On the mechanism of salt tolerance

As glycerol was suggested as an osmotic agent in the salt tolerantDebaryomyces hansenii the concentrations of total, intracellular, and extracellular glycerol produced by this yeast was followed during growth in 4 mM, 0.68 M, and 2.7 M NaCl media. The total amount of glycerol was not directly proportional to biomass production but to the cultural salinity with maximum concentrations just prior to or at the beginning of the stationary phase. In all cultures the cells lost some glycerol to the media, at 2.7 M NaCl the extracellular glycerol even amounted maximally to 80% of the total. A distinct maximum of intracellular glycerol, related to dry weight or cell number, appeared during the log phase at all NaCl concentrations. As the intracellular calculated glycerol concentrations amounted to 0.2 M, 0.8 M, and 2.6 M in late log phase cells at 4 mM, 0.68 M, and 2.7 M NaCl, respectively, whereas the corresponding analysed values for the glycerol concentrations of the media were 0.7 mM, 2.5 mM, and 3.0 mM, glycerol contributes to the osmotic balance of the cells.During the course of growth all cultures showed a decreasing heat production related to cell substance produced, most pronounced at 2.7 M NaCl. At 2.7 M NaCl the total heat production amounted to-1690 kJ per mole glucose consumed in contrast to-1200 and-1130 kJ at 4 mM and 0.68 M NaCl, respectively. TheY _m -values were of an inverse order, being 129, 120, and 93 at 4 mM, 0.68 M, and 2.7 M NaCl, respectively.     

Polyhydric alcohol production and intracellular amino acid pool in relation to halotolerance of the yeast Debaryomyces hansenii

Changes in polyol production and the intracellular amino acid pool were followed during the growth cycle of Debaryomyces hansenii in 4 mM and 2.7 M NaCl media. The intracellular levels of polyols were markedly enhanced by high salinity, the dominant solutes being glycerol in log phase cells and arabinitol in stationary phase cells. At low salinity arabinitol was the most prominent intracellular solute throughout the growth cycle. There were no major changes in the composition of the total amino acid pool with changes in cultural salinity. The amount of total free amino acids related to cell dry weight was 15–50% lower in cells cultured in 2.7 M NaCl as compared to 4 mM NaCl media.After subtraction of contributions from intracellular polyols the calculated cellular C/N ratio was found to be unaffected by cultural age and salinity during the late log and early stationary phase. On prolonged incubation of stationary phase cells, this ratio decreased, particularly at high salinity. The sensitivity of cells towards exposure to high salinity was measured in terms of the length of the lag phase after transference to 2.7 M NaCl media. This lag phase decreased with increasing intracellular polyol concentrations. At a given polyol content, stationary phase cells were considerably less sensitive than were log phase cells.When cultured at high salinity the mutant strain, 26-2b, grew more slowly and retained less of the total polyol produced during the early growth stages than did the wildtype. Exogenously supplied mannitol, arabinitol, and glycerol stimulated the growth of the mutant in saline media. Erythritol was without effect.Abbreviations GLC gas-liquid chromatography - TCA trichloroacetic acid     

On the mechanism of salt tolerance. Production of glycerol and heat during growth of Debaryomyces hansenii.

As glycerol was suggested as an osmotic agent in the salt tolerant Debaryomyces hansenii the concentrations of total, intracellular, and extracellular glycerol produced by this yeast was followed during growth in 4 mM, 0.68 M, and 2.7 M NaCl media. The total amount of glycerol was not directly proportional to biomass production but to the cultural salinity with maximum concentrations just prior to or at the beginning of the stationary phase. In all cultures the cells lost some glycerol to the media, at 2.7 M NaCl the extracellular glycerol even amounted maximally to 80% of the total. A distinct maximum of intracellular glycerol, related to dry weight or cell number, appeared during the log phase at all NaCl concentrations. As the intracellular calculated glycerol concentrations amounted to 0.2 M, 0.8 M, and 2.6 M in late log phase cells at 4mM, 0.68 M, and 2.7 M NaCl, respectively, whereas the corresponding analysed values for the glycerol concentrations of the media were 0.7 mM, 2.5 mM, and 3.0 mM, glycerol contributes to the osmotic balance of the cells. During the course of growth all cultures showed a decreasing heat production related to cell substance produced, most pronounced at 2.7 M NaCl. At 2.7 M NaCl the total heat production amounted to--1690 kJ per mole glucose consumed in contrast to--1200 and--1130 kJ at 4 mM and 0.68 M NaCl, respectively. The Ym-values were of an inverse order, being 129, 120, and 93 at 4 mM, 0.68 M, and 2.7 M NaCl respectively.     

Glycerol production in relation to the ATP pool and heat production rate of the yeasts Debaryomyces hansenii and Saccharomyces cerevisiae during salt stress

Changes in glycerol production and two parameters related to energy metabolism i. e. the heat production rate and the ATP pool, were assayed during growth of Saccharomyces cerevisiae and Debaryomyces hansenii in 4 mM and 1.35 M NaCl media. For both of the yeasts, the specific ATP pool changed during the growth cycle and reached maximum values around 10 nmol per mg dry weight in both types of media. The levels of glycerol were markedly enhanced by high salinity. In the presence of 1.35 M NaCl, D. hansenii retained most of its glycerol produced intracellularly, while S. cerevisiae extruded most of the glycerol to the environment. The intracellular glycerol level of S. cerevisiae equalled or exceeded that of D. hansenii, however, with values never lower than 3 mol per mg dry weight at all phases of growth. When D. hansenii was grown at this high salinity the intracellular level of glycerol was found to correlate with the specific heat production rate. No such correlation was found for S. cerevisiae. We concluded that during salt stress, D. hansenii possesses the capacity to regulate the metabolism of glycerol to optimize growth, while S. cerevisiae may not be able to regulate when exposed to different demands on the glycerol metabolism.     

Osmoregulation of the salt-tolerant yeast Debaryomyces hansenii grown in a chemostat at different salinities.

The intracellular solute composition of the salt-tolerant yeast Debaryomyces hansenii was studied in glucose-limited chemostat cultures at different concentrations of NaCl (4 mM, 0.68 M, and 1.35 M). A strong positive correlation between the total intracellular polyol concentration (glycerol and arabinitol) and medium salinity was demonstrated. The intracellular polyol concentration was sufficient to balance about 75% of the osmotic pressure of the medium in cultures with 0.68 and 1.35 M NaCl. The intracellular concentration of K+ and Na+, which at low external salinity gave a considerable contribution to the intracellular water potential, was only slightly enhanced with raised medium salinity. However, the ratio of intracellular K+ to Na+ decreased; but this decrease was less drastic in the cells than in the surrounding medium, i.e., the cells were able to select for K+ in favor of Na+. The turgor pressure, which was estimated on the basis of intracellular solute concentrations, was 2,200 kPa in cultures with 4 mM NaCl and decreased when the external salinity was raised, resulting in a value of about 500 kPa in cultures with 1.35 M NaCl. The maintenance of a positive turgor pressure at high salinity was mainly due to an increased production and accumulation of glycerol.     

Effect of 24-epibrassinolide on biomass, growth and free proline concentration in Spirulina platensis (Cyanophyta) under NaCl stress

In this study, biomass, growth and free proline concentration were investigated in Spirulina platensis treated with different concentrations of NaCl (50, 100, 150, and 200 mM) and 24-epibrassinolide (24-epiBL) hormone (0.5, 1.0, and 3.0 μM) over 5 days. As a result of analysing the cultures under salinity stress, it was determined that biomass and growth rate decreased significantly, while proline concentration increased considerably under salinity stress. The increase in the concentration of proline suggests a role in response to NaCl stress. Among the cultures treated with different concentrations of 24-epiBL, maximum growth was determined at the cultures at 1.0 μM 24-epiBL. Algal growth was also greater at the 0.5 and 3.0 μM concentrations of 24-epiBL with respect to control cultures. With respect to control, 24-epiBL affected growth rate and biomass positively, but proline concentration did not change. Among the cultures supplied with different combinations of NaCl and 24-epiBL, growth rate increased in 150/0.5 and 150/3.0 mM/μM concentrations, but was maximal for the culture containing 150/1.0 mM/μM combination. The increase in algal growth suggests a role for 24-epiBL in partially alleviated to NaCl stress. These results suggest that 24-epiBL may have a protective role for S. platensis reducing the inhibitor effects of salinity stress.     

Halotolerance studies onChlamydomonas reinhardtii: glycerol excretion by free and immobilized cells

The freshwater green algaChlamydomonas reinhardtii can tolerate a maximum saline concentration of 200 mM NaCl. In response to this osmotic shock, the cells accumulated during the first 24 h 15% of the total glycerol synthesized as osmoregulatory metabolite, to provide the corresponding osmotic balance. After this period all glycerol synthesized was excreted to the medium, 4 g L^-1 at 120 h in optimal conditions, before cell degradation occurred. This excretion was about 2-fold higher in Ca-alginate entrapped cells in the presence of 250 mM NaCl. It was concluded that immobilized cells may be of biotechnological interest for continuous glycerol photoproduction in air-lift bioreactors.     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

Microalgal biotechnology: Carotenoid and glycerol production by the green algae <Emphasis Type="Italic">Dunaliella</Emphasis> isolated from the Gave-Khooni salt marsh,Iran

In this study, carotenoid and glycerol production in two unicellular green algae (Dunaliella salina and D. viridis) isolated from the Gave-Khooni salt marsh grown in media containing five different salt concentrations (0.17, 1, 2, 3, and 4 M NaCl) were evaluated under sterile conditions. Algae growth decreased as the medium salinity increased. Optimum growth of D. salina and D. viridis were obtained at 2 and 1 M NaCl, respectively. As salinity increased, glycerol and carotenoid production were increased in D. salina, whereas lower values for these products were produced in D. viridis under the same conditions. Furthermore, the cell color of D. salina changed from green to orange-red following accumulation of carotenoid, but the color of D. viridis was not changed. Thereby, it seems that the Iranian D. salina may be suitable for carotenoid production (betacarotene) on a large scale. In addition, since carotenoid compounds enhance the efficiency of photosynthesis and glycerol synthesis, it appears that the pathway for glycerol production and mechanisms of salt tolerance in D. viridis are unique from those of D. salina.     

Cell turgor: A critical factor for the proliferation of cyanobacteria at unfavorable salinity

We employed chlorophyll a fluorometry in order to measure the evolution of turgor threshold (intracellular osmolality) during the adaptation of two genetic transformants of the freshwater cyanobacterium Synechococcus sp. PCC7942 to unfavorable external salinity: PAMCOD cells which oxidize imported choline and accumulate approx. 0.06–0.08 M glycine betaine; and PAM cells which do not oxidize choline [Deshnium et al. (1995a) Plant Mol Biol 29: 897–909]. Turgor thresholds increased linearly (a) with the NaCl concentration in the culture, and (b) with the molar sucrose/chlorophyll a ratio in the cell. PAMCOD cells could proliferate in culture medium containing 0.4 M NaCl (external osmolality, 0.815 Osm kg⁻¹), after a lag period, during which intracellular sucrose rose to 10 mol (mol Chl a)⁻¹, or more, and turgor threshold (cytoplasmic osmolality) exceeded 1 Osm kg⁻¹. At comparative conditions, PAM cells accumulated approx. half as much sucrose, and attained approx. half as high turgor thresholds as the PAMCOD cells, but they did not proliferate. These results indicate that glycine betaine improved the salinity tolerance of the PAMCOD cells synergistically, by means of two effects that implicate sucrose, the main organic osmolyte of Synechocccus: enhancement of sucrose biosynthesis, and/or alleviation of sucrose toxicity. This revised version was published online in June 2006 with corrections to the Cover Date.     

The influence of NaCl on the degradation rate of dichloromethane byHyphomicrobium sp.

The degradation of dichloromethane by the pure strainHyphomicrobium GJ21 and by an enrichment culture, isolated from a continuously operating biological trickling filter system, as well as the corresponding growth rates of these organisms were investigated in several batch experiments. By fitting the experimental data to generally accepted theoretical expressions for microbial growth, the maximum growth rates were determined. The effect of NaCl was investigated at salt concentrations varying from 0 to 1000 mM. Furthermore the dichloromethane degradation was investigated separately in experiments in which a high initial biomass concentration was applied. The results show that microbial growth is strongly inhibited by increased NaCl concentrations (50% reduction of _max at 200–250 mM NaCl), while a certain degree of adaptation has taken place within an operational system eliminating dichloromethane. A critical NaCl concentration for growth of 600 mM was found for the microbial culture isolated from an operational trickling filter, while a value of 375 mM was found for the pure cultureHyphomicrobium GJ21. The substrate degradation appears to be much less susceptible to inhibition by NaCl. Even at 800 mM NaCl relatively high substrate degradation rates are still observed, although this process is again dependent on the NaCl concentration. Here the substrate elimination is due to the maintenance requirements of the microorganisms. The inhibition of the dichloromethane elimination was also investigated in a laboratory scale trickling filter. The results of these experiments confirmed those obtained in the batch experiments. At NaCl concentrations exceeding 600 mM a considerable elimination of dichloromethane was still observed for during several months of operation. These observations indicate that the inhibition of microbial growth offers a significant control parameter against excessive biomass growth in biological trickling filters for waste gas treatment.     

Response to increasing rates of boron and NaCl on shoot proliferation and chemical composition of in vitro kiwifruit shoot cultures

The in vitro response of kiwifruit (Actinidia deliciosa) to increasing concentrations of boron (B) and NaCl in the culture medium was studied. Kiwifruit shoot cultures were grown in vitro for 12 weeks on an MS medium containing two B concentrations (0.1 and 2 mM) combined with five NaCl concentrations (0, 10, 20, 40 and 80 mM). Kiwifruit produced the longest shoots with 2 mM B when NaCl concentration was 0--20 mM. More shoots were produced with 2 mM B for all NaCl treatments. More shoots were produced with 2 mM B and 10 and 20 mM NaCl. High B concentrations in the culture medium significantly increased shoot proliferation. Explants exhibited a moderate chlorotic appearance with 40 mM NaCl and shoots died with 80 mM NaCl. With 2 mM B, the B concentration of explants was 5--9X greater for the various NaCl treatments compared to the control. Increasing the NaCl concentration from 10 to 80 mM, resulted in higher Na and Cl concentrations in explants for all B treatments, while K and Ca concentrations decreased. Phosphorus concentration in the explants was significantly increased by increasing the NaCl concentration reaching a maximum value at 80 mM NaCl for the two B concentrations.     

Counteraction of Salinity Stress on Wheat Plants by Grain Soaking in Ascorbic Acid, Thiamin or Sodium Salicylate

The interactive effects of salinity stress (40, 80, 120 and 160 mM NaCl) and ascorbic acid (0.6 mM), thiamin (0.3 mM) or sodium salicylate (0.6 mM) were studied in wheat (Triticum aestivum L.). The contents of cellulose, lignin of either shoots or roots, pectin of root and soluble sugars of shoots were lowered with the rise of NaCl concentration. On the other hand, the contents of hemicellulose and soluble sugars of roots, starch and soluble proteins of shoots, proline of either shoots or roots, and amino acids of roots were raised. Also, increasing NaCl concentration in the culture media increased Na⁺ and Ca²⁺ accumulation and gradually lowered K⁺ and Mg²⁺ concentration in different organs of wheat plant. Grain soaking in ascorbic acid, thiamin or sodium salicylate could counteract the adverse effects of NaCl salinity on the seedlings of wheat plant by suppression of salt stress induced accumulation of proline.     

The intracellular Na+ and K+ composition of the moderately halophilic bacterium, Paracoccus halodenitrificans

The intracellular concentrations of Na+ and K+ in exponentially growing Paracoccus halodenitrificans were independent of the NaCl concentration of the growth medium. The observed values were approximately 100 and 300 mM for Na+ and K+, respectively. In stationary phase cells, the ultimate values for Na+ depended on the NaCl concentration of the growth medium. With cells grown in the presence of 1 M NaCl, the value was about 500 mM; for cells grown in the presence of 3 M NaCl, the value was about 1.1 M. The K+ concentration in stationary phase cells was unaffected by the NaCl concentration in the growth medium. The final value was about 100 mM. Associated with these changes were changes in the ATP pool and decreases in the activities of the NADH oxidase system and the membrane-bound ATPase. It is proposed that the decrease in the activities of these enzymes may account for the ion flows observed in stationary phase cells.     

Glycerol fomation inDunaliella cells under non-growing conditions with hypertonic lithium or magnesium media

Glycerol formation ofDunaliella cells in non-growing media was investigated.Dunaliella tertiolecta andD. bioculata grew well in a NaCl medium but not at all in a LiCl or a MgCl₂ medium. When the cells originally suspended in a medium containing 0.5 M NaCl were transferred to media which contained one of 1 M NaCl, 1 M LiCl or 0.7 M MgCl₂, the intracellular glycerol content increased.D. tertiolecta cultured in either a 1 M LiCl or a 0.7 M MgCl₂ medium did not multiply, but maintained abilities to evolve O₂ in the light and absorb O₂ in thedark even after about a 5 day culture. From these results, it can be concluded that the halotolerance ofDunaliella to different kinds of salts is not directly related to osmoregulation by the glycerol formation.     

Physiological characterization and stress-induced metabolic responses of Dunaliella salina isolated from salt pan

A Dunaliella strain was isolated from salt crystals obtained from experimental salt farm of the institute (latitude 21.46 N, longitude 72.11 degrees E). The comparative homology study of amplified molecular signature 18S rRNA, proves the isolated strain as D. salina. The growth pattern and metabolic responses such as proline, glycine betaine, glycerol, total protein and total sugar content to different salinity (from 0.5 to 5.5 M NaCl) were studied. The optimum growth was observed at 1.0 M NaCl and thereafter it started to decline. Maximum growth was obtained on 17th day of inoculation in all salt concentrations except 0.5 M NaCl, whereas maximum growth was observed on 13th day. There were no significant differences (P < 0.01) in chlorophyll a/b contents (1.0-1.16 +/- 0.05 mug chl. a and 0.2-0.29 +/- 0.01 mug chl. b per 10(6) cells) up to 2.0 M NaCl, however at 3.0 M NaCl a significant increase (2.5 +/- 0.12 mug chl. a and 0.84 +/- 0.4 mug chl. b per 10(6) cells) was observed which declined again at 5.5 M NaCl concentration (2.0 +/- 0.1 mug chl. a and 0.52 +/- 0.03 mug chl. b per 10(6) cells). Stress metabolites such as proline, glycine betaine, glycerol and total sugar content increased concomitantly with salt concentration. Maximum increase in proline (1.4 +/- 0.07 mug), glycine betaine (5.7 +/- 0.28 mug), glycerol (3.7 +/- 0.18 ml) and total sugar (250 +/- 12.5 mug) per 10(5) cells was observed in 5.5 M NaCl. A decrease in total protein with reference to 0.5 M NaCl was observed up to 3.0 M NaCl, however, a significant increase (P < 0.01) was observed at 5.5 M NaCl (0.19 +/- 0.01 mug per 10(5) cells). Inductive coupled plasma (ICP) analysis shows that intracellular Na(+) remained unchanged up to 2.0 M NaCl concentration and thereafter a significant increase was observed. No relevant increase in the intracellular level of K(+) and Mg(++) was observed with increasing salt concentration. Evaluation of physiological and metabolic attributes of Dunaliella salina can be used to explore its biotechnological and industrial potential.     

The role of salinity in seed maturation of the euhalophyte Suaeda salsa

Controlled conditions were used to investigate how salinity maintains the salt tolerance of seeds and seedlings of the euhalophyte Suaeda salsa. Seeds were harvested from S. salsa plants that had been treated with 1 or 500 mM NaCl for 113 days in a glasshouse. The results showed that high salinity (500 mM NaCl) increased chlorophyll concentration and oxygen production in embryos of maturing seeds. At 500 mM NaCl, the phosphatidylglycerol and sulfoquinovosyldiacylglycerol levels and the digalactosyldiacylglycerol/monogalactosyldiacylglycerol ratio were higher in young seedlings derived from seeds whose source plants were cultured in 500 mM rather than in 1 mM NaCl. When seeds were incubated with 600 mM NaCl, the conductivity and malondialdehyde concentration in the embryos was greater if the source plants had been cultured in 1 mM rather than in 500 mM NaCl. The opposite pattern was evident for seedling survival and shoot weight. In conclusion, salinity during seed maturation may increase the salt tolerance of seeds and seedlings by increasing the oxygen production in the embryos of the maturing seeds and by changing the lipid composition of membranes in the seedlings.     

Photosystem II photochemistry and physiological parameters of three fodder shrubs, <Emphasis Type="Italic">Nitraria retusa</Emphasis>, <Emphasis Type="Italic">Atriplex halimus</Emphasis> and <Emphasis Type="Italic">Medicago arborea</Emphasis> under salt stress

Nitraria retusa and Atriplex halimus (xero-halophytes) plants were grown in the range 0–800 mM NaCl while Medicago arborea (glycophyte) in 0–300 mM NaCl. Salt stress caused a marked decrease in osmotic potential and a significant accumulation of Na⁺ and Cl⁻ in leaves of both species. Moderate salinity had a stimulating effect on growth rate, net CO₂ assimilation, transpiration and stomatal conductance for the xero-halophytic species. At higher salinities, these physiological parameters decreased significantly, and their percentages of reduction were higher in A. halimus than in N. retusa whereas, in M. arborea they decreased linearly with salinity. Nitraria retusa PSII photochemistry and carotenoid content were unaffected by salinity, but a reduction in chlorophyll content was observed at 800 mM NaCl. Similar results were found in A. halimus, but with a decrease in the efficiency of PSII (F′v/F′m) occurred at 800 mM. Conversely, in M. arborea plants we observed a significant reduction in pigment concentrations and chlorophyll fluorescence parameters. The marked toxic effect of Na⁺ and/or Cl⁻ observed in M. arborea indicates that salt damage effect could be attributed to ions’ toxicity, and that the reduction in photosynthesis is most probably due to damages in the photosynthetic apparatus rather than factors affecting stomatal closure. For the two halophyte species, it appears that there is occurrence of co-limitation of photosynthesis by stomatal and non-stomatal factors. Our results suggest that both N. retusa and A. halimus show high tolerance to both high salinity and photoinhibition while M. arborea was considered as a slightly salt tolerant species.     

Regulation of intracellular osmotic pressure during the initial stages of salt stress in a salt-tolerant yeast, Zygosaccharomyces rouxii.

The accumulation of glycerol and inorganic ions as it related to osmotic pressure, and the regulation of intracellular osmotic pressure in a salt-tolerant yeast, Zygosaccharomyces rouxii, were examined for several hours after salt stress. Intracellular contents of glycerol increased for up to 6 h in media supplemented with 1 M and 2 M NaCl and did not increase in medium containing 3 M NaCl. Intracellular contents of Na+ and Cl- reached a maximum value within 1 and 3 h, respectively, in all NaCl-containing media and increases were proportional to the concentration of NaCl in the medium. As glycerol was accumulated in cells, the intracellular contents of Na+ and Cl- gradually decreased in media containing 1 M and 2 M NaCl. After salt stress, cell volume decreased within 1 h and the original volume was re-established for 3 to 6 h in media with 1 M and 2 M NaCl but not in medium with 3 M NaCl. Intracellular concentrations of solutes, which were calculated from the total contents of glycerol and inorganic ions and the cell volume, became almost equivalent to the external osmotic pressure within 1 h after salt stress. Experiments using various inhibitors showed that a large amount of ATP was required not only for the synthesis and accumulation of glycerol but also for the exclusion of Na+ and Cl- from cells under salt-stressed conditions.     

Physiological responses of Zygosaccharomyces rouxii to osmotic stress

When cell suspensions of Zygosaccharomyces rouxii were subjected to osmotic shock with NaCl, the cell volume decreased sharply and plasmolysis was observed. The cell subsequently recovered and volumes similar to those of cells growing at the respective water activity (a_w) values were found. Cycloheximide prevented cell recovery, indicating the involvement of protein synthesis in the recovery process. The intracellular glycerol concentration of Z. rouxii incubated in the presence of [¹⁴C]glycerol increased from 13 to 96 mmol/l during the initial 20 min after an upshock from 0.998 a_w to 0.96 a_w. All the intracellular glycerol was labelled and therefore derived from the medium. Labelled glycerol was subsequently utilized and replaced by unlabelled glycerol produced by the cell within 90 min. The initial increase in glycerol concentration following the upshock was confirmed by ¹³C-nuclear magnetic resonance (NMR) spectroscopic studies of cell extracts. The combined dihydroxyacetone and dihydroxyacetone phosphate concentrations fluctuated during this period, whereas glycerol-3-phosphate initially increased and then remained constant. This indicates that the production of glycerol is regulated. Decreases in ATP and polyphosphate levels were observed following osmotic upshock and may reflect a greater demand for ATP during the period of adjustment to decreased a_w. The changes in cell volume and in ATP concentration following osmotic upshock may serve as osmoregulatory signals in Z. rouxii, as suggested previously for other microorganisms. Correspondence to: S. G. Kilian