Possible roles of vacuolar H+-ATPase and mitochondrial function in tolerance to air-drying stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains

Yeasts used in bread making are exposed to air-drying stress during dried yeast production processes. To clarify the genes required for air-drying tolerance, we performed genome-wide screening using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 278 gene deletions responsible for air-drying sensitivity. These genes were classified based on their cellular function and on the localization of their gene products. The results showed that the genes required for air-drying tolerance were frequently involved in mitochondrial functions and in connection with vacuolar H(+)-ATPase, which plays a role in vacuolar acidification. To determine the role of vacuolar acidification in air-drying stress tolerance, we monitored intracellular pH. The results showed that intracellular acidification was induced during air-drying and that this acidification was amplified in a deletion mutant of the VMA2 gene encoding a component of vacuolar H(+)-ATPase, suggesting that vacuolar H(+)-ATPase helps maintain intracellular pH homeostasis, which is affected by air-drying stress. To determine the effects of air-drying stress on mitochondria, we analysed the mitochondrial membrane potential under air-drying stress conditions using MitoTracker. The results showed that mitochondria were extremely sensitive to air-drying stress, suggesting that a mitochondrial function is required for tolerance to air-drying stress. We also analysed the correlation between oxidative-stress sensitivity and air-drying-stress sensitivity. The results suggested that oxidative stress is a critical determinant of sensitivity to air-drying stress, although ROS-scavenging systems are not necessary for air-drying stress tolerance.     

Quantitative proteomics of Lactococcus lactis F44 under cross-stress of low pH and lactate

Lactococcus lactis encounters 3 environmental stimuli, H⁺, lactate, and undissociated lactic acid, because of the accumulation of lactic acid—the predominant fermentation product. Few studies have examined how these stimuli synergistically affect the bacteria. Herein, we analyzed the dissociation degree of lactic acid at different pH and investigated the cellular response to cross-stress in L. lactis ssp. lactis F44 through quantitative proteomic analysis using isobaric tags for relative and absolute quantitation of 3 groups: 0% lactic acid with pH 4.0 and 0% lactic acid with pH 5.0 for acid stress; 2% lactic acid with pH 7.0 and 3% lactic acid with pH 7.0 for lactate stress; and 2% lactic acid with pH 4.0, 2% lactic acid with pH 5.0, 3% lactic acid with pH 4.0, and 3% lactic acid with pH 5.0 for cross-stress. We observed that the metabolisms of carbohydrate and energy were inhibited, which might be due to the feedback inhibition of lactic acid. The arginine deiminase pathway was improved to maintain the stability of intracellular pH. Additionally, some differentially expressed genes associated with the general stress response, amino acid metabolism, cell wall synthesis, and regulatory systems played significant roles in stress response. Overall, we highlighted the response mechanisms to lactic acid stress and provided a new opportunity for constructing robust industrial strains.     

Production of Kefir from Soymilk With or Without Added Glucose, Lactose, or Sucrose

ABSTRACT: The effects of added glucose, lactose, and sucrose on microbial growth, acid, and ethanol production, and galactosidase activity in soymilk fermented with kefir grains were studied. Immediately after the addition of kefir grains to soymilk, the lactic-acid bacterial counts were higher, but the yeast counts were lower than in milk kefir. After fermentation for 32 h, the concentrations of yeast, lactic acid, and ethanol in soymilk were significantly lower than those in milk kefir. Addition of 1% glucose to soymilk stimulated growth of lactic-acid bacteria and yeast, the production of lactic acid and ethanol, and the β-galactosidase activity. Nevertheless α- galactosidase activity was suppressed by 1% glucose.     

Phenotypic analysis of gene deletant strains for sensitivity to oxidative stress

Ascertaining the impact of inhibitors on the growth phenotype of yeast mutants can be useful in elucidating the function of genes within the cell. Microtitre plates and robotics have been used to screen over 600 deletions from EUROSCARF, constructed in an FY1679 strain background, for sensitivity to various oxidants. These included the inorganic hydroperoxide, H(2)O(2), an organic peroxide (cumene hydroperoxide) and a lipid hydroperoxide (linoleic acid hydroperoxide). These produce within the cell several different reactive oxygen species that can cause damage to DNA, proteins and lipids. Approximately 14% of deletants displayed sensitivity to at least one of the oxidants and there was also a distribution of deletants that showed sensitivity to all or different combinations of the oxidants. Deletants included genes encoding proteins involved in stress responses, heavy metal homeostasis and putative cell wall proteins. Although global mechanisms have been identified that provide general stress responses, these results imply that there are also distinct mechanisms involved in the protection of the cell against specific damage caused by different oxidants. Further analysis of these genes may reveal unknown mechanisms protecting the cell against reactive oxygen species.     

酵母菌对重金属的吸附与抗性和解毒重金属的胞内分子机制研究进展

利用有益的酵母菌去除食品基质、动物及人体的重金属污染是近年的研究热点。本文概述了多种酵母菌吸附及抗重金属的情况,并对酵母菌在重金属胁迫下的胞内解毒机制进行分析,包括谷胱甘肽合成的解毒机制、与酵母菌解毒重金属相关的基因和蛋白、转运蛋白介导的细胞内重金属的排出和液泡隔离机制及金属硫蛋白和植物螯合肽对重金属的螯合作用,重点从分子角度分析了酵母菌对重金属的解毒机制,归纳了解毒过程中关键性的基因和蛋白质以及它们的功能作用,旨在为酵母菌作为生物吸附剂应用于生态环境、被重金属污染的发酵食品及动物和人体提供依据。     

Isolation and characterization of a novel yeast gene,ATH1, that is required for vacuolar acid trehalase activity

We have isolated a plasmid containing a gene, ATH1, that results in eight- to ten-fold higher acid trehalase activity in yeast cells when present in high copy. The screening procedure was based on overproduction-induced mislocalization of acid trehalase activity; overproduction of vacuolar enzymes that transit through the secretory pathway leads to secretion to the cell surface. A DNA fragment that confers cell surface expression of acid trehalase activity was cloned and sequenced. The deduced amino acid sequence displayed no homology to known proteins, indicating that we have identified a novel gene. A deletion in the genomic copy of the ATH1 gene eliminates vacuolar acid trehalase activity. These results suggest that ATH1 may be the structural gene encoding vacuolar acid trehalase or that the gene product may be an essential regulatory component involved in control of trehalase activity. The sequence has been deposited in the GenBank data library under Accession Number X84156 S. cerevisiae ATH1 gene.     

牛源性大肠杆菌O26热休克-酸应激响应

为探讨热休克-酸应激对大肠杆菌O26存活及相关基因表达的影响，以本实验收集的22 株牛源性O26为对象，首先进行乳酸和盐酸耐受实验，进而选取乳酸耐受性能不同的菌株混合进行热休克-酸应激存活实验，最后选取1 株代表菌株采用实时聚合酶链式反应分析应激2 h和4 h时一般应激基因rpoS、酸应激基因（asr、ycfR、gadA）、热应激基因（rpoH、dnaK、clpB和groEL）的表达差异。结果表明，乳酸或盐酸处理2 h后22 株O26存活菌数均显著下降（P＜0.05），耐受程度呈现菌株差异，且同一菌株对乳酸、盐酸的耐受有差异。与正常胰蛋白胨大豆肉汤对照组相比，5 株O26混菌酸应激和热休克-酸应激均导致存活菌数逐渐下降，而热休克-酸应激组存活菌数高于酸应激组，表明热休克发挥交叉保护效应，增强了O26抗酸能力。酸应激导致菌株G10Z1应激相关基因表达基本下调，而热休克-酸应激组与酸应激组相比上述基因的表达基本上调，表明热休克的交叉保护作用与上述基因表达水平的增加有关。提示食品实际生产加工中，当非致死性热处理与酸化联合使用时应注意交叉保护可能导致的食品安全风险增加的现象。     

ZMVHA-B1, the gene for subunit B of vacuolar H+-ATPase from the eelgrass Zostera marina L. Is able to replace vma2 in a yeast null mutant

A vacuolar H(+)-ATPase (VHA) gene (ZMVHA-B1) was isolated from an eelgrass (Zostera marina) leaf cDNA library and was characterized to be approximately 1.4 kbp in length and to encode the B subunit protein of VHA comprising 488 amino acids. ZMVHA-B1 was highly expressed in all organs of eelgrass; the expression level was highest in the leaves. On transformation of a yeast vma2 null mutant with ZMVHA-B1, yeast cells became able to grow at pH 7.5, accompanied by the vesicular accumulation of LysoSensor green DND-189. Thus, ZMVHA-B1 expressed in yeast cells produced a functional B subunit that was efficiently incorporated into the VHA complex and eventually restored vacuolar morphology and activity. This success expedites the application of heterologous expression in yeast mutant cells to the screening of eelgrass genes involved in salt-resistance mechanisms, which are to be utilized in improving important crops.     

Biosynthetic regulation of phytochelatins, heavy metal-binding peptides

Phytochelatins (PCs) are heavy metal-binding peptides that play important roles in the detoxification of toxic heavy metals and the regulation of intracellular concentrations of essential metals in eukaryotes, including higher plants, fungi, and microalgae. Recently, PC synthase genes in higher plants and fission yeast have been identified and characterized, enabling molecular biological studies to unravel the mechanisms underlying PC synthesis. Moreover, recent routine database searches have unexpectedly identified genes that are similar to plant PC synthase genes in the genomes of worms and some prokaryotes. In this review, we introduce these recent advances in our understanding of the molecular mechanisms for PC biosynthesis and functions in order to supply basic information about the unique and attractive peptides applicable to various fields.     

The Acetic Acid Tolerance Response induces cross-protection to salt stress in Salmonella typhimurium

Salmonella typhimurium induces an Acid Tolerance Response (ATR) upon exposure to mildly acidic conditions in order to protect itself against severe acid shock. This response can also induce cross-protection to other stresses such as heat and salt. We investigated whether both the acetic acid induced and lactic acid induced ATR in S. typhimurium provided cross-protection to a salt stress at 20 degrees C. Acid-adapted cells were challenged with both a sodium chloride (NaCl) and potassium chloride (KCl) shock and their ability to survive ascertained. Acetic acid adaptation provided cells with protection against both NaCl and KCl stress. However, lactic acid adaptation did not protect against either osmotic stressor and rendered cells hypersensitive to NaCl. These results have implications for the food industry where hurdle technology means multiple sub-lethal stresses such as mild pH and low salt are commonly used in the preservation of products.     

Lactococcus lactis, a bacterial model for stress responses and survival

The dairy organism, Lactococcus lactis, is continuously exposed to stress conditions generated during industrial processes. To identify the mechanisms that confer resistance to the lethal effects of oxygen and thermal stress, we isolated resistant strains by insertional mutagenesis. Mutated genes were identified and mutations were shown to confer resistance to multiple stresses (including non-selected stresses such as carbon starvation). Our results revealed that metabolic flux plays an important role in L. lactis stress response, and suggested that phosphate and guanine pools may be intracellular stress sensors. As previously shown, we also observed an increase of stress resistance during the stationary phase. We have evidence that stationary phase actually initiates very early during growth. Taken together, these data show that the stationary phase is a very complex system with multiple participants interacting altogether. These results reinforce the idea of the interdependence of stress response and the intimate relation between metabolic flux and stress responses in L. lactis.     

Osmotic stress response in the wine yeast Dekkera bruxellensis

Dekkera bruxellensis is mainly associated with lambic beer fermentation and wine production and may contribute in a positive or negative manner to the flavor development. This yeast is able to produce phenolic compounds, such as 4-ethylguaiacol and 4-ethylphenol which could spoil the wine, depending on their concentration. In this work we have investigated how this yeast responds when exposed to conditions causing osmotic stress, as high sorbitol or salt concentrations. We observed that osmotic stress determined the production and accumulation of intracellular glycerol, and the expression of NADH-dependent glycerol-3-phosphate dehydrogenase (GPD) activity was elevated. The involvement of the HOG MAPK pathway in response to this stress condition was also investigated. We show that in D. bruxellensis Hog1 protein is activated by phosphorylation under hyperosmotic conditions, highlighting the conserved role of HOG MAP kinase signaling pathway in the osmotic stress response.