Genome-wide analysis of longevity in nutrient-deprived Saccharomyces cerevisiae reveals importance of recycling in maintaining cell viability.

Although typically cosseted in the laboratory with constant temperatures and plentiful nutrients, microbes are frequently exposed to much more stressful conditions in their natural environments where survival and competitive fitness depend upon both growth rate when conditions are favourable and on persistence in a viable and recoverable state when they are not. In order to determine the role of genetic heterogeneity in environmental fitness we present a novel approach that combines the power of fluorescence-activated cell sorting with barcode microarray analysis and apply this to determining the importance of every gene in the Saccharomyces cerevisiae genome in a high-throughput, genome-wide fitness screen. We have grown > 6000 heterozygous mutants together and exposed them to a starvation stress before using fluorescence-activated cell sorting to identify and isolate those individual cells that have not survived the stress applied. Barcode array analysis of the sorted and total populations reveals the importance of cellular recycling mechanisms (autophagy, pexophagy and ribosome breakdown) in maintaining cell viability during starvation and provides compelling evidence for an important role for fatty acid degradation in maintaining viability. In addition, we have developed a semi-batch fermentor system that is a more realistic model of environmental fitness than either batch or chemostat culture. Barcode array analysis revealed that arginine biosynthesis was important for fitness in semi-batch culture and modelling of this regime showed that rapid emergence from lag phase led to greatly increased fitness. One hundred and twenty-five strains with deletions in unclassified proteins were identified as being over-represented in the sorted fraction, while 27 unclassified proteins caused a haploinsufficient phenotype in semi-batch culture. These methods thus provide a screen to identifying other genes and pathways that have a role in maintaining cell viability.

Phenomic and transcriptomic analyses reveal that autophagy plays a major role in desiccation tolerance in Saccharomyces cerevisiae.

Saccharomyces cerevisiae can survive extreme desiccation, but the molecular mechanisms are poorly understood. To define genes involved in desiccation tolerance, two complementary genome-wide approaches, phenomics and transcriptomics, have been used, together with a targeted analysis of gene deletion mutants tested individually for their ability to survive drying. Genome-wide phenotypic analyses carried out on a pooled library of single-gene deletion mutants subjected to three cycles of desiccation and re-growth to post-diauxic phase identified about 650 genes that contributed to strain survival in the drying process. Air-drying desiccation-tolerant post-diauxic phase cells significantly altered transcription in 12% of the yeast genome, activating expression of over 450 genes and down-regulating 330. Autophagy processes were significantly over-represented in both the phenomics study and the genes up-regulated on drying, indicating the importance of the clearance of protein aggregates/damaged organelles and the recycling of nutrients for the survival of desiccation in yeast. Functional carbon source sensing networks governed by the PKA, Tor and Snf1 protein kinase complexes were important for the survival of desiccation, as indicated by phenomics, transcriptomics, and individual analyses of mutant strains. Changes in nitrogen metabolism were evident during the drying process and parts of the environmental stress response were activated, repressing ribosome production and inducing genes for coping with oxidative and osmotic stress.

Identification and characterization of high-flux-control genes of yeast through competition analyses in continuous cultures.

Using competition experiments in continuous cultures grown in different nutrient environments (glucose limited, ammonium limited, phosphate limited and white grape juice), we identified genes that show haploinsufficiency phenotypes (reduced growth rate when hemizygous) or haploproficiency phenotypes (increased growth rate when hemizygous). Haploproficient genes (815, 1,194, 733 and 654 in glucose-limited, ammonium-limited, phosphate-limited and white grape juice environments, respectively) frequently show that phenotype in a specific environmental context. For instance, genes encoding components of the ubiquitination pathway or the proteasome show haploproficiency in nitrogen-limited conditions where protein conservation may be beneficial. Haploinsufficiency is more likely to be observed in all environments, as is the case with genes determining polar growth of the cell. Haploproficient genes seem randomly distributed in the genome, whereas haploinsufficient genes (685, 765, 1,277 and 217 in glucose-limited, ammonium-limited, phosphate-limited and white grape juice environments, respectively) are over-represented on chromosome III. This chromosome determines a yeast's mating type, and the concentration of haploinsufficient genes there may be a mechanism to prevent its loss.

Application of the comprehensive set of heterozygous yeast deletion mutants to elucidate the molecular basis of cellular chromium toxicity.

BACKGROUND: The serious biological consequences of metal toxicity are well documented, but the key modes of action of most metals are unknown. To help unravel molecular mechanisms underlying the action of chromium, a metal of major toxicological importance, we grew over 6,000 heterozygous yeast mutants in competition in the presence of chromium. Microarray-based screens of these heterozygotes are truly genome-wide as they include both essential and non-essential genes. RESULTS: The screening data indicated that proteasomal (protein degradation) activity is crucial for cellular chromium (Cr) resistance. Further investigations showed that Cr causes the accumulation of insoluble and toxic protein aggregates, which predominantly arise from proteins synthesised during Cr exposure. A protein-synthesis defect provoked by Cr was identified as mRNA mistranslation, which was oxygen-dependent. Moreover, Cr exhibited synergistic toxicity with a ribosome-targeting drug (paromomycin) that is known to act via mistranslation, while manipulation of translational accuracy modulated Cr toxicity. CONCLUSION: The datasets from the heterozygote screen represent an important public resource that may be exploited to discover the toxic mechanisms of chromium. That potential was validated here with the demonstration that mRNA mistranslation is a primary cause of cellular Cr toxicity.