubated on a rotary wheel for one week at room temperature, before dissecting tetrads on a Singer Instruments MSM400 (Somerset, UK). Recovered spores were grown to saturation in liquid YPD before testing on GLY + GlcN media.

One mg/mL of sodium hyaluronate (Sigma-Aldrich, MO, USA) (previously dried in a vacuum oven with phosphorus pentoxide (Sigma-Aldrich, MO, USA) for 48 h) stock solution was diluted before use with an equal volume of 0.02 M phosphate buffer solution (pH 6.3, dissolved 2.5 g of monobasic sodium phosphate (Sigma-Aldrich, MO, USA), 1.0 g of anhydrous dibasic sodium phosphate, and 8.2 g of sodium chloride in water to make 1 L). Then, 10 mg/mL of BSA solution was diluted with 4 volumes of 0.5 M acetate buffer solution (dissolve 14 g of potassium acetate (Sigma-Aldrich, MO, USA) and 20.5 mL of glacial acetic acid in water to make 1 L, and then adjust with 4 M hydrochloric acid to pH 3.1). The standard solution was prepared immediately before use by dissolving standard hyaluronidase in cold hydrolyzed gelatin solution (mix 250 mL of phosphate buffer solution with 250 mL of water and dissolve 330 mg of hydrolyzed gelatin in the mixture) to obtain a solution of 10 U/mL. The sample solution was also properly diluted with cold hydrolyzed gelatin before use. The sample detection reaction temperature was 42 °C [55 (link)].

Distilled water (DW), dimethyl sulfoxide (DMSO), methanol (MeOH), ethyl acetate (EA), standard antifungals (amphotericin B and terbinafine), nutrient agar, and HPLC-grade polyphenols (vanillic acid, rutin, plumbagin, thymoquinone, gallic acid, catechin, syringic acid, coumaric acid, emodin, gentisic acid, caffeic acid, ferulic acid, luteolin, apigenin, myricetin, quercetin and kaempferol), 2,2-diphenyl-1-picryhydrazyl (DPPH), ascorbic acid, sulfuric acid, gallic acid, potassium acetate, quercetin, ammonium molybdate, aluminum chloride, trichloroacetic acid (TCA), potassium ferricyanide, and ferric chloride were purchased from Sigma Aldrich, United States. Phosphate buffer and Folin–Ciocalteu (FC) reagent were procured from Riedel-de Haen, Germany. Sabouraud dextrose agar (SDA) was purchased from Oxoid, England, Tween-80 from Merck-Schuchardt, United States, and RPMI-1640 was obtained from United traders, Rawalpindi, Pakistan.

Diploid strains were inoculated into 5 mL of YP plus 1% potassium acetate (Sigma-Aldrich) and grown overnight at 30°C with shaking. For tetrad enrichment, 1 mL of the overnight culture was harvested by centrifugation (0.4 g × 3 min at room temperature), washed twice with 1 mL dH₂O (0.4 g × 3 min at room temperature), and finally resuspended in 2 mL 2% potassium acetate in a 50 mL Falcon tube. Monad enrichment was performed similarly but with the following changes: 2 mL of overnight culture were harvested, and cells were finally resuspended in 10 mL of dH₂O in a 250 mL Erlenmeyer flask. The cells were then incubated for 48 h at 30°C with shaking. Harvested spores were stored at 4°C.