Project description:Iron is an essential cofactor for enzymes involved in numerous cellular processes. We analyzed the metabolomes and transcriptomes of yeast grown in iron-rich and iron-poor media to determine which biosynthetic processes are altered when iron availability falls.
Project description:RNA-seq was used to assess mRNA transcript abundance in wild type and fra2Δ S. cerevisiae (BY4741) cells treated with 2-(6-benzyl-2-pyridyl)quinazoline (BPQ) and CuSO4. BPQ potentiates copper toxicity and in yeast, in common with other organisms, a major cause of copper toxicity is damage of iron-sulphur clusters. Iron sensing within yeast relies on mitochondrial iron-sulphur cluster biosynthesis and therefore treatment with BPQ and copper can be used to mimic iron deficiency. Fra2 is known to be a key component of the iron sensing mechanism; however, this mechanism can operate, to an extent, independently of Fra2. BPQ (+CuSO4) treatment was used with the aim of probing the regulation of the iron regulon of S. cerevisiae and the role of Fra2 in the suppression of the low iron response. This study has uncovered nine new Cth2 target-transcripts, plus a new Aft1 target-gene and paralogous non-target. Fra2 dominates basal repression of the iron regulon in iron-replete cultures, however, Fra2-independent control of the iron regulon is also observed with CTH2 appearing to be atypically Fra2-dependent. Transcripts from untreated and CuSO4 treated cells were included as controls.
Project description:Iron is an essential cofactor for enzymes involved in numerous cellular processes. We analyzed the metabolomes and transcriptomes of yeast grown in iron-rich and iron-poor media to determine which biosynthetic processes are altered when iron availability falls. Saccharomyces cerevisiae DBY7286 strain was grown from very low density to mid-log phase (A600 = 0.5, approximately 18 hrs.) in defined-iron SD minimal medium containing only the supplements necessary to meet auxotrophic requirements. Defined-iron SD minimal media were prepared with yeast nitrogen base lacking iron and copper, supplemented with 1 µM copper sulfate, 25 mM MES pH 6.1, 1 mM Ferrozine (Fluka), and the indicated concentrations of ferrous ammonium sulfate 10 µM (low iron) or 300 µM (high iron). All cells were grown at 30°C with shaking and four independent cultures were prepared for each growth condition
Project description:RNA-seq was used to assess mRNA transcript abundance in wild type and fra2M-NM-^T S. cerevisiae (BY4741) cells treated with 2-(6-benzyl-2-pyridyl)quinazoline (BPQ) and CuSO4. BPQ potentiates copper toxicity and in yeast, in common with other organisms, a major cause of copper toxicity is damage of iron-sulphur clusters. Iron sensing within yeast relies on mitochondrial iron-sulphur cluster biosynthesis and therefore treatment with BPQ and copper can be used to mimic iron deficiency. Fra2 is known to be a key component of the iron sensing mechanism; however, this mechanism can operate, to an extent, independently of Fra2. BPQ (+CuSO4) treatment was used with the aim of probing the regulation of the iron regulon of S. cerevisiae and the role of Fra2 in the suppression of the low iron response. This study has uncovered nine new Cth2 target-transcripts, plus a new Aft1 target-gene and paralogous non-target. Fra2 dominates basal repression of the iron regulon in iron-replete cultures, however, Fra2-independent control of the iron regulon is also observed with CTH2 appearing to be atypically Fra2-dependent. Transcripts from untreated and CuSO4 treated cells were included as controls. Three independent biological replicates were analysed for each condition (BPQ and CuSO4 treated wild type and fra2M-NM-^T cells, CuSO4 treated wild type and fra2M-NM-^T cells and untreated wild type and fra2M-NM-^T cells)
Project description:Iron-resistant Saccharomyces cerevisiae mutant was obtained by evolutionary engineering selection strategy. The mutant obtained M-bM-^@M-^\M8FEM-bM-^@M-^] is much more resistant to iron stress than the reference strain which was used to select this mutant. Mutant can resist up to 35mM Iron* stress whereas the reference strain cannot. Whole-genome microarray analysis might be promising to identify the iron resistance mechanisms and stress response upon high levels of iron in the yeast cells. Iron-resistant mutant is also cross resistant to Cobalt, Chromium and Nickel but sensitive to Zinc. * refers to [NH4]2[Fe][SO4]2 and FeCl2. The reference Saccharomyces cerevisiae strain and the iron-resistant mutant were grown in minimal medium to an Optical Density (OD600) of 1.00 which correspond to the logarithmic growth phase of the yeast cells. Cultures were harvested and whole RNA isolation was carried out. The experiment was repeated three times.
Project description:Eupolauridine and liriodenine are plant-derived aporphinoid alkaloids that exhibit potent inhibitory activity against the opportunistic fungal pathogens Candida albicans and Cryptococcus neoformans. However, the molecular mechanism of this antifungal activity is unknown. In this study, we show that eupolauridine 9591 (E9591), a synthetic analog of eupolauridine, and liriodenine methiodide (LMT), a methiodide salt of liriodenine, mediate their antifungal activities by disrupting mitochondrial iron-sulfur (Fe-S) cluster synthesis. Several lines of evidence supported this conclusion. First, both E9591 and LMT elicited a transcriptional response indicative of iron imbalance, causing the induction of genes that are required for iron uptake and for the maintenance of cellular iron homeostasis. Second, a genome-wide fitness profile analysis showed that yeast mutants with deletions in iron homeostasis–related genes were hypersensitive to E9591 and LMT. Third, treatment of wild-type yeast cells with E9591 or LMT generated cellular defects that mimicked deficiencies in mitochondrial Fe-S cluster synthesis, including an increase in mitochondrial iron levels, a decrease in the activities of Fe-S cluster enzymes, a decrease in respiratory function, and an increase in oxidative stress. Collectively, our results demonstrate that E9591 and LMT perturb mitochondrial Fe-S cluster biosynthesis; thus, these two compounds target a cellular pathway that is distinct from the pathways commonly targeted by clinically used antifungal drugs. Therefore, the identification of this pathway as a target for antifungal compounds has potential applications in the development of new antifungal therapies.