Background
Combinatorial effects of environmental parameters on transcriptional regulation in Saccharomyces cerevisiae: a quantitative analysis of a compendium of chemostat-based transcriptome data.
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View SamplesAddition of 3 new arrays made from carbon limited chemostat of CENPK113-7D and 3 new arrays made from aerobic carbon limited chemostat of CENPK113-7D Complmentary data to the data of the serie GSE1723.
Exploiting combinatorial cultivation conditions to infer transcriptional regulation.
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View SamplesThe Saccharomyces cerevisiae SFP1 is required for proper regulation of ribosome biogenesis and cell size in response to nutrients. A mutant deleted for SFP1 shows specific traits among which a slow growth phenotype, which is particularly evident during growth on glucose. To assess the effects of nutrients on the activity of Sfp1 independent by growth rate related feedback we grew an sfp1 mutant and its isogenic reference strain in chemostat cultures, at the same specific growth rate, under glucose/ethanol-limitation. Our data show that Sfp1 is involved in the modulation of cell size and RiBi gene expression and that these two functions are differently influenced by nutrients.
Saccharomyces cerevisiae SFP1: at the crossroads of central metabolism and ribosome biogenesis.
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View SamplesThe goal of this study was to study this interaction by analyzing genome-wide transcriptional responses to four different nutrient-limitation regimes under aerobic and anaerobic conditions in chemostat cultures of S. cerevisiae. This two-dimensional approach resulted in a new, robust set of anaerobic and aerobic signature transcripts for S. cerevisiae, as well as to a refinement of previous reports on nutrient-responsive genes. Moreover, the identification of genes regulated both by nutrient and oxygen availability provided new insight in cross-regulated network and hierarchy in the control of gene expression.
Two-dimensional transcriptome analysis in chemostat cultures. Combinatorial effects of oxygen availability and macronutrient limitation in Saccharomyces cerevisiae.
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View SamplesDespite the scientific and applied interest in anaerobic metabolism of Saccharomyces cerevisiae, not all genes whose transcription is up-regulated under anaerobic conditions have yet been linked to known transcription factors. Experiments with a reporter construct in which the promoter of the anaerobically up-regulated TIR1 gene was fused to LacZ revealed a complete loss of anaerobic up-regulation in a snf7 mutant. Anaerobic up-regulation was restored by expression of a truncated allele of RIM101 that encodes for a constitutively active Rim101p transcription factor. Analysis of LacZ expression in several deletion mutants confirmed that the effect of Snf7p on anaerobic up-regulation of TIR1 involved Rim101p and did not require a functional multi-vesicular body sorting pathway (in which Snf7p also participates). Transcriptome analysis in anaerobic chemostat cultures revealed that 26 additional genes exhibited a Snf7p/Rim101p dependent anaerobic up-regulation. Since, in its activated form, Rim101p is generally known as a transcriptional repressor, its role in anaerobic up regulation of TIR1 and other anaerobic yeast genes must involve additional factors. Further studies with deletion mutants in NRG1, NRG2 and SMP1, which were previously shown to be regulated by Rim101p, showed that these genes were not involved in the regulation of TIR1. However, the aerobic repression mechanism of TIR1 involved the general repressor Ssn6p-Tup1p complex. The physiological relevance of Snf7p/Rim101p-mediated transcriptional up-regulation of several genes in anaerobic yeast cultures was evident from reduced growth of a snf7 under anaerobic conditions.
Involvement of Snf7p and Rim101p in the transcriptional regulation of TIR1 and other anaerobically upregulated genes in Saccharomyces cerevisiae.
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View SamplesIn Saccharomyces cerevisiae, glycogen and trehalose are important reserve carbohydrates that accumulate under nutrient limitation in batch cultures. An inherent draw-back of batch studies is that specific growth rate and substrate and product concentrations are variable over time and between cultures. The aim of this present study was to identify the nutritional requirements associated with high accumulation of reserve carbohydrates at a fixed specific growth rate (0.10 h-1) in anaerobic chemostat cultures that were limited by one of five different nutrients (carbon, nitrogen, sulfur, phosphorus or zinc). Reserve carbohydrates accumulation is not a general response to nutrient limitation. Over the conditions tested, accumulation occurs essentially under nitrogen (and to a lesser extent carbon) limited conditions. This was confirmed by the transcriptional profile of the genes involved in trehalose biosynthesis. We show that the transcriptional induction of both glycogen and trehalose biosynthesis genes was to a large extent driven by the regulator Msn2/4. However, the main regulatory control of glycogen biosynthesis was post-translational. Under nitrogen limitation, the ratio of glycogen synthase over glycogen phosphorylase increased up to eight-fold, thus enabling an increased flux towards glycogen biosynthesis.
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View SamplesSaccharomyces cerevisiae is currently widely used as a model to study chronological aging of metazoan cells. Chronological aging is typically studied in aerobic stationary phase (SP) cultures, i.e. the final stage of batch cultures in which growth is arrested due to exogenous carbon source exhaustion. Survival of yeast cells in SP defines their chronological lifespan (CLS). S. cerevisiae SP cultures have strongly contributed to the understanding of cellular mechanisms involved in aging and indicated a key role for oxygen. Oxygen is the natural starting point for reactive oxygen species (ROS) that may both have malignant and beneficial effects on aging. In addition, oxygen allows yeast to grow on ethanol and organic acids formed during the initial respiro-fermentative growth phase on glucose. This post-diauxic phase is hallmarked by reduced growth rates, increased expression of genes involved in SP survival, and increased stress resistance. To date, the role of oxygen and respiration in aging has mostly been studied using respiratory deficient mutants, and respiration repressing agents. However, genetic or chemical interventions may result in unwanted side effects that influence survival in SP. We therefore followed a different approach to evaluate the impact of oxygen availability on yeast robustness in SP, i.e. its CLS and stress resistance, by using the capability of S. cerevisiae to grow under anaerobic conditions. A thorough physiological comparison of strictly anaerobic and aerobic SP cultures revealed that the presence of oxygen during growth and aging of S. cerevisiae strongly affects its energetic status, longevity and stress tolerance in a positive way. Combining the physiological data with genome-wide expression analysis revealed that the oxygen-dependent diauxic growth phase enabled the full induction of robustness in S. cerevisiae, and points to appropriate pre-conditioning of cells as a crucial factor to survive starvation. These findings highlight the importance of exogenous energy availability in the conditions leading to growth arrest, and bring new insight on the role of oxygen in the aging of eukaryotes.
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View SamplesPhysiological effects of carbon dioxide and impact on genome-wide transcript profiles were analysed in chemostat cultures of Saccharomyces cerevisiae. In anaerobic, glucose-limited chemostat cultures grown at atmospheric pressure, cultivation under CO2-saturated conditions had only a marginal (<10%) impact on the biomass yield. Conversely, a 25% decrease of the biomass yield was found in aerobic, glucose-limited chemostat cultures aerated with a mixture of 79% CO2 and 21% O2. This observation indicated that respiratory metabolism is more sensitive to CO2 than fermentative metabolism. Consistent with the more pronounced physiological effects of CO2 in respiratory cultures, the number of CO2-responsive transcripts was higher in aerobic cultures than in anaerobic cultures. Many genes involved in mitochondrial functions showed a transcriptional response to elevated CO2 concentrations. This is consistent with an uncoupling effect of CO2 and/or intracellular bicarbonate on the mitochondrial inner membrane. Other transcripts that showed a significant transcriptional response to elevated CO2 included NCE103 (probably encoding carbonic anhydrase), PCK1 (encoding PEP carboxykinase) and members of the IMD gene family (encoding isozymes of inosine monophosphate dehydrogenase
Physiological and genome-wide transcriptional responses of Saccharomyces cerevisiae to high carbon dioxide concentrations.
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View SamplesDiurnal temperature cycling is an intrinsic characteristic of many exposed microbial ecosystems. However, its influence on yeast physiology and transcriptome has not been studied in detail. In this study, 24-h sinoidal temperature cycles, oscillating between 12 and 30C, were imposed on anaerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. After three diurnal temperature cycles (DTC), concentrations of glucose, and extracellular metabolites, as well as CO2-production rates showed regular, reproducible circadian rhytms. DTC also led to waves of transcriptional activation and repression, which involved one sixth of the yeast genome. A substantial fraction of these DTC-responsive genes appeared to primarily respond to changes in glucose concentration. Elimination of known glucose-responsive genes revealed overrepresentation of previously identified temperature-responsive genes as well as genes involved in cell cycle and de novo purine biosynthesis. Analyses of budding index and flow cytomery demonstrated that DTC led to a partial synchronization of the cell cycle of the yeast populations in the chemostat cultures, which was lost upon release from DTC. Comparison of DTC results with data from steady-state cultures showed that DTC was sufficiently slow to allow S. cerevisiae chemostat cultures to almost completely acclimatize their transcriptome and physiology at the DTC temperature maximum, and to approach acclimation at the DTC temperature minimum.
Physiological and transcriptional responses of anaerobic chemostat cultures of Saccharomyces cerevisiae subjected to diurnal temperature cycles.
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View SamplesSucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-H+ symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9 % higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5 coding sequences of SUC2, resulting in relocation of invertase to the cytosol. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 95 generations of anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11 % higher ethanol yield on sucrose in chemostat and batch cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. Deletion of AGT1, which had been duplicated during laboratory evolution, restored the growth characteristics of the unevolved iSUC2 strain. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.
Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase.
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