Effect of salt stress on sugar uptake in osmotolerant yeasts

Abstract

Among yeasts there are many species considered osmotolerant because of their ability to grow in media of reduced water activity. Debaryomyces hansenii, which belongs to this group, is frequently isolated fxom salt-rich habitats [ 1, 2]. In order to enhance the osmotolerance of the industrial S. cerevisiae, a hybrid (also halotolerant) was obtained by protoplast fusion between D. hansenii NRRL Y-7393 and S. cerevisiae UCD 522 (industrial wine yeast; [3]). Both the halotolerant parent strain and the hybrid strain accumulate glycerol and arabinitol as compatible solutes during growth under salt stress conditions [3-5]. In terms of sugar uptake, both strains accumulate D-xylose and 2-deoxy-D-glucose (2-dGlc). However, in the presence of 2 mol/L NaC1 the uptake of D-xylose was virtually suspended. Glucose uptake, on the other hand, proceeded, though with a considerably lower velocity, until all added hexose was completely consumed [6]. This communication deals with substrate specificity of the sugar uptake system(s). Yeast cultures were maintained on complete YEPD solid medium (yeast extract 1%, peptone 2 %, glucose 2 % and agar 1.5 %). For uptake assays, the cells were grown in a liquid medium, YEPD or YEP + 2 mol/L NaC1. The culture flasks were inoculated with a preculture to give a cell concentration of 5 x 106/mL. The cultures were incubated at 30 ~ at 3.3 Hz. The cells were harvested in the early stationary phase, washed twice with distilled water and resuspended to a 5 % aqueous suspension. The cell suspension was aerated on a magnetic stirrer for 4 h at room temperature (cell starvation). Following the aeration, the cells were washed once again and resuspended as before. After mixing with 100 mmol/L KH2PO4 buffer (pH 6.5), the suspension was shaken in a water bath at 30 ~ and the experiment was started by the addition of the appropriate monosaccharide (D-xylose or D-glucose). Samples were taken at intervals. The uptake of D-glucose was measured enzymically in the supernatant after centrifugation using hexokinase and glucose-6-phosphate dehydrogenase in the presence of ATP and NAD +. The uptake of 14C-D-xylose and 3H-deoxy-D-glucose was determined by measuring the intracellular radioactivity in a liquid scintillation counter (Packard Tri-Carb 1600CA Scintillation Analyzer). Samples of the yeast suspension (1 mL) were taken at intervals and the cells were separated by filtration [7]. With the aim to distinguish between the inhibitory effect of NaC1 and an osmotic effect on sugar uptake, NaC1 was replaced by choline chloride or sodium benzenesulfonate (both at 2 mol/L final concentration). Glucose consumption was measured in D. hansenii and in the hybrid HM92 cell suspensions. The results showed that the presence of Na + is the principal factor responsible for the inhibition of glucose transport (Fig. 1). Among the cations, Ca 2+ and Mg 2+ were the most efficient inhibitors followed by Na +, Li + and K + (Fig. 2). Tetraphenylphosphonium (TPP +) was shown to depolarize yeast plasma membranes when added at concentrations of mmol/L [8]. Thirty mmol/L TPP + completely inhibited the accumulation of D-xylose and 2-dGlc in cell suspensions of both D. hansenii and the hybrid HM92 (Fig. 3). The inhibition of sugar accumulation by TPP + is a strong evidence for the participation of membrane potential in energization of sugar transport in the two tested strains. The inhibition of glucose consumption by cations (see above) is also in accordance with this conclusion since high cation concentrations also depolarize the yeast plasma membrane. So 30

DOI: 10.1007/BF02816515

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Cite this paper

@article{Loray1998EffectOS, title={Effect of salt stress on sugar uptake in osmotolerant yeasts}, author={M. A. Loray and L. I. C. de Figueroa and Milan Hoefer}, journal={Folia Microbiologica}, year={1998}, volume={43}, pages={204-206} }