Study of the adaptation of - PowerPoint Presentation

1. Study of the adaptation of S. cerevisiae strains to winemaking conditions by means of directed evolution and competition experiments with bar-coded YKO strains

2. Long-term objectiveComprehensive identification of genes involved in the adaptation of Saccharomyces cerevisiae to the winemaking environment.

3. Some limitations of transcriptomic approachesGenes relevant for many biological processes are not subject to transcriptional regulation in response to environmental conditions that influence these processes (Birrell et al. 2002; PNAS). Not all genes showing a transcriptional change in response to a given culture condition are required for fitness under these conditions (Tai et al., 2007; Microbiology SGM).

4. Some alternative genome-wide approaches (wine)ProteomicsComplementary informationUsually no direct correlation with transcription dataComparative genomics by hybridization(aCGH or low coverage sequencing)Strains showing different fermentation phenotypesWine vs. non-wine strainsWhole genome sequencingHorizontal transfer¿New mobile elements?

5. HaploInsuficiency Profiling/HOmozygous ProfilingHIP/HOPConstruction of YKO S. cerevisiae collectionsBY4743BY4742BY4741XHeterozygous strainsx6000Homozygous strainsx4500

6. HaploInsuficiency Profiling/HOmozygous ProfilingHIP/HOP

7. Some considerations about HIP/HOP analysis of wine fermentationEnvironmental conditions experiment dramatic changesLow number of generations in similar conditionsAlternative approachContinuous cultureO.D.glucoseethanolCO2·t-1

8. Simulation of wine fermentation in continuous culture

9. Simulation of first step of wine fermentation in continuous culture10 generation times for homozygous competition (SM)20 generation times for heterozygous competition (SM)Controls for 10 and 20 generation times in YPD3 biological replicates for each of the above

10. HIP-HOP results for the first step of alcoholic fermentationAt least 150 heterozygous deleted strains showed deficient growth in synthetic must after 20 generations(>2-fold reduced fitness as compared to fitness in YPD)At least 126 homozygous deleted strains showed deficient growth in synthetic must after 10 generations(>2-fold reduced fitness as compared to fitness in YPD)

11. Individual phenotypic characterization of selected strains.

12. Relevant functions from HIP analysisVacuolar functions, including autophagyDifferent functions in the “DNA-to-protein” pathwaymRNA processing and stabilityProtein synthesisSecretion (ER functions)Adenine and lysine biosynthesisInositol biosynthesisBiosynthesis of phospholipidsRelevant functions from HOP analysis

13. Apparent limitations of the HIP/HOP approachLimited to loss-of-function phenotypesDifficulty to estimate wine-related phenotypes in a BY4743 backgroundComplementary approachesDirected evolution of laboratory strainsQTL mapping by high throughput methods

14. Directed evolution of laboratory strainsHaploid laboratory strain (BY4741)Continuous culture in conditions emulating the first step of alcoholic fermentationWorking volume 40-50 ml150-250 generations (three biological replicates)Verification of the “evolved” phenotypeWhole genome sequence analysis of the evolved strains

15. Phenotype of evolved strains. Batch cultureStrainGrowth rateBY 47410,1683AV 80,2254BV 190,2569E180,2760AV 160,2779Adaptation to first steps does not involve improved overall fermentation performance, rather the opposite

16. Phenotype of evolved strains. Continuous cultureOD 600D=0,20 h-1BY47410.69Av161.28D=0,25 h-1BY47410.28Av160.74

17. Summary of mutations already identifiedSNPs in non-coding regionsNonsense mutationsMissense mutations50% mutations Mutations requiring further confirmationDeletions/insertionsChanges in copy-numberChromosomal rearrangementsStrainSNPs50%E1844BV1921AV862AV16*10

18. RSP5: E3 Ubiquitin ligaseBY4741E18BV19AV16

19. Ubiquitin-proteasome pathway mutantsGeneStrainMutationRSP5E18Asn>LysE3 Ubiquitin ligaseBV19Glu>AspE3 Ubiquitin ligaseAV16Asn>ThrE3 Ubiquitin ligaseCDC4E18Ser>Leu (50%)Part of a complex with ubiquitin ligase activity on a CDK inhibitorBRE5E18Glu>STOPUbiquitin protease cofactorUBC6BV19Small deletion*Ubiquitin-conjugating enzyme BUL1AV8Asp>HisUbiquitin-binding component of the Rsp5p E3-ubiquitin ligase complex

20. Pilar MoralesMaite NovoManuel QuirósZoel SalvadóMartijn WapenaarAna Mangadowww.icvv.es/winehiphop

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22. 19 overlapping HIP genes28 overlapping HOP genes3 overlapping HIP genesCoincidences with previous studies

23. Previous reports of HIP/HOP analysis of wine fermentationDelneri et al. 2008Commercial grape must (100 g/L sugar) (among several other media)ChemostatNot supplemented with uridine. Aerobic.Only HIP analysisNo unstressed contrastConcluded all nutritional requirements were provided by must

24. Previous reports of HIP/HOP analysis of wine fermentationPiggott et al. 2011Synthetic must (200 g/L sugar)Single biological replicate each (HIP and HOP analyses)Time-courseYPD amplification of samplesNo unstressed contrastAutophagy and ubiquitin-proteasome functions requiredProficient deleted strains also identified (ribosomal and peroxisomal functions)FUR4

25. Some genes to watchFrom the HIP analysisSAM1 and SAM2; URE2; DUR1,2; MAL12, OCA6; CDC19; genes involved in Gap1p sorting; genes involved in chromatin remodeling and histone modificationFrom the HOP analysisNPR2, NPR3 and RTC1; CAR1 and CAN1; GPD1 y GPD2; UBR1; STB5; BCK1; BUL2; ADH3; AQR1; genes coding for ribosomal proteins; genes involved in protein folding in the ER

26. SAM1S-adenosylmethionine synthetase, catalyzes transfer of the adenosyl group of ATP to the sulfur atom of methionine; one of two differentially regulated isozymes (Sam1p and Sam2p)SAM2S-adenosylmethionine synthetase, catalyzes transfer of the adenosyl group of ATP to the sulfur atom of methionine; one of two differentially regulated isozymes (Sam1p and Sam2p)URE2Nitrogen catabolite repression transcriptional regulator that acts by inhibition of GLN3 transcription in good nitrogen source; has glutathione peroxidase activity and can mutate to acquire GST activity; altered form creates [URE3] prionDUR1,2Urea amidolyase, contains both urea carboxylase and allophanate hydrolase activities, degrades urea to CO2 and NH3; expression sensitive to nitrogen catabolite repression and induced by allophanate, an intermediate in allantoin degradationMAL12Maltase (alpha-D-glucosidase), inducible protein involved in maltose catabolism; encoded in the MAL1 complex locus; hydrolyzes the disaccharides maltose, turanose, maltotriose, and sucroseOCA6Cytoplasmic protein required for replication of Brome mosaic virus in S. cerevisiae, which is a model system for studying positive-strand RNA virus replication; null mutation confers sensitivity to tunicamycin and DTTGTR1Cytoplasmic GTP binding protein and negative regulator of the Ran/Tc4 GTPase cycle; component of GSE complex, which is required for sorting of Gap1p; involved in phosphate transport and telomeric silencing; similar to human RagA and RagBGTR2Putative GTP binding protein that negatively regulates Ran/Tc4 GTPase cycle; activates transcription; subunit of EGO and GSE complexes; required for sorting of Gap1p; localizes to cytoplasm and to chromatin; homolog of human RagC and RagDSWD3Essential subunit of the COMPASS (Set1C) complex, which methylates histone H3 on lysine 4 and is required in transcriptional silencing near telomeres; WD40 beta propeller superfamily member and ortholog of mammalian WDR5SGF29Component of the HAT/Core module of the SAGA, SLIK, and ADA complexes; HAT/Core module also contains Gcn5p, Ngg1p, and Ada2p; binds methylated histone H3K4; involved in transcriptional regulation through SAGA recruitment to target promoters and H3 acetylationRPH1JmjC domain-containing histone demethylase; specifically demethylates H3K36 tri- and dimethyl modification states; associates with actively transcribed (RNA polymerase II) regions in vivo and specifically targets H3K36 in its trimethylation state as its substrate; transcriptional repressor of PHR1; Rph1p phosphorylation during DNA damage is under control of the MEC1-RAD53 pathwayNHP6BHigh-mobility group (HMG) protein that binds to and remodels nucleosomes; involved in recruiting FACT and other chromatin remodelling complexes to the chromosomes; functionally redundant with Nhp6Ap; homologous to mammalian HMGB1 and HMGB2ITC1Subunit of the ATP-dependent Isw2p-Itc1p chromatin remodeling complex, required for repression of a-specific genes, repression of early meiotic genes during mitotic growth, and repression of INO1; similar to mammalian Acf1p, the regulatory subunit of the mammalian ATP-utilizing chromatin assembly and modifying factor (ACF) complexGTR2Putative GTP binding protein that negatively regulates Ran/Tc4 GTPase cycle; activates transcription; subunit of EGO and GSE complexes; required for sorting of Gap1p; localizes to cytoplasm and to chromatin; homolog of human RagC and RagDCAC2Subunit of chromatin assembly factor I (CAF-1), with Rlf2p and Msi1p; chromatin assembly by CAF-1 is important for multiple processes including silencing at telomeres, mating type loci, and rDNA; maintenance of kinetochore structure; deactivation of the DNA damage checkpoint after DNA repair; and chromatin dynamics during transcriptionRLF2Largest subunit (p90) of the Chromatin Assembly Complex (CAF-1); chromatin assembly by CAF-1 is important for multiple processes including silencing at telomeres, mating type loci, and rDNA; maintenance of kinetochore structure; deactivation of the DNA damage checkpoint after DNA repair; and chromatin dynamics during transcriptionHIP

27. GPD1NAD-dependent glycerol-3-phosphate dehydrogenase, key enzyme of glycerol synthesis, essential for growth under osmotic stress; expression regulated by high-osmolarity glycerol response pathway; homolog of Gpd2pGPD2NAD-dependent glycerol 3-phosphate dehydrogenase, homolog of Gpd1p, expression is controlled by an oxygen-independent signaling pathway required to regulate metabolism under anoxic conditions; located in cytosol and mitochondriaNPR2Subunit of SEA (Seh1-associated), Npr2/3, and Iml1p complexes; Npr2/3 complex mediates downregulation of TORC1 activity upon amino acid limitation; SEA complex is a coatomer-related complex that associates dynamically with the vacuole; Iml1p complex (Iml1p-Npr2p-Npr3p) is required for non-nitrogen-starvation (NNS)-induced autophagy; Iml1p interacts primarily with phosphorylated Npr2p; homolog of human NPRL2; target of Grr1p; required for growth on urea and prolineNPR3Subunit of SEA (Seh1-associated), Npr2/3, and Iml1p complexes; Npr2/3 complex mediates downregulation of TORC1 activity upon amino acid limitation; SEA complex is a coatomer-related complex that associates dynamically with the vacuole; Iml1p complex (Iml1p-Npr2p-Npr3p) is required for non-nitrogen-starvation (NNS)-induced autophagy; required for Npr2p phosphorylation and Iml1p-Npr2p interaction; null mutant shows delayed meiotic DNA replication and double-strand break repairRTC1Subunit of the SEA (Seh1-associated) complex, a coatomer-related complex that associates dynamically with the vacuole; null mutation suppresses cdc13-1 temperature sensitivity; has N-terminal WD-40 repeats and a C-terminal RING motifCAR1Arginase, responsible for arginine degradation, expression responds to both induction by arginine and nitrogen catabolite repression; disruption enhances freeze toleranceCAN1Plasma membrane arginine permease, requires phosphatidyl ethanolamine (PE) for localization, exclusively associated with lipid rafts; mutation confers canavanine resistanceUBR1E3 ubiquitin ligase (N-recognin), forms heterodimer with Rad6p to ubiquitinate substrates in the N-end rule pathway; regulates peptide transport via Cup9p ubiquitination; mutation in human UBR1 causes Johansson-Blizzard Syndrome (JBS)STB5Transcription factor, involved in regulating multidrug resistance and oxidative stress response; forms a heterodimer with Pdr1p; contains a Zn(II)2Cys6 zinc finger domain that interacts with a pleiotropic drug resistance element in vitroSTB5Transcription factor, involved in regulating multidrug resistance and oxidative stress response; forms a heterodimer with Pdr1p; contains a Zn(II)2Cys6 zinc finger domain that interacts with a pleiotropic drug resistance element in vitroBUL2Component of the Rsp5p E3-ubiquitin ligase complex, involved in intracellular amino acid permease sorting, functions in heat shock element mediated gene expression, essential for growth in stress conditions, functional homolog of BUL1ADH3Mitochondrial alcohol dehydrogenase isozyme III; involved in the shuttling of mitochondrial NADH to the cytosol under anaerobic conditions and ethanol productionAQR1Plasma membrane multidrug transporter of the major facilitator superfamily, confers resistance to short-chain monocarboxylic acids and quinidine; involved in the excretion of excess amino acidsHOP