Project description:Alkaline pH stress invokes in S. cerevisiae a potent and fast transcriptional response that includes many genes repressed by glucose. Certain mutants in the glucose-sensing and response pathways, such as those lacking the Snf1 kinase, are sensitive to alkalinization. We show that addition of glucose to the medium improves growth of wild type cells at high pH, fully abolish the snf1 alkali-sensitive phenotype and attenuates high pH-induced Snf1 phosphorylation at Thr210. The elm1 mutant, lacking one of the three upstream Snf1 kinases (tos3, elm1 and sak1), is markedly alkali sensitive, whereas the phenotype of the tos3 elm1 sak1 strain is even stronger than that of snf1 cells and it is not fully rescued by glucose supplementation. DNA microarray analysis reveals that about 75% of genes induced at short term by high pH are also induced by glucose scarcity. Snf1 mediates, in full or in part, the activation of a significant subset (38%) of short-term alkali-induced genes, including those coding high-affinity hexose transporters and phosphorylating enzymes. Induction of genes encoding enzymes involved in glycogen (but not trehalose) metabolism is largely dependent of the presence of Snf1. Therefore, the function of Snf1 in adaptation to glucose scarcity appears crucial for alkaline pH tolerance. Incorporation of micromolar amounts of iron and copper to a glucose-supplemented medium result in an additive effect and allows near normal growth at high pH, thus indicating that these three nutrients are key limiting factors for growth in an alkaline environment.
Project description:Alkaline pH stress invokes in S. cerevisiae a potent and fast transcriptional response that includes many genes repressed by glucose. Certain mutants in the glucose-sensing and response pathways, such as those lacking the Snf1 kinase, are sensitive to alkalinization. We show that addition of glucose to the medium improves growth of wild type cells at high pH, fully abolish the snf1 alkali-sensitive phenotype and attenuates high pH-induced Snf1 phosphorylation at Thr210. The elm1 mutant, lacking one of the three upstream Snf1 kinases (tos3, elm1 and sak1), is markedly alkali sensitive, whereas the phenotype of the tos3 elm1 sak1 strain is even stronger than that of snf1 cells and it is not fully rescued by glucose supplementation. DNA microarray analysis reveals that about 75% of genes induced at short term by high pH are also induced by glucose scarcity. Snf1 mediates, in full or in part, the activation of a significant subset (38%) of short-term alkali-induced genes, including those coding high-affinity hexose transporters and phosphorylating enzymes. Induction of genes encoding enzymes involved in glycogen (but not trehalose) metabolism is largely dependent of the presence of Snf1. Therefore, the function of Snf1 in adaptation to glucose scarcity appears crucial for alkaline pH tolerance. Incorporation of micromolar amounts of iron and copper to a glucose-supplemented medium result in an additive effect and allows near normal growth at high pH, thus indicating that these three nutrients are key limiting factors for growth in an alkaline environment. We identified the changes in the expression profiles caused by alkalinization of the medium (pH8 vs. pH5.5 for 10 min) in several strains: wild type cells (4 chips), snf1 mutant cells (4 chips) We also identified the transcriptomic changes that occur after glucose deprivation (0.05% vs 2% for 15 min) in: wild type cells (2 chips) snf1 mutant cells (2 chips) Total: 12 chips
Project description:Industrial bioethanol production may involve a low pH environment,improving the tolerance of S. cerevisiae to a low pH environment caused by inorganic acids may be of industrial importance to control bacterial contamination, increase ethanol yield and reduce production cost. Through analysis the transcriptomic data of Saccharomyces cerevisiae with different ploidy under low pH stress, we hope to find the tolerance mechanism of Saccharomyces cerevisiae to low pH.
Project description:Exposure of Saccharomyces cerevisiae to alkaline pH represents a stress condition that generates a compensatory reaction. Here we examine a possible role of the protein kinase-A (PKA) pathway in this response. The phenotypic analysis reveals that mutations that activate the PKA pathway (ira1 ira2, bcy1) tend to cause sensitivity to alkaline pH, whereas its deactivation develops tolerance to this stress. We observe that alkalinization causes a transient decrease in cAMP, the main regulator of the pathway. Alkaline pH causes rapid nuclear localization of the PKA-regulated Msn2 transcription factor which, together with Msn4, mediates a general stress response by binding to STRE sequences in many promoters. Consequently, a synthetic STRE-LacZ reporter shows a rapid induction in response to alkaline stress. An msn2 msn4 mutant is sensitive to alkaline pH, and transcriptomic analysis reveals that after 10 minutes of alkaline stress, the expression of many induced genes (47%) depends, at least in part, on the presence of Msn2 and Msn4. Taken together, these results demonstrate that inhibition of the PKA pathway by alkaline pH represents a substantial part of the adaptive response to this kind of stress and that this response involves Msn2/Msn4-mediated gene remodeling. However, the relevance of attenuation of PKA in high pH tolerance is not restricted to regulation of Msn2 function.
Project description:Cultivation methods used to investigate microbial calorie restriction often result in carbon and energy starvation. This study aims to dissect cellular responses to calorie restriction and starvation in Saccharomyces cerevisiae by using retentostat cultivation. In retentostats, cells are continuously supplied with a small, constant carbon and energy supply, sufficient for maintenance of cellular viability and integrity but insufficient for growth. When glucose-limited retentostats cultivated under extreme calorie restriction were subjected to glucose starvation, calorie-restricted and glucose-starved cells were found to share characteristics such as increased heat-shock tolerance and expression of quiescence-related genes. However, they also displayed strikingly different features. While calorie-restricted yeast cultures remained metabolically active and viable for prolonged periods of time, glucose starvation resulted in rapid consumption of reserve carbohydrates, population heterogeneity due to appearance of senescent cells and, ultimately, loss of viability. Moreover, during starvation, calculated rates of ATP synthesis from storage carbohydrates were 2-3 orders of magnitude lower than steady-state ATP-turnover rates calculated under extreme calorie restriction in retentostats. Stringent reduction of ATP turnover during glucose starvation was accompanied by a strong down-regulation of genes involved in protein synthesis. These results demonstrate that extreme calorie restriction and carbon starvation represent different physiological states in S. cerevisiae.
Project description:A universal feature of the response to stress and nutrient limitation is transcriptional upregulation of genes encoding proteins important for survival. Interestingly, under many of these conditions overall protein synthesis levels are reduced, thereby dampening the stress response at the level of protein expression. For example, during glucose starvation in yeast, translation is rapidly and reversibly repressed, yet transcription of many stress- and glucose-repressed genes is increased. Using ribosome profiling and microscopy, we found that this transcriptionally upregulated gene set consists of two classes: (1) one producing mRNAs that are preferentially translated during glucose limitation and are diffusely localized in the cytoplasm – this class includes many heat shock protein mRNAs; and (2) another producing mRNAs that are poorly translated during glucose limitation, have high rates of translation initiation, and are concentrated in foci that co-localize with P bodies and stress granules – this class is enriched for glucose metabolism mRNAs. Remarkably, the information specifying differential localization and translation of these two classes of mRNAs is encoded in the promoter sequence – promoter responsiveness to heat shock factor (Hsf1) specifies diffuse cytoplasmic localization and preferential translation upon glucose starvation, whereas different promoter elements upstream of genes encoding poorly translated glucose metabolism mRNAs direct these mRNAs to RNA granules under glucose starvation. Thus, promoter sequences and transcription factor binding can influence not only mRNA levels, but also subcellular localization of mRNAs and the efficiency with which they are translated, enabling cells to tailor protein production to environmental conditions. Examination of mRNA translation in S. cerevisiae upon glucose starvation.
Project description:Purpose: High carbonate and bicarbonate concentrations of calcareous soils with high pH can affect crop performance due to different constraints. The goal of this study is to perform a comparative transcriptomic analysis using demes moderate-tolerance and sensitive under alkaline stress ( high pH 8.3 and 10 mM NaHCO3) Methods: Transcriptomic analysis was performed on two naturally selected Arabidopsis thaliana demes. Carbon soil tolerant A1(c+) and the sensitive T6(c-). Plants 15 day-old were exposed for 3 or 48 h to either pH stress alone (pH 5.9 vs pH 8.3 adjusted by BTP and MES buffers) or to alkaline stress (pH 8.3) caused by 10 mM of Results Shoot transcriptome analysis revealed that bicarbonate quickly (3 h) induced Fe-deficiency related genes in T6(c-) leaves, while in A1 (c+) main initial changes were found in receptor-like proteins (RPL), jasmonate (JA) and salicylate (SA) pathways, methionine-derived glucosinolate (GS), Sulphur starvation, starch degradation, and cell cycle. Conclusions: Our results suggest that leaves of carbonate tolerant plants do not sense iron deficiency as fast as sensitive ones. This is in line with the ability to translocate more iron to aerial parts, producing a higher biomass and maintaining silique production. In leaves of A1(c+) plants, the activation of other genes related to apoplastic stress perception, signal transduction, GS, sulphur acquisition, and cell cycle regu-lation precedes the induction of iron homeostasis mechanisms yielding an efficient response to bicarbonate stress
Project description:Cultivation methods used to investigate microbial calorie restriction often result in carbon and energy starvation. This study aims to dissect cellular responses to calorie restriction and starvation in Saccharomyces cerevisiae by using retentostat cultivation. In retentostats, cells are continuously supplied with a small, constant carbon and energy supply, sufficient for maintenance of cellular viability and integrity but insufficient for growth. When glucose-limited retentostats cultivated under extreme calorie restriction were subjected to glucose starvation, calorie-restricted and glucose-starved cells were found to share characteristics such as increased heat-shock tolerance and expression of quiescence-related genes. However, they also displayed strikingly different features. While calorie-restricted yeast cultures remained metabolically active and viable for prolonged periods of time, glucose starvation resulted in rapid consumption of reserve carbohydrates, population heterogeneity due to appearance of senescent cells and, ultimately, loss of viability. Moreover, during starvation, calculated rates of ATP synthesis from storage carbohydrates were 2-3 orders of magnitude lower than steady-state ATP-turnover rates calculated under extreme calorie restriction in retentostats. Stringent reduction of ATP turnover during glucose starvation was accompanied by a strong down-regulation of genes involved in protein synthesis. These results demonstrate that extreme calorie restriction and carbon starvation represent different physiological states in S. cerevisiae. The yeast was first grown for 14 days under extreme calorie restriction in anaerobic, glucose-limited retentostats (Boender et al., 2009, Appl.Environ.Microbiol., 75: 5607-5614.). Subsequently, starvation was started by terminating the glucose feed. Yeast transcriptional reprogramming in response to calorie restriction and starvation was monitored by microarray analysis. Independent duplicate retentostat cultures, and subsequently starvation, were sampled for transcriptome analysis using Affymetrix microarrays. One time-point was sampled during calorie restriction (T0) and four time points were sampled during the starvation phase 10, 30, 60 and 120 minutes after switching of the feed, resulting in a dataset of 10 arrays.
Project description:Exposure of Saccharomyces cerevisiae to alkaline pH represents a stress condition that generates a compensatory reaction. Here we examine a possible role of the protein kinase-A (PKA) pathway in this response. The phenotypic analysis reveals that mutations that activate the PKA pathway (ira1 ira2, bcy1) tend to cause sensitivity to alkaline pH, whereas its deactivation develops tolerance to this stress. We observe that alkalinization causes a transient decrease in cAMP, the main regulator of the pathway. Alkaline pH causes rapid nuclear localization of the PKA-regulated Msn2 transcription factor which, together with Msn4, mediates a general stress response by binding to STRE sequences in many promoters. Consequently, a synthetic STRE-LacZ reporter shows a rapid induction in response to alkaline stress. An msn2 msn4 mutant is sensitive to alkaline pH, and transcriptomic analysis reveals that after 10 minutes of alkaline stress, the expression of many induced genes (47%) depends, at least in part, on the presence of Msn2 and Msn4. Taken together, these results demonstrate that inhibition of the PKA pathway by alkaline pH represents a substantial part of the adaptive response to this kind of stress and that this response involves Msn2/Msn4-mediated gene remodeling. However, the relevance of attenuation of PKA in high pH tolerance is not restricted to regulation of Msn2 function. Eight samples were analyzed: WT and the MCY5278 mutant strain, lacking both Msn2 and Msn4, in the presence of 20 mM KOH (pH 8) and in the presence of 20 mM KCl (non-induced conditions) for 10 and 30 min of stress. 2 biological replicates were analyzed for each condition, and dye-swapping was carried out for each comparison of samples. We compared the expression profiles of: 1) WT +KOH vs. WT +KCl after 10 min 2) msn2 msn4 mutant +KOH vs. msn2 msn4 +KCl after 10 min 3)WT +KOH vs. WT +KCl after 30 min 4) msn2 msn4 mutant +KOH vs. msn2 msn4 +KCl after 30 min Total number of chips analyzed: 16.