Investigation Title Transcription profiling of S. cerevisiae after prolonged selection in aerobic, glucose-limited chemostat cultures Comment[Submitted Name] Prolonged selection in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae Experimental Design unknown_experiment_design_type transcription profiling by array Experimental Design Term Source REF EFO Comment[AEMIAMESCORE] 3 Comment[ArrayExpressReleaseDate] 2008-06-16 Comment[SecondaryAccession] GSE8898 Comment[ArrayExpressAccession] E-GEOD-8898 Comment[MAGETAB TimeStamp_Version] 2010-08-09 11:34:11 Last Changed Rev: 13058 Experimental Factor Name Experimental Factor Type Experimental Factor Term Source REF Person Last Name Daran Person First Name Jean-Marc Person Mid Initials Person Email j.m.daran@tnw.tudelft.nl Person Phone Person Fax Person Address Kluyver centre for genomics of industrial organisms,Department of Biotechnology,TU Delft,Julianalaan 67,Delft,2628BC,The Netherlands Person Affiliation TU Delft Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2008-06-16 PubMed ID 15870473 Publication DOI 15870473 Publication Author List Mickel L A Jansen, Jasper A Diderich, Mlawule Mashego, Adham Hassane, Johannes H de Winde, Pascale Daran-Lapujade, Jack T Pronk Publication Title Prolonged selection in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae causes a partial loss of glycolytic capacity. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description Prolonged cultivation of Saccharomyces cerevisiae in aerobic, glucose-limited chemostat cultures (dilution rate, 0·10 h–1) resulted in a progressive decrease of the residual glucose concentration (from 20 to 8 mg l–1 after 200 generations). This increase in the affinity for glucose was accompanied by a fivefold decrease of fermentative capacity, and changes in cellular morphology. These phenotypic changes were retained when single-cell isolates from prolonged cultures were used to inoculate fresh chemostat cultures, indicating that genetic changes were involved. Kinetic analysis of glucose transport in an ‘evolved’ strain revealed a decreased Km, while Vmax was slightly increased relative to the parental strain. Apparently, fermentative capacity in the evolved strain was not controlled by glucose uptake. Instead, enzyme assays in cell extracts of the evolved strain revealed strongly decreased capacities of enzymes in the lower part of glycolysis. This decrease was corroborated by genome-wide transcriptome analysis using DNA microarrays. In aerobic batch cultures on 20 g glucose l–1, the specific growth rate of the evolved strain was lower than that of the parental strain (0·28 and 0·37 h–1, respectively). Instead of the characteristic instantaneous production of ethanol that is observed when aerobic, glucose-limited cultures of wild-type S. cerevisiae are exposed to excess glucose, the evolved strain exhibited a delay of 90 min before aerobic ethanol formation set in. This study demonstrates that the effects of selection in glucose-limited chemostat cultures extend beyond glucose-transport kinetics. Although extensive physiological analysis offered insight into the underlying cellular processes, the evolutionary ‘driving force’ for several of the observed changes remains to be elucidated Experiment Overall Design: A crucial feature of bakers' yeast is its capacity to produce CO2, referred to as fermentative capacity (van Hoek et al., 1998). After prolonged glucose-limited cultivation of S. cerevisiae, in addition to an increased affinity for glucose, we observed a dramatic decrease in fermentative capacity. Consequently, the aim of the present study was to perform an integral analysis of the long-term adaptation of S. cerevisiae during prolonged glucose-limited, aerobic cultivation in chemostat cultures, with special emphasis on the regulation of glucose transport and glycolytic capacity. To this end, we applied an integrated approach that combined transcriptome analysis, measurement of fermentative capacity and activities of glucose transport and glycolytic enzymes, and characterization of cellular morphology. Protocol Name P-G8898-6 P-G8898-2 P-G8898-14 P-G8898-10 P-G8898-5 P-G8898-1 P-G8898-13 P-G8898-9 P-G8898-8 P-G8898-4 P-G8898-16 P-G8898-12 P-G8898-7 P-G8898-3 P-G8898-15 P-G8898-11 Affymetrix:Protocol:Hybridization-EukGE-WS2v4 Affymetrix:Protocol:Hybridization-Mini_Euk1 Affymetrix:Protocol:Hybridization-EukGE-WS2 P-AFFY-6 Protocol Type specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action grow grow grow grow nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction labeling labeling labeling labeling hybridization hybridization feature_extraction Protocol Description Liquid N2 Quenching liquid N2 Quenching Liquid nitrogen quenching Liquid N2 quenching Chemostat Cultivation Steady-state chemostat cultures were grown in Applikon laboratory fermentors of 1-liter working volume as described in detail elsewhere [van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953-28959]. In brief, the cultures were fed with a defined mineral medium containing glucose as the growth-limiting nutrient [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616-630]. The dilution rate (which equals the specific growth rate) in the steady-state cultures was 0.10 h_1, the temperature was 30 °C, and the culture pH was 5.0. Aerobic conditions were maintained by sparging the cultures with air (0.5 liter_min_1). The dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe, remained above 80% of air saturation. Chemostat Cultivationâ€" Steady-state chemostat cultures were grown in Applikon laboratory fermentors of 1-liter working volume as described in detail elsewhere [van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953â€"28959]. In brief, the cultures were fed with a defined mineral medium containing glucose as the growth-limiting nutrient [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616â€"630]. The dilution rate (which equals the specific growth rate) in the steady-state cultures was 0.10 h_1, the temperature was 30 °C, and the culture pH was 5.0. Aerobic conditions were maintained by sparging the cultures with air (0.5 liter_min_1). The dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe, remained above 80% of air saturation. Media. Synthetic medium containing mineral salts and vitamins was prepared and sterilized as described by Verduyn et al. (1992). For chemostat cultivation, the glucose concentration in reservoir media was 7·5 g lâ€"1 (0·25 mol C lâ€"1). This medium composition has previously been demonstrated to sustain glucose-limited cultivation of S. cerevisiae CEN.PK113-7D (Lange & Heijnen, 2001; Verduyn et al., 1992). For batch cultivation, the initial glucose concentration was 20 g lâ€"1. Chemostat cultivation. Aerobic chemostat cultivation was performed at a dilution rate of 0·10 hâ€"1 at 30 °C in 1·5 l laboratory fermenters (Applikon) at a stirrer speed of 800 r.p.m. The working volume of the cultures was kept at 1·0 l by a peristaltic effluent pump coupled to an electrical level sensor. This set-up ensured that under all growth conditions, biomass concentrations in samples taken directly from the culture differed by <1 % from those in samples taken from the effluent line . The exact working volume was measured after each experiment. The pH was kept at 5·0±0·1 by an ADI 1030 biocontroller (Applikon), via the automatic addition of 2 mol KOH lâ€"1. The fermenter was flushed with air at a flow rate of 0·5 l minâ€"1 using a Brooks 5876 mass-flow controller. The dissolved-oxygen concentration was continuously monitored with an oxygen electrode (model 34 100 3002; Ingold), and it remained above 60 % of air saturation. Chemostat cultures were routinely checked for potential bacterial and fungal infection by phase-contrast microscopy. Verduyn, C., Postma, E., Scheffers, W. A. & van Dijken, J. P. (1992). Effect of benzoic acid on metabolic fluxes in yeasts: a continuous study on regulation of respiration and alcoholic fermentation. Yeast 8, 501â€"517 Chemostat Cultivation Steady-state chemostat cultures were grown in Applikon laboratory fermentors of 1-liter working volume as described in detail elsewhere [van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953-28959]. In brief, the cultures were fed with a defined mineral medium containing glucose as the growth-limiting nutrient [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616-630]. The dilution rate (which equals the specific growth rate) in the steady-state cultures was 0.10 h_1, the temperature was 30°C, and the culture pH was 5.0. Aerobic conditions were maintained by sparging the cultures with air (0.5 liter_min_1). The dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe, remained above 80% of air saturation. Sampling and RNA Isolation Samples from the chemostat cultures were taken as rapidly as possible to limit any potential changes in transcript profiles during the procedure. 40-60 ml of culture broth was sampled directly from the chemostat into a beaker containing 200 ml of liquid nitrogen. With vigorous stirring, the sample froze instantly. The frozen sample was then broken into small fragments and transferred to a 50-ml centrifuge tube. The sample was then thawed at room temperature, ensuring that it remained as close to zero as possible. Cells were pelleted (5000 rpm at 0 °C for 4 min), resuspended in 2 ml of ice-cold AE buffer (50 mM sodium acetate, 10 mM EDTA, pH 5.0) and aliquoted into 5 Eppendorf tubes. This corresponded to _20 mg of dry weight per tube. For each array, total RNA was extracted from a single tube using the hot-phenol method (32) or the FastRNA kit, Red (BIO 101, Inc., Vista, CA). Sampling and RNA Isolationâ€" Samples from the chemostat cultures were taken as rapidly as possible to limit any potential changes in transcript profiles during the procedure. 40â€"60 ml of culture broth was sampled directly from the chemostat into a beaker containing 200 ml of liquid nitrogen. With vigorous stirring, the sample froze instantly. The frozen sample was then broken into small fragments and transferred to a 50-ml centrifuge tube. The sample was then thawed at room temperature, ensuring that it remained as close to zero as possible. Cells were pelleted (5000 rpm at 0 °C for 4 min), resuspended in 2 ml of ice-cold AE buffer (50 mM sodium acetate, 10 mM EDTA, pH 5.0) and aliquoted into 5 Eppendorf tubes. This corresponded to _20 mg of dry weight per tube. For each array, total RNA was extracted from a single tube using the hot-phenol method (32) or the FastRNA kit, Red (BIO 101, Inc., Vista, CA). Cells were rapidly (within 3 s) transferred from the chemostat culture into liquid nitrogen to immediately quench the metabolism. The frozen cell suspension (about 40 g cell broth) was thawed gently on ice. After complete thawing, the cell suspension was centrifuged at 0 °C, 5000 g, for 5 min. Total RNA extraction from the pellets was performed using the hot-phenol method Probe Preparation and Hybridization to Arrays—mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix users’ manual (1). Briefly, poly(A)_ RNA was enriched from total RNA in a single round using the Qiagen Oligotex kit. Double-stranded cDNA synthesis was carried out incorporating the T7 RNA-polymerase promoter in the first round. This cDNA was then used as template for in vitro transcription (ENZO BioArray High Yield IVT kit), which amplifies the RNA pool and incorporates biotinylated ribonucleotides required for the staining procedures after hybridization. 15 mg of fragmented, biotinylated cRNA was hybridized to Affymetrix yeast S98 arrays at 45 °C for 16 h as described in the Affymetrix users’ manual (1). Washing and staining of arrays were performed using the GeneChip Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner. (1) Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual, Santa Clara, CA Probe Preparation and Hybridization to Arrays—mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix users manual (1). Briefly, poly(A)_ RNA was enriched from total RNA in a single round using the Qiagen Oligotex kit. Double-stranded cDNA synthesis was carried out incorporating the T7 RNA-polymerase promoter in the first round. This cDNA was then used as template for in vitro transcription (ENZO BioArray High Yield IVT kit), which amplifies the RNA pool and incorporates biotinylated ribonucleotides required for the staining procedures after hybridization. 15 mg of fragmented, biotinylated cRNA was hybridized to Affymetrix yeast S98 arrays at 45 °C for 16 h as described in the Affymetrix users™ manual (1). Washing and staining of arrays were performed using the GeneChip Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner. (1) Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual, Santa Clara, CA Probe Preparation and Hybridization to Arraysâ€"mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix users’ manual (1). Briefly, poly(A)_ RNA was enriched from total RNA in a single round using the Qiagen Oligotex kit. Double-stranded cDNA synthesis was carried out incorporating the T7 RNA-polymerase promoter in the first round. This cDNA was then used as template for in vitro transcription (ENZO BioArray High Yield IVT kit), which amplifies the RNA pool and incorporates biotinylated ribonucleotides required for the staining procedures after hybridization. 15 mg of fragmented, biotinylated cRNA was hybridized to Affymetrix yeast S98 arrays at 45 °C for 16 h as described in the Affymetrix users’ manual (1). Washing and staining of arrays were performed using the GeneChip Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner. (1) Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual, Santa Clara, CA The results for each growth condition were derived from three independently cultured replicates. Sampling of cells from chemostats, probe preparation, and hybridization to Affymetrix GeneChip microarrays, as well as data acquisition and analysis, were performed as previously described (Daran-Lapujade et al., 2004; Piper et al., 2002). Piper, M. D., Daran-Lapujade, P., Bro, C., Regenberg, B., Knudsen, S., Nielsen, J. & Pronk, J. T. (2002). Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 277, 37001â€"37008 Daran-Lapujade, P., Jansen, M. L., Daran, J. M., Van Gulik, W., de Winde, J. H. & Pronk, J. T. (2004). Role of transcriptional regulation in controlling fluxes in central carbon metabolism of Saccharomyces cerevisiae: a chemostat culture study. J Biol Chem 279, 9125â€"9138 Probe Preparation and Hybridization to Arrays—mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix users’ manual (1). Briefly, poly(A)_ RNA was enriched from total RNA in a single round using the Qiagen Oligotex kit. Double-stranded cDNA synthesis was carried out incorporating the T7 RNA-polymerase promoter in the first round. This cDNA was then used as template for in vitro transcription (ENZO BioArray High Yield IVT kit), which amplifies the RNA pool and incorporates biotinylated ribonucleotides required for the staining procedures after hybridization. 15 mg of fragmented, biotinylated cRNA was hybridized to Affymetrix yeast S98 arrays at 45 °C for 16 h as described in the Affymetrix users’ manual (1). Washing and staining of arrays were performed using the GeneChip Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner. (1) Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual, Santa Clara, CA Title: Fluidics Station Protocol. Description: Title: Affymetrix CEL analysis. Description: Protocol Parameters Protocol Hardware Protocol Software MicroArraySuite 5.0 MicroArraySuite 5.0 Protocol Contact Protocol Term Source REF SDRF File E-GEOD-8898.sdrf.txt Term Source Name The MGED Ontology ArrayExpress EFO The MGED Ontology Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version