Saccharomyces cerevisiae
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PROVIDER: PRJNA105887 | ENA |
REPOSITORIES: ENA
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Project description:Background We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we demonstrated the transferability of this respiratory phenotype in the presence of up to 100 g/L glucose to a yeast strain in which only HXT1-7 had been deleted. In this study, we wanted to examine the basis of the respiratory phenotype of the resultant strain, V5.TM6*P, by comparing its transcriptome with that of its parent, V5, at different glucose concentrations. Results cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. Highly complete collections of known genes of the TCA cycle, glyoxylate cycle and respiratory chain were identified, consistent with a respiratory metabolism. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4, Cat8 and Mig1, and using this in combination with verified binding consensus sequences, to identify genes likely to be regulated by one or more of these transcription factors. Of the induced genes of our dataset, 77 % had binding sites for Hap2/3/5 (Hap4 is an activator of this complex), with 72 % having at least two (the latter set being more induced than the former). This is relevant since Hap4 is known to be involved in the transcriptional activation of respiratory genes and other mitochondrial functions. In addition, 13 % of genes were found to have a binding site for Cat8, which together with its complexes with Sip4 have previously been identified as mediating de-repression of a number of genes during the diauxic shift. Finally, 21 % of genes had a binding site for Mig1 which is a transcriptional repressor involved in glucose repression. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This important result indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell. Conclusions Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88 % of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three transcription factors; Hap2/3/5, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations.
2008-06-20 | GSE11799 | GEO
Project description:Deletion mutants of S. cerevisiae 4743 were grown in glucose-limited continuous chemostats (D = 0.2h-1). The deletion mutants used were: Homozygous deletion mutants of HAP4, OXA1, BCS1, RIP1, MBA1 and MIG1. Heterozygous deletion mutants of HAP4, RIP1 and MIG1.
2008-03-21 | E-MEXP-727 | biostudies-arrayexpress
Project description:The opportunistic pathogen Cryptococcus neoformans causes fungal meningoencephalitis in immunocompromised individuals. In previous studies, we found that the Hap complex in this pathogen represses genes encoding mitochondrial respiratory functions and TCA cycle components under low-iron conditions. The orthologous Hap2/3/4/5 complex in Saccharomyces cerevisiae exerts a regulatory influence on mitochondrial functions and Hap4 is subject to glucose repression via the carbon catabolite repressor Mig1. In this study, we explored the regulatory link between a candidate ortholog of the Mig1 protein and the HapX component of the Hap complex in C. neoformans. This analysis revealed repression of MIG1 by HapX and activation of HAPX by Mig1 in low iron conditions, and Mig1 regulation of mitochondrial functions including respiration, tolerance for reactive oxygen species, and expression of genes for iron-consuming and iron-acquisition functions. Consistent with these regulatory functions, a mig1Δ mutant had impaired growth on inhibitors of mitochondrial respiration and ROS inducers. Furthermore, deletion of MIG1 provoked a dysregulation in nutrient sensing via the TOR pathway and impacted the pathway for cell wall remodeling. Importantly, loss of Mig1 increased susceptibility to fluconazole thus further establishing a link between azole antifungal drugs and mitochondrial function. Mig1 and HapX were also required together for survival in macrophages, but Mig1 alone had a minimal impact on virulence in mice. Overall, these studies provide novel insights into a HapX/Mig1 regulatory network and reinforce an association between mitochondrial dysfunction and drug susceptibility that may provide new targets for the development of antifungal drugs.
2015-12-17 | GSE76063 | GEO
Project description:The opportunistic pathogen Cryptococcus neoformans causes fungal meningoencephalitis in immunocompromised individuals. In previous studies, we found that the Hap complex in this pathogen represses genes encoding mitochondrial respiratory functions and TCA cycle components under low-iron conditions. The orthologous Hap2/3/4/5 complex in Saccharomyces cerevisiae exerts a regulatory influence on mitochondrial functions and Hap4 is subject to glucose repression via the carbon catabolite repressor Mig1. In this study, we explored the regulatory link between a candidate ortholog of the Mig1 protein and the HapX component of the Hap complex in C. neoformans. This analysis revealed repression of MIG1 by HapX and activation of HAPX by Mig1 in low iron conditions, and Mig1 regulation of mitochondrial functions including respiration, tolerance for reactive oxygen species, and expression of genes for iron-consuming and iron-acquisition functions. Consistent with these regulatory functions, a mig1Î mutant had impaired growth on inhibitors of mitochondrial respiration and ROS inducers. Furthermore, deletion of MIG1 provoked a dysregulation in nutrient sensing via the TOR pathway and impacted the pathway for cell wall remodeling. Importantly, loss of Mig1 increased susceptibility to fluconazole thus further establishing a link between azole antifungal drugs and mitochondrial function. Mig1 and HapX were also required together for survival in macrophages, but Mig1 alone had a minimal impact on virulence in mice. Overall, these studies provide novel insights into a HapX/Mig1 regulatory network and reinforce an association between mitochondrial dysfunction and drug susceptibility that may provide new targets for the development of antifungal drugs. In this study, transcription profiles of WT and mig1D mutant strains of Cryptococcus neoformans were compared in a dye-swap experiment following 6hr exposure to Low Iron Medium (LIM) or LIM + 100mM FeCl3.
2015-12-17 | E-GEOD-76063 | biostudies-arrayexpress
Project description:To get a global view of the impact of Hap4 and Hap5 on gene expression in the human pathogen Candida glabrata, we compared the transcriptomes of wild type starins with those of mutant strains in which either HAP4 or HAP5 were deleted. We performed these experiments in two different growth conditions: glycerol as sole carbon source (as Hap4 is supposed to be involved in the induction of respiratory genes in non-fermentative growth conditions) or glucose with iron excess (2 mM FeSO4) as Hap5 is supposed to be involved in the high iron response independently of Hap4.
2021-11-19 | E-MTAB-10544 | biostudies-arrayexpress
Project description:When the yeast Saccharomyces cerevisiae is subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from being purely respiratory to mixed respiratory and fermentative. This shift is characterized by ethanol production, a phenomenon known as the Crabtree effect due to its analogy with lactate overflow in cancer cells. It is well known that at high glycolytic fluxes there is glucose repression of respiratory pathways resulting in a decrease in the respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect (or overflow metabolism) is due to a limited respiratory capacity or caused by glucose-mediated repression of respiration. We addressed this issue by increasing respiration in S. cerevisiae by introducing a heterologous alternative oxidase, and observed reduced aerobic ethanol formation. In contrast, increasing non-respiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, while NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, while alternative oxidase is directed to the mitochondria. The onset of aerobic ethanol formation is demonstrated to be a consequence of an imbalance in mitochondrial redox balancing. In addition to answering fundamental physiological questions, our findings are relevant for all biomass derived applications of S. cerevisiae. Keywords: Genetic Modification
2007-02-01 | GSE6277 | GEO
Project description:S. cerevisiae cells (homozygous deletion mutants of BY4743) grown in chemostats, sampled at steady state. Glucose and ammonium limitation, dilution rates 0.1 and 0.2 hr<sup>-1</sup>, gene deletions HO and HAP4 applied.
2005-12-31 | E-MEXP-442 | biostudies-arrayexpress
Project description:We performed RNAseq with Saccharomyces cerevisiae cells under both transient and steady-state conditions to study the regulation of genes by two pulsatile transcription factors, Msn2 and Mig1. The transient data allowed us to identify combinatorial targets while the steady-state data was used to study target expression dependence on the relative pulse timing between the two TFs.
2015-10-15 | GSE71712 | GEO
Project description:When the yeast Saccharomyces cerevisiae is subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from being purely respiratory to mixed respiratory and fermentative. This shift is characterized by ethanol production, a phenomenon known as the Crabtree effect due to its analogy with lactate overflow in cancer cells. It is well known that at high glycolytic fluxes there is glucose repression of respiratory pathways resulting in a decrease in the respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect (or overflow metabolism) is due to a limited respiratory capacity or caused by glucose-mediated repression of respiration. We addressed this issue by increasing respiration in S. cerevisiae by introducing a heterologous alternative oxidase, and observed reduced aerobic ethanol formation. In contrast, increasing non-respiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, while NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, while alternative oxidase is directed to the mitochondria. The onset of aerobic ethanol formation is demonstrated to be a consequence of an imbalance in mitochondrial redox balancing. In addition to answering fundamental physiological questions, our findings are relevant for all biomass derived applications of S. cerevisiae. Experiment Overall Design: Heterologous gene expression in chemostats using Affymetrix Yeast Genome 2.0 arrays. Total RNA extraction and sample preparation, hybridization was done according to the manufacturer's protocol.
2008-06-14 | E-GEOD-6277 | biostudies-arrayexpress
Project description:The highly conserved heterotrimeric protein kinase SNF1 is important for metabolic adaptations in the pathogenic yeast Candida albicans. A key function of SNF1 is to inactivate the repressor protein Mig1 and thereby allow the expression of genes that are required for the the utilization of alternative carbon sources when the preferred carbon source glucose is absent or becomes limiting. However, how SNF1 controls Mig1 activity in C. albicans has remained elusive. Using a phosphoproteomic approach, we found that Mig1 is phosphorylated at multiple serine residues. Replacement of these serine residues by nonphosphorylatable alanine residues strongly increased the repressor activity of Mig1 in cells lacking a functional SNF1 complex, indicating that additional protein kinases are involved in the regulation of Mig1. Unlike wild-type Mig1, whose levels strongly decreased when the cells were grown on sucrose or glycerol instead of glucose, the levels of a mutant Mig1 protein lacking nine phosphorylation sites remained high under these conditions. Despite the increased protein levels and the absence of multiple phosphorylation sites, cells with a functional SNF1 complex could still sufficiently inhibit the hyperactive Mig1 to enable wild-type growth on alternative carbon sources. In line with this, phosphorylated forms of the mutant Mig1 were still detected in the presence and absence of a functional SNF1, demonstrating that Mig1 contains additional, unidentified phosphorylation sites and that downstream protein kinases are involved in the control of Mig1 activity by SNF1.
2023-10-27 | PXD045393 | Pride