Strains and growth conditions
The yeast Saccharomyces cerevisiae strain VKM Y-1173 was grown in a shaker in the Reader medium with 0.2% yeast extract, 2% glucose (120 r.p.m.), or 1% ethanol (200 r.p.m.). The medium contained (g l-1): (NH4)2SO4, 3; MgSO4, 0.7; Ca(NO3)2, 0.4; NaCl, 0.5; KH2PO4, 1; K2HPO4, 0.1; (NH4)2SO4. FeSO4. 6H2O, 0.00025; and trace elements (Vagabov et al.2000).
Polyphosphate (PolyP) biosynthesis was studied using the cells with low PolyP levels grown in a phosphate-free medium as described in (Vagabov et al.2000). Then the cells were cultivated in complete medium for 0.5 h. Biomass samples were harvested at 3000 g for 10 min, washed twice with distilled water at 4°C, and used for PolyP extraction. Dry cell mass was determined after drying cell aliquots at 85°C under vacuum.
Purification of cellobiose lipids
The cellobiose lipid of the yeast Cryptococcus humicola 9-6 (All-Russian Collection of Microorganisms, VKM) was obtained as described (Kulakovskaya et al.2009). After the cultivation, the culture supernatant was separated by centrifugation at 5000 g for 40 min, filtered through a Whatman glass fiber filter GF/A from Sigma-Aldrich Rus (Moscow, Russia), and lyophilized. The residue was extracted with methanol for four to five days at 5°C and filtered. The filtrate was evaporated at 50°C, and the resulting product was suspended in deionized water. The suspension was kept for 24 h at 5°C, and the resulting precipitate was separated by filtration through a glass filter, washed twice with cooled deionized water, and dissolved in methanol. The concentration of glycolipids was determined by weighing after methanol evaporation. In the course of purification, the antifungal activity was assayed by placing the aliquots of preparations on glucose-peptone agar (GPA) containing 0.5% glucose, 0.2% yeast extract, 0.25% peptone, 2% agar, 0.04 M citrate-phosphate buffer, pH 4.0 and inoculated with S. serevisiae.
The ESI-MS spectra were recorded with a Finnigan MAT LCQ (San Jose, CA, USA) mass spectrometer as described earlier for positive ions (Kulakovskaya et al.20052009. For direct (syringe) inlet, the methanol solution of a sample was injected at 10 µL/min. MS spectra were measured in positive mode.
Inorganic polyphosphate (PolyP) assay
Five separate polyP fractions differ in the chain length were obtained from Saccharomyces cerevisiae cells as described in (Vagabov et al.2000). Acid-soluble polyphosphates (PolyP1) were extracted with 0.5N HClO4. Salt-soluble polyphosphates (PolyP2) were extracted with saturated NaClO4 solution. Two fractions of alkali-soluble polyphosphates (PolyP3 and PolyP4) were extracted with the weak NaOH solution (pH was adjusted to 9–10) and 0.05 M NaOH (pH 12), respectively. All extractions were performed twice at 0°C under stirring for 10 min. The PolyP contents in the fractions PolyP1, PolyP2, PolyP3 and PolyP4 were quantified as a difference in Pi amounts before and after hydrolysis of the samples in 1N HCl for 10 min at 100°C. The level of PolyP5 fraction was determined by treating residual material with 0.5N HClO4 at 90°C twice for 20 min and assaying the released Pi. Pi was determined according to (Vagabov et al.2000). The data in the tables and figures are the average values of three experiments.
The chain length of PolyP from different fractions was determined by electrophoresis in polyacrylamide gel according to (Kumble and Kornberg 1995). For PolyP5 electrophoresis, residual biomass was extracted with distilled water for 12 h (Vagabov et al.2008). PolyP standards with the average chain lengths of 15, 25, 45, 75 phosphate residues were from Sigma (St Louis, USA), and with the average chain lengths of 208 phosphate residues were from Monsanto (St Louis, USA).
The ATP content in the cells was assayed after treating biomass samples with dimethylsulfoxide (0.2 ml / 25–50 mg of wet biomass). The effects of cellobiose lipids on ATP leakage from the cells was assayed as described (Kulakovskaya et al.2003). ATP was assayed by the luciferin-luciferase method using a Sigma assay kit and a LKB 1250 Luminometer (Sweden).
Potassium ion leakage
The leakage of K+ from the yeast cells was registered with a K+-selective electrode (Orion, USA). The measurements were made in a thermostatically controlled 2.5 ml cell at 25°C under stirring. The measuring medium containing 0.01 M citrate-phosphate buffer, pH 4.0, was injected with 50 µl of cell suspension to a final cell concentration of 6–6.5 · 108 ml-1. The maximum quantity of K+ found in the medium was taken as 100%.
Measurement of EKP (electrokinetic potential)
The cells were suspended in 0.01 M citrate buffer, pH 4.0, to a concentration of 107 – 5 × 107 cells ml-1. The EKP of yeast cells was measured with a Zetasizez nano ZS (Malvern, Great Britain) by the method of laser Doppler spectroscopy at 25°C. The average EKP value was calculated from three repeated measurements in each population of yeast cells.
The assay of cell viability
Yeast cell viability assay was performed as follows. The starting cell suspension was diluted in distilled water (1:100). Then the cells were treated with different cellobiose lipid concentrations (0.025 to 0.8 mg ml-1). The incubation mixture contained 0.5 ml of 0.04 M citrate-phosphate buffer, pH 4.0, and 0.1 ml of cell suspension. The mixture without cellobiose lipid was used as a control (100% viability). After the treatment, the cells were incubated at 30°C for 1 h. Then the cell suspensions were diluted in the citrate-phosphate buffer to different ratios and deposited on Petri dishes. The Petri dishes were incubated at 28°C for 3 days and the number of colonies was calculated.
All experiments were performed in triplicate. The biochemicals except those which are listed separately were obtained from Sigma-Aldrich-Rus (Moscow, Russia).