Identification and classification of genes required for tolerance to freeze-thaw stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains

Yeasts used in bread making are exposed to freeze-thaw stress during frozen-dough baking. To clarify the genes required for freeze-thaw tolerance, genome-wide screening was performed using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 58 gene deletions that conferred freeze-thaw sensitivity. These genes were then classified based on their cellular function and on the localization of their products. The results showed that the genes required for freeze-thaw tolerance were frequently involved in vacuole functions and cell wall biogenesis. The highest numbers of gene products were components of vacuolar H(+)-ATPase. Next, the cross-sensitivity of the freeze-thaw-sensitive mutants to oxidative stress and to cell wall stress was studied; both of these are environmental stresses closely related to freeze-thaw stress. The results showed that defects in the functions of vacuolar H(+)-ATPase conferred sensitivity to oxidative stress and to cell wall stress. In contrast, defects in gene products involved in cell wall assembly conferred sensitivity to cell wall stress but not to oxidative stress. Our results suggest the presence of at least two different mechanisms of freeze-thaw injury: oxidative stress generated during the freeze-thaw process, and defects in cell wall assembly.     

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.     

Isolation of salt-tolerant mutants of <Emphasis Type="Italic">Mesorhizobium ciceri</Emphasis> strain Rch125 and identification of genes involved in salt sensitivity

Mesorhizobium ciceri Rch125 is a salt-sensitive strain isolated from root nodules of chickpea (Cicer arietinum L.). The aim of this work was to investigate the genes responsible for the sensitivity to salinity. Twelve Rch125 salt-tolerant mutants were isolated after random Tn5 mutagenesis and selected using a medium containing 300 mM NaCl, where growth of the wild-type is totally inhibited. In addition to this NaCl tolerance, the mutants also displayed higher tolerance to LiCl, CaCl₂ and sucrose. Genes that were disrupted in the salt-tolerant mutants were in one of three functional categories: membrane transporters, outer membrane proteins, and genes of unknown function. Genetic complementation experiments demonstrated that the genes identified were involved in the salt sensitivity of the Rch125 strain. In most cases, disruption of the salt-sensitivity genes did not negatively affect the free-living or the symbiotic capabilities of Rch125 under non-saline conditions.     

Regulation of chronological aging in Schizosaccharomyces pombe by the protein kinases Pka1 and Sck2

Budding yeast shows a progressive decline in viability after entering stationary phase, a phenomenon known as chronological aging. We show here that the fission yeast Schizosaccharomyces pombe also undergoes chronological aging and that the process is regulated by genes controlling two related nutrient signalling pathways. The first pathway includes the serine/threonine cAMP-activated protein kinase Pka1 and the second pathway comprises the serine/threonine kinase Sck2, a homologue of Saccharomyces cerevisiae SCH9. A double mutant for pka1 and sck2 displayed an additive effect on prolonging the fission yeast lifespan, suggesting that these genes regulate related but independent pathways. These long-lived mutants also accumulated less reactive oxygen species and had a delayed initiation of apoptosis compared with wild-type cells. We also found that strains carrying pka1 deletion but not those with sck2 deletion gained resistance to oxidative stress due to exposure to H(2)O(2) or menadione. On the other hand, the additional increase in lifespan shown by the Deltapka1Deltasck2 double-mutant strain correlated with an increased resistance to both oxidative stress and heat shock. These results underscore the importance of nutrient signalling pathways and reactive oxygen species on organismal lifespan and establish S. pombe as a new model organism to study the molecular mechanisms underlying aging.     

Genome-wide identification of genes involved in tolerance to various environmental stresses inSaccharomyces cerevisiae

During fermentation, yeast cells are exposed to a number of stresses — such as high alcohol concentration, high osmotic pressure, and temperature fluctuation — so some overlap of mechanisms involved in the response to these stresses has been suggested. To identify the genes required for tolerance to alcohol (ethanol, methanol, and 1-propanol), heat, osmotic stress, and oxidative stress, we performed genome-wide screening by using 4828 yeast deletion mutants. Our screens identified 95, 54, 125, 178, 42, and 30 deletion mutants sensitive to ethanol, methanol, 1-propanol, heat, NaCl, and H₂O₂, respectively. These deleted genes were then classified based on their cellular functions, and cross-sensitivities between stresses were determined. A large number of genes involved in vacuolar H⁺-ATPase (V-ATPase) function, cytoskeleton biogenesis, and cell wall integrity, were required for tolerance to alcohol, suggesting their protective role against alcohol stress. Our results revealed a partial overlap between genes required for alcohol tolerance and those required for thermotolerance. Genes involved in cell wall integrity and the actin cytoskeleton are required for both alcohol tolerance and thermotolerance, whereas the RNA polymerase II mediator complex seems to be specific to heat tolerance. However, no significant overlap of genes required for osmotic stress and oxidative stress with those required for other stresses was observed. Interestingly, although mitochondrial function is likely involved in tolerance to several stresses, it was found to be less important for thermotolerance. The genes identified in this study should be helpful for future research into the molecular mechanisms of stress response.     

Physiological and genetic characterisation of osmosensitive mutants of Saccharomyes cerevisiae

The screening of 20,000 Saccharomyces cerevisiae random mutants to identify genes involved in the osmotic stress response yielded 14 mutants whose growth was poor in the presence of elevated concentrations of NaCl and glucose. Most of the mutant strains were more sensitive to NaCl than to glucose at the equivalent water activity (a_w) and were classified as salt-sensitive rather than osmosensitive. These mutants fell into 11 genetic complementation groups and were designated osr1–osr11 (osmotic stress response). All mutations were recessive and showed a clear 2⁺ : 2^– segregation of the salt-stress phenotype upon tetrad analysis when crossed to a wild-type strain. The complementation groups osr1, osr5 and osr11 were allelic to the genes PBS2, GPD1 and KAR3, respectively. Whereas intracellular and extracellular levels of glycerol increased in the wild-type strains when exposed to NaCl, all mutants demonstrated some increase in extracellular glycerol production upon salt stress, but a number of the mutants showed little or no increase in intracellular glycerol concentrations. The mutants had levels of glycerol-3-phosphate dehydrogenase, an enzyme induced by osmotic stress, either lower than or similar to those of the parent wild-type strain in the absence of osmotic stress. In the presence of NaCl, the increase in glycerol-3-phosphate dehydrogenase activity in the mutants did not match that of the parent wild-type strain. None of the mutants had defective ATPases or were sensitive to heat stress. It is evident from this study and from others that a wide spectrum of genes is involved in the osmotic stress response in S. cerevisiae. Received: 5 January 1998 / Accepted: 24 March 1998     

Novel mitogen-activated protein kinase MpkC of Aspergillus fumigatus is required for utilization of polyalcohol sugars

The genome of Aspergillus fumigatus has four genes that encode mitogen-activated protein kinases (MAPKs), sakA/hogA, mpkA, mpkB, and mpkC. The functions of the MpkB and MpkC MAPKs are unknown for A. fumigatus and the closely related and genetically amenable species Aspergillus nidulans. mpkC deletion mutants of A. fumigatus were made and their phenotypes characterized. The mpkC deletion mutants were viable and had normal conidial germination and hyphal growth on minimal or complete media. This is in contrast to deletion mutants with deletions in the closely related MAPK gene sakA/hogA that we previously reported had a nitrogen source-dependent germination phenotype. Similarly, the growth of the mpkC deletion mutants was wild type on high-osmolarity medium. Consistent with these two MAP kinase genes regulating different cellular responses, we determined that the mpkC deletion mutants were unable to grow on minimal medium with sorbitol or mannitol as the sole carbon source. This result implicates MpkC signaling in carbon source utilization. Changes in mRNA levels for sakA and mpkC were measured in response to hypertonic stress, oxidative stress, and a shift from glucose to sorbitol to determine if there was overlap in the SakA and MpkC signaling pathways. These studies demonstrated that SakA- and MpkC-dependent patterns of change in mRNA levels are distinct and have minimal overlap in response to these environmental stresses.     

Comprehensive phenotypic analysis for identification of genes affecting growth under ethanol stress in Saccharomyces cerevisiae

We quantified the growth behavior of all available single gene deletion strains of budding yeast under ethanol stress. Genome-wide analyses enabled the extraction of the genes and determination of the functional categories required for growth under this condition. Statistical analyses revealed that the growth of 446 deletion strains under stress induced by 8% ethanol was defective. We classified these deleted genes into known functional categories, and found that many were important for growth under ethanol stress including several categories that have not been characterized, such as peroxisome. We also performed genome-wide screening under osmotic stress and identified 329 osmotic-sensitive strains. We excluded these strains from the 446 ethanol-sensitive strains to extract the genes whose deletion caused sensitivity to ethanol-specific (359 genes), osmotic-specific (242 genes), and both stresses (87 genes). We also extracted the functional categories that are specifically important for growth under ethanol stress. The genes and functional categories identified in the analysis might provide clues to improving ethanol stress tolerance among yeast cells.     

Degradation of pyrene by indigenous fungi from a former gasworks site

Salt tolerance in Saccharomyces cerevisiae is a complex trait, involving regulation of membrane polarization, Na(+) efflux and sequestration of Na(+) in the vacuole. Since transmembrane transport energized by H(+)-adenosine triphosphatases (ATPases) is common to all of these tolerance mechanisms, the objective of this study was to characterize the responses of the plasma membrane H(+)-ATPase, vacuolar H(+)-ATPase and mitochondrial F(1)F(0)-ATPase to NaCl stress. We hypothesized that since the vacuolar ATPase is responsible for generating the proton motive force required for import of cations (such as Na(+)) into the vacuole, strains lacking this activity should be hypersensitive to NaCl. We found that strains lacking vacuolar ATPase activity were in fact hypersensitive to NaCl, while strains lacking ATP synthase were not. This effect was specific to the ionic component of NaCl stress, since the mutant strains were indistinguishable from wild-type and complemented strains in the presence of sorbitol.     

Involvement of a putative response regulator FgRrg-1 in osmotic stress response, fungicide resistance and virulence in Fusarium graminearum

Response regulator (RR) proteins are core elements of the high-osmolarity glycerol (HOG) pathway, which plays an important role in the adaptation of fungi to a variety of environmental stresses. In this study, we constructed deletion mutants of two putative RR genes, FgRRG-1 and FgRRG-2, which are orthologues of Neurospora crassa RRG-1 and RRG-2, respectively. The FgRRG-1 deletion mutant (ΔFgRrg1-6) showed increased sensitivity to osmotic stress mediated by NaCl, KCl, sorbitol or glucose, and to metal cations Li(+) , Ca(2+) and Mg(2+) . The mutant, however, was more resistant than the parent isolate to dicarboximide and phenylpyrrole fungicides. Inoculation tests showed that the mutant exhibited decreased virulence on wheat heads. Quantitative real-time polymerase chain reaction assays indicated that the expression of FgOS-2, the putative downstream gene of FgRRG-1, was decreased significantly in ΔFgRrg1-6. All of the defects were restored by genetic complementation of ΔFgRrg1-6 with the wild-type FgRRG-1 gene. Different from the FgRRG-1 deletion mutant, FgRRG-2 deletion mutants were morphologically indistinguishable from the wild-type progenitor in virulence and in sensitivity to the dicarboximide fungicide iprodione and osmotic stresses. These results indicate that the RR FgRrg-1 of F. graminearum is involved in the osmotic stress response, pathogenicity and sensitivity to dicarboximide and phenylpyrrole fungicides and metal cations.     

在全基因组范围内筛选酵母中砷抗性相关基因

砷化物是广泛应用的抗癌药物,特别是对白血病有显著疗效.然而,治疗过程中病人会因对砷化物具有耐药性而影响治疗效果,而目前对于砷抗性机制尚缺全面深入的研究.利用酵母作为模式生物,使用不同浓度的砷对由4757个酵母缺失型突变体组成的菌株库进行筛查.共鉴定出104个基因/开放阅读框(ORF),其缺失导致酵母对砷的抗性增加.生物信息学分析结果提示,这些基因与mRNA分解代谢、应激反应、组蛋白乙酰化和蛋白质合成及分解代谢等功能有关.同时这些基因中多于半数具有哺乳动物同源类似物.所以,进一步研究这些基因有望为人类砷化物的耐药性及毒性机制研究提供富有价值的新线索.     

Expression Analysis of Proline Metabolism-related Genes From Halophyte Arabis stelleri under Osmotic Stress Conditions

Arabis stelleri var.japonica evidenced stronger osmotic stress tolerance than Arabidopsis thaliana.Using an A.thaliana microarray chip,we determined changes in the expression of approximately 2 800genes between A.stelleri plants treated with 0.2 M mannitol versus mock-treated plants.The most significant changes in the gene expression patterns were in genes defining cellular components or in genes associated with the endomembrane system,stimulus response,stress response,chemical stimulus response,and defense response.The expression patterns of three de novo proline biosynthesis enzymes were evaluated in A.stelleri var.japonica seedlings treated with 0.2 M mannitol,0.2 M sorbitol,and 0.2 M NaCl.The expression of Δ1-pyrroline-5-carboxylate synthetase was not affected by NaCl stress but was similarly induced by mannitol and sorbitol.The proline dehydrogenase gene,which is known to be repressed by dehydration stress and induced by free L-proline,was induced at an early stage by mannitol treatment,but the level of proline dehydrogenase was increased later by treatment with both mannitol and NaCl.The level of free L-proline accumulation increased progressively in response to treatments with mannitol,sorbitol,and NaCl.Mannitol induced L-proline accumulation more rapidly than NaCl or sorbitol.These findings demonstrate that the osmotic tolerance of the novel halophyte,Arabis stelleri,is associated with the accumulation of L-proline.     

Expression analysis of proline metabolism-related genes from halophyte Arabis stelleri under osmotic stress conditions

Arabis stelleri var.japonica evidenced stronger osmotic stress tolerance than Arabidopsis thaliana.Using an A.thaliana microarray chip,we determined changes in the expression of approximately 2 800genes between A.stelleri plants treated with 0.2 M mannitol versus mock-treated plants.The most significant changes in the gene expression patterns were in genes defining cellular components or in genes associated with the endomembrane system,stimulus response,stress response,chemical stimulus response,and defense response.The expression patterns of three de novo proline biosynthesis enzymes were evaluated in A.stelleri var.japonica seedlings treated with 0.2 M mannitol,0.2 M sorbitol,and 0.2 M NaCl.The expression of Δ1-pyrroline-5-carboxylate synthetase was not affected by NaCl stress but was similarly induced by mannitol and sorbitol.The proline dehydrogenase gene,which is known to be repressed by dehydration stress and induced by free L-proline,was induced at an early stage by mannitol treatment,but the level of proline dehydrogenase was increased later by treatment with both mannitol and NaCl.The level of free L-proline accumulation increased progressively in response to treatments with mannitol,sorbitol,and NaCl.Mannitol induced L-proline accumulation more rapidly than NaCl or sorbitol.These findings demonstrate that the osmotic tolerance of the novel halophyte,Arabis stelleri,is associated with the accumulation of L-proline.