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Study of the adaptation of S. cerevisiae strains to winemaking conditions by means of directed evolution and competition experiments with bar-coded YKO strains Long-term objective Comprehensive identification of genes involved in the adaptation of Saccharomyces cerevisiae to the winemaking environment. Some limitations of transcriptomic approaches •Genes 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). Some alternative genome-wide approaches (wine) Proteomics •Complementary information •Usually no direct correlation with transcription data Comparative genomics by hybridization (aCGH or low coverage sequencing) •Strains showing different fermentation phenotypes •Wine vs. non-wine strains Whole genome sequencing •Horizontal transfer •¿New mobile elements? HaploInsuficiency Profiling/HOmozygous Profiling HIP/HOP Construction of YKO S. cerevisiae collections X Heterozygous strains Homozygous strains x6000 x4500 HaploInsuficiency Profiling/HOmozygous Profiling HIP/HOP Some considerations about HIP/HOP analysis of wine fermentation •Environmental conditions experiment dramatic changes •Low number of generations in similar conditions Alternative approach Continuous culture Simulation of wine fermentation in continuous culture Continuo EC1118 CENIT 24% 25,00 600,00 CTR3 VCT3 500,00 20,00 300,00 10,00 200,00 5,00 100,00 0,00 0:00:00 0,00 4:48:00 9:36:00 14:24:00 19:12:00 24:00:00 Inoculation Time 28:48:00 33:36:00 38:24:00 43:12:00 VCT [mM] CTR [mM/h] 400,00 15,00 Simulation of first step of wine fermentation in continuous culture •10 generation times for homozygous competition (SM) •20 generation times for heterozygous competition (SM) •Controls for 10 and 20 generation times in YPD 3 biological replicates for each of the above HIP-HOP results for the first step of alcoholic fermentation At 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) Individual phenotypic characterization of selected strains. Relevant functions from HIP analysis •Vacuolar functions, including autophagy •Different functions in the “DNA-to-protein” pathway omRNA processing and stability oProtein synthesis oSecretion (ER functions) Relevant functions from HOP analysis •Adenine and lysine biosynthesis •Inositol biosynthesis •Biosynthesis of phospholipids Apparent limitations of the HIP/HOP approach •Limited to loss-of-function phenotypes •Difficulty to estimate wine-related phenotypes in a BY4743 background Complementary approaches •Directed evolution of laboratory strains •QTL mapping by high throughput methods Directed evolution of laboratory strains •Haploid laboratory strain (BY4741) •Continuous culture in conditions emulating the first step of alcoholic fermentation •Working volume 40-50 ml •150-250 generations (three biological replicates) •Verification of the “evolved” phenotype •Whole genome sequence analysis of the evolved strains Phenotype of evolved strains. Batch culture Strain Growth rate BY 4741 0,1683 AV 8 0,2254 BV 19 0,2569 E18 0,2760 AV 16 0,2779 1,4 1,2 w t-1 w t-2 w t-3 1 AV16.1 AV 16.2 AV 16.3 0,8 AV 18.1 AV 18.2 AV 18.3 0,6 AV 21.1 AV 21.2 AV 21.3 0,4 AV 22.1 AV 22.2 AV 23.3 0,2 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Adaptation to first steps does not involve improved overall fermentation performance, rather the opposite Phenotype of evolved strains. Continuous culture OD 600 D=0,20 h-1 D=0,25 h-1 BY4741 0.69 Av16 1.28 BY4741 0.28 Av16 0.74 Summary of mutations already identified •SNPs in non-coding regions Strain SNPs 50% •Nonsense mutations E18 4 4 •Missense mutations BV19 2 1 •50% mutations AV8 6 2 AV16* 1 0 Mutations requiring further confirmation •Deletions/insertions •Changes in copy-number •Chromosomal rearrangements RSP5: E3 Ubiquitin ligase Ubiquitin-proteasome pathway mutants Gene Strain Mutation RSP5 E18 Asn>Lys E3 Ubiquitin ligase BV19 Glu>Asp E3 Ubiquitin ligase AV16 Asn>Thr E3 Ubiquitin ligase CDC4 E18 Ser>Leu (50%) Part of a complex with ubiquitin ligase activity on a CDK inhibitor BRE5 E18 Glu>STOP Ubiquitin protease cofactor UBC6 BV19 Small deletion* Ubiquitin-conjugating enzyme BUL1 AV8 Asp>His Ubiquitin-binding component of the Rsp5p E3-ubiquitin ligase complex Pilar Morales Maite Novo Manuel Quirós Zoel Salvadó Martijn Wapenaar Ana Mangado www.icvv.es/winehiphop Coincidences with previous studies 28 overlapping HOP genes 19 overlapping HIP genes 3 overlapping HIP genes Previous reports of HIP/HOP analysis of wine fermentation Delneri et al. 2008 •Commercial grape must (100 g/L sugar) (among several other media) •Chemostat •Not supplemented with uridine. Aerobic. •Only HIP analysis •No unstressed contrast •Concluded all nutritional requirements were provided by must Previous reports of HIP/HOP analysis of wine fermentation Piggott et al. 2011 •Synthetic must (200 g/L sugar) •Single biological replicate each (HIP and HOP analyses) •Time-course •YPD amplification of samples •No unstressed contrast •Autophagy and ubiquitin-proteasome functions required •Proficient deleted strains also identified (ribosomal and peroxisomal functions) •FUR4 Some genes to watch From the HIP analysis SAM1 and SAM2; URE2; DUR1,2; MAL12, OCA6; CDC19; genes involved in Gap1p sorting; genes involved in chromatin remodeling and histone modification From the HOP analysis NPR2, 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 HIP SAM1 SAM2 URE2 DUR1,2 MAL12 OCA6 GTR1 GTR2 SWD3 SGF29 RPH1 NHP6B ITC1 GTR2 CAC2 RLF2 S-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) S-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) Nitrogen 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] prion Urea 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 degradation Maltase (alpha-D-glucosidase), inducible protein involved in maltose catabolism; encoded in the MAL1 complex locus; hydrolyzes the disaccharides maltose, turanose, maltotriose, and sucrose Cytoplasmic protein required for replication of Brome mosaic virus in S. cerevisiae, which is a model system for studying positivestrand RNA virus replication; null mutation confers sensitivity to tunicamycin and DTT Cytoplasmic 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 RagB Putative 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 RagD Essential 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 WDR5 Component 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 acetylation JmjC 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 pathway High-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 HMGB2 Subunit 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) complex Putative 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 RagD Subunit 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 transcription Largest 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 transcription HOP GPD1 GPD2 NPR2 NPR3 RTC1 CAR1 CAN1 UBR1 STB5 STB5 BUL2 ADH3 AQR1 NAD-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 Gpd2p NAD-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 mitochondria Subunit 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 proline Subunit 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 repair Subunit 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 motif Arginase, responsible for arginine degradation, expression responds to both induction by arginine and nitrogen catabolite repression; disruption enhances freeze tolerance Plasma membrane arginine permease, requires phosphatidyl ethanolamine (PE) for localization, exclusively associated with lipid rafts; mutation confers canavanine resistance E3 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) Transcription 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 vitro Transcription 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 vitro Component 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 BUL1 Mitochondrial alcohol dehydrogenase isozyme III; involved in the shuttling of mitochondrial NADH to the cytosol under anaerobic conditions and ethanol production Plasma membrane multidrug transporter of the major facilitator superfamily, confers resistance to short-chain monocarboxylic acids and quinidine; involved in the excretion of excess amino acids