The respirative breakdown of glucose by Saccharomyces cerevisiae: an assessment of a physiological state

Cells of Saccharomyces cerevisiae exhibiting respirative glucose metabolism in continuous culture were able to use ethanol as a co-substrate. The ethanol uptake rate was dependent on the residual respirative capacity of the cells. The activities of gluconeogenic enzymes and of malate dehydrogenase were higher in cells degrading glucose respiratively than in cells metabolizing glucose respiro-fermentatively, but were lower than in cells growing on ethanol only. The pattern of distribution of the mitochondrial cytochromes was similar but the differences were less distinct. In synchronously growing cells, the activities of gluconeogenic enzymes and of malate dehydrogenase oscillated, with activities increasing during the budding phase. The increase was preceded by the appearance of ethanol in the culture medium.     

Changes in activities of several enzymes involved in carbohydrate metabolism during the cell cycle of Saccharomyces cerevisiae.

Activity changes of a number of enzymes involved in carbohydrate metabolism were determined in cell extracts of fractionated exponential-phase populations of Saccharomyces cerevisiae grown under excess glucose. Cell-size fractionation was achieved by an improved centrifugal elutriation procedure. Evidence that the yeast populations had been fractionated according to age in the cell cycle was obtained by examining the various cell fractions for their volume distribution and their microscopic appearance and by flow cytometric analysis of the distribution patterns of cellular DNA and protein contents. Trehalase, hexokinase, pyruvate kinase, phosphofructokinase 1, and fructose-1,6-diphosphatase showed changes in specific activities throughout the cell cycle, whereas the specific activities of alcohol dehydrogenase and glucose-6-phosphate dehydrogenase remained constant. The basal trehalase activity increased substantially (about 20-fold) with bud emergence and decreased again in binucleated cells. However, when the enzyme was activated by pretreatment of the cell extracts with cyclic AMP-dependent protein kinase, no significant fluctuations in activity were seen. These observations strongly favor posttranslational modification through phosphorylation-dephosphorylation as the mechanism underlying the periodic changes in trehalase activity during the cell cycle. As observed for trehalase, the specific activities of hexokinase and phosphofructokinase 1 rose from the beginning of bud formation onward, finally leading to more than eightfold higher values at the end of the S phase. Subsequently, the enzyme activities dropped markedly at later stages of the cycle. Pyruvate kinase activity was relatively low during the G1 phase and the S phase, but increased dramatically (more than 50-fold) during G2. In contrast to the three glycolytic enzymes investigated, the highest specific activity of the gluconeogenic enzyme fructose-1, 6-diphosphatase 1 was found in fractions enriched in either unbudded cells with a single nucleus or binucleated cells. The observed changes in enzyme activities most likely underlie pronounced alterations in carbohydrate metabolism during the cell cycle.     

Biosynthesis and regulation of fructose-1,6-bisphosphatase and phosphofructokinase in Saccharomyces cerevisiae grown in the presence of glucose and gluconeogenic carbon sources.

The mode of synthesis and the regulation of fructose-1,6-bisphosphatase (Fbpase), a gluconeogenic enzyme, and phosphofructokinase (PFK), a glycolytic enzyme, were investigated in Saccharomyces cerevisiae after growth in the presence of different concentrations of glucose or various gluconeogenic carbon sources. The activity of FBPase appeared in the cells after the complete disappearance of glucose from the growth medium with a concomitant increase of the pH and no significant change in the levels of accumulated ethanol. The appearance of FBPase activity following glucose depletion was dependent upon the synthesis of protein. The FBPase PFK were present in glucose-, ethanol-, glycerol-, lactate-, or pyruvate-grown cells; however, the time of appearance and the levels of both these enzymes varied. The FBPase activity was always higher in 1% glucose-grown cells than in cells grown in the presence of gluconeogenic carbon sources. Phosphoglucose isomerase activity did not vary significantly. Addition of glucose to an FBPase and PFK synthesizing culture resulted in a complete loss, followed by a reappearance, of PFK activity. In the presence of cycloheximide the disappearance of glucose and the changes in the levels of FBPase and PFK were decreased significantly. It is concluded that S. cerevisiae exhibits a more efficient synthesis of FBPase after the exhaustion of glucose compared to the activity present in cells grown in the presence of exogenous gluconeogenic carbon sources. Two metabolically antagonistic enzymes, FBPase and PFK, are present during the transition phase, but not during the exponential phase, of growth, and the decay or inactivation of these enzymes in vivo may be dependent upon a glucose-induced protease activity.     

Control of pyruvate kinase activity during glycolysis and gluconeogenesis in Propionibacterium shermanii

The concentrations of glycolytic intermediates and ATP and the activities of certain glycolytic and gluconeogenic enzymes were determined in Propionibacterium shermanii cultures grown on a fully defined medium with glucose, glycerol or lactate as energy source. On all three energy sources, enzyme activities were similar and pyruvate kinase was considerably more active than the gluconeogenic enzyme pyruvate, orthophosphate dikinase, indicating the need for regulation of pyruvate kinase activity. The intracellular concentration of glucose 6-phosphate, a specific activator of pyruvate kinase in this organism, changed markedly according to both the nature and the concentration of the growth substrate: the concentration (7-10 mM) during growth with excess glucose or glycerol was higher than that (1-2 mM) during growth with lactate or at growth-limiting concentrations of glycerol or glucose. Other glycolytic intermediates, apart from pyruvate, were present at concentrations below 2 mM. Glucose 6-phosphate overcame inhibition of pyruvate kinase activity by ATP and inorganic phosphate. With 1 mM-ATP and more than 10 mM inorganic phosphate, a change in glucose 6-phosphate concentration from 1-2 mM was sufficient to switch pyruvate kinase from a strongly inhibited to a fully active state. The results provide a plausible mechanism for the regulation of glycolysis and gluconeogenesis in P. shermanii.     

Enzyme Pattern and Aerobic Growth of Saccharomyces cerevisiae Under Various Degrees of Glucose Limitation

The enzyme pattern of Saccharomyces cerevisiae was followed during batch growth and in continuous culture in a synthetic medium limited for glucose under aerobic conditions. Seven enzymes were measured: succinate-cytochrome c oxidoreductase, malate dehydrogenase, nicotinamide adenine dinucleotide-linked glutamate dehydrogenase, malate synthase, isocitrate lyase, aldolase, and nicotinamide adenine dinucleotide phosphate (NADP(+))-linked glutamate dehydrogenase. During fermentation of glucose and high growth rate (mu) during the first log phase in batch experiments, the first five enzymes (group I) were repressed, and aldolase and NADP(+)-linked glutamate dehydrogenase (group II) were derepressed. During growth on the accumulated ethyl alcohol and lower mu, the group I enzymes were preferentially formed and the other two were repressed. A sequence of derepression of the group I enzymes was found during the shift from glucose to ethyl alcohol metabolism, which can be correlated with a strong increase in the percentage of single (nonbudding) cells in the population. A correlation between the state of cells in the budding cycle and enzyme repression and derepression is suggested. In continuous culture, the enzyme pattern was shown to be related to the growth rate. The group I enzymes were repressed at high growth rates, while the group II enzymes were derepressed. Each enzyme exhibits a different dependence. The enzyme pattern is shown to depend on the rate of substrate consumption as well as on the type of metabolism and to be correlated with the budding cycle. The enzyme pattern is considered to be controlled by changes of intracellular catabolic or metabolic conditions inherent in the division cycle.     

Properties of the Invertases of Cultured Sycamore Cells and Changes in Their Activity during Culture Growth

When cultured sycamore cells are homogenised in a phosphate-citrate buffer at pH 7.0 and the homogenate centrifuged two fractions are obtained both of which show the presence of an acid (opt. pH 4.0–4.5) and a neutral (opt. pH 7.0–7.4) invertase. The activity of the insoluble pellet appears to be located in its cell wall fragments. The acid and neutral invertases of the soluble fraction can be separated by fractional precipitation with (NH₄SO₄. The activities of these enzymes are low in stationary phase cells but they increase following subculture to reach peaks of activity towards the end of the period of most active cell growth and division and then decline again as the cells begin to enter stationary phase. The activities of both enzymes are higher in the cell wall than in the soluble fraction and the acid invertase reaches higher levels of activity than the neutral enzyme in both fractions. When cells are subcultured there occurs within a few hours an increase in the acid invertase and a decline in the neutral invertase activity in the cell wall fraction and a decline in the acid invertase of the soluble fraction prior to the large net increases in the activities of both enzymes in both locations which occurs as the cells embark upon cell division. The pattern of changes in the invertase activities through the growth cycle of batch propagated cultures is similar whether the cells are grown in sucrose, or glucose, or sucrose plus glucose; the highest levels of activities were recorded in the glucose-grown cells. The total yield of invertase activities and the distribution of activities between the soluble and cell wall fractions of the homogenates are affected by the pH of the extraction medium (within the range pH 4.0–8.0). It has not proved possible to completely remove the invertases from the cell wall fraction; upwards of 50 % of the acid invertase was recovered from this fraction by treatment with Triton-X followed by urea, but these treatments inactivated a high proportion of the neutral enzyme. These findings are compared with other studies on the activity and intra-cellular distribution of plant invertases and the possible roles of these enzymes discussed.     

Expression of key substrate cycle enzymes in rat spermatogenic cells: fructose 1,6 bisphosphatase and 6 phosphofructose 1-kinase

A substrate cycle composed of phosphofructo 1-kinase I (PFK) and fructose 1,6 bisphosphatase I (FBPase) has been proposed in rat spermatids. This substrate cycle can explain the ability of glucose to induce a decrease in intracellular ATP, a phenomenon that was related to regulation of [Ca(2+)]i in these cells. In spite of the importance of this metabolic cycle, the expression and activities of the enzymes that compose such cycle have not been systematically studied in spermatogenic cells. Here, we show that PFK and FBPase activities were present in pachytene spermatocytes and round spermatids extracts. Expression of PFK at the mRNA and protein levels showed a relatively similar expression in spermatogenic cells, but a stronger expression in Sertoli cells. Instead, expression of FBPase at the mRNA and protein levels was stronger in round and elongating spermatids as compared to other spermatogenic cells. A similar pattern was observed when evidencing FBPase activity by a NADPH-nitroblue tetrazolium-linked cytochemical assay in isolated pachytene spermatocytes and round spermatids. Rat spermatids also showed the ability to convert lactate to fructose- and glucose-6-P, indicating that both glycolytic and gluconeogenic fluxes are present in these cells. Our results indicate that a coordinated expression of key substrate cycle enzymes, at the level of PFK/FBPase, appear in the last stages of spermatogenic cell differentiation, suggesting that the co-regulation of these enzymes are required for the ability of these cells to respond to glucose and induce metabolic and Ca(2+) signals that can be important for sperm development and function.     

在不同碳源培养条件下酿酒酵母的蛋白质组解析

为了分析酿酒酵母在不同培养条件下的代谢调控过程的差异,采用固相pH梯度-SDS聚丙烯酰胺双向凝胶电泳对其利用不同碳源时细胞的总蛋白进行了分离,银染显色,使用2D蛋白质图像分析系统Image Master-2D Elite对双向电泳图谱进行分析,查询SWISS-2D-PAGE蛋白质组数据库,识别了约500个蛋白质点。对与糖酵解途径、磷酸戊糖途径、三羧酸循环和几种回补反应相关的大部分关键的蛋白质进行了差异分析。探讨了酿酒酵母利用不同碳源时及生长的不同阶段代谢机理的变化和在蛋白质水平的调控。     

Glycogen synthesis by rat hepatocytes.

1. Hepatocytes from starved rats or fed rats whose glycogen content was previously depleted by phlorrhizin or by glucagon injections, form glycogen at rapid rates when incubated with 10mM-glucose, gluconeogenic precursors (lactate, glycerol, fructose etc.) and glutamine. There is a net synthesis of glucose and glycogen. 14C from all three types of substrate is incorporated into glycogen, but the incorporation from glucose represents exchange of carbon atoms, rather than net incorporation. 14C incorporation does not serve to measure net glycogen synthesis from any one substrate. 2. With glucose as sole substrate net glucose uptake and glycogen deposition commences at concentrations of about 12--15mM. Glycogen synthesis increases with glucose concentrations attaining maximal values at 50--60mM, when it is similar to that obtained in the presence of 10mM glucose and lactate plus glutamine. 3. The activities of the active (a) and total (a+b) forms of glycogen synthase and phosphorylase were monitored concomitant with glycogen synthesis. Total synthase was not constant during a 1 h incubation period. Total and active synthase activity increased in parallel with glycogen synthesis. 4. Glycogen phosphorylase was assayed in two directions, by conversion of glycose 1-phosphate into glycogen and by the phosphorylation of glycogen. Total phosphorylase was assyed in the presence of AMP or after conversion into the phosphorylated form by phosphorylase kinase. Results obtained by the various methods were compared. Although the rates measured by the procedures differ, the pattern of change during incubation was much the same. Total phosphorylase was not constant. 5. The amounts of active and total phosphorylase were highest in the washed cell pellet. Incubation in an oxygenated medium, with or without substrates, caused a prompt and pronounced decline in the assayed amounts of active and total enzyme. There was no correlation between phosphorylase activity and glycogen synthesis from gluconeogenic substrates. With fructose, active and total phosphorylase activities increased during glycogen syntheses. 6. In glycogen synthesis from glucose as sole substrate there was a decline in phosphorylase activities with increased glucose concentration and increased rates of glycogen deposition. The decrease was marked in cells from fed rats. 7. To determine whether phosphorolysis and glycogen synthesis occur concurrently, glycogen was prelabelled with [2-3H,1-14C]-galactose. During subsequent glycogen deposition there was no loss of activity from glycogen in spite of high amounts of assayable active phosphorylase.     

Changes in the enzyme activities of Saccharomyces cerevisiae during aerobic growth on different carbon sources

1. The activities of the enzymes of the citric acid cycle, the glyoxylate by-pass and some other enzymes acting on the substrates of these cycles have been measured at the pH of the yeast cell during the aerobic growth of yeast on different carbon sources and in different growth media. 2. Sugars induced an anaerobic type of metabolism as measured by ethanol production. Glucose was much more effective in inducing the anaerobic pathways than was galactose. The production of ethanol by cells grown on pyruvate was very small. 3. Glucose was also a more effective repressor than was galactose of the citric acid-cycle enzymes but both were equally effective in repressing almost completely the enzymes of the glyoxylate by-pass. 4. Disappearance of the sugars from the growth medium resulted in an increase in the activities of the enzymes of the citric acid cycle and in the appearance of substantial activities of the enzymes of the glyoxylate cycle. By contrast, the activities of purely biosynthetic enzymes (glutamate-oxaloacetate transaminase, NADP(+)-linked glutamate dehydrogenase) and of pyruvate decarboxylase were decreased. 5. The 2-oxoglutarate-oxidase system was found to be the least active enzyme of the citric acid cycle. 6. The regulatory control at the levels of pyruvate and acetaldehyde and the control of the citric acid cycle are discussed.     

Development of gluconeogenic enzymes in fetal sheep liver and kidney

In the sheep, the system of enzymes necessary for conversion of nonhexose substrates to glucose becomes active during late fetal life. Glucose-6-phosphatase and fructose-1,6-diphosphatase, two of the four key gluconeogenic enzymes, appear in significant amounts between 100 and 120 days gestation. Phosphoenolpyruvate carboxykinase activity is comparable to mature animals as early as 45 days gestation. Two aminotransferases, necessary to allow amino acid access to the gluconeogenic pathway, likewise have substantial activity as early as 45 days gestation. Hence, the surge of glucose-6-phosphatase and fructose-1,6-diphosphatase at 100-120 days gestation makes possible the endogenous production of new glucose by fetal sheep at a time when the amount of glucose transferred from the maternal circulation is less than the total aerobic substrate utilized by the fetus. Both renal cortex and liver have similar developmental patterns for the gluconeogenic enzymes, although renal cortex generally shows greater activity than liver. This observation holds true for tissue from both fetal and mature animals.     

Alterations in the levels of deoxyuridine triphosphatase, uracil-DNA glycosylase and AP endonuclease during the cell cycle

Deoxyuridine triphosphatase and uracil-DNA glycosylase were assayed in K12 cells, a Chinese hamster temperature-sensitive mutant known to arrest in G1 when shifted to the non-permissive temperature. Stimulation of cell division resulted in an increase of both enzyme activities. Levels of both enzymes were minimal during G0 and maximal in S phase. By contrast, apurinic/apyrimidinic site endonuclease activity, which incises the DNA at the apyrimidinic site generated by the glycosylase, was constant throughout the cell cycle.     

Expression of differentiated function by hepatocytes in primary culture: variable effects of glucagon and insulin on gluconeogenesis during cell growth

Hormonal effects on gluconeogenesis from lactate were studied during the growth cycle of adult rat parenchyma liver cells using a primary monolayer culture system previously described [25]. Basal and glucagon-stimulated gluconeogenic ability were found to decline rapidly during log phase, insulin-stimulated growth. A progressive recovery of gluconeogenesis activity was observed after cell division subsided. Rates of lactate-gluconeogenesis were found also to decline in the absence of prior insulin exposure. This decline was not as rapid as the loss observed in cells cultured with insulin. However, in insulin-deficient cultures gluconeogenesis was completely abolished after 12 days and did not reelevate with further incubation unless cells were washed and exposed to glucagon. Decreasing growth rates of insulin-supplemented cultures by decreasing serum concentrations resulted in comparatively higher gluconeogenic activity. The results presented here are consistent with previous observations of hepatic parenchymal expression of 'differentiated function' during cellular growth phases in culture (i.e., differentiated functions are generally lost during rapid growth and regained as cells become quiescent). The present study, however, presents unexpected effects of insulin on the apparent growth-state dependent gluconeogenic recovery. Our data imply that although insulin has long been known to inhibit gluconeogenesis, its presence in culture may facilitate long-term basal maintenance of gluconeogenic enzyme activity. Insulin also functions as a growth factor whose initial mitogenic effect correlates with decreased gluconeogenic function. These changes show no simple or predictive correlation with cyclic nucleotide metabolism.     

Thymidine as synchronizing agent. 3. Persistence of cell cycle patterns of phosphatase activities and elevation of nuclease activity during inhibition of DNA synthesis

Synchronous cultures of HeLa cells were obtained by selective detachment of cells in mitosis and fluctuations in enzyme activity were followed during the subsequent cell cycle. The enzymes measured were alkaline and acid phosphatases and a nuclease active on denatured DNA at alkaline pH (alkaline DNase). Each of these enzymes showed a different pattern of activity in the cell cycle, but a temporal relationship to the DNA synthetic phase was apparent in each case. Treatment of the cultures at the beginning of the cell cycle with 15 mM thymidine did not alter the subsequent pattern of fluctuations in activity of alkaline phosphatase or of acid phosphatase, although DNA synthesis was fully inhibited by this treatment. This indicates that the pattern of activity of some enzymes is not linked to DNA replication. On the other hand, the pattern of fluctuations in the activity of alkaline DNase was abolished by thymidine treatment, and elevation of the activity of this enzyme was observed. These results suggest complex and variable relationships between phases of the cell cycle and enzyme activity, and show that inhibition of DNA synthesis is not a suitable procedure for induction of culture synchrony if enzyme activities are to be studied.     

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.     

Cell volume changes affect gluconeogenesis in the perfused liver of the catfish<Emphasis Type="Italic">Clarias batrachus</Emphasis>

In addition to lactate and pyruvate, some amino acids were found to serve as potential gluconeogenic substrates in the perfused liver ofClarias batrachus. Glutamate was found to be the most effective substrate, followed by lactate, pyruvate, serine, ornithine, proline, glutamine, glycine, and aspartate. Four gluconeogenic enzymes, namely phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase (PC), fructose 1,6-bisphosphatase (FBPase) and glucose 6-phosphatase (G6Pase) could be detected mainly in liver and kidney, suggesting that the latter are the two major organs responsible for gluconeogenic activity in this fish. Hypo-osmotically induced cell swelling caused a significant decrease of gluconeogenic efflux accompanied with significant decrease of activities of PEPCK, FBPase and G6Pase enzymes in the perfused liver. Opposing effects were seen in response to hyperosmotically induced cell shrinkage. These changes were partly blocked in the presence of cycloheximide, suggesting that the aniso-osmotic regulations of gluconeogenesis possibly occurs through an inverse regulation of enzyme proteins and/or a regulatory protein synthesis in this catfish. In conclusion, gluconeogenesis appears to play a vital role inC. batrachus in maintaining glucose homeostasis, which is influenced by cell volume changes possibly for proper energy supply under osmotic stress.     

Metabolic control of hepatic gluconeogenesis during exercise.

Prolonged exercise increased the concentrations of the hexose phosphates and phosphoenolpyruvate and depressed those of fructose 1,6-bisphosphate, triose phosphates and pyruvate in the liver of the rat. Since exercise increases gluconeogenic flux, these changes in metabolite concentrations suggest that metabolic control is exerted, at least, at the fructose 6-phosphate/fructose 1,6-bisphosphate and phosphoenolpyruvate/pyruvate substrate cycles. Exercise increased the maximal activities of glucose 6-phosphatase, fructose 1,6-bisphosphatase, pyruvate kinase and pyruvate carboxylase in the liver, but there were no changes in those of glucokinase, 6-phosphofructokinase and phosphoenolpyruvate carboxykinase. Exercise changed the concentrations of several allosteric effectors of the glycolytic or gluconeogenic enzymes in liver; the concentrations of acetyl-CoA, ADP and AMP were increased, whereas those of ATP, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate were decreased. The effect of exercise on the phosphorylation-dephosphorylation state of pyruvate kinase was investigated by measuring the activities under conditions of saturating and subsaturating concentrations of substrate. The submaximal activity of pyruvate kinase (0.5 mM-phosphoenolpyruvate), expressed as percentage of Vmax., decreased in the exercised animals to less than half that found in the controls. These changes suggest that hepatic pyruvate kinase is less active during exercise, possibly owing to phosphorylation of the enzyme, and this may play a role in increasing the rate of gluconeogenesis.     

Intensification of synthesis of the exopolysaccharide ethapolan by Acinetobacter sp. 12S grown on a mixture of substrates

Enhanced synthesis of the exopolysaccharide (EPS) ethapolan by Acinetobacter sp. 12S was observed when the bacterium was grown on a mixture of two energetically nonequivalent substrates (ethanol and glucose) taken in a molar proportion of 3.1:1. The efficiency of carbon transformation into EPSs was maximum when sodium ions were absent in the medium, the concentration of nitrogen source was reduced to 0.3-0.45 g/l, and the inoculum was grown on ethanol. Such conditions provided an increase in the maximum specific growth rate and its attainment in earlier cultivation terms. Molasses as a substitution for glucose was inefficient. The activities of the key enzymes of C2-metabolism in Acinetobacter sp. 12S cells grown on the substrate mixture was 1.1 to 1.7 times lower than they were during growth on ethanol alone. The activity of isocitrate lyase in cells grown on the substrate mixture declined to an even greater extent (by 4 to 7 times), indicating that the role of the glyoxylate cycle in such cells is insignificant.     

Ethanol catabolism in Corynebacterium glutamicum

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h(-1) and biomass yields up to 0.47 g dry weight (g ethanol)(-1). Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.