Media for chemostat cultivation. The synthetic medium composition was based on that described by Verduyn (1). The modifications introduced for carbon, nitrogen, sulphur, phosphorus and zinc limited growth were determined previously (1-3) and are listed in Table 2. In all chemostats except for those limited by carbon, the residual glucose concentration was targeted to 17 g/l (95 mM) in order to have the same degree of glucose repression. The reservoir medium was supplemented with vitamins and the anaerobic growth factors Tween-80 and ergosterol as previously described (1). Chemostat cultivation. Chemostat cultures were fed with synthetic medium that limited growth by carbon, nitrogen, sulphur, phosphorus or zinc with all other growth requirements in excess and at constant residual concentration. The dilution rate was set at 0.10 h-1, the pH was measured on-line and kept constant at 5.0 by the automatic addition of 2 M KOH as previously described (3). Cultures were assumed to be in steady-state when, after at least 5 volume changes, culture dry-weight, glucose concentration, carbon-dioxide production rate and oxygen consumption rate varied by less than 2 % during one additional volume change (4). Steady-state samples were taken after 10 generations at the latest to avoid strain adaptation due to long-term cultivation (5). Each cultivation condition was performed in triplicate. Reference List 1.Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) J. Gen. Microbiol. 136, 405-412 2.De Nicola, R., Hazelwood, L. A., De Hulster, E. A., Walsh, M. C., Knijnenburg, T. A., Reinders, M. J., Walker, G. M., Pronk, J. T., Daran, J. M., and Daran-Lapujade, P. (2007) Appl. Environ. Microbiol. 73, 7680-7692 3.Tai, S. L., Boer, V. M., Daran-Lapujade, P., Walsh, M. C., de Winde, J. H., Daran, J. M., and Pronk, J. T. (2005) J. Biol. Chem. 280, 437-447 4.Ferea, T. L., Botstein, D., Brown, P. O., and Rosenzweig, R. F. (1999) Proc. Natl. Acad. Sci. U. S. A 96, 9721-9726 5.Jansen, M. L., Daran-Lapujade, P., de Winde, J. H., Piper, M. D., and Pronk, J. T. (2004) Appl. Environ. Microbiol. 70, 1956-1963 6.Boer, V. M., de Winde, J. H., Pronk, J. T., and Piper, M. D. (2003) J. Biol. Chem. 278, 3265-3274
Extracted molecule
total RNA
Extraction protocol
Microarrays, data acquisition and statistical analysis. Sampling of cells from chemostats and total RNA extraction was performed as previously described (2). Results for each growth condition were derived from three independent culture replicates. Acquisition and quantification of array images and data filtering were performed using Affymetrix GeneChip® Operating Software version 1.2. To eliminate insignificant variations, genes with expression values below 12 were set to 12 as previously described (6). Reference List 1. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) J. Gen. Microbiol. 136, 405-412 2. De Nicola, R., Hazelwood, L. A., De Hulster, E. A., Walsh, M. C., Knijnenburg, T. A., Reinders, M. J., Walker, G. M., Pronk, J. T., Daran, J. M., and Daran-Lapujade, P. (2007) Appl. Environ. Microbiol. 73, 7680-7692 3. Tai, S. L., Boer, V. M., Daran-Lapujade, P., Walsh, M. C., de Winde, J. H., Daran, J. M., and Pronk, J. T. (2005) J. Biol. Chem. 280, 437-447 4. Ferea, T. L., Botstein, D., Brown, P. O., and Rosenzweig, R. F. (1999) Proc. Natl. Acad. Sci. U. S. A 96, 9721-9726 5. Jansen, M. L., Daran-Lapujade, P., de Winde, J. H., Piper, M. D., and Pronk, J. T. (2004) Appl. Environ. Microbiol. 70, 1956-1963 6. Boer, V. M., de Winde, J. H., Pronk, J. T., and Piper, M. D. (2003) J. Biol. Chem. 278, 3265-3274
Label
biotin
Label protocol
Microarrays, data acquisition and statistical analysis. Sampling of cells from chemostats and total RNA extraction was performed as previously described (2). Results for each growth condition were derived from three independent culture replicates. Acquisition and quantification of array images and data filtering were performed using Affymetrix GeneChip® Operating Software version 1.2. To eliminate insignificant variations, genes with expression values below 12 were set to 12 as previously described (6). Reference List 1. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) J. Gen. Microbiol. 136, 405-412 2. De Nicola, R., Hazelwood, L. A., De Hulster, E. A., Walsh, M. C., Knijnenburg, T. A., Reinders, M. J., Walker, G. M., Pronk, J. T., Daran, J. M., and Daran-Lapujade, P. (2007) Appl. Environ. Microbiol. 73, 7680-7692 3. Tai, S. L., Boer, V. M., Daran-Lapujade, P., Walsh, M. C., de Winde, J. H., Daran, J. M., and Pronk, J. T. (2005) J. Biol. Chem. 280, 437-447 4. Ferea, T. L., Botstein, D., Brown, P. O., and Rosenzweig, R. F. (1999) Proc. Natl. Acad. Sci. U. S. A 96, 9721-9726 5. Jansen, M. L., Daran-Lapujade, P., de Winde, J. H., Piper, M. D., and Pronk, J. T. (2004) Appl. Environ. Microbiol. 70, 1956-1963 6. Boer, V. M., de Winde, J. H., Pronk, J. T., and Piper, M. D. (2003) J. Biol. Chem. 278, 3265-3274
Hybridization protocol
According to manufacturer's procedures
Scan protocol
Data acquisition was performed using the Affymetrix scanner 3000, quantification of array images and data filtering were performed with the Affymetrix software packages Microarray Suite v5.0, MicroDB v3.0 and Data Mining Tool v3.0.
