Glucose-repressed growth of was analysed in a nitrogen-limited continuous culture at different dilution rates (D). The glucose consumption of the yeast decreased from 3.4 g g h to 3.0 g g h when was decreased from 0.3 h to 0.15 h. No transcripts of the and genes, encoding, respectively, invertase and hexokinase isoenzyme 1, could be detected. Because both genes are regulated by glucose repression at the transcriptional level, this confirmed that the culture was glucose repressed at every . During the decrease in , no change in the activities or mRNA levels of key enzymes in carbon metabolism was observed, except for alcohol dehydrogenases I and II and phosphoglucomutase. These enzymes increased in activity and/or mRNA level when was decreased, which was also observed in glucose- and galactose-limited continuous cultures. This demonstrates that the expression levels of alcohol dehydrogenases I and II, and also phosphoglucomutase, are coupled to the growth rate of the organism. A comparison between the alcohol dehydrogenase II activity in glucose- and nitrogen-limited continuous cultures demonstrated that the growth rate contributes as much to repression of alcohol dehydrogenase II activity as does glucose. Both the glucose consumption and the activity of the glycolytic enzymes were relatively constant when was decreased and, as a consequence, the concentrations of intracellular metabolites remained constant. A slight decrease in the glucose 6-phosphate concentration was observed, which could be caused by the slight decrease in glucose consumption at low values. The 2-oxoglutarate and cAMP concentrations increased twofold when was decreased. The first probably reflects the increased NH consumption at high values, while the latter is caused by the high amount of extracellular cAMP compared with the amount of intracellular cAMP. The decrease in growth rate raised the amount of biomass, while nitrogen was limiting in all cases. Analysis of the biomass composition at the different values revealed that the amount of nitrogen per gram dry weight was constant, while the amounts of carbon, hydrogen and oxygen were higher at low values. In addition, higher concentrations of trehalose and glycogen were found at low values. This demonstrates that the glucose which is used at high values for the production of biomass is converted into storage carbohydrates, e.g. trehalose and glycogen, at low values. A nitrogen pulse did not result in a dramatic response of except for a rapid change in the free amino acid pool. Thus is not able rapidly to enhance growth upon a nitrogen pulse, which is in contrast with the response to a glucose pulse of a carbon-limited continuous culture of .


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