Effect of proline and arginine metabolism on freezing stress of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the PUT1-encoded proline oxidase and the PUT2-encoded delta1-pyrroline-5-carboxylate dehydrogenase are required to convert proline to glutamate. We recently showed that a put1 disruptant accumulated higher levels of proline intracellularly and conferred higher resistance to freezing stress. Here, we determined the effect of put2 disruption on yeast cell viability under freezing stress. When grown on arginine as the sole nitrogen source, the put2 disruptant showed a significant decrease in cell viability after freezing despite the high proline and arginine contents. This result suggests that delta1-pyrroline-5-carboxylate or glutamate-gamma-semialdehyde, a proline catabolism intermediate, is toxic to yeast cells under freezing stress. In contrast, the survival rate of the wild-type and the put1-disruptant strains was found to increase after freezing in proportion to their arginine contents. This indicates that arginine has a cryoprotective function in yeast. Furthermore, the yeast cells accumulated proline as well as arginine in the vacuole, suggesting that there is a system for the transport of excess proline to the vacuole and that this vacuolar accumulation may be important in the freezing resistance of yeast cells.     

Endocytosis and vacuolar morphology in Saccharomyces cerevisiae are altered in response to ethanol stress or heat shock

The vital lipophilic dye N‐(3‐triethylammoniumpropyl)‐4‐[6‐(4‐(diethylamino)phenyl]hexatrienyl) pyridinium dibromide (FM 4‐64) was used to study the effect of ethanol stress and heat shock on endocytosis in the yeast Saccharomyces cerevisiae. Yeast cells stained with FM 4‐64 were placed in a culture chamber and the internalization of the dye was monitored by fluorescence microscopy during perfusion of the cells with fresh growth medium. In the absence of ethanol in the perfusion medium, the internalization of FM 4‐64 from the plasma membrane to the vacuolar membrane by yeast cells harvested from the exponential phase of growth was completed in 30 min. The presence of 6% (v/v) ethanol in the perfusion medium had no obvious effect on the internalization of FM 4‐64 from the plasma membrane, but did lead to an accumulation of the dye in endocytic intermediates. Consequently, vacuolar membrane staining was delayed. Cells stained with FM 4‐64 and subjected to heat shock displayed a similar effect, with endocytic intermediates becoming more prominent with the severity of the heat shock. For both ethanol stress and heat shock, vacuolar morphology altered from segregated structures to a single, large organelle. The findings of this study reinforce previous observations that ethanol stress and heat shock induce similar responses in yeast. Copyright © 1999 John Wiley & Sons, Ltd.     

Removal and recovery of lithium using various microorganisms

The accumulation of lithium by microorganisms was examined. Among the 70 strains of the 63 species tested (20 bacteria, 18 actinomycetes, 18 fungi, and 14 yeasts), a high lithium accumulating ability was exhibited by strains of the bacteria, Arthrobacter nicotianae and Brevibacterium helovolum. Lithium accumulation by A. nicotianae cells was strongly affected by the pH of the solution. The amount of accumulated lithium was maximum at pH 6. Cells immobilized with polyacrylamide gel also adsorbed lithium. They could be reused during repeated adsorptions, and adsorbed 548 micromol of lithium/g dry wt. cells. The adsorbed lithium was quantitatively and easily desorbed with 1 M hydrochloric acid using a column system.     

Production of L-talitol from L-psicose by Metschnikowia koreensis LA1 isolated from soy sauce mash

A strain LA1 that can convert L-psicose to L-talitol was isolated from soy sauce mash and identified as Metschnikowia koreensis. The cells grown on L-arabitol were found to have relatively high conversion potential. Addition of D-sorbitol to the reaction mixture considerably accelerated the conversion rate of L-psicose to L-talitol. During the conversion reaction, D-sorbitol was added to the reaction mixture at 12-h intervals to maintain the concentration of D-sorbitol at 1.0%. The final conversion ratios were 81.4%, 75.2%, 73.0%, 60.4% and 43.5% using washed cells when the concentrations of L-psicose were 0.5%, 1.0%, 2.0%, 3.0% and 5.0%, respectively. The product from L-psicose was identified as L-talitol by HPLC analysis, and infrared spectroscopy, optical rotation and melting point measurements.     

In situ study of K+ transport into the vacuole of Saccharomyces cerevisiae

Permeable spheroplasts were prepared from two strains of Saccharomyces cerevisiae by incubating with zymolyase without a permeabilizing agent. The loss of the plasma membrane barrier was confirmed by the nucleotide release, the activity of glucose 6-phosphate dehydrogenase with external substrates and by the effects on respiration of mitochondrial substrates and ADP. Mitochondrial integrity was maintained, as shown by respiration with lactate, pyruvate, glucose and ethanol, and its acceleration by ADP showed a coupled respiration. Potassium uptake into the vacuole was measured with a selective electrode and found to be taken up effectively by spheroplasts only in the presence of Mg-ATP; it was reverted by CCCP and PCP and inhibited by bafilomycin A1, but not by sodium vanadate or sodium azide. Potassium ions did not alter DeltaPsi of the vacuole, followed with oxonol V, but caused vacuolar alkalinization, as followed with pyranine. The increase of vacuolar pH was non-selective and observed at 50-200 mM of several monovalent cations. Isolated vacuoles with pyranine inside showed similar changes of the internal pH in the presence of KCl. Results indicate that some strains do not require a permeabilizing agent to directly access the vacuole in spheroplasts prepared with zymolyase. The hypothesis about the existence of a K+/H+ antiporter in the vacuolar membrane of S. cerevisiae is discussed.     

Expression of the Drosophila 70,000 Dalton heat shock protein is translationally controlled in yeast

Plasmid pPW229, containing the 2.25 kilobase transcribed sequence for the 70,000 Dalton heat shock protein of Drosophila, was integrated into plasmid CV13 and used to transform Saccharomyces cerevisiae. Upon a heat shock, at 41 degrees C for 20 min, a new 70,000 Dalton protein appeared in the transformants. This protein was not detected in transformants grown at 23 degrees C, nor in transformants carrying the hybrid plasmid from which the structural gene for the 70,000 Dalton protein had been deleted. RNA was isolated from transformants grown at 23 degrees C and from transformants heat shocked at 41 degrees C. RNA complementary to the Drosophila heat shock gene was present in the transformants, grown either at 23 degrees C or heat shocked. No complementary RNA was detected in yeast cells transformed with the hybrid plasmid from which the structural gene had been deleted. The Drosophila heat shock gene in yeast appears to be transcribed constitutively but translated only under heat shock conditions.     

Production of L-sorbitol from L-fructose by Aureobasidium pullulans LP23 isolated from soy sauce mash

A strain LP23 that can convert L-fructose to L-sorbitol was isolated from soy sauce mash and identified as Aureobasidium pullulans. The cells grown on L-arabinose were found to have relatively high L-fructose to L-sorbitol conversion potential. Addition of erythritol to the reaction mixture considerably accelerated the conversion rate of L-fructose to L-sorbitol. During the conversion reaction, erythritol was added to the reaction mixture at 8-h intervals to maintain the concentration of erythritol at 1.0%. The final conversion ratios were 82.8%, 95.3%, 92.4%, and 42.6% using washed cells when the concentrations of L-fructose were 1.0%, 2.0%, 5.0% and 10.0%, respectively. The product from L-fructose was identified as L-sorbitol by HPLC analysis, infrared spectroscopy, optical rotation and melting point measurements.     

Therapeutic use of phage cocktail for controlling Escherichia coli O157:H7 in gastrointestinal tract of mice

To investigate the therapeutical use of phage mixture for controlling gastrointestinal Escherichia coli O157:H7 cells, in vitro and in vivo experiments were conducted. Three phages, SP15, SP21, and SP22 were selected from 26 phage stock screened from feces of stock animals and sewage influent. Addition of single or binary phage to the E. coli cell batch-culture reduced the turbidity of the culture. However, reascend of the turbidity due to the appearance of phage resistance cell was observed. On the other hand, addition of three phage mixture (SP15-21-22) did not produce reascend of culture turbidity under aerobic condition. Under anaerobic condition, slight reascend of culture turbidity was observed after SP15-21-22 addition. Chemostat continuous culture was operated under anaerobic condition to optimize the titer of phage cocktail and frequency of the addition for controlling E. coli cells. Five-log decrease of E. coli cell concentration after addition of phage cocktail of 10(9) Plaque forming unit (PFU)/ml was observed. However, reascend of cell concentration was observed after 1 d incubation. Repeated addition of phage cocktail was effective to reduce the cell concentration. Suspension of phage cocktail in the buffer containing 0.25% CaCO3 neutralized 9 times much more buffer of pH 2. Based on this in vitro experiment, phage cocktail (SP15-21-22) suspended in the buffer containing 0.25% CaCO3 was orally administrated to the mice in which E. coli O157:H7 cells was administrated in 2-d advance. E. coli and phage concentration in the feces was monitored for 9 d after phage addition. High titer of phage was detected in the feces when the phage cocktail administrated daily. E. coli O157:H7 concentration in the feces has been reduced according to the time period. However, difference of E. coli concentration in the feces of mice administrates with phage and in the control mice without phage addition became slight after 9-d test period. High titer of the phage settled down in the gastrointestinal tracts and reduced the concentration of E. coli cell. Repeated oral administration of SP15-21-22 was effective for rapid evacuation of E. coli O157:H7 from the feces and gastrointestinal tract of mice.     

Cloning and expression of NAD+-dependent L-arabinitol 4-dehydrogenase gene (ladA) of Aspergillus oryzae

A gene of Aspergillus oryzae, ladA, which encodes L-arabinitol 4-dehydrogenase (EC 1.1.1.12), and its cDNA were cloned in Escherichia coli. The gene consisted of a 1209-bp coding region, interrupted by a 59-bp intron, which encoded a 382-amino-acid polypeptide (40,812 Da). The protein showed 67% identity to a well-studied L-arabinitol 4-dehydrogenase (Lad1) of Hypocrea jecorina. The cell-free extract of E. coli, which expressed ladA cDNA, showed L-arabinitol dehydrogenase activity with NAD+. It was also reactive for ribitol and xylitol.     

Lactic-acid stress causes vacuolar fragmentation and impairs intracellular amino-acid homeostasis in Saccharomyces cerevisiae

To gain more insight into adaptation response to lactic-acid stress in yeast, a genome-wide screening for genes whose disruption caused hypersensitivity to 4.0% l-lactic acid (pH 2.8) was performed using the gene deletion collection of Saccharomyces cerevisiae. We identified 107 genes that contributed significantly to the ability of yeast cells to adapt lactic-acid stress. More than 30% of the genes identified in this screening were newly identified to be involved in mechanisms for adaptation response to lactic acid. We found that protein urmylation by Uba4 and N-terminal acetylation by Nat3 were involved in lactic acid adaptation mechanisms. Functional categorization of the genes followed by microscopic analysis revealed that a variety of cellular functions were involved in adaptation response to lactic acid and function associated with vacuolar transport played important roles in adaptation response to lactic acid. We also found that vacuole fragmented immediately upon exposure to lactic- and hydrochloric-acid stress. In addition, our analysis revealed that lactic-acid stress significantly reduced the amount of intracellular amino acids. Amino acid supplementation recovered the adaptation deficiency to lactic acid, suggesting that intracellular amino-acid homeostasis plays important roles in adaptation response to lactic-acid stress. These data suggest that enhancing vacuolar integrity, as well as maintaining intracellular amino-acid homeostasis may be an efficient approach to confer resistance to lactic-acid stress.     

Sterol glycosides and cerebrosides accumulate in Pichia pastoris, Rhynchosporium secalis and other fungi under normal conditions or under heat shock and ethanol stress

The occurrence of glycolipids such as sterol glycosides, acylated sterol glycosides, cerebrosides and glycosyldiacylglycerols was examined in the three yeast species Candida albicans, Pichia pastoris and Pichia anomala, as well as in the six fungal species Sordaria macrospora, Pyrenophora teres, Ustilago maydis, Acremonium chrysogenum, Penicillium olsonii and Rhynchosporium secalis. Cerebroside was found in all organisms tested, whereas acylated sterol glycosides and glycosyldiacylglycerols were not found in any organism. Sterol glycosides were detected in P. pastoris strain GS115, U. maydis, S. macrospora and R. secalis. This glycolipid occurred in both yeast and filamentous forms of U. maydis but in neither form of C. albicans. This suggests that sterol glycoside is not correlated with the separately grown dimorphic forms of these organisms. Cerebrosides and sterol glycosides from P. pastoris and R. secalis were purified and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The cerebrosides are beta-glucosyl ceramides consisting of a saturated alpha-hydroxy or non-hydroxy fatty acid and a Delta4,8-diunsaturated, C9-methyl-branched sphingobase. Sterol glycoside from P. pastoris was identified as ergosterol-beta-D-glucopyranoside, whereas the sterol glucosides from R. secalis contain two derivatives of ergosterol. The biosynthesis of sterol glucoside in P. pastoris CBS7435 and GS115 depended on the culture conditions. The amount of sterol glucoside in cells grown in complete medium was much lower than in cells from minimal medium and a strong increase in the content of sterol glucoside was observed when cells were subjected to stress conditions such as heat shock or increased ethanol concentrations. From these data we suggest that, in addition to Saccharomyces cerevisiae, new yeast and fungal model organisms should be used to study the physiological functions of glycolipids in eukaryotic cells. This suggestion is based on the ubiquitous and frequent occurrence of cerebrosides and sterol glycosides, both of which are rarely detected in S. cerevisiae. We suggest P. pastoris and two plant pathogenic fungi to be selected for this approach.     

Vacuole Segregation in the Saccharomyces cerevisiae vac2-1 Mutant: Structural and Biochemical Quantification of the Segregation Defect and Formation of New Vacuoles

The conditional vacuolar segregation mutant vac2-1 [Shaw and Wickner (1991) EMBO J. 10, 1741–1748] shifted to non-permissive temperature (37°C), forms large-budded cells without a vacuole in the bud, and daughter cells without an apparent vacuole. Some cells still contain normal segregation structures. Structural and biochemical quantification of the segregation defect showed that (i) about 10% of the full-grown buds did not contain a vacuole, (ii) about 15% of the small cells washed out of a population growing in an elutriation chamber at 37°C, did not contain a visible vacuole, and (iii) 15% of the cells per generation lost carboxypeptidase Y activity after proteinase A depletion. Thus, 10–15% of the daughter cells did not inherit vacuolar structures or vacuolar proteolytic activity from the mother cell. To investigate the fate of vacuole-less daughters, these cells were isolated by optical trapping. The isolated cells formed colonies on agar plates that consisted of cells with normal vacuoles, both at 23 and 37°C. Thus, the vacuole-less cells that failed to inherit proteolytic activities from the mother cell apparently give rise to progeny containing structurally normal vacuoles. Time-lapse experiments showed that vacuole-less daughter cells formed vacuolar vesicles that fused into a new vacuole within 30 min. Although new buds only emerged after a vacuole had formed in the mother cell, the temporary lack of a vacuole had little effect on growth rate. The results suggest that an alternative pathway for vacuole formation exists, and that yeast cells may require a vacuole of some minimal size to initiate a new round of budding. © 1997 by John Wiley & Sons, Ltd.     

Cell shape and growth of budding yeast cells in restrictive microenvironments

Effects of limited growth space on the cell morphology and cell growth are investigated by creating rigid outside environments. The cube-shaped holes big enough for a single cell of the budding yeast Saccharomyces cerevisiae were prepared with a focused ion-beam (FIB), commonly used for processing semiconductors. We demonstrated that the outline of the cells changes their ellipsoidal morphology into a cubic form when the daughter cells are grown in the holes, indicating that yeast cells change their shape in response to external limited space. The yeast cells grown in the microenvironments exhibit neither bud formation nor nuclear division. Although restricted growth caused by the physical barriers leads to the block of cell cycle progression in the wild-type cells, swe1Delta cells defective in the morphogenesis checkpoint become binucleate after being grown in the microenvironments. These results suggest that yeast cells under spatial restriction arrest cell cycle progression in a Swelp-dependent manner.     

Saccharomyces cerevisiae and Zygosaccharomyces mellis exhibit different hyperosmotic shock responses

The effect of hyperosmotic shock on cell volume, vacuole volume, and intracellular pH (pH(i)) of individual cells of Saccharomyces cerevisiae and Zygosaccharomyces mellis was investigated. After transfer from a high water activity (a(w)) medium to low a(w) media, the growth latency periods of Z. mellis were shorter than those of S. cerevisiae. These results demonstrate that Z. mellis manages hyperosmotic shock better than S. cerevisiae. As a response to acute hyperosmotic shock, i.e. the first minute of perfusion with hypertonic buffers, the vacuoles shrank and the pH(i) decreased in both yeasts. Furthermore, in the presence of glucose, vacuole shrinkage and intracellular acidification were more pronounced in S. cerevisiae than in Z. mellis. These results may be explained by the fact that the S. cerevisiae cells shrank more than the Z. mellis cells as a response to acute hyperosmotic shock. In the presence of glucose, the vacuoles and the cells of both S. cerevisiae and Z. mellis shrank simultaneously and in proportion to a minimum level during acute hyperosmotic shock, and remained constant at this level throughout the experiment (11 min). These results indicate that vacuoles do not act as water reserves in yeasts after acute hyperosmotic shock. Finally, Z. mellis was able to maintain its pH(i) near normal physiological levels after acute hyperosmotic shock, whereas S. cerevisiae was not. These results suggest that pH(i) regulation may be important for the ability of yeasts to manage hyperosmotic shock.     

The determinants of heat-shock element-directed lacZ expression in Saccharomyces cerevisiae.

Heat-shock induction of heat-shock protein genes is due to a specific promoter element (the heat-shock element, HSE). This study used lacZ under HSE control (HSE-lacZ) to characterize HSE activity in Saccharomyces cerevisiae cells of different physiological states and differing genetic backgrounds. In batch fermentations HSE-lacZ induction by heat shock was maximal in exponential growth, and showed marked decline with the approach to stationary phase. Expression in the absence of heat shock was unaffected by growth phase, indicating that the growth-dependent expression of many yeast heat-shock genes uses promoter elements in addition to the HSE. Heat-induced expression was strongly influenced by the temperature at which cultures were grown. While basal, uninduced expression was constant during growth at different temperatures to 30 degrees C, induction by transfer to 39 degrees C was reduced by increases in growth temperature as low as 18-24 degrees C. Maximal HSE-lacZ induction (30- to 50-fold) was in cultures grown at low temperatures (18-24 degrees C), then heat shocked at 39 degrees C. Ethanol was a poor inducer. Mutations having little effect on HSE-lacZ expression included a respiratory petite; ubi4 (which inactivates the poly-ubiquitin gene); also ubc4 and ubc5 (which each inactivate one of the ubiquitin ligases involved in degradation of aberrant protein). pep4-3 increased both basal and induced beta-galactosidase about two-fold, probably because of slower turnover of this enzyme in pep4-3 strains.     

Proline accumulation in baker's yeast enhances high-sucrose stress tolerance and fermentation ability in sweet dough

During bread-making processes, yeast cells are exposed to various baking-associated stresses. High-sucrose concentrations exert severe osmotic stress that seriously damages cellular components by generation of reactive oxygen species (ROS). Previously, we found that the accumulation of proline conferred freeze-thaw stress tolerance and the baker's yeast strain that accumulated proline retained higher-level fermentation abilities in frozen doughs than the wild-type strain. In this study, we constructed self-cloning diploid baker's yeast strains that accumulate proline. These resultant strains showed higher cell viability and lower intracellular oxidation levels than that observed in the wild-type strain under high-sucrose stress condition. Proline accumulation also enhanced the fermentation ability in high-sucrose-containing dough. These results demonstrate the usefulness of proline-accumulating baker's yeast for sweet dough baking.     

The Yeast Vacuole ‐A Scanning Electron Microscopy Study During High Gravity Wort Fermentations

The yeast vacuole has been shown to exhibit morphological responses to environmental conditions when exposed to worts of different gravity during fermentation. Marked effects of high gravity wort (20° Plato) on yeast morphology compared to more conventional wort gravity (12° Plato) were observed. High gravity worts caused vacuolar enlargement compared to conventional gravity wort. These results suggested that yeast cells experienced severe alterations with the vacuolar tonoplast when exposed to high osmotic pressure and elevated levels of ethanol.     

Performance of a styrene-degrading biofilter inoculated with Pseudomonas sp. SR-5

Styrene removal was studied for 3 months in a laboratory-scale biofilter packed with a mixed packing material of peat and ceramic at a ratio of 1 to 1 on a dry-weight basis and inoculated with Pseudomonas sp. SR-5. More than 90% removal efficiency (RE) was attained at 1-140 g/m3/h styrene loads under nitrogen-source limitation. When RE decreased to 70% after 30 d with an increase in styrene load, readdition of SR-5 and washing of the filter packing material restored the RE to more than 90% by maintaining the population of SR-5 at 1-10% of the total cell number. The maximum elimination capacity (EC) by kinetic analysis was estimated to be 290 g/m3/h. High conversion of the removed styrene carbon to CO2, and significantly small production of cell mass from the removed carbon were confirmed.     

高耐性酿酒酵母的筛选及其耐受性研究

优良的耐逆性菌株的添加能有效提高酱油生产效率和产品品质风味,该研究筛选出两株耐高温、耐高渗和耐高酸的酵母菌株用于酱油发酵。通过对酵母菌株高温热激之后稀释点板,对比各稀释度的菌落数量和形态,以及通过在高渗板和高酸板上各个菌的生长情况和在抗性培养基中菌的生长曲线测定来对比各菌株的耐受性。稀释点板实验以及生长曲线结果都显示,酿酒酵母L-19和L-38在55 ℃热激条件下以及在分别含有6%NaCl、0.6%乙酸和5%乳酸固体平板上菌落形态和大小都优于酱油酵母,而且在含有高盐和高酸的液体培养基中生长速率均高于酱油酵母。因此,成功筛选出两株具有高耐性的酿酒酵母。