The haploid prototrophic S. cerevisiae strain CEN.PK 113-7D (MATa) was obtained from Dr. P. Kötter, Frankfurt, Germany. Zinc-depleted cells were obtained by four serial transfers of yeast cells in shake flasks containing synthetic medium from which zinc was omitted, and subsequently mixed with glycerol (final concentration 20%), aliquoted and stored at -80 °C.
Biomaterial provider
L Hazelwood
Treatment protocol
Liquid nitrogen
Growth protocol
Chemostat cultivation Steady-state chemostat cultures of S. cerevisiae CEN.PK113-7D strain were performed in a 2-liter fermenter (Applikon) with a working volume of 1 liter (van den Berg et al 1996) . These cultures were fed with synthetic medium (see below) which limited growth by carbon, nitrogen or zinc with all other growth requirements in excess and at constant residual concentration (Boer et al 2003). The dilution rate was set at 0.1 h-1. The pH was measured on-line and kept constant at 5.0 by the automatic addition of 2 M KOH using an Applikon ADI 1030 Biocontroller. The stirrer speed was set at 800 rpm. Anaerobic conditions were maintained by sparging the medium reservoir (0.05 liter.min-1) and the fermenter with pure nitrogen gas (0.5 liter.min-1). Norprene tubing and butyl septa were used to minimize oxygen diffusion into the anaerobic cultures (Visser et al 1990). The off-gas was cooled by a condenser connected to a cryostat set at 2 °C. Oxygen and carbon dioxide were measured off-line with an NGA 2000 Rosemont gas analyzer. Cultures were assumed to be in steady-state when, after 4 to 5 volume changes, culture dry-weight, glucose concentration, carbon-dioxide production rate and oxygen consumption rate varied by less than 2 % during one volume change (Ferea et al, 1999; Jansen etal 2004). Steady-state samples were taken after 10 generations at the latest to avoid strain adaptation due to long-term cultivation. Each cultivation condition was performed in triplicate. Growth media The synthetic media composition was based on that described by Verduyn (1992). In all chemostats except for those limited by carbon, residual glucose concentration was targeted to 17 g.liter-1 in order to sustain glucose repression at the same level. For anaerobic cultivations, Tween 80 (420 mg.l-1) and ergosterol (10 mg.l-1) were supplemented to the media as described previously (Verduyn et al, 1990) to compensate for the inability of S. cerevisiae to synthesise fatty acids in the absence of oxygen. These media contained the following components: for zinc-limited cultivation, 5.0 g.liter-1 (NH4)2SO4, 3.0 g.liter-1 KH2PO4, 0.5 g.liter-1 MgSO4•7H2O and 58 g.liter-1 glucose. ZnSO4•7H2O was omitted from the anaerobic zinc-limited media. van den Berg, M. A., P. de Jong-Gubbels, C. J. Kortland, J. P. van Dijken, J. T. Pronk, and H. Y. Steensma. 1996. The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation. J. Biol. Chem. 271:28953-28959. van Dijken, J. P., J. Bauer, L. Brambilla, P. Duboc, J. M. Francois, C. Gancedo, M. L. Giuseppin, J. J. Heijnen, M. Hoare, H. C. Lange, E. A. Madden, P. Niederberger, J. Nielsen, J. L. Parrou, T. Petit, D. Porro, M. Reuss, R. N. van, M. Rizzi, H. Y. Steensma, C. T. Verrips, J. Vindelov, and J. T. Pronk. 2000. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb. Technol. 26:706-714. Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1990. Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J. Gen. Microbiol. 136:405-412. Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1992. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501-517. Visser, W., W. A. Scheffers, Batenburg-van der Vegte WH, and J. P. van Dijken. 1990. Oxygen requirements of yeasts. Appl. Environ. Microbiol. 56:3785-3792. Boer, V. M., J. H. de Winde, J. T. Pronk, and M. D. Piper. 2003. The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J. Biol. Chem. 278:3265-3274. Ferea, T. L., D. Botstein, P. O. Brown, and R. F. Rosenzweig. 1999. Systematic changes in gene expression patterns following adaptive evolution in yeast. Proc. Natl. Acad. Sci. U. S. A 96:9721-9726. Jansen, M. L., P. Daran-Lapujade, J. H. de Winde, M. D. Piper, and J. T. Pronk. 2004. Prolonged maltose-limited cultivation of Saccharomyces cerevisiae selects for cells with improved maltose affinity and hypersensitivity. Appl. Environ. Microbiol. 70:1956-1963.
Extracted molecule
total RNA
Extraction protocol
Microarray analysis. Sampling of cells from chemostats and total RNA extraction was performed as previously described in Abbott et al. (2007). Probe preparation and hybridization to Affymetrix Genechip® microarrays were performed following Affymetrix instructions. The one-cycle eukaryotic target labelling assay was used, starting with 15 g of total RNA. The quality of total RNA, cDNA, cRNA and fragmented cRNA were checked using the Agilent Bioanalyzer 2100 (Agilent Technologies). Results for each growth condition were derived from three independent culture replicates. Abbott DA, Knijnenburg TA, de Poorter LM, Reinders MJ, Pronk JT, van Maris AJ. (2007) Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae. FEMS Yeast Res. doi:10.1111/j.1567-1364.2007.00242.x
Label
biotin
Label protocol
EukGE-ws2v4 Microarray analysis. Sampling of cells from chemostats and total RNA extraction was performed as previously described in Abbott et al. (2007). Probe preparation and hybridization to Affymetrix Genechip® microarrays were performed following Affymetrix instructions. The one-cycle eukaryotic target labelling assay was used, starting with 15 g of total RNA. The quality of total RNA, cDNA, cRNA and fragmented cRNA were checked using the Agilent Bioanalyzer 2100 (Agilent Technologies). Results for each growth condition were derived from three independent culture replicates. Abbott DA, Knijnenburg TA, de Poorter LM, Reinders MJ, Pronk JT, van Maris AJ. (2007) Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae. FEMS Yeast Res. doi:10.1111/j.1567-1364.2007.00242.x
Hybridization protocol
Microarray analysis. Sampling of cells from chemostats and total RNA extraction was performed as previously described in Abbott et al. (2007). Probe preparation and hybridization to Affymetrix Genechip® microarrays were performed following Affymetrix instructions. The one-cycle eukaryotic target labelling assay was used, starting with 15 g of total RNA. The quality of total RNA, cDNA, cRNA and fragmented cRNA were checked using the Agilent Bioanalyzer 2100 (Agilent Technologies). Results for each growth condition were derived from three independent culture replicates. Abbott DA, Knijnenburg TA, de Poorter LM, Reinders MJ, Pronk JT, van Maris AJ. (2007) Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae. FEMS Yeast Res. doi:10.1111/j.1567-1364.2007.00242.x
Scan protocol
Affymetrix Scanner 3000
Description
LH13 Establishment of Zn-free culture vessels Glassware and fermenters employed in this study were all acid-washed and deionised according to the following procedure: overnight soak in 2 % nitric acid, two washes with deionised water, one wash with 0.1 M EDTA and four washes with deionised water. The glassware was dried prior to use. This procedure was fundamental to avoid any contamination of zinc from all glassware including the fermenter
