Regulation of glycerol metabolism in Zygosaccharomyces rouxii in response to osmotic stress

Summary Enzyme analyses indicated that the metabolism of glycerol by Zygosaccharomyces rouxii occurred via either glycerol-3-phosphate (G3P) or dihydroxyacetone (DHA). The route via DHA is significant in osmoregulation. The specific activities of glycerol dehydrogenase (GDHG) and DHA kinase, which metabolize glycerol via DHA, increased nine- and fourfold respectively during osmotic stress [0.960 water activity (a_w) adjusted with NaCl] when compared to non-stressed conditions (0.998 a_w). Both pathways are under metabolic regulation. Glycerol kinase, mitochondrial G3P dehydrogenase and DHA kinase are induced by glycerol while the latter is also repressed by glucose. Cells treated with cycloheximide prior to osmotic upshock showed significantly lower DHA kinase and GDHG levels and lower intracellular glycerol concentrations when compared to untreated control cells. Thus protein synthesis is essential for osmotic adaptation. Offprint requests to: B. A. Prior     

Physiological and genetic responses of bacteria to osmotic stress.

The capacity of organisms to respond to fluctuations in their osmotic environments is an important physiological process that determines their abilities to thrive in a variety of habitats. The primary response of bacteria to exposure to a high osmotic environment is the accumulation of certain solutes, K+, glutamate, trehalose, proline, and glycinebetaine, at concentrations that are proportional to the osmolarity of the medium. The supposed function of these solutes is to maintain the osmolarity of the cytoplasm at a value greater than the osmolarity of the medium and thus provide turgor pressure within the cells. Accumulation of these metabolites is accomplished by de novo synthesis or by uptake from the medium. Production of proteins that mediate accumulation or uptake of these metabolites is under osmotic control. This review is an account of the processes that mediate adaptation of bacteria to changes in their osmotic environment.     

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.     

A temperature-sensitive osmophilic mutant of Zygosaccharomyces rouxii

Summary A temperature-sensitive osmophilic mutant of Zygosaccharomyces rouxii, OS15, was isolated, which required high salt or sugar concentration for growth above 30°C. Cell viability at 35°C in the presence of NaCl was higher than in the absence of NaCl, and a survival ratio of the mutant cells after incubation at 55°C was also higher in the presence of NaCl than NaCl-free condition. Furthermore, resistance to UV light, hygromycin B and geneticin was improved in the presence of NaCl. There was no difference between the parent and the mutant in fatty acid saturation and microscopic cell shape under NaCl condition.     

Microbial responses to salt-induced osmotic stress

Summary The construction and assembly of a model root region is described. The model was used to manipulate the soil matrix, soil microorganisms, and to simulate release of root exudates. The design of the apparatus facilitated long-term, direct microscopic observations of microbial activity in soil and on artificial roots. Preliminary studies indicate that microbial responses to osmotic stress and to changes in components of exudate solutions are easily monitored.     

Microbial responses to salt-induced osmotic stress

Summary Soil columns were exposed to balanced (low Na⁺) or unbalanced (high Na⁺) high-salt solutions for a period of 7 days followed by 7 days of stress reflief. Total numbers of bacteria released into the perfusates rose under both types of stress, but the proportion of displaced bacteria that were viable fell significantly. Relief from both types of stress stimulated rapid increases in the number of viable micro-organisms released from soil. Examination of the soils at the end of the relief periods revealed that soils exposed to stress contained more viable bacteria than the non-stressed controls. However, high levels of balanced stress led to a significant decrease in species diversity within the microbial population, but a similar effect was not observed in soils exposed to unbalanced, high Na⁺ stress. These results suggest that, while salt stress may cause a significant reduction in the number of microorganisms in a soil, a large portion of the microbial population can rapidly adapt to marked changes in salinity.     

Tools for the genetic manipulation of Zygosaccharomyces rouxii

A set of tools for the genetic manipulation of the osmotolerant yeast Zygosaccharomyces rouxii was developed. Auxotrophic mutants (ura3 leu2, ura3 ade2, ura3 leu2 ade2) derived from the CBS 732 type strain were prepared. Centromeric and episomal Z. rouxii/Escherichia coli shuttle plasmids with different marker genes (ScURA3, ZrLEU2, ZrADE2) and with multiple cloning sites were constructed, together with a plasmid enabling green fluorescent protein-tagging. A system for repeatable targeted gene deletion in Z. rouxii was established, involving first the integration of a PCR-generated loxP-kanMX-loxP cassette and second the removal of kanMX from the genome using a Z. rouxii plasmid harbouring cre recombinase.     

鲁氏酵母胞内海藻糖积累过程的代谢特征分析

海藻糖合成是微生物对抗环境逆境的一种重要途径。研究10L发酵罐中的分批、分批补料及分批补料控温三种不同的海藻糖发酵调控策略下酱油风味形成微生物鲁氏酵母CCTCC M2013310的代谢特征。色谱结果表明,乳酸、丙酮酸和α-酮戊二酸受到不同发酵调控模式的显著影响,但谷氨酸和谷氨酰胺总含量在三种发酵调控模式间却无显著差异。这些结果表明,细胞还原力平衡途径和碳氮调控代谢均对胞内海藻糖的积累产生影响。研究结果为鲁氏酵母CCTCC M2013310的高浓度内源性海藻糖细胞代谢工程改造提供了新思路。     

Regulation of aspartate-derived amino-acid metabolism in Zygosaccharomyces rouxii compared to Saccharomyces cerevisiae

To elucidate the growth inhibitory effect of threonine, the regulation of the aspartate-derived amino-acid metabolism in Zygosaccharomyces rouxii, an important yeast for the flavor development in soy sauce, was investigated. It was shown that threonine inhibited the growth of Z. rouxii by blocking the methionine synthesis. It seemed that threonine blocked this synthesis by inhibiting the conversion of aspartate. In addition, it was shown that the growth of Z. rouxii, unlike that of Saccharomyces cerevisiae, was not inhibited by the herbicide sulfometuron methyl (SMM). From enzyme assays, it was concluded that the acetohydroxy acid synthase in Z. rouxii, unlike that in S. cerevisiae, was not sensitive to SMM. Furthermore, the enzyme assays demonstrated that the activity of threonine deaminase in Z. rouxii, like in S. cerevisiae, was strongly inhibited by isoleucine and stimulated by valine. From this work, it is clear that the aspartate-derived amino-acid metabolism in Z. rouxii only partly resembles that in S. cerevisiae.     

Novel actin ring structure in sporulation of Zygosaccharomyces rouxii

The filamentous actin (F-actin) during sporulation of Zygosaccharomyces rouxii was visualized with rhodamine-phalloidin, and then the behavior was observed using confocal laser scanning microscopy. During spore formation, we found a novel actin ring structure that has not been reported in other yeasts and molds in sporulation. The ring surrounded each meiotic nucleus at the peripheral regions of spores. Three-dimensional observation suggested that the ring was not an artificial structure produced by spherical structure sectioning. The period and location of the rings appearance suggest that the ring may have some relation to the spore membrane or wall development. In addition, this ring structure was more stable than other F-actin structures against latrunculin A, an F-actin disrupting agent.     

Synthesis and Secretion of Acid Phosphatase in Halotolerant Zygosaccharomyces rouxii

Changes in the contents of defensive substances against the active oxygen in water-stressed spinach plants were examined. The contents of ascorbate peroxidase (AP; EC 1.11.1.7), glutathione reductase (GR; EC 1.6.4.2) and α-tocopherol increased remarkably in water-stressed spinach leaves, while those of Superoxide dismutase (SOD; EC 1.15.1.1), dehydroascorbate reductase (EC 1.8.5.1), ascorbate and glutathione changed little. The content of α-tocopherol in chloroplast thylakoid membranes isolated from water-stressed leaves was higher than that from normal leaves. It is, therefore, conceivable that GR, AP and α-tocopherol might be related to the tolerance of plants to water deficiency.     

Efficient transformation of the osmotolerant yeast Zygosaccharomyces rouxii by electroporation

A rapid and simple electroporation method to transform osmotolerant yeast Zygosaccharomyces rouxii has been developed and conditions for efficient transformation of mutants derived from different Z. rouxii wild-type strains optimized.     

Saccharomyces cerevisiae forms actin ring structures in sporulation, similarly to Zygosaccharomyces rouxii

Yeast filamentous actin (F-actin) exists mainly as patches and cables. Previously, we investigated the behavior of F-actin during sporulation of Zygosaccharomyces rouxii and found a novel actin ring localized around the spore periphery in zygotic asci at a late stage of sporulation. To clarify whether the actin rings are also formed in sporulation in the model yeast Saccharomyces cerevisiae, we observed the distribution of F-actin in sporulating S. cerevisiae by rhodamine-phalloidin staining and confocal laser scanning microscopy. Ringlike actin structures were detected at the peripheral regions of S. cerevisiae spores in globose asci. When asci of S. cerevisiae were induced to become zygotic, actin rings were more obvious than those in globose asci. These results indicate that S. cerevisiae forms characteristic actin ring structures at a late stage of sporulation, similarly to Z. rouxii.     

Escherichia coli and Lactobacillus plantarum responses to osmotic stress

Escherichia coli and Lactobacillus plantarum were subjected to final water potentials of −5.6 MPa and −11.5 MPa with three solutes: glycerol, sorbitol and NaCl. The water potential decrease was realized either rapidly (osmotic shock) or slowly (20 min) and a difference in cell viability between these conditions was only observed when the solute was NaCl. The cell mortality during osmotic shocks induced by NaCl cannot be explained by a critical volume decrease or by the intensity of the water flow across the cell membrane. When the osmotic stress is realized with NaCl as the solute, in a medium in which osmoregulation cannot take place, the application of a slow decrease in water potential resulted in the significant maintenance of cell viability (about 70–90%) with regard to the corresponding viability observed after a sudden step change to same final water potential (14–40%). This viability difference can be explained by the existence of a critical internal free Na⁺ concentration. Received: 20 May 1998 / Received revision: 31 July 1998 / Accepted: 31 July 1998     

Physiological responses of Daphnia pulex to acid stress

Background  

Acidity exerts a determining influence on the composition and diversity of freshwater faunas. While the physiological implications of freshwater acidification have been intensively studied in teleost fish and crayfish, much less is known about the acid-stress physiology of ecologically important groups such as cladoceran zooplankton. This study analyzed the extracellular acid-base state and CO₂ partial pressure (P _CO2), circulation and ventilation, as well as the respiration rate of Daphnia pulex acclimated to acidic (pH 5.5 and 6.0) and circumneutral (pH 7.8) conditions.     

瓜多竹(Guadua amplexifolia)对NaCl胁迫的生理响应

为了解瓜多竹（Guadua amplexifolia）的耐盐性,对2年生盆栽苗进行了不同浓度NaCl胁迫处理,结果表明：以处理40d的叶片脱落率将盐害程度分为无盐害（〈10％）、轻度盐害（10％-50％）和重度盐害（〉50％）3级;随着胁迫时间的延长,离子渗漏和MDA含量均呈上升趋势,40d时0．30％及以上浓度的NaCl处理水平相对电导率均〉50％,MDA含量显著高于低浓度处理;POD和SOD活性随胁迫时间的延长呈先升后降规律,40d时0．20％NaCl处理的POD活性最高,0．30％NaCl处理的SOD活性最高;游离脯氨酸和可溶性蛋白等渗透调节物质也呈先升后降规律,40d时0．30％及以上浓度的NaCl处理的可溶性蛋白和游离脯氨酸含量显著下降,0．20％及以下浓度的NaCl处理则继续上升。综合上述生理指标的反应,土壤含盐量0．30％及以上时不适合该竹种生长。