Project description:Dynamic transcriptional response of S. cerevisiae cells to copper impulse was investigated in both HO deletion strain used as the reference strain and the mutant strain lacking CCC2 gene which were grown in continuous cultures using a copper-deficient defined medium. Copper was introduced into the medium as an impulse so as to reach a copper sulfate concentration of 0.5 mM. Samples were collected within the first two hours following the copper addition (1st, 5th, 10th, 15th, 20th, 25th, 30th, 60th, 120th minutes), in addition to the steady-state sampling.
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:In frozen dough baking technology, baker’s yeast Saccharomyces cerevisiae encounter freeze-thaw injury. After thawing, dramatically decrease in cell viability and fermentation activity is caused by freeze-thaw injury. The freezing period is critical factor in freeze-thaw injury, thus we focused and investigated time-dependent gene expression profiles in recovery process from freeze injury. First, changes in gene expression profiles in S. cerevisiae in recovery process from freeze-thaw injury were analyzed using a DNA microarray. The results showed the genes which were involved in homeostasis of metal ions were time-dependent up-regulated 2-fold or more in a series. Then we examined whether these genes were related to tolerance in freeze-thaw injury by using deletion strain. The results showed that deletion of MAC1, CTR1, and PCA1 genes which involved in copper ion transport exhibited freeze-thaw sensitivity in compared with wild type. These genes are involved in copper ion uptake to a cell under a copper deficiency condition or in copper ion homeostasis, suggesting that it may be related between freeze-thaw injury and copper ion transport. To determine the effect of supplementation of copper ion on cells after freeze-thaw treatment, cell viability, intracellular superoxide dismutase (SOD) activity, and intracellular levels of reactive oxygen species (ROS) were examined by various copper ion condition medium. The results showed that intracellular SOD activity was increased and intracellular levels of ROS were decreased by supplementation of copper ion, but there was no significant difference in cell viability. These results of the present study may suggest that copper ion concentration in yeast cell after freeze-thaw treatment is important to recovery from freeze-thaw injury due to redox control of intracellular levels of ROS, but copper ion did not directly affect cell viability.
Project description:A 3 x 2 factorial design was used to elucidate the genome-wide transcriptional response to the deletion of yeast ortholog of Wilson and Menkes disease causing gene; CCC2, at changing copper levels. Homozygous deletion mutant of CCC2, which encodes Cu+2 transporting P-type ATPase required to export copper from the cytosol into the extracytosolic compartment, and the reference strain were cultivated in fully controlled fermenters in duplicates in glucose-rich defined medium containing three different levels of copper. The three different copper concentrations were selected such that; copper deficient condition, which was prepared by excluding the CuSO4.7H2O from the defined medium, low copper or adequate copper concentration, which is the standard amount of copper in defined medium (0.04 ?M) and high copper concentration (0.5 mM), which was able to restore respiration deficiency in ccc2?/ccc2? strain.
Project description:Copper tolerance and sulfite tolerance are two well-studied phenotypic traits of Saccharomyces cerevisiae. The genetic bases of these traits are derived from allelic expansion at the CUP1 locus and reciprocal translocation at the SSU1 locus, respectively. Previous work identified a negative association between sulfite and copper tolerance in S. cerevisiae wine yeasts. Here we probe the relationship between sulfite and copper tolerance and show that an increase in CUP1 copy number does not impart copper tolerance in all S. cerevisiae wine yeast. Bulk-segregant QTL analysis was used to identify variance at SSU1 as a causative factor in copper sensitivity, which was verified by reciprocal hemizygosity analysis in a strain carrying 20 copies of CUP1. Transcriptional and proteomic analysis demonstrated that SSU1 over-expression did not suppress CUP1 transcription or constrain protein production but suggested that SSU1 overexpression induced sulfur limitation during exposure to copper. Finally, an SSU1 over-expressing strain exhibited increased sensitivity to moderately elevated copper concentrations in sulfur-limited medium, demonstrating that SSU1 over-expression burdens the sulfate assimilation pathway. Over-expression of MET 3/14/16, genes upstream of H2S production in the sulfate assimilation pathway increased the production of SO2 and H2S but did not improve copper sensitivity in an SSU1 overexpressing background. We conclude that copper and sulfite tolerance are conditional traits in S. cerevisiae and provide evidence of the metabolic basis for their mutual exclusivity. These findings suggest an evolutionary basis for the extreme amplification of CUP1 observed in some yeasts.
Project description:Catfish farming remains number one among U.S. aquaculture production. Flavobacterium columnare, the causative agent of columnaris disease, produces substantial mortality during the production of freshwater farmed finfish species. F. columnare is ubiquitous in the aquatic environment, and outbreaks of disease are often triggered during the spring and summer months of the catfish production cycle. As food fish production continues to increase, the frequency of columnaris disease will only continue to rise within the aquaculture industry and thus new preventative measures will be required. Copper sulfate is the most common algaecide used in aquaculture. Copper sulfate has also been suggested as a therapeutant for columnaris disease, as we have previously discovered copper sulfate treatment effects the survivability and gill histology of channel catfish during an infection with F. columnare. Therefore to evaluate the functional effects of treatment with copper sulfate on columnaris disease, we performed RNA sequencing on gill samples from control and copper sulfate-treated catfish either immediately (more susceptible) or 24 hours after (more resistant) copper treatment followed by a challenge with F. columnare. We evaluated the differential gene expression patterns over the time-course of infection between groups with a focus on identifying functional differences in immune function.
Project description:We studied the influence of copper in physiological and morphological differentiation of Streptomyces coelicolor. We demonstrate differences in phenotype (germination, growth rate, antibiotic production) and genetic expression between a strain mutated at copper chaperone CopZ (SCO2730::Tn5062), the wild-type strain and a wild-type strain sporulated in a media with 80µM CuSO4. These differences are correlated with the cytosolic copper. Our results demonstrate a pleiotropic effect of copper modulating S. coelicolor development.