Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide study of the adaptation of Saccharomyces cerevisiae to the proliferative stages of wine fermentation

ABSTRACT: This work was designed to identify yeast cellular functions specifically affected by the stress factors predominating during the first stages of wine fermentation and genes required for optimal growth under these conditions. The main experimental method used was quantitative fitness analysis by means of competition experiments of whole genome barcoded yeast knock-out collections in continuous culture. This methodology allowed the identification of haploinsufficient genes and homozygous deletions resulting in growth impairment in synthetic must. However, genes identified as haploproficient or homozygous deletions resulting in fitness advantage were of little predictive power concerning optimal growth in this medium. The relevance of these functions for enological performance of yeast was assessed in batch cultures with single strains. Previous studies addressing yeast adaptation to winemaking conditions by quantitative fitness analysis, were not specifically focused on the proliferative stages, and their results were greatly dependent on the effects of gene deletions on yeast survival during stationary phase. Since biomass production has a great influence on the whole fermentation kinetics, focusing on the proliferative stages of the fermentation process has practical implications. In some instances our results highlight the importance of genes not previously linked to winemaking. In other cases our results are complementary to those reported in previous studies concerning, for example, the relevance of some genes involved in vacuolar, peroxisomal, or ribosomal functions. Transport processes and glucose signaling seem to be negatively affected by the stress factors encountered by yeast in synthetic must. Vacuolar activity is important for continued growth during the transition to stationary phase. Finally, reduced biogenesis of peroxisomes also seems to be advantageous. However, in contrast to what was described for later stages, reduced protein synthesis is not advantageous for the first stages of the fermentation, when most cell proliferation takes place. Competition experiments of the genome-wide collections of mutants were performed in triplicate using conditions that mimicked Phases I or II of a batch fermentation (equivalent to around 14 and 22 hours after inoculation of a batch fermentation, respectively), as well as on YPD (complete medium) for reference purposes. To this end, different feed formulations were used. One mL of either the homozygote or heterozygote pool stored at -80 ºC was added to 50 mL of YPD broth supplemented with 200 µg/mL G418 and incubated overnight at 28 °C and 150 rpm to serve as inoculum for the competition experiments. Samples were taken before the onset of the continuous culture as t=0 (preculture) references. Variations in pool composition were always estimated against the cognate preculture (most often a single preculture served as inoculum for several competitions). After 10 or 20 generations in continuous culture, samples of cells were used for the genomic DNA extraction. lification of the barcodes (uptags and downtags), hybridization, and scanning were performed according to the protocol described elsewhere [Pierce SE, Davis RW, Nislow C, Giaever G (2007) Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures. Nat Protocols 2: 2958-2974]. Slight modifications concerning the hybridization of Tag4 microarrays by using reagents and protocols from the GeneChip Hybridization, Wash and Stain Kit (Affymetrix) were made.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Ramon Gonzalez

PROVIDER: E-GEOD-46145 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:This work was designed to identify yeast cellular functions specifically affected by the stress factors predominating during the first stages of wine fermentation and genes required for optimal growth under these conditions. The main experimental method used was quantitative fitness analysis by means of competition experiments of whole genome barcoded yeast knock-out collections in continuous culture. This methodology allowed the identification of haploinsufficient genes and homozygous deletions resulting in growth impairment in synthetic must. However, genes identified as haploproficient or homozygous deletions resulting in fitness advantage were of little predictive power concerning optimal growth in this medium. The relevance of these functions for enological performance of yeast was assessed in batch cultures with single strains. Previous studies addressing yeast adaptation to winemaking conditions by quantitative fitness analysis, were not specifically focused on the proliferative stages, and their results were greatly dependent on the effects of gene deletions on yeast survival during stationary phase. Since biomass production has a great influence on the whole fermentation kinetics, focusing on the proliferative stages of the fermentation process has practical implications. In some instances our results highlight the importance of genes not previously linked to winemaking. In other cases our results are complementary to those reported in previous studies concerning, for example, the relevance of some genes involved in vacuolar, peroxisomal, or ribosomal functions. Transport processes and glucose signaling seem to be negatively affected by the stress factors encountered by yeast in synthetic must. Vacuolar activity is important for continued growth during the transition to stationary phase. Finally, reduced biogenesis of peroxisomes also seems to be advantageous. However, in contrast to what was described for later stages, reduced protein synthesis is not advantageous for the first stages of the fermentation, when most cell proliferation takes place.

Project description:The combination of genetic analysis and genome-wide expression profiles make Saccharomyces cerevisiae the cradle of Systems Biology. The way S. cerevisiae uses carbon sources has long been a landmark for studies concerning cellular responses to environmental conditions. S. cerevisiae conserves the entire machinery for utilization of fatty acids, yet with a different compartmentalization compared to higher eukaryotes. We propose S. cerevisiae as a model to understand how perixosomal compartmentalization of oleate metabolism co-evolved with the transcriptional regulatory networks of FA metabolism. With this aim, we performed growth competition experiments in oleate on the pooled collection of all viable yeast null mutants. We integrated results of competitive fitness with transcriptional profiles in oleate, identifying two genes previously not directly associated to FA metabolism: OAR1 and FET3. For the first time we show the importance of iron metabolism not only in maintaining functional mitochondria, but also in influencing peroxisome wellness. An aliquot of the Yeast Homozygous Diploid Deletion Pool (95401.H1Pool, Invitrogen), containing equal amounts of each barcoded viable knock-out mutant from the Saccharomyces Genome Deletion project, was grown in the presence of G418 in YPD medium for 24h, then aliquots of 5x10^7 cells were harvested and stored at -80M-BM-0C. From the same pooled culture, equal amounts of cells were washed and transferred to YP medium added with oleic acid 0.1% and 5%. We collected samples of cells 24h and 72h after carbon source metabolic shift, and genomic DNA was extracted. Each experiment was performed in biological duplicate. The upTAG and downTAG regions were amplified separately in PCR reactions, one for each experimental condition, using specific primers. Amplified upTAG and downTAG sequences were combined in equal amounts and used to probe high-density oligonucleotide arrays (Affymetrix GeneChip_DNA_Tag3). Data analysis was performed as previously described (Pierce et al., 2007). In order to adapt this data analysis protocol to our experimental design, and to the use of GeneChip_DNA_Tag3 arrays, some changes to the procedure have been improved.

Project description:In many animals living in groups the reproductivestatus of individuals is determined by their social status. In specieswith social hierarchies, the death of dominant individuals typicallyupheaves the social hierarchy and provides an opportunity for subordinateindividuals to improve their social status. Such a phenomenon occursin the monogyne form of the fire ant\emph{, Solenopsis invicta, }wherecolonies typically contain a single wingless reproductive queen, thousandsof workers and hundreds of winged non-reproductive virgin queens.Upon the death of the mother queen, many virgin queens shed theirwings and initiate reproductive development instead of departing ona mating flight. Workers progressively execute almost all of themover the following weeks. The workers base their collective decisionon pheromonal cues associated with the onset of reproductive developmentof the virgin queens which occurs after orphaning. To examine the factors that determine which virgin queens are executed and which survive, we set up artificial competitions between queens from different colonies. Using microarrays, we found that queens from winning colonies showed higher mitochondrial as well as organ development activities 24 hours after orphaning than did queens from colonies that lost the competitions. Furthermore, queens from colonies where queens shed their wings faster after orphaning were more likely to survive competitions. Finally, higher wing shedding speed is linked to higher mitochondrial activity. Eight competitions were initially conducted between queens form pairs of colonies. Six competitions clearly identified one winning colony and one losing colony. We thus have microarray data for six competitions (ie biological replicates), with one winning and one losing colony within each competition. Thus 12 colonies total. For each colony, RNA extracted from five queens was pooled, and hybridized against an unrelated common reference RNA that had been made from a pool of many S. invicta individuals of all castes and developmental stages. Lab work was conducted in blocks (by competition) with randomized order within competition to avoid introducing bias. No technical replication was performed.

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Genome-wide study of the adaptation of Saccharomyces cerevisiae to the proliferative stages of wine fermentation

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