Unknown,Transcriptomics,Genomics,Proteomics

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Growth competition experiment on the Yeast Homozygous Diploid Deletion Pool to investigate which ORFs conferred an advantage/disadvantage when growing on both standard and high concentration of oleic acid

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Clelia Peano

PROVIDER: E-GEOD-21620 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Biofilm formation is an important virulence trait of the pathogenic yeast Candida albicans. Large-scale genetics strategies aimed at identifying genes involved in biofilm development are hampered by lack of a complete sexual cycle in this diploid yeast in addition to the tedious generation of homozygous gene-deletion mutants. Gene overexpression is an attractive alternative strategy for large-scale phenotypic analyses and gene-function studies. We combined gene overexpression, strain barcoding and microarray profiling to screen a library of 531 C. albicans conditional overexpression strains (~10% of the genome) for genes affecting i) planktonic cell fitness and ii) biofilm development in mixed-population experiments. We found 5 genes whose overexpression affects planktonic strain fitness, including RAD53, RAD51, PIN4, orf19.2781, all encoding (or predicted to encode) regulators of DNA-damage response or cell-cycle progression and SFL2, involved in filamentous growth. We identified 16 and 4 genes (out of 531) whose overexpression respectively increases and decreases strain occupancy within the multi-strain biofilm. Strikingly, strains with increased abundance in the multi-strain biofilm were significantly enriched for genes encoding cell wall proteins (10 genes), including the glycosylphosphatidylinositol (GPI)-anchored proteins Pga15, Pga19, Pga22, Pga59, Pga32 and Pga41. Data validation experiments using either individually- or competitively-grown overexpression and/or the respective knock-out strains revealed that the identified genes differently contribute to biofilm formation during specific stages of biofilm development, including increased or decreased substrate adherence (IHD1, PGA32, PGA37, PGA15, PGA22, PGA59 and PGA19) or biofilm biomass growth and cohesion (PGA15, PGA59 and PGA22). In line with the hypothesis that cell wall genes contribute to biofilm development, we show that strains overexpressing PGA15 or PGA22 display altered cell wall structures. Our study reveals that cell wall proteins are important actors during C. albicans biofilm formation and illustrates the powerful use of signature tagging in conjunction with gene overexpression for the identification of genes involved in processes pertaining to C. albicans virulence. A total of 8 samples are included in this study. For fitness profiling of planktonic cells, 2 biological replicates were analyzed (samples Pool_Fitness_planktonic_rep1 and Pool_Fitness_planktonic_rep2). For quantification of strain abundance during biofilm formation, 6 biological replicates were analyzed (Pool_Biofilm_rep1, Pool_Biofilm_rep2, Pool_Biofilm_rep3, Pool_Biofilm_rep4, Pool_Biofilm_rep5, Pool_Biofilm_rep6). Genomic DNA was purified and used as template to PCR-amplify barcodes, which were then used as probes for microarray hybridization. This experiment was done twice independently. For quantification of strain abundance during multi-strain biofilm formation, strain pools were grown in minimal GHAUM medium with or without doxycycline, each inoculum was then diluted to an OD600 of 1 in fresh minimal GHAUM medium with or without doxycycline and left at room temperature for 30 min, to allow further overexpression. Plastic slides (ThermanoxM-bM-^DM-"; Nunc) were immersed in the inoculum for 30 min at room temperature to allow adhesion of cells to the plastic substrate. The plastic slides were then transferred to the glass vessel of a 40-mL incubation chamber. This vessel has two glass tubes inserted to drive the entry of medium and air, while used medium is evacuated through a third tube. The flow of medium is controlled by a recirculation pump (IsmatecM-BM-.) set at 0.6 mL.min-1 and pushed by pressured air supplied at 105 Pa, conditions minimizing planktonic phase growth and promoting biofilm formation. The chambers with the plastic substrate were incubated at 37C and biofilms were grown for 40h followed by genomic DNA extraction, barcode amplification and differential labeling (dox-treated samples with Cy5, untreated samples with Cy3) and hybridization to barcode microarrays.

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Growth competition experiment on the Yeast Homozygous Diploid Deletion Pool to investigate which ORFs conferred an advantage/disadvantage when growing on both standard and high concentration of oleic acid

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