Uracil

Haloferax volcanii strain H26 was kindly provided by Thorsten Allers (University of Nottingham, UK). It was and grown in complex medium [36] (link) or in synthetic medium [37] (link) supplemented with 8 µM FeSO₄ (Roth, P015.1), 0,1% (v/v) SL-6 trace element solution [38] (link) (all from Roth), 1 ml vitamin solution (Sigma Aldrich, B6891), 50 µg ml⁻¹ uracil (Applichem, A0667) and 100 mM MOPS pH 7.2 (Sigma Aldrich, M3183). All components of the synthetic medium were of the grade “per analysis” and thus free of phosphate, e.g. K₂HPO₄ (Roth, 6878.2), NH₄Cl (Applichem, A0988), glucose (Merck, 1083441000), NaCl (Roth, 3957.5), MgCl₂ (Roth, 2189.1), MgSO₄ (Applichem, A1037), KCl (Roth, 6781,1), CaCl₂ (Applichem, A3587), and Tris (A1086). If not otherwise stated, the synthetic medium was also supplemented with 0.5% (w/v) glucose as a C source, 10 mM NH₄Cl as a N source, and 1 mM K₂HPO₄ as a P source. For growth experiments with DNA as a source of P K₂HPO₄ was omitted and genomic DNA was added to a final concentration of 250 µg/ml. Cultures were grown in Erlenmeyer flasks in a rotary shaker at 42 °C and 250 rpm or in microtiter plates as described below.

S. cerevisiae strains deleted for multiple genes were generated using PCR deletion cassettes and marker rescue strategies as described before (Darwiche et al., 2017 (link)). Strains were grown in a minimal defined medium (containing 0.67% yeast nitrogen base without amino acids (US Biological, Salem, MA, United States), 0.73 g/L of an amino acid/nucleobase mixture, and 2% glucose) at 30°C under shaking condition at 180 rpm. The amino acid/nucleobase mixture contained 1.0 g adenine, 1.0 g argenine, 1.0 g histidine, 3.0 g leucine, 11.5 g lysine, 1.0 g methionine, 15.0 g threonine, 1.0 g tryptophan, and 2.0 g uracil (all from AppliChem GmbH, Darmstadt, Germany). For fatty acid analyses, pre-cultures were grown overnight in either SC-Ura or SC-Leu medium. The next morning, the main cultures were inoculated at 0.1 OD_600nm and after 24 h of growth, 3 or 5 OD_600nm units of cells and the corresponding culture medium were collected.

Media components were purchased from BD (Difco), USA. Amino acids (LAA21 set) were purchased from Sigma-Aldrich Chemie GmbH Munich, Germany. Uracil was purchased from AppliChem GmbH, Germany. Geneticin (G418) and phleomycin were purchased from InvivoGen, USA. γ-EV and VG were purchased from Bachem AG, Switzerland, and γ-EVG was synthesized by Kokusan Chemical Co., Ltd., Japan. Culture media preparation and yeast strain manipulation were carried out according to standard procedures [60 (link)]. For transformation of yeast cells, the lithium acetate method was used [61 ]. The culture media used in this work are listed in Table 1. For selection for geneticin or phleomycin resistance after yeast transformation, YPD agar plates were supplemented with 200 mg/L geneticin or 7.5 mg/L phleomycin, respectively. For selection of the Ura⁺ phenotype, SD agar plates were used. For cultivation of Ura^- strains, the synthetic media were supplemented with 20 mg/L Uracil. The yeast strains used in this study were derivatives of S288C. The construction of these strains is described in S1 File. The MATa yeast deletion collection (95401.H2) was purchased from Invitrogen Life Technologies Ltd., Carlsbad, USA.