Project description:The transcriptome from a S. cerevisiae tup1 deletion mutant was one of the first comprehensive yeast transcriptomes published. Subsequent transcriptomes from tup1 and cyc8 mutants firmly established the Tup1-Cyc8 complex as predominantly acting as a repressor of gene transcription. However, transcriptomes from tup1/cyc8 gene deletion or conditional mutants would all have been influenced by the striking flocculation phenotypes that these mutants display. In this study, we have separated the impact of flocculation from the transcriptome in a cyc8 conditional mutant to reveal those genes (i) subject solely to Cyc8p dependent regulation, (ii) regulated by flocculation only, and (iii) regulated by Cyc8p and further influenced by flocculation. We reveal an improved Cyc8p transcriptome that includes newly identified Cyc8p-regulated genes that were masked by the flocculation phenotype and excludes genes which were indirectly influenced by flocculation and not regulated by Cyc8p. Furthermore, we show evidence to suggest that flocculation exerts a complex and potentially dynamic influence upon global gene transcription. These data should be of interest to future studies into the mechanism of action of the Tup1-Cyc8 complex and to those involved in understanding the development of flocculation and its impact upon cell function.
Project description:In oenological conditions, yeasts are exposed to several stresses and Saccharomyces cerevisiae strains respond modifying their transcriptional program. Yeast flocculation is a social trait that permits to the cells to escape to hostile conditions by sedimentation. This behaviour is mainly due to the presence in the genome of FLO greenbeards genes. In order to understand the role of flocculation in stress response a flocculent S. cerevisiae wine strain and his FLO5 deleted strain were submitted to standard and second fermentation. At the middle of these two processes a genome wide expression analysis was performed. Results showed that while the FLO5 deleted strain was engaged to a re-organization of cell wall and to find a different way to adhere, flocculent yeast presented a proteic stress response and an up-regulation of genes involved in superior alcohols production. Obtained data confirmed the importance of FLO5 in flocculation phenotype and highlight the role of flocculation in stress response.
Project description:The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1-expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1+ cells avoid exploitation by non-expressing flo1 cells by self/non-self recognition: FLO1+ cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known “green beard genes”, which direct cooperation towards other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly-evolving social trait. This dataset contains raw transcriptome data of flocculating cells (that express FLO1 driven by the GAL1 promoter) and non-flocculating cells (that do not express FLO1).
Project description:D-lactic acid is a three-carbon organic acid with a chiral structure and can improve the thermostability of polylactic acid. Microorganisms such as the methylotrophic yeast Pichia pastoris, which lack the natural ability to produce or accumulate high amounts of D-lactic acid, have been engineered to produce it in high titers. However, tolerance to D-lactic acid remains a challenge. In this study, we demonstrate that cell flocculation improves tolerance to D-lactic acid and leads to increased D-lactic acid production in Pichia pastoris. By incorporating a flocculation gene from Saccharomyces cerevisiae (ScFLO1) into P. pastoris KM71, we created a strain (KM71-ScFlo1) that demonstrated up to a 1.6-fold improvement in specific growth rate at high D-lactic acid concentrations. Furthermore, integrating a D-lactate dehydrogenase gene from Leuconostoc pseudomesenteroides (LpDLDH) into KM71-ScFlo1 resulted in an engineered strain (KM71-ScFlo1-LpDLDH) that can produce D-lactic acid at a titer of 5.12 0.35 g/L in 48 hours , a 2.6-fold improvement over the control strain lacking ScFLO1 expression. Transcriptomics analysis of this strain provided insights into the mechanism of increased tolerance to D-lactic acid including the upregulations of genes involved in lactate transport and iron metabolism. Overall, our work represents an advancement in the efficient microbial production of D-lactic acid by manipulating yeast flocculation.
Project description:The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1-expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1+ cells avoid exploitation by non-expressing flo1 cells by self/non-self recognition: FLO1+ cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known â??green beard genesâ??, which direct cooperation towards other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly-evolving social trait. This dataset contains raw transcriptome data of flocculating cells (that express FLO1 driven by the GAL1 promoter) and non-flocculating cells (that do not express FLO1). Experiment Overall Design: Cultures of flocculating and non-flocculating cells were grown for 24 hours in YPGal medium. Subsequently, RNA was isolated from these cultures, converted into cDNA and analyzed using commercially available Afymetrix S98 arrays. For each culture, two biological replicates were analyzed.
Project description:Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeast Schizosaccharomyces pombe. Genome-wide studies of the Crz1 and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This paper identified an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2, and tunicamycin treatment, as well as a ∆pmr1 genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of the Saccharomyces cerevisiae ortholog Crz1. These genes were functionally enriched for Crz1-conserved processes such as cell wall biosynthesis. Overexpression of prz1+ increased resistance to the cell wall degradation enzyme zymolyase, likely from upregulation of the O-mannosyltransferase encoding gene omh1+. Loss of omh1+ abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss of prz1+ resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the ∆prz1 strain was abrogated by the loss of gsf2+ or cbf12+. This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes between Crz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional-regulatory network.
Project description:Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeast Schizosaccharomyces pombe. Genome-wide studies of the Crz1 and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This paper identified an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2, and tunicamycin treatment, as well as a âpmr1 genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of the Saccharomyces cerevisiae ortholog Crz1. These genes were functionally enriched for Crz1-conserved processes such as cell wall biosynthesis. Overexpression of prz1+ increased resistance to the cell wall degradation enzyme zymolyase, likely from upregulation of the O-mannosyltransferase encoding gene omh1+. Loss of omh1+ abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss of prz1+ resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the âprz1 strain was abrogated by the loss of gsf2+ or cbf12+. This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes between Crz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional-regulatory network. We generated 3 overexpression microarrays and 2 deletion microarrays with dye swaps, and 2 deletion microarrays with drug treatments with dye swaps, each with a biological replicate performed as a dye swap. The effect of the mutant strains were all compared to wild type or empty vector strains, except for one experiment where the prz1 deletion mutant was compared to the pmr1 deletion mutant.
Project description:Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins can bind directly to AU-rich elements in mRNAs and promote transcript deadenylation and decay. The yeast Schizosaccharomyces pombe expresses a single TTP family member, Zfs1p, that has been linked to the mating response pathway and septum formation. We showed previously that Zfs1p can bind to and promote the destabilization of AU-rich element-containing transcripts. In this study, we identified additional target transcripts by comparing transcript levels in wild type and zfs1 mutant yeast, using deep sequencing and microarray approaches. We also used direct RNA sequencing to determine the locations of the polyA tails in both wild type and mutant strains, and to confirm the presence of potential Zfs1p target sequences within the mRNA. These studies identified a set of transcripts containing potential Zfs1p binding sites that accumulated significantly in the zfs1 mutants; a subset of these turned over more slowly in the zfs1 mutant strain, and bound directly to Zfs1p in co-immunoprecipitations. One apparent direct target encodes the transcription factor Cbf12p, which is known to increase cell-cell adhesion and flocculation when over-expressed. Studies of zfs1 and cbf12 double mutants demonstrated that the increased flocculation seen in zfs1 mutants is due, at least in part, to a direct effect on the turnover of cbf12 mRNA, leading in turn to changes in the levels of its transcriptionally regulated genes. These data suggest that Zfs1p can both directly and indirectly regulate the levels of transcripts involved in cell-cell adhesion in this species.
Project description:Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins can bind directly to AU-rich elements in mRNAs and promote transcript deadenylation and decay. The yeast Schizosaccharomyces pombe expresses a single TTP family member, Zfs1p, that has been linked to the mating response pathway and septum formation. We showed previously that Zfs1p can bind to and promote the destabilization of AU-rich element-containing transcripts. In this study, we identified additional target transcripts by comparing transcript levels in wild type and zfs1 mutant yeast, using deep sequencing and microarray approaches. We also used direct RNA sequencing to determine the locations of the polyA tails in both wild type and mutant strains, and to confirm the presence of potential Zfs1p target sequences within the mRNA. These studies identified a set of transcripts containing potential Zfs1p binding sites that accumulated significantly in the zfs1 mutants; a subset of these turned over more slowly in the zfs1 mutant strain, and bound directly to Zfs1p in co-immunoprecipitations. One apparent direct target encodes the transcription factor Cbf12p, which is known to increase cell-cell adhesion and flocculation when over-expressed. Studies of zfs1 and cbf12 double mutants demonstrated that the increased flocculation seen in zfs1 mutants is due, at least in part, to a direct effect on the turnover of cbf12 mRNA, leading in turn to changes in the levels of its transcriptionally regulated genes. These data suggest that Zfs1p can both directly and indirectly regulate the levels of transcripts involved in cell-cell adhesion in this species.
Project description:We investigated the effects of AICAr and SAICAr accumulation on the global transcriptome of yeast, comparing the ade3 mutant to the WT strain.