Sip18 hydrophilin prevents yeast cell death during desiccation stress

Aims: For this study, we performed a genetic screen of S. cerevisiae’s deletion library for mutants sensitive to dehydration stress, with which we aimed to discover cell dehydration–tolerant genes. Methods and Results: We used a yeast gene deletion set (YGDS) of 4850 viable mutant haploid strains to perform a genome‐wide screen for the identification of desiccation stress modifiers. SIP18 is among the genes identified as essential for cells surviving to drying/rehydration process. Deletion of SIP18 promotes the accumulation of reactive oxygen species and enhances apoptotic and necrotic cell death in response to dehydration/rehydration process. Conclusions: SIP18p acts as an inhibitor of apoptosis in yeast under dehydration stress, as suggested by its antioxidative capacity through the ROS accumulation reduction after an H₂O₂ attack. Significance and Impact of the Study: To our knowledge, this is the first systematic screen for the identification of putative genes essential to overcoming cell dehydration process. A broad range of identified genes could help to understand why some strains of high biotechnological interest cannot cope with the drying and rehydration treatments. Dehydration sensitivity makes these strains not suitable to be commercialized by yeast manufactures.     

Stress co-tolerance and trehalose content in baking strains of Saccharomyces cerevisiae

Fourteen wild-type baking strains of Saccharomyces cerevisiae were grown in batch culture to true stationary phase (exogenous carbon source exhausted) and tested for their trehalose content and their tolerance to heat (52°C for 4.5 min), ethanol (20% v/v for 30 min), H₂O₂ (0.3 M for 60 min), rapid freezing (−196°C for 20 min, cooling rate 200°C min⁻¹), slow freezing (−20°C for 24 h, cooling rate 3°C min⁻¹), salt (growth in 1.5 M NaCl agar) or acetic acid (growth in 0.4% w/v acetic acid agar) stresses. Stress tolerance among the strains was highly variable and up to 1000-fold differences existed between strains for some types of stress. Compared with previously published reports, all strains were tolerant to H₂O₂ stress. Correlation analysis of stress tolerance results demonstrated relationships between tolerance to H₂O₂ and tolerance to all stresses except ethanol. This may imply that oxidative processes are associated with a wide variety of cellular stresses and also indicate that the general robustness associated with industrial yeast may be a result of their oxidative stress tolerance. In addition, H₂O₂ tolerance might be a suitable marker for the general assessment of stress tolerance in yeast strains. Trehalose content failed to correlate with tolerance to any stress except acetic acid. This may indicate that the contribution of trehalose to tolerance to other stresses is either small or inconsistent and that trehalose may not be used as a general predictor of stress tolerance in true stationary phase yeast. Received 10 October 1995/ Accepted in revised form 10 September 1996     

Migratory behaviour and desiccation tolerance of protostrongylid nematode first-stage larvae

Migration of first-stage larvae (L1) from faeces to soil is a crucial stage in the life-history of protostrongylids transmitted via land snails. Migration of Muellerius cf. capillaris and a Cystocaulus sp. L1 from fresh Nubian ibex (Capra ibex nubiana) faeces (48–50% water content, W.C.) to substrate soils (at 100% r.h., 26°C) was measured experimentally using dry (3 ± 1% W.C.), wet (31 ± 0.43% W.C.) and flooded (48.4 ± 2.45% W.C.) soils. The highest migration rates (90.4 ± 1.6% migration) in both species occurred on flooded soils when the faecal pellet W.C. reached 90%. The next highest migration rates (43.2 ± 3.6% migration, at 60% faecal W.C.) were on the wet soils and no migration occurred on dry soil or dry-substrate papers. Migration rates did not differ significantly (P > 0.05) between species. Active Theba pisana were not infected by M. cf. capillaris L1 on dry infested soils, but were infected following rehydration of the same soils. By day 10, L1 of M. cf. capillaris demonstrated lower survival rates in water and in 97% and 76% r.h. (74.5%, 15.2% and 1.9%, respectively) than the Cystocaulus sp. (97.5%, 43.8%, 43.3%) and Protostrongylus sp. (97.9%, 43.2%, 23.8%, P < 0.05). All three nematodes had a remarkably high survival rate (> 99% overall survival, by day 10) when exposed directly to 0% r.h. at 23°C, Results demonstrate the ability of L1 to survive extreme desiccation through anhydrobiosis. Migration of L1 from facces to soil can take place only during rains which coincide, with peak activity of land snails in desert habitat.     

Genetic basis of sodium exclusion and sodium tolerance in yeast. A model for plants

A yeast strain carrying disruptions in TRK1 and ENA genes was very sensitive to Na⁺ because uptake discriminated poorly between K⁺ and Na⁺, and Na⁺ efflux was insignificant. Transformation with TRK1 and ENA1 restored discrimination, Na⁺ efflux and Na⁺ tolerance. Increasing external Ca²⁺ increased Na⁺ tolerance almost in the same proportion in TRK1 enal cells and in trkl ENAI cells, suggesting an unspecific effect of this cation. By using a vacuolar ATPase mutant, the role of the vacuole in Na⁺ tolerance was also demonstrated. The yeast model of Na⁺ exclusion and Na⁺ tolerance may be extended to plants.     

Acquisition of thermotolerant yeast Saccharomyces cerevisiae by breeding via stepwise adaptation

A thermotolerant Saccharomyces cerevisiae yeast strain, YK60‐1, was bred from a parental strain, MT8‐1, via stepwise adaptation. YK60‐1 grew at 40°C, a temperature at which MT8‐1 could not grow at all. YK60‐1 exhibited faster growth than MT8‐1 at 30°C. To investigate the mechanisms how MT8‐1 acquired thermotolerance, DNA microarray analysis was performed. The analysis revealed the induction of stress‐responsive genes such as those encoding heat shock proteins and trehalose biosynthetic enzymes in YK60‐1. Furthermore, nontargeting metabolome analysis showed that YK60‐1 accumulated more trehalose, a metabolite that contributes to stress tolerance in yeast, than MT8‐1. In conclusion, S. cerevisiae MT8‐1 acquired thermotolerance by induction of specific stress‐responsive genes and enhanced intracellular trehalose levels. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1116–1123, 2013     

Nitric oxide is neither necessary nor sufficient for resolution of Plasmodium chabaudi malaria in mice

Malaria is a life-threatening re-emerging disease, yet it is still not clear how bloodstage malarial parasites are killed. Nitric oxide (NO), which has potent anti-microbial activity, may represent an important killing mechanism. The production of NO during descending Plasmodium chabaudi parasitemia, a period when parasites are killed by the immune response, supports this concept. However, NOS20/0 and NOS30/0 mice as well as mice treated with NO synthase 2 (NOS2) inhibitors do not develop exacerbated malaria, indicating that NO production is not necessary for the suppression of P. chabaudi parasitemia. It is possible due to the plasticity in the immune response during malaria that Ab-mediated immunity is enhanced in the absence of NO, thereby explaining the lack of exacerbated malaria in NOS-deficient mice even though NO may function in protection. However, NOS2- and B cell-deficient mice, which cannot use Ab-mediated immunity, suppress their parasitemia with a similar time course as B cell-deficient controls. C57BL/6 mice treated with Propionibacterium acnes to elicit high levels of macrophage-derived NO have a similar time course of P. chabaudi parasitemia as P. acnes-treated NOS20/0 mice, which do not produce NO; this indicates that NO is not sufficient for parasite killing. Collectively, these results indicate that NO is not necessary or sufficient to resolve P. chabaudi malaria.     

Effects of trehalose on stress tolerance and biocontrol efficacy of Cryptococcus laurentii

AIMS: To investigate the effects of internal trehalose on viability and biocontrol efficacy of antagonistic yeast Cryptococcus laurentii under stresses of low temperature (LT), controlled atmosphere (CA) and freeze drying. METHODS AND RESULTS: The content of trehalose in C. laurentii was increased by culturing the yeast in trehalose-containing medium. Compared with yeast cells with low trehalose level, the yeast cells with high level of internal trehalose not only obtained higher viability, but also showed higher population and better biocontrol efficacy against Penicillium expansum on apple fruit both at 1 degrees C and in CA condition (5% O(2), 5% CO(2), 1 degrees C). After freeze drying, survival of the yeast with high trehalose level was markedly increased when stored at 25 degrees C for 0, 15 and 30 days. Meanwhile, high integrity of plasma membrane was detected in the freeze-dried yeast with high trehalose level by propidium iodide staining. CONCLUSIONS: Induced accumulation of internal trehalose could improve viability and biocontrol efficacy of C. laurentii under stresses of LT and CA. Moreover, survival of the yeast was also increased as internal trehalose accumulation after freeze drying, and one of the reasons might be that trehalose gave an effective protection to plasma membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this experiment show a promising way to improve the biocontrol performance of antagonistic yeasts under the commercial conditions.     

A group 13 homeodomain is neither necessary nor sufficient for posterior prevalence in the mouse limb

Posterior prevalence is the general property attributed to HOX proteins describing the dominant effect of more posterior HOX proteins over the function of anterior orthologs in common areas of expression. To explore the HOX group 13 protein domains required for this property, we used the mouse Prx-1 promoter to drive transgenic expression of Hox constructs throughout the entire limb bud during development. This system allowed us to conclusively demonstrate a hierarchy of Hox function in developing limbs. Furthermore, by substituting the HOXD11 or HOXA9 homeodomain for that of HOXD13, we show that a HOXD13 homeodomain is not necessary for posterior prevalence. Proximal expression of these chimeric proteins unexpectedly caused defects consistent with wild-type HOXD13 mediated posterior prevalence. Moreover, group 13 non-homeodomain residues appear to confer the property as proximal expression of HOXA9 containing the HOXD13 homeodomain did not result in limb reductions characteristic of HOXD13. These data are most compatible with models of posterior prevalence based on protein-protein interactions and support examination of the N-terminal non-homeodomain regions of Hox group 13 proteins as necessary agents for posterior prevalence.     

酵母内源性海藻糖代谢调控研究进展

作为一种天然稳定剂的双糖,海藻糖(Trehalose)在逆境下对生物体活性的保护功能既吸引了广泛的研究兴趣,也使其具有良好的应用价值和潜力。本文聚焦重要模式微生物和工业应用微生物酵母,结合组学研究最新进展,从海藻糖代谢途径、应激条件下的海藻糖代谢和转录特征以及提高胞内海藻糖含量策略等方面,对内源性海藻糖研究新进展进行了综述。     

产海藻糖酿酒酵母培养基优化及生理学研究

采用单因子和均匀试验对酿酒酵母产海藻糖的培养基组成进行初步研究,并对发酵过程作了分析。结果表明,培养基组成、碳源浓度以及氮源的浓度比等都对细胞生物量和海藻糖积累有影响。在初步优化培养基条件下,酒酵母产海藻糖的细胞干重8g／L,海藻糖的含量为10％。通过观测发酵过程生理现象的变化,认为海藻糖的产生是对数生长期后期的稳定期,呈现不规律变化。     

Differential pattern of trehalose accumulation in wine yeast strains during the microvinification process

Trehalose accumulation in wine yeast strains growing under microvinification conditions was determined and compared to that obtained under laboratory conditions. Industrial strains accumulate 10-fold more trehalose than laboratory strains. Contrary to batch-culture growth, under microvinification conditions trehalose accumulation is not consequence of glucose exhaustion. Physiological relevance of trehalose during the process of wine making and their use for potential improvements of alcoholic fermentation are discussed.     

拟南芥MT-II过量表达提高抗旱性(英文)

富含巯基的植物II型金属硫蛋白(MT)对植物抵抗重金属胁迫具有重要作用,其中一个可能机制是金属硫蛋白可能猝灭重金属引起的氧化胁迫。利用转MT-II基因和野生型拟南芥(Arabidopsis thaliana)植株来对比研究MT在胁迫过程中通过清除氧自由基,特别是H2O2而对植物抗旱性的影响。研究表明,转基因型拟南芥能有效维持体内氧化—还原势,减少MDA的产生,从而缓解干旱胁迫引起的伤害,提高抗旱性。     

酿酒酵母乙醇耐性的分子机制及基因工程改造

提高工业微生物对毒性代谢产物及高温等环境胁迫因素的耐受性对工业生产具有重要的意义。发酵过程中产生的乙醇对酵母细胞的生长和代谢都具有较强的抑制作用,是酿酒酵母的重要环境胁迫因素之一。对酿酒酵母乙醇耐性的分子机制的研究可为选育具有较强乙醇耐受性的酵母菌种提供理论基础。近年来,通过细胞全局基因转录分析和基因功能分析,对酿酒酵母乙醇耐性的分子机制有了更多新的认识,揭示了很多新的与乙醇耐性相关的基因,并在此基础上,通过对相关基因进行过量表达或敲除,成功提高了酵母菌的乙醇耐性。以下综述了近年来酵母菌乙醇耐性的生物化学与分子生物学机制的研究进展,以及构建具有较高乙醇耐性的酵母菌的基因工程操作。这些研究不仅加深了对酿酒酵母乙醇耐性的机理认识,也可为高效进行生物转化生产生物质能源奠定理论基础。     

The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols

A set of homozygous diploid deletion mutants of the yeast Saccharomyces cerevisiae was screened for the genes required for tolerance to aliphatic alcohols. The screen identified 137, 122 and 48 deletion mutants sensitive to ethanol, 1-propanol and 1-pentanol, respectively. A number of the genes required for ethanol tolerance were those also required for tolerance to other alcohols. Numerous mutants with defective genes encoding for vacuolar H+ -ATPase (V-ATPase) were cosensitive to these alcohols. A global screening approach of yeast deletion library mutants was useful in elucidating the mechanisms of alcohol tolerance based on different lipophilicities.     

The effect of selection for desiccation resistance on cold tolerance of Drosophila melanogaster

Abstract.  Low temperature and desiccation stress are thought to be mechanistically similar in insects, and several studies indicate that there is a degree of cross-tolerance between them, such that increased cold tolerance results in greater desiccation tolerance and vice versa . This assertion is tested at an evolutionary scale by examining basal cold tolerance, rapid cold-hardening (RCH) and chill coma recovery in replicate populations of Drosophila melanogaster selected for desiccation resistance (with controls for both selection and concomitant starvation) for over 50 generations. All of the populations display a RCH response, and there is no effect of selection regime on RCH or basal cold tolerance, although there are differences in basal cold tolerance between sampling dates, apparently related to inter-individual variation in development time. Flies selected for desiccation tolerance recover from chill coma slightly, but significantly, faster than control and starvation-control flies. These findings provide little support for cross-tolerance between survival of near-lethal cold and desiccation stress in D. melanogaster .     

Saccharomyces cerevisiae Bor1p is a boron exporter and a key determinant of boron tolerance

Boron is toxic to living organisms when present in excess. Saccharomyces cerevisiae Bor1p is a plasma membrane protein that decreases the intracellular concentration of boron and confers boron tolerance in yeasts. We investigated the detailed characteristics of boron transport by Bor1p and its roles in boron tolerance. Boron transport assays showed that the bor1 deletion mutant (bor1Delta) accumulates higher intracellular concentrations of boron and has a lower rate of boron export. The bor1Delta showed greater susceptibility to high concentrations of boron than the wild-type strain, and the growth rates of both strains were negatively correlated with the intracellular concentrations of boron. With normal to toxic levels of external boron, green fluorescent protein (GFP)-tagged Bor1p localized to the plasma membrane irrespective of the concentration of boron in the medium. Taken together, these results establish Bor1p as a plasma membrane boron exporter and a key determinant of boron tolerance.     

转新疆雪莲去饱和酶基因sikSAD重组酵母低温和酒精耐受性分析

根据GenBank收录的sikSAD基因序列, 采用反转录PCR技术从新疆雪莲(Sasussured involucrata Kar. et Kir)中克隆了sikSAD基因, 并构建了pYES2-sikSAD大肠杆菌/酵母穿梭表达载体, 通过电击法转化酿酒酵母288C菌株, 并利用PCR和SDS-PAGE对转化酿酒酵母进行鉴定, 最后通过低温胁迫和酒精胁迫进行抗性初步分析及方差分析。结果表明: 低温胁迫实验中, 转sikSAD基因酿酒酵母在低温条件下仍能存活, 并且在温度恢复到28 °C时能迅速生长, 生长状态良好, 不饱和脂肪酸油酸的含量有明显的变化。酒精胁迫实验中, 其能耐受一定浓度的酒精, 并且耐受能力比非转基因酿酒酵母提高了十几个百分点。可见, 在低温胁迫和高浓度酒精条件下, 转新疆雪莲sikSAD基因酿酒酵母表现出了优良的活性和生长优势, 显示出较强的抗性特征, 用分子手段改造酿酒酵母, 为工业生产提供高质量的酿酒酵母奠定实验基础。