The Accumulation of Glycerol and Inorganic Ions Were Examined in A

J. Gen. Appl. Microbiol., 42, 201-212 (1996)

REGULATION OF INTRACELLULAR OSMOTIC PRESSURE AND SOME FACTORS THAT INFLUENCE THE PROMOTION OF GLYCEROL SYNTHESIS IN A RESPIRATION-DEFICIENT MUTANT OF THE SALT-TOLERANT YEAST ZYGOSACCHAROMYCES ROUXH DURING SALT STRESS

KYOUICHI OHSHIRO AND TADASHI YAGI*

Department of Biology, Faculty of Science, Osaka City University, Sumiyoshi-ku, Osaka 558, Japan

(Received December 5, 1995; Accepted March 6, 1996)

The accumulation of glycerol and inorganic ions were examined in a respiration-deficient (RD) mutant isolated from the salt-tolerant yeast Zygosaccharomyces rouxii for 3 h after salt stress due to 1 M NaCI. After the start of salt stress, intracellular levels of glycerol continued to increase for up to 3 h, while the levels of Na+ and Cl- ions in cells reached maximum values within 1 h and then decreased gradually. Increases in intracellular concentrations of solutes resulted in an osmotic pressure that was almost equivalent to the external osmotic pressure within 2 h after salt stress. The RD strain had the same ability to tolerate salt as the wild-type strain. Therefore, we used the RD strain to examine the mechanism in the glycolytic pathway that is responsible for the promotion of glycerol synthesis that is induced by NaCI. When exposed to medium with 1 M NaCI, RD cells diverted about one-sixteenth of the amount of ethanol that was produced in the medium without NaCI to the production of glycerol. This result suggests the presence of factors that mediate a change from the normal metabolism of glucose to the promotion of glycerol synthesis in response to external NaCI. The specific activities of glycerol-3-phosphate dehydrogenase (GPDH) in extracts of cells grown with and without 1 M NaCI were very low in reaction mixtures with NADH or NADPH, although the cellular activity of alcohol dehydrogenase (ADH) was high and was repressed by external NaCI. This result indicates that the pathway involving GPDH makes only a small contribution to the synthesis

* Address reprint requests to: Dr . Tadashi Yagi, Department of Biology, Faculty of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558, Japan.

201 202 OHsHIR0 and YAGI VOL. 42

of glycerol and that an alternative pathway functions for the synthesis in Z. rouxii. The addition of sodium sulfite, which binds to acetaldehyde, and of glycidol, an inhibitor of triosephosphate isomerase (TPI), to the medium promoted the synthesis of glycerol in RD strain. These results suggest the possibility that the extra NADH resulted from the binding of sulfite to acetaldehyde, or the inhibition of ADH and/or TPI under the NaCI-stressed condition lead to the promotion of glycerol synthesis by Z. rouxii.

Zygosaccharomyces rouxii is a salt-tolerant yeast that can grow over a wide range of salt concentrations (13, 20). This yeast produces and accumulates high levels of glycerol in response to increases in the concentration of NaCI in the growth medium (4). The accumulated glycerol functions both as an osmoregulator and as an osmoprotector of enzymatic activities under salt-stressed conditions (4). In Z. rouxii, the promotion of the production and the intracellular accumulation of glycerol begin immediately after the start of salt stress, and the intracellular osmotic pressure is regulated in such a way that it becomes almost equal to the external osmotic pressure within 1 h (21, 22). Some unknown mechanism(s), triggered at high concentrations of NaCI, diverts the ordinary metabolism of glucose via the tricarboxylic acid cycle or ethanol fermentation to the production of glycerol. However, the mechanism(s) responsible for the promotion of the synthesis of glycerol upon exposure of cells to high levels of NaCI remains unknown. As part of an attempt to characterize this mechanism(s), we isolated a respiration-deficient (RD) strain of Z. rouxii (23). The RD strain grows at almost the same rate as the wild-type strain in media containing NaCI up to 2.5 M NaCI, although the mass of RD cells at the stationary phase is smaller than that of the wild-type cells in all NaCI-containing media. In this study, we examine the regulation of intracellular osmotic pressure and the factors that influence the promotion of glycerol synthesis in the RD strain of the salt-tolerant yeast Z, rouxii under the NaCI-stressed condition.

MATERIALS AND METHODS

Organism, media and culture conditions. Zygosaccharomyces rouxii (Bou- trox) Yarrow (IFO 0533) and the RD strain derived from it (23) were used in this study. The wild-type and RD strains were maintained on YPD-30 agar slants with and without 1 M NaCI, respectively. YPD-30 medium contained yeast extract (0.5%), peptone (0.5%), KH2PO4 (0.5%), MgSOe7H2O (0.2%) and glucose (3%). For the RD strain, the levels of yeast extract and peptone were increased to 1 and 2%, respectively, and 5 ml of Tween 80 and 20 mg of ergosterol were added to each liter of YPD-30 agar medium. For preculture and basal growth media, YPD-30 and Burkholder's synthetic (BS) medium were used. BS medium con- 1996 Glycerol Synthesis of Respiration-Deficient Yeast 203 tamed glucose (3%), KH2PO4 (0.15%), CaCl2.2H2O (0.033%), MgSO4.7H2O (0.05%), (NH4)25O4 (0.2%), asparagine (0.2%) and trace amounts of six ele- ments and eight vitamins. The cells were precultured aerobically for 2 days in YPD-30 medium in test tubes and then inoculated at a density of 5 X 105 cells/ml into 80 ml of YPD-30 or BS medium in 500-ml flasks. Cells were cultured to the late logarithmic phase at 26°C on a reciprocating shaker operated at 120 strokes/min. Then, for determina- tions of intracellular contents of glycerol and inorganic ions under non-prolife- rating conditions, these cells were inoculated at a density of 5 X 10' cells/ml into YPD-30 or BS medium with or without 1 M NaCI. The flasks were incubated aerobically at 26°C for 3 h on the reciprocating shaker. Determination of cell number and cell volume. The numbers of cells in cultures, diluted with distilled water or a 1 M solution of NaCI, were counted in a Thoma haematocytometer. For determination of cell volume, cytocrit tubes (total volume about 3 ml, capillary tubing 200 icl) filled with 3 ml of sample culture were centrifuged at 3,000 X g for 5 min and the packed cell volume was measured. An interstitial volume of 23% was assumed on the basis of previously reported experimental data for yeast cells (5). Quantification of glycerol and ethanol. The methods for the extraction of glycerol and ethanol from cells have been described previously (12). Glycerol in cells and in the culture medium was quantified enzymatically by the glycerokinase method (F-kit glycerol; Boehringer Mannheim, Mannheim, Germany), and ethanol was quantified by the alcohol dehydrogenase method (332 alcohol kit; Sigma Chemical Co., St. Louis, MO, U.S.A.). For the quantification of extracellular solutes, a sample from fresh medium was used as a blank. Total production was estimated from the sum of the amount of each solute in cells and in the culture medium. Quantification of intracellular inorganic ions. Intracellular Nat, K+ and Cl- ions were extracted twice with hot 0.5 N HNO3 from the cells (12). The Na+ and K+ ions were quantified by flame photometry in an atomic absorption spectrophotometer (model 208; Hitachi Ltd., Tokyo, Japan), and Cl- ions were quantified colorimetrically (7, 12) . Preparation of cell extracts for assays of enzymatic activity. Cells that had been incubated for 3 h in BS medium with or without 1 M NaCI were harvested and washed twice with a solution of 1 M NaCI or distilled water, respectively. The cells were suspended in a cold 10 mM triethanolamine/HC1 buffer (pH 7.0) containing 1 mM EDTA, 1 mM dithiothreitol and three inhibitors of proteinase, l /IM pepstatin, l /IM leupeptin and 200,u M phenylmethylsulfonyl fluoride. The suspension of cells was homogenized with glass beads (0.3 mm in diameter) at 4°C for 10 min in Vibrogen cell mill (Edmund Bi hler, Tubingen, Germany). After removal of the beads by low-speed centrifugation, the crude extract was centrifuged at 10,000 X g for 20 min. The supernatant was desalted by passage through a column of Sephadex G-25 (PD-10; Pharmacia Fine Chemicals, Uppsala, Sweden) and then assayed for 204 OHsHIRO and YAGI VOL. 42 various enzymatic activities. The concentration of protein was determined by the method of Lowry et al. (8) with bovine serum albumin as the standard. Assays of enzymatic activity. The assays were performed at 25°C in 3-ml reaction mixtures. Depletion or production of NADH or NADPH was monitored by following changes in absorption at 340 nm in a spectrophotometer (model 200; Hitachi Ltd.). Blank solution in cuvettes contained all constituents of the reaction mixture except that the substrate was replaced by water. The reactions were started by the simultaneous addition of a cell extract to each cuvette. To calculate the enzymatic activities, the rate of the reaction between 30 and 90s and an absorption coefficient for NADH and NADPH of 6.22><103 M-1 cm-1 were used. One unit of enzymatic activity was defined as the amount of enzyme that consumed 1 nmol of NAD(P)H or NAD per minute, and specific activities are given as U mg -1 protein. Glycerol-3-phosphate dehydrogenase (NAD+) (GPDH; EC 1.1.1.8) was assayed in a buffer containing 10 mM triethanolamine-HC1 (pH 7.0), 0.1 mM NADH or NADPH, 1 mM dithiothreitol, 1 mM EDTA and 1.2 mM dihydroxyacetone phosphate (DHAP). Alcohol dehydrogenase (ADH; EC 1.1.1.1) was assayed in a buffer containing 50mM Tris-HC1 (pH 8.5), 0.5 mM NAD+ and 0.7 M ethanol. The data represent average results from two or three independent experiments.

RESULTS AND DISCUSSION

Production and intracellular accumulation of glycerol in the RD strain Figure 1 shows the rate of production and accumulation of glycerol by the RD strain incubated for 3 h in YPD-30 medium with or without 1 M NaCI, in compar- ison with the results for the wild-type strain. The total production of glycerol by the RD strain was higher in both media with and without 1 M NaCI than that by the wild-type strain (Fig. la). Both strains produced a considerable amount of glycerol

Fig. 1. Synthesis and intracellular accumulation of glycerol by the wild-type and the respiratory-deficient (RD) strains during incubation for 3 h in YPD-30 medium with or without 1 M NaCI. a, total glycerol/culture; b, intracellular glycerol/total cells in culture; c, extracellular glycerol/80 ml culture medium. 0, •, wild-type strain; LII,•, RD strain. Open and closed symbols indicate the results for medium without and with 1 M NaCI, respectively. 1996 Glycerol Synthesis of Respiration-Deficient Yeast 205 even in the medium without NaCI, although they produced less than in the medium with NaCI (Fig. la). However, a large amount of glycerol accumulated intracellularly under the salt-stressed conditions but not under non-stressed conditions (Fig. ib). Additional glycerol leaked into the extracellular medium from both wild-type and the RD strains (Fig. lc). Such leakage was also observed in BS medium (data not shown). These results indicated that the ability of the RD strain to produce glycerol and accumulate intracellularly was almost equal to that of the wild-type strain.

Changes in intracellular levels of glycerol and inorganic ions, and the regulation of intracellular osmotic pressure in the RD strain during salt stress We examined whether the RD strain had the ability to regulate the intracellular osmotic pressure in response to salt stress. Figure 2 shows the time course of changes in intracellular levels of glycerol and inorganic ions, such as Nat, K+ and Cl-, in cells of the RD strain incubated in BS medium with and without 1 M NaCI. No increase in cell numbers occurred over the course of 3 h in either media (data not shown). In the medium with 1 M NaCI, intracellular levels of glycerol increased linearly up to 3 h (Fig. 2a), but the levels of Na+ and Cl- ions decreased gradually after reaching maximum values at 1 h (Fig. 2b, c). The intracellular levels of K+ ions varied within the limits of 3.0 and 4.5, and the limits of 0.5 and 3.0 fmol/cell during incubation for 3 h in salt-free and 1 M NaCI-containing medium, respectively, but they did not change very much in either medium (data not shown). The total amounts of intracellular glycerol, Nat, K+ and Cl- ions

Fig. 2.. Changes in intracellular levels of glycerol and Na+, K+ and Cl- ions in the RD strain during incubation for 3 h in ]BS medium with or without 1 M NaCI. 0, no addition; •, 1 M NaCI. 206 OHSHIR0 and YAGI VOL. 42 increased rapidly during the first one hour and then continued to increase gradually to reach maximum values (Fig. 2d). This result indicated that, after the start of salt stress, the cells increased the intracellular osmotic pressure by accumulating glycerol and ions in response to an external increase in osmotic pressure due to NaCI in the medium. During this process, the intracellular levels of Na+ and Cl- ions decreased (Fig. 2b, c) and, thus, the ratio of the amount of glycerol to the total amount of intracellular glycerol and inorganic ions increased. This phenomenon is considered to be due to the operation of the Na+/H+-antiporter that extrudes Na+ ions from the cells in exchange for protons (12). Changes in cell volume of the RD strain during the 3 h after salt stress were examined (Fig. 3a). The mean volume of individual cells inoculated was about 27 ,um3and decreased by about 10% over 3 h, even in salt-free medium. This decrease was considered to be due to the difference in osmotic pressure between the fresh medium and the spent medium in which the inoculated cells had been grown. In medium with 1 M NaCI, the cell volume decreased to 20 tm3 during the first two hours and then tended to return to the original value at 3 h. We estimated the changes in intracellular osmotic pressure during the 3-h experiment. Figure 3b shows the changes in intracellular concentrations of solutes, as calculated from the total contents of glycerol and inorganic ions (Fig. 2d) and cell volume (Fig. 3a). The concentration of solutes in cells of the RD strain that were incubated in the medium with 1 M NaCI reached a maximum value of 2.7 mold cell volume within 2 h. This estimated value was 1.2 times as high as the total

Fig. 3. Changes in cell volume and in the intracellular concentration of glycerol and Nat, K+ and C1 ions in cells of the RD strain during incubation for 3 h in BS medium with or without 1 M NaCI. 0, no addition; •, 1 M NaCI. 1996 Glycerol Synthesis of Respiration-Deficient Yeast 207 concentrations of 0.5 osm in salt-free medium and 1.8 osm yielded by the addition of 1 M NaCI. This result demonstrates that, upon exposure to salt stress, the RD strain had the ability to produce and accumulate a large amount of glycerol and to maintain intracellular levels of Na+ and Cl- ions lower than those in the external medium. In this way, the cells maintained a balanced intracellular osmotic pressure to that of the external medium. Therefore, the RD strain seems to be an appropriate strain with which to examine the mechanism(s) responsible for the change from the normal metabolism of glucose to the production of glycerol that is induced by NaCI in the medium.

Production of glycerol and ethanol, and related enzymatic activities during salt stress The RD strain obtains energy only by the fermentation of glucose to ethanol, even under aerobic conditions, and it is unable to utilize non-fermentable sources of carbon such as glycerol as a consequence of defective mitochondrial activity (24). We examined the production of glycerol and ethanol by cells of the RD strain incubated for 3 h in BS medium with or without 1 M NaCI (Fig. 4). Production of glycerol increased from 165 to 240,amol/80 ml culture at 3 h by the addition of 1 M NaCI. By contrast, the production of ethanol in the medium with NaCI decreased by an amount equivalent to the increased amount of glycerol, and consequently the total production of glycerol and ethanol was equal in the media with and without 1 M NaCI. This result indicated that, when exposed to the medium with 1 M NaCI, the RD cells diverted about one-sixteenth of the amount of ethanol (1,200,umol/80 ml culture) that was produced in the medium without NaCI to the production of glycerol. This result also suggested that the mechanism responsible for the change from the normal metabolism of glucose to the promotion of glycerol synthesis by

Fig. 4. Changes in the rates of production of glycerol and ethanol in the RD strain during incubation for 3 h in BS medium with or without 1 M NaCI. 0, •, glycerol; 0, •, ethanol. Open and closed symbols indicate the data in medium without and with 1 M NaCI, respectively. 208 OHsHIR0 and YAGI VOL. 42

Table 1. Specific activities of glycerol-3-phosphate dehydrogenase and alcohol dehydrogenase in desalted extracts of the RD strain of Zygosaccharomyces rouxii after incubation for 3 h in BS medium with or without 1 M NaCI.

external NaCI in the growth medium was operative in the RD strain as well as in the wild-type strain. The activities of GPDH and ADH in desalted cell extracts from the RD strain that was incubated for 3 h in BS medium with or without 1 M NaCI were examined (Table 1). The specific activity of ADH in extracts of cells grown with 1 M NaCI was two-fifths of that in extracts of cells grown under salt-free conditions, suggest- ing that the NaCI-stressed conditions inhibited ADH activity. By contrast, the activity of GPDH in extracts of cells from media with and without NaCI was unexpectedly low in reaction mixtures with NADH usually utilized as the hydrogen donor. Although the addition of NADPH instead of NADH to reaction mixtures increased the GPDH activity ten- to thirteen-fold, the activity was still low and little difference in activity was observed between the cells incubated with and without 1 M NaCI (Table 1). The activity of NADH-dependent GPDH in extracts of Saccharomyces cerevisiae incubated for 3 h in BS medium without NaCI was 56.6 Umg-I protein when the same method was used (data not shown). The specific activity of GPDH in cell extract of Debaryomyces hansenii (1) and S. cerevisiae (3) grown under the NaCI-stressed condition also increased, and the production of GPDH increased (2). These results coincide with the report of van Zyl et al. (19) that the activity of GPDH was low and was the same in cells of Z. rouxii grown in medium with or without NaCI. Thus, the pathway that involves GPDH may make only a small contribution to the synthesis of glycerol and that an alternative pathway involving glycerol dehydrogenase (EC 1.1.1.6) via dihydroxyacetone may function in Z. rouxii, even though the pathway involving GPDH and a phosphatase has been described in Z. rouxii (17). Further studies are necessary to reexamine the assay method and stability of GPDH activity, and to investigate the synthetic pathway of glycerol in Z. rouxii.

Factors influencing the promotion of glycerol synthesis in the RD strain To evaluate the factors that influence the promotion of glycerol synthesis, we examined the effects of changes in the concentration of glucose on the RD strain incubated in BS medium with or without 1 M NaCI (Table 2). Removal of glucose from the BS medium caused a significant decrease in the production of glycerol and in the intracellular accumulation in medium both with and without NaCI, suggest- 1996 Glycerol Synthesis of :Respiration-D eficient Yeast 209

Table 2. Effects of changes in the concentration of glucose in BS medium on the production and intracellular accumulation of glycerol in the RD strain during a 3-h incubation with or without 1 M NaCI.

ing that, in this case, glycerol was synthesized from glucose (Table 1). The presence of glucose at levels above 10 g/l in the medium was necessary for main- tenance of the production and intracellular level of glycerol in response to the external osmotic pressure. The effects of inhibitors of the glycolytic pathway on the production of glycerol were examined in RD cells incubated in salt-free BS medium. In perme- abilized RD cells, in which the permeability barrier to small molecules had been destroyed by vigorous shaking in the presence of a low-concentration of toluene (16), the addition of ADP, ATP, NAD or NADH to the medium did not affect the production of glycerol (data not shown). The addition of sulfite to cultures of fermenting baker's yeast is known to induce the production of large amounts of glycerol (6, 9). The added sulfite binds to acetaldehyde, such that acetaldehyde can no longer serve as a hydrogen acceptor in the normal re-oxidation of NADH. To restore the redox balance, the yeast utilizes NADH for the production of glycerol via glycerol-3-phosphate. We examined the effects of various concentrations of sodium sulfite on the production of glycerol and ethanol in RD cells incubated for 3 h in medium without 1 M NaCI (Fig. 5). The total production of glycerol increased with increases in the concentration of sodium sulfite in the medium. The production of glycerol in salt-free medium containing from 5 to 20 mM sodium sulfite was nearly the same as that in the 1 M NaCI-containing medium without sodium sulfite (Fig. la). However, the production of ethanol was strongly inhibited by the addition of 5 mM sodium sulfite. Thus, the sum of the glycerol and ethanol produced was reduced by the addition of sodium sulfite even though the production of glycerol was promoted. This result suggests that sulfite has some inhibitory effect on the production of ethanol in addition to its effect on the supply of NADH for glycerol synthesis. This inhibitory action of sulfite was reported to be due, in S. cerevisiae, to a rapid decrease in the level of ATP and to the subsequent interruption of ATP-dependent processes (15). Intracellular levels of glycerol were unaffected by the addition of sodium sulfite and 210 OH5HIR0 and YAGI VOL. 42

Fig. 5. Effects of sodium sulfite on the production of glycerol and ethanol in the RD strain during a 3-h incubation in BS media without 1 M NaCI. 0, total glycerol; C], ethanol; 0, the sum of the amounts of ethanol and glycerol produced. were nearly as high as the levels in the medium without sulfite (data not shown). These results suggest the possibility that the extra NADH that resulted from the binding of sulfite to acetaldehyde or the inhibition of ADH activity under NaCI- stressed conditions leads to the promotion of the synthesis of glycerol by Z. rouxii. Triose phosphate isomerase (TPI) acts mainly to catalyze the conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (GAP) in the glycolytic pathway. DHAP is also converted to glycerol via glycerol-3- phosphate. Therefore, to examine whether the activity of TPI affects the production of glycerol, an inhibitor of TPI, glycidol (14), was added to the salt-free medium (Fig. 6). The total production of glycerol increased at 100 mM glycidol but this effect was not seen above 150 mM glycidol. This result also suggests the possibility that inhibition of the conversion of DHAP to GAP increases the rate of synthesis of glycerol. These results suggest the possibility that the inactivation of ADH or TPI might be involved in the promotion of the synthesis of glycerol to increase the intracellular osmotic pressure in response to a high external level of NaCI. Further studies of the enzymes involved in the promotion of glycerol synthesis are in progress. For growth in medium that contains a high concentration of NaCI, the wild-type and RD strains of Z, rouxii must, at least, do the following in addition to increasing the rate of glycerol synthesis. They must eliminate large amounts of Na+ and Cl- ions that have entered the cells as a consequence of the concentration gradient of NaCI, and they must retain the glycerol that is synthesized in the cells. The cells usually leak the greater part of the amount of glycerol that is con- stitutively synthesized in the salt-free medium (Fig. 1). Upon salt stress, the cells 1996 Glycerol Synthesis of Respiration-Deficient Yeast 211

Fig. 6. Effects of glycidol on the production of glycerol in the RD strain during a 3-h incubation in BS media without 1 M NaCI. must stop leaking glycerol or they must take up the leaked glycerol from the medium to increase the intracellular osmotic pressure. Indeed, van Zyl et al. (18) reported that glycerol is actively transported into Z. rouxii cells by a carrier- mediated system with a high degree of specificity for glycerol, and we have obtained a similar result (unpublished data). When the cells are exposed to the medium with 1 M NaCI, cAMP is synthesized immediately and then the plasma membrane ATPase is activated (10,11). The activated plasma membrane ATPase rapidly extrudes protons from cells, the invading Na+ ions are extruded via the Na+/Htantiporter from the cells in exchange for protons (12). As a result, the intracellular level of Na+ ions is lowered and is maintained at a lower level. Thus, as a consequence of the promotion of the synthesis and the intracellular accumulation of glycerol, as well as the elimination of Na+ ions from the cells, the cells of Z. rouxii are able to grow in medium that contains a high concentration of NaCI.

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