Regulation and genetic enhancement of beta-amylase production in Clostridium thermosulfurogenes.

We studied the general mechanism for regulation of beta-amylase synthesis in Clostridium thermosulfurogenes. beta-Amylase was expressed at high levels only when the organism was grown on maltose or other carbohydrates containing maltose units. Three kinds of mutants altered in beta-amylase production were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained starch-glucose agar plates. beta-Amylase was produced only when maltose was added to cells growing on sucrose in wild-type and catabolite repression-resistant mutant strains, but the differential rate of enzyme synthesis in constitutive mutants was constant regardless of the presence of maltose. In carbon-limited chemostats of wild-type and catabolite repression-resistant mutant stains, beta-amylase was expressed on maltose but not on glucose or sucrose. beta-Amylase synthesis was immediately repressed by the addition of glucose. Therefore, we concluded that beta-amylase synthesis in C. thermosulfurogenes was inducible and subject to catabolite repression. The addition of cAMP did not eliminate the repressive effect of glucose. The mutants were generally characterized in terms of beta-amylase production, growth properties, fermentation product formation, and alterations in glucose isomerase and glucoamylase activities. A hyperproductive mutant produced eightfold more beta-amylase on starch medium than the wild type and more rapidly fermented starch to ethanol.     

Use of catabolic repression in the selection of the glucoamylase-producer Aspergillus niger

The effect of various carbon sources and cAMP on the glucoamylase synthesis in Aspergillus niger was studied to find carbon sources repressed the enzyme synthesis and conditions for the selection of catabolite stable mutants. Maltose at a concentration of 0.5% stimulated the glucoamylase synthesis, but at a concentration of 4% it repressed not only the enzyme synthesis but the growth of the parental strain on the agar medium. The more active mutant 66 was obtained as a result of treatment of Asp. niger st 6 with NG. This mutant is able to grow on the Czapek's medium containing maltose at concentrations 4 or 6%. The mutant 66 produced about 2.9 times more glucoamylase than its parent when maltose was added at 0.5% concentration to the medium. The glucoamylase synthesis in the parental strain was completely repressed under repressing conditions, while the level of the mutant strain activity was 35% from the level of enzyme activity on the medium without the repressor. The addition of cAMP (5.10(-5] resulted in a partial release of maltose (4%) repression of the glucoamylase synthesis in both strains. The results obtained indicate a possibility to select Asp niger mutants with the partially derepressed glucoamylase synthesis. Other regulation mechanisms in addition to catabolite repression may be involved in the regulation of the glucoamylase synthesis.     

Differential amylosaccharide metabolism of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum.

Clostridium thermosulfurogenes displayed faster growth on either glucose, maltose, or starch than Clostridium thermohydrosulfuricum. Both species grew faster on glucose than on starch or maltose. The fermentation end product ratios were altered based on higher ethanol and lactate yields on starch than on glucose. In C. thermohydrosulfuricum, glucoamylase, pullulanase, and maltase were mainly responsible for conversion of starch and maltose into glucose, which was accumulated by a putative glucose permease. In C. thermosulfurogenes, beta-amylase was primarily responsible for degradation of starch to maltose, which was accumulated by a putative maltose permease and then hydrolyzed by glucoamylase. Regardless of the growth substrate, the rates of glucose, maltose, and starch transformation were higher in C. thermosulfurogenes than in C. thermohydrosulfuricum. Both species had a functional Embden-Meyerhof glycolytic pathway and displayed the following catabolic activities: ferredoxin-linked pyruvate dehydrogenase, acetate kinase, NAD(P)-ethanol dehydrogenase, NAD(P)-ferredoxin oxidoreductase, hydrogenase, and fructose-1,6-diphosphate-activated lactate dehydrogenase. Ferredoxin-NAD reductase activity was higher in C. thermohydrosulfuricum than NADH-ferredoxin oxidase activity, but the former activity was not detectable in C. thermosulfurogenes. Both NAD- and NADP-linked ethanol dehydrogenases were unidirectional in C. thermosulfurogenes but reversible in C. thermohydrosulfuricum. The ratio of hydrogen-producing hydrogenase to hydrogen-consuming hydrogenase was higher in C. thermosulfurogenes. Two biochemical models are proposed to explain the differential saccharide metabolism on the basis of species enzyme differences in relation to specific growth substrates.     

Simultaneous uptake and utilisation of glucose and xylose by Clostridium thermohydrosulfuricum

Abstract Growth studies of Clostridium thermohydrosulfuricum Rt8.B1 demonstrated that glucose and xylose were used simultaneously when supplied together at nonlimiting concentrations in pH-controlled batch culture. Under conditions of hyperbolic growth, both catabolite repression and inducer exclusion were absent. Glucose did not repress xylose metabolism (i.e. xylose permease and xylose isomerase genes were expressed in the presence of glucose and were not subject to catabolite inhibition when glucose was added to cultures growing on high concentrations of xylose). The kinetics of glucose and xylose utilisation indicated that separate systems were present for the uptake of these substrates when supplied together. Glucose utilisation was biphasic, indicating high- and low-affinity systems for glucose uptake. Xylose utilisation was directly proportional to the xylose concentration, suggesting a facilitated diffusion mechanism was operative for uptake.     

A constitutive catabolite repression mutant of a recombinantSaccharomyces cerevisiae strain improves xylose consumption during fermentation

Efficient xylose utilisation by microorganisms is of importance to the lignocellulose fermentation industry. The aim of this work was to develop constitutive catabolite repression mutants in a xylose-utilising recombinantSaccharomyces cerevisiae strain and evaluate the differences in xylose consumption under fermentation conditions.S. cerevisiae YUSM was constitutively catabolite repressed through specific disruptions within theMIG1 gene. The strains were grown aerobically in synthetic complete medium with xylose as the sole carbon source. Constitutive catabolite repressed strain YCR17 grew four-fold better on xylose in aerobic conditions than the control strain YUSM. Anaerobic batch fermentation in minimal medium with glucose-xylose mixtures and N-limited chemostats with varying sugar concentrations were performed. Sugar utilisation and metabolite production during fermentation were monitored. YCR17 exhibited a faster xylose consumption rate than YUSM under high glucose conditions in nitrogen-limited chemostat cultivations. This study shows that a constitutive catabolite repressed mutant could be used to enhance the xylose consumption rate even in the presence of high glucose in the fermentation medium. This could help in reducing fermentation time and cost in mixed sugar fermentation.     

Construction of industrial baker's yeast strains able to assimilate maltose under catabolite repression conditions

Spore progeny from an industrial baker's yeast strain were mutagenized with UV and mutants resistant to 2-deoxyglucose isolated. One of these mutants (10a12–13) showed high levels of maltase (-glucosidase) and external invertase, and assimilated maltose when growing under catabolite repression conditions. This mutant was not allelic to any of the catabolite repression mutants tested cat4, cat80, cid1, cyc8, hex2, hxk2 and tup1. Mutant 10a12–13 was crossed with appropriate strains to construct hybrids that were also able to assimilate maltose in the presence of glucose. These hybrids may be useful in fermentation processes where both glucose and maltose are present.     

Glucose kinase-dependent catabolite repression in Staphylococcus xylosus.

By transposon Tn917 mutagenesis, 16 mutants of Staphylococcus xylosus were isolated that showed higher levels of beta-galactosidase activity in the presence of glucose than the wild-type strain. The transposons were found to reside in three adjacent locations in the genome of S. xylosus. The nucleotide sequence of the chromosomal fragment affected by the Tn917 insertions yielded an open reading frame encoding a protein with a size of 328 amino acids with a high level of similarity to glucose kinase from Streptomyces coelicolor. Weaker similarity was also found to bacterial fructokinases and xylose repressors of gram-positive bacteria. The gene was designated glkA. Immediately downstream of glkA, two open reading frames were present whose deduced gene products showed no obvious similarity to known proteins. Measurements of catabolic enzyme activities in the mutant strains grown in the presence or absence of sugars established the pleiotropic nature of the mutations. Besides beta-galactosidase activity, which had been used to detect the mutants, six other tested enzymes were partially relieved from repression by glucose. Reduction of fructose-mediated catabolite repression was observed for some of the enzyme activities. Glucose transport and ATP-dependent phosphorylation of HPr, the phosphocarrier of the phosphoenolpyruvate:carbohydrate phosphotransferase system involved in catabolite repression in gram-positive bacteria, were not affected. The cloned glkA gene fully restored catabolite repression in the mutant strains in trans. Loss of GlkA function is thus responsible for the partial relief from catabolite repression. Glucose kinase activity in the mutants reached about 75% of the wild-type level, indicating the presence of another enzyme in S. xylosus. However, the cloned gene complemented an Escherichia coli strain in glucose kinase. Therefore, the glkA gene encodes a glucose kinase that participates in catabolite repression in S. xylosus.     

Glucose transport and catabolite repression in Endomycopsis fibuligera yeasts

The role of systems for glucose transport in the manifestation of carbon catabolite repression of glucoamylase synthesis was studied in the yeast Endomycopsis fibuligera. Experimentas were conducted with its mutant AB-192 defective in the system of transport universal for glucose and 2-deoxy-D-glucose (2-DG). The nature of the mutation was established from the following data: (1) transport of labeled glucose into the mutant cells was twice as low in comparison with the parent culture 20-9; (2) transport of labeled 2-DG was suppressed almost entirely; (3) no competition was found between glucose and 2-DG for penetration into the mutant cells. Glucoamylase synthesis in the mutant AB-192 was not sensitive to catabolite repression by glucose. This was confirmed by the resistance of the AB-192 cells to the inhibition by glucose and their complete resistance to the repression by 2-DG. Moreover, an addition of cAMP did not stimulate glucoamylase synthesis by the mutant culture in the presence of glucose and 2-DG. It can be concluded therefore that the resistance of the yeast to catabolite repression by the glucose is caused by the mutation in the system for carbohydrate transport. The results suggest that the system of glucose transport plays an important role in the manifestation of carbon catabolite repression in the yeast Endomycopsis fibuligera.     

Derepressed synthesis of cellulase by Cellulomonas.

A Cellulomonas sp. was isolated from the soil which hydrolyzed cellulose, as shown by clear-zone formation on cellulose agar medium. Catabolite repression of cellulase synthesis occurred when moderate levels of glucose were added to the medium. A stable mutant that no longer exhibits catabolite repression was produced through treatment of the wild-type organism with N-methyl-N'-nitro-N-nitrosoguanidine. Both enzyme concentration and specific activity, as determined by the rate of hydrolysis of carboxymethylcellulose, were greater with the mutant than with the wild-type organism under various test conditions. The wild type had no measurable cellulase activity when grown in the presence of either 1.0% glucose or cellobiose. Cellobiose, but not glucose, inhibited enzyme activity towards both cellulose and carboxymethylcellulose. Cellobiose, cellulose, and sophorose at low concentrations induced cellulase synthesis in both the wild-type and the mutant organism. Cellulase regulation appears to depend upon a complex relationship involving catabolite repression, inhibition, and induction.     

Ethanol fermentation on glucose/xylose mixture by co-cultivation of restricted glucose catabolite repressed mutants of Pichia stipitis with respiratory deficient mutants of Saccharomyces cerevisiae

Restricted glucose catabolite repressed mutants of P. stipiti CCY 39501 were selected using UV irradiation. Four mutants were obtained which assimilated glucose slower than the native strain of P. stipitis and the degree of glucose repression was about 2-fold lower for P5-90-133 and P5-200-16 mutants and about 10-fold lower for P5-80-7 and P5-80-35 mutants. P5-80-7 and P5-80-35 produced very small amounts of ethanol from glucose and xylose, whereas P5-90-133 and P5-200-16 fermented sugars at the wild-type level. These two mutants were selected for co-fermentation process with native strain of S. cerevisiae V30 or Ja(a), as well as with their respiratory deficient mutants. During co-culture process of P. stipitis mutants with native strains of S. cerevisiae the ethanol yields obtained ranged from 0.38 to 0.45 g/g, and this alcohol was produced mainly from glucose. But, when also xylose, besides glucose was fermented to ethanol during co-fermentation of both mutant strains, lower yields of ethanol (0.28-0.40 g/g) were obtained.     

Transient Repression of the lac Operon

Severe transient repression of constitutive or induced beta-galactosidase synthesis occurs upon the addition of glucose to cells of Escherichia coli growing on glycerol, succinic acid, or lactic acid. Only mutants particularily well adapted to growth on glucose exhibit this phenomenon when transferred to a glucose-containing medium. No change in ribonucleic acid (RNA) metabolism was observed during transient repression. We could show that transient repression is pleiotropic, affecting all products of the lac operon. It occurs in a mutant insensitive to catabolite repression. It is established much more rapidly than catabolite repression, and is elicited by glucose analogues that are phosphorylated but not further catabolized by the cell. Thus, transient repression is not a consequence of the exclusion of inducer from the cell, does not require catabolism of the added compound, and does not involve a gross change in RNA metabolism. We conclude that transient repression is distinct from catabolite repression.     

In-depth analysis of the Aspergillus niger glucoamylase (glaA) promoter performance using high-throughput screening and controlled bioreactor cultivation techniques

An in-depth characterization of the Aspergillus niger glucoamylase (glaA) promoter performance was carried out on defined medium employing multi-well high-throughput screening as well as controlled batch and fed-batch bioreactor culture techniques with GFP as a fluorescent reporter protein. A variety of metabolizable carbon substrates and non-metabolizable analogs were screened with regard to their effect on the glaA expression system. The results clearly demonstrate that only starch and its hydrolytic products, including glucose, act as inducers. However, induction of the glaA expression system through the monosaccharide glucose is significantly lower compared to starch and the higher molecular weight starch degradation products. All other 26 carbon substrates tested do not induce, or even, as in the case of the easily metabolizable monosaccharide xylose, repress glaA-promoter controlled gene expression in the presence of the inducing disaccharide maltose with an increase of repression strength by increasing xylose concentrations. The complex effect of glucose on glaA-promoter controlled expression was also analyzed using non-metabolizable glucose analogs, namely 5-thio-glucose and 2-deoxyglucose, which were identified as novel and potent inducers of the glaA expression system. The results show that the induction strength depends on the inducer concentration with a maximum at defined concentrations and lower induction or even repression at concentrations above. Moreover, controlled fed-batch cultivations using a high maltose feed rate with concomitant extracellular accumulation of glucose resulted in lower levels of the reporter protein compared to cultures with a low-maltose feed rate without extracellular glucose accumulation, thus supporting the conclusion that increasing the glucose concentration beyond a critical point reduces the induction strength or may even cause repression. This way, the speed of polymer hydrolysis, glucose uptake and intracellular breakdown can be fine-tuned for optimal fungal growth and the metabolic burden for glucoamylase synthesis can be limited adequately in response to nutrient availability.     

Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system

Due to catabolite repression in microorganisms, sugar mixtures cannot be metabolized in a rapid and efficient manner. Therefore, the development of mutant strains that avoid this regulatory system is of special interest to fermentation processes. In the present study, the utilization of sugar mixtures by an Escherichia coli mutant strain devoid of the phosphotransferase system (PTS) was characterized. This mutant can transport glucose (PTS- Glucose+ phenotype) by a non-PTS mechanism as rapidly as its wild-type parental strain. In cultures grown in minimal medium supplemented with glucose-xylose or glucose-arabinose mixtures, glucose repressed arabinose- or xylose-utilization in the wild-type strain. However, under the same culture conditions with the PTS- Glucose+ mutant, glucose and arabinose were co-metabolized, but glucose still exerted a partial repressive effect on xylose consumption. In cultures growing with a triple mixture of glucose-arabinose-xylose, the wild-type strain sequentially utilized glucose, arabinose and finally, xylose. In contrast, the PTS- Glucose+ strain co-metabolized glucose and arabinose, whereas xylose was utilized after glucose-arabinose depletion. As a result of glucose-arabinose co-metabolism, the PTS- Glucose+ strain consumed the total amount of sugars contained in the culture medium 16% faster than the wild-type strain. [14C]-Xylose uptake experiments showed that in the PTS- Glucose+ strain, galactose permease increases xylose transport capacity and the observed partial repression of xylose utilization depends on the presence of intracellular glucose.     

Pullulanase synthesis in Klebsiella (Aerobacter) aerogenes strains growing in continuous culture

1. Pullulanase synthesis was studied in 16 classified (N.C.I.B.) strains and in an industrial strain (R) of Klebsiella aerogenes grown in chemostats containing maltose as inducer and sole carbon source. 2. Maximum synthesis was associated with carbon-limited growth at a low dilution rate (about 0.2h(-1)). The enzyme remained firmly cell-bound and seemed to be located on the cell surface. 3. Three strains had high activity (R, N.C.I.B. 5938, 8017), twelve were intermediate, and two (N.C.I.B. 8153, 9146) had negligible activity but were inducible with pullulan. 4. Pullulan similarly induced low, but adequate, activity in the other strains in conditions (nutrient limitation other than carbon-limitation) in which pullulanase was otherwise very seriously repressed. Nevertheless, in carbon limitation pullulan induced no more enzyme than did maltose, maltotriose or oligosaccharide mixtures, and ;hyperactivity' never developed on protracted culture. 5. Cyclic AMP relieved the transient repression produced by adding glucose to maltose-limited cultures and a further change to glucose-limited conditions led to constitutive pullulanase synthesis. 6. Amylomaltase and alpha-glucosidase activities were also examined but in less detail. 7. The presence of pullulanase in maltose-limited growth is discussed, but no clear function can be assigned to it at present. The molar growth yields for all the strains were very similar, and no correlation was found between the overgrowth of one strain by another and pullulanase activity. Further, any function as a general branching enzyme in polysaccharide synthesis seems unlikely.     

Relaxation of catabolite repression in streptomycin-dependent Escherichia coli

Acetohydroxy acid synthetase, which is sensitive to catabolite repression in wild-type Escherichia coli B, was relatively resistant to this control in a streptomycin-dependent mutant. The streptomycin-dependent mutant was found to be inducible for beta-galactosidase in the presence of glucose, although repression of beta-galactosidase by glucose occurred under experimental conditions where growth of the streptomycin-dependent mutant was limited. Additional glucose-sensitive enzymes of wild-type E. coli B (citrate synthase, fumarase, aconitase and isocitrate dehydrogenase) were found to be insensitive to the carbon source in streptomycin-dependent mutants: these enzymes were formed by streptomycin-dependent E. coli B in equivalent quantities when either glucose or glycerol was the carbon source. Two enzymes, glucokinase and glucose 6-phosphate dehydrogenase, that are glucose-insensitive in wild-type E. coli B were formed in equivalent quantity on glucose or glycerol in both streptomycin-sensitive and streptomycin-dependent E. coli B. The results indicate a general decrease or relaxation of catabolite repression in the streptomycin-dependent mutant. The yield of streptomycin-dependent cells from glucose was one-third less than that of the streptomycin-sensitive strain. We conclude that the decreased efficiency of glucose utilization in streptomycin-dependent E. coli B is responsible for the relaxation of catabolite repression in this mutant.     

Characterization of sugar transport in 2-deoxy-d-glucose resistant mutants of yeast

Summary A number of 2-deoxy-d-glucose (2-DOG) resistant mutants exhibiting resistance to glucose repression were isolated from variousSaccharomyces yeast strains. Most of the mutants isolated were observed to have improved maltose uptake ability in the presence of glucose. Fermentation studies indicated that maltose was taken up at a faster rate and glucose taken up at a slower rate in the mutant strains compared to the parental strains, when these sugars were fermented together. When these sugars were fermented separately, only the 2-DOG resistant mutant obtained fromSaccharomyces cerevisiae strain 1190 exhibited alterations in glucose and maltose uptake compared to the parental strain. Kinetic analysis of sugar transport employing radiolabelled glucose and maltose indicated that both glucose and maltose were transported with higher rates in the mutant strain. These results suggested that the high affinity glucose transport system was regulated by glucose repression in the parental strain but was derepressed in the mutant.     

Physiology and enzymology of thermophilic anaerobic bacteria degrading starch

Abstract The capability of secreting thermoactive enzymes exhibiting α-amylase and pullulanase with debraching activity, seems to be widely distributed amongst anaerobic thermophilic bacteria. Interestingly, pullulanase formed by these bacteria displays dual specificity by attacking α-1,6- as well as α-1,4-glycosidic linkages in branched glucose polymers. Unlike the enzyme system of aerobic microorganisms the majority of starch hydrolysing enzymes of anaerobic bacteria is metal indepedent and is extremely thermostable. This enzyme system is controlled by substrate induction and catabolite repression; enzyme expression is accomplished when maltose or maltose-containing carbohydrates are used as substrates. By developing a process in continuous culture we were able to greatly enhance enzyme synthesis and release by anaerobic thermophilic bacteria. An elevation in the specific activities of cell-free amylases and pullulanases could also be achieved by entrapping of bacteria in calcium alginate beads. The unique properties of extracellular enzymes of thermophilic anaerobic bacteria makes this group of organisms suitable candidates for inductrial application.     

A defect in carbon catabolite repression associated with uncontrollable and excessive maltose uptake

Summary The previously isolated recessive mutant allele hex2-3 of Saccharomyces cerevisiae caused a defect in carbon catabolite repression of maltase, invertase, malate dehydrogenase, and respiration but at the same time led to an extreme sensitivity to maltose (Zimmermann and Scheel, 1977; Entian and Zimmermann, 1980). Addition of maltose to a growing culture of a hex2-3 mutant resulted within 60 to 90 min in an inhibition of growth, glycolysis, and de novo protein synthesis. This was not accompanied by any abnormal levels of glycolysis metabolites or glycolytic enzyme activities. However, inhibitory effects coincided with a dramatic increase in intracellular glucose up to 150 mM relative to cell water as opposed to 2.5 mM in wild-type cells. This abnormal behavior is interpreted as a result of an uncontrolled maltose uptake in hex2 mutants, which in combination with increasing maltase activity results in an accumulation of intracellular glucose. Obviously the amount of available glucose surpassed glycolytic capacity in hex2 mutants.Properties of mutant alleles hex2 and hex1 (see Entian and Zimmermann, 1980) clearly show, that specific gene functions are involved in adapting the rate of sugar uptake into the cell to the actual glycolytic capacity.     

Regulation of beta-glucosidase in Bacteroides ruminicola by a different mechanism: growth rate-dependent derepression

Bacteroides ruminicola B(1)4, a predominant ruminal and cecal bacterium, was grown in batch and continuous cultures, and beta-glucosidase activity was measured by following the hydrolysis of p-nitrophenyl-beta-glucopyranoside. Specific activity was high when the bacterium was grown in batch cultures containing cellobiose, mannose, or lactose (greater than 286 U/g of protein). Activity was reduced approximately 90% when the organism was grown on glucose, sucrose, fructose, maltose, or arabinose. The specific activity of cells fermenting glucose was initially low but increased as glucose was depleted. When glucose was added to cultures growing on cellobiose, beta-glucosidase synthesis ceased immediately. Catabolite repression by glucose was not accompanied by diauxic growth and was not relieved by cyclic AMP. Since glucose-grown cultures eventually exhibited high beta-glucosidase activity, cellobiose was not needed as an inducer. Catabolite repression explained beta-glucosidase activity of batch cultures and high-dilution-rate chemostats where glucose accumulated, but it could not account for activity at slow dilution rates. Maximal beta-glucosidase activity was observed at a dilution rate of approximately 0.35 h-1, and cellobiose-limited chemostats showed a 15-fold decrease in activity as the dilution rate declined. An eightfold decline was observed in glucose-limited chemostats. Since inducer availability was not a confounding factor in glucose-limited chemostats, the growth rate-dependent derepression could not be explained by other mechanisms.     

Regulation of beta-glucosidase in Bacteroides ruminicola by a different mechanism: growth rate-dependent derepression.

Bacteroides ruminicola B(1)4, a predominant ruminal and cecal bacterium, was grown in batch and continuous cultures, and beta-glucosidase activity was measured by following the hydrolysis of p-nitrophenyl-beta-glucopyranoside. Specific activity was high when the bacterium was grown in batch cultures containing cellobiose, mannose, or lactose (greater than 286 U/g of protein). Activity was reduced approximately 90% when the organism was grown on glucose, sucrose, fructose, maltose, or arabinose. The specific activity of cells fermenting glucose was initially low but increased as glucose was depleted. When glucose was added to cultures growing on cellobiose, beta-glucosidase synthesis ceased immediately. Catabolite repression by glucose was not accompanied by diauxic growth and was not relieved by cyclic AMP. Since glucose-grown cultures eventually exhibited high beta-glucosidase activity, cellobiose was not needed as an inducer. Catabolite repression explained beta-glucosidase activity of batch cultures and high-dilution-rate chemostats where glucose accumulated, but it could not account for activity at slow dilution rates. Maximal beta-glucosidase activity was observed at a dilution rate of approximately 0.35 h-1, and cellobiose-limited chemostats showed a 15-fold decrease in activity as the dilution rate declined. An eightfold decline was observed in glucose-limited chemostats. Since inducer availability was not a confounding factor in glucose-limited chemostats, the growth rate-dependent derepression could not be explained by other mechanisms.