Involvement of the lac regulatory genes in catabolite repression in Escherichia coli

1. Acute transient catabolite repression of beta-galactosidase synthesis, observed when glucose is added to glycerol-grown cells of Escherichia coli (Moses & Prevost, 1966), requires the presence of a functional operator gene (o) in the lactose operon. Total deletion of the operator gene abolished acute transient repression, even in the presence of a functional regulator gene (i). 2. Regulator constitutives (i(-)) also show transient repression provided that the operator gene is functional. Regulator deletion mutants (i(del)), with which to test specifically the role of the i gene, have not so far been available. 3. The above mutants, showing various changes in the lactose operon, show no alteration in the effect of glucose on induced tryptophanase synthesis. Glucose metabolism, as measured in terms of the release of (14)CO(2) from [1-(14)C]glucose and [6-(14)C]glucose, also showed no differences between strains exhibiting or not exhibiting transient repression. This suggests no change in the operation of the pentose phosphate cycle, a metabolic activity known to be of paramount importance for glucose repression of beta-galactosidase synthesis (Prevost & Moses, 1967). 4. Chronic permanent repression by glucose of beta-galactosidase synthesis (less severe in degree than acute transient repression) persists in strains in which transient repression has been genetically abolished. Constitutive alkaline-phosphatase synthesis, which shows no transient repression, also demonstrates chronic permanent repression by glucose. 5. Chloramphenicol repression also persists in mutants with no transient repression, and also affects alkaline phosphatase. It is suggested that chronic permanent repression and chloramphenicol repression are non-specific, and that they do not influence beta-galactosidase synthesis via the regulatory system of the lactose operon.     

Two types of glucose effects on beta-galactosidase synthesis in a membrane fraction of Escherichia coli: correlation with repression observed in intact cells.

A membrane fraction obtained from an osmotic lysate of Escherichia coli spheroplasts retains capability to synthesize beta-galactosidase. The system also retains cellular regulatory functions, one of which is known as catabolite repression. Two types of repression of beta-galactosidase synthesis were observed in this membrane system: one was caused by the addition of 2-deoxyglucose or glucose at a low concentration (3 times 10- minus 4 M), and the other was caused by glucose-6-phosphate or glucose at a high concentration (3 times 10- minus 2 M). In the presence of cyclic adenosine 3',5'-monophosphate (10 mM), repression caused by the former was completely reversed, whereas repression by the latter was only partially reversed. Conditions in intact cells causing transient and permanent repression were also investigated. Upon addition of 2-deoxyglucose or glucose at a low concentration to intact cells, only transient repression of beta-galactosidase synthesis was observed. Glucose at a high concentration caused both transient and subsequent permanent repression, and intensity of permanent repression depended upon glucose concentration, whereas duration and intensity of transient repression were independent of glucose concentration. Mutants deficient in phosphoenolpyruvate-phosphotransferase system (Hpr minus and enzyme I minus) showed transient repression but failed to show permanent repression. In mutants deficient in glucose catabolism beyond glucose-6-phosphate, both transient and permanent repression were observed. Correlation between the observations in the membrane system and in intact cells is discussed. The results obtained here strongly suggest that transient repression is caused by glucose itself, and that permanent repression is caused by glucose-6-phosphate of high intracellular levels of glucose.     

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.     

Catabolite repression of tryptophanase in Escherichia coli

Catabolite repression of tryptophanase was studied in detail under various conditions in several strains of Escherichia coli and was compared with catabolite repression of beta-glactosidase. Induction of tryptophanase and beta-galactosidase in cultures grown with various carbon sources including succinate, glycerol, pyruvate, glucose, gluconate, and arabinose is affected differently by the various carbon sources. The extent of induction does not seem to be related to the growth rate of the culture permitted by the carbon source during the course of the experiment. In cultures grown with glycerol as carbon source, preinduced for beta-galactosidase or tryptophanase and made permeable by ethylenediaminetetraacetic acid (EDTA) treatment, catabolite repression of tryptophanase was not affected markedly by the addition of cAMP (3',5'-cyclic adenosine monophosphate). Catabolite repression by glucose was only partially relieved by the addition of cAMP. In contrast, under the same conditions, cAMP completely relieved catabolite repression of beta-galactosidase by either pyruvate or glucose. Under conditions of limited oxygen, induction of tryptophanase is sensitive to catabolite repression; under the same conditions, beta-galactosidase induction is not sensitive to catabolite repression. Induction of tryptophanase in cells grown with succinate as carbon source is sensitive to catabolite repression by glycerol and pyruvate as well as by glucose. Studies with a glycerol kinaseless mutant indicate that glycerol must be metabolized before it can cause catabolite repression. The EDTA treatment used to make the cells permeable to cAMP was found to affect subsequent growth and induction of either beta-galactosidase or tryptophanase much more adversely in E. coli strain BB than in E. coli strain K-12. Inducation of tryptophanase was reduced by the EDTA treatment significantly more than induction of beta-galactosidase in both strains. Addition of 2.5 x 10(-3)m cAMP appeared partially to reverse the inhibitory effect of the EDTA treatment on enzyme induction but did not restore normal growth.     

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.     

Nature of the effector of catabolite repression of beta-galactosidase in Escherichia coli

Loomis, William F., Jr. (Massachusetts Institute of Technology, Cambridge, Mass.), and Boris Magasanik. Nature of the effector of catabolite repression of beta-galactosidase in Escherichia coli. J. Bacteriol. 92:170-177. 1966.-Many carbon sources were found to give rise to catabolite repression of beta-galactosidase in a mutant strain of Escherichia coli lacking hexose phosphate isomerase activity. Compounds containing glucose or galactose cannot be formed from several of these carbon sources in this mutant strain, and, therefore, appear not to be required for catabolite repression of beta-galactosidase. Glucose was observed to elicit catabolite repression of beta-galactosidase in another mutant strain under conditions in which the formation of compounds of the citric acid cycle is inhibited. If catabolite repression of the lac operon is mediated by a single compound, it appears that the compound is related to the pentoses and trioses of intermediary metabolism. The repression of beta-galactosidase by galactose in galactokinase negative strains was shown to be independent of the gene, CR, which determines catabolite sensitivity of the lac operon, and to be dependent on a functional i gene.     

Elucidation of enzyme control mechanisms using macroscopic measurements in a mixed substrate fermentation system

The objective of this work was to relate macroscopically measurable on-line fermentation parameters such as dissolved oxygen, off-gas oxygen and carbon dioxide, and cell mass, to the controlled production of key intracellular enzymes under carbon limited conditions. Both batch and perturbed batch aerobic fermentations were performed using two different strains of Escherichia coli, with glucose and lactose as the sole carbon sources. The two strains differed from each other only in the lac operon region of their genome. The parent strain, E. coli 3000, was inducible for the enzyme beta-galactosidase. The other strain, E. coli 3300, was a constitutive mutant in the production of beta-galactosidase. In all experiments, off-line assays of sugars and beta-galactosidase activity were performed. It was observed that there is a clear relationship between the macroscopic on-line measurements, dissolved oxygen tension, carbon dioxide evolution rate and oxygen uptake rate, and the microscopic control phenomena of catabolite repression, catabolite inhibition, and inducer repression.     

Transient repression of beta-galactosidase synthesis by glucose-6-phosphate in a mutant of Escherichia coli lacking enzyme II specific for glucose in the phosphoenolpyruvate-sugar phosphotransferase system.

The effects of glucose and glucose-6-phosphate in initiating the repression of beta-galactosidase synthesis were studied using a mutant of Escherichia coli K12 which lacks glucose-specific enzyme II of the phosphoenolpyruvate-sugar phosphotransferase system. It was found that glucose-6-phosphate causes transient repression of beta-galactosidase synthesis but glucose does not cause transient repression in this mutant. Evidence was obtained that both the presence of an active transport system for glucose-6-phosphate in the cells and glucose-6-phosphate in the medium are necessary for the initiation of transient repression. No metabolism of glucose-6-phosphate is required. Upon depletion of glucose-6-phosphate in the medium the transient repression was reversed. After the reversal the rate of enzyme synthesis was high in the cells which had been exposed to a high concentration of glucose-6-phosphate. It was concluded that the translocation of glucose-6-phosphate across the membranes is the primary event which affects both the initiation of and the recovery from the transient repression. During the transient repression the cellular content of cyclic adenosine 3',5'-monophosphate decreased significantly.     

Effect of growth conditions on catabolite repression and cyclic AMP synthesis in Escherichia coli 3000A1

Catabolite repression of beta-galactosidase synthesis in E. coli 3000A1 (adenine-) was studied under a variety of growth conditions. The differential rate of induced beta-galactosidase synthesis was maximal at the growth rate of 0.75 division per h, irrespective of whether growth conditions were aerobic or anaerobic. The addition of cyclic AMP (cAMP) to the medium partly restored the repressed synthesis of beta-galactosidase under some growth conditions, but showed little or no effect on the enzyme synthesis under other conditions. Although growth rate and profile of beta-galactosidase synthesis in glucose-grown cells were similar to those in arabinose-grown cells, the acceleration of beta-galactosidase synthesis upon the addition of cAMP was found only in glucose-grown cells. The cells aerobically grown in the presence of glycerol, xylose, or arabinose showed a high synthetic rate of cAMP and were insensitive to exogenously supplied cAMP as regards beta-galactosidase synthesis. Although the cells grown with glucose showed similar rates of cAMP synthesis under aerobic and anaerobic conditions, the differential rate of beta-galactosidase synthesis was much higher in the anaerobic state than in the aerobic state. These findings support the idea that catabolite repression found in the strain is caused through two mechanisms, i.e., cAMP-mediated and cAMP-independent ones.     

Evidence against the involvement of adenosine 3',5'-cyclic monophosphate in glucose inhibition of beta-galactosidase induction in Bacillus megaterium.

When Bacillus megaterium cells are grown on D-galactose as the sole carbon source, the cells actively synthesize beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23). However, D-galactose, when added to a glucose-grown culture, did not induce beta-galactosidase, apparently because of the glucose inhibition of the transport of galactose. On the other hand, when glucose was added to a galactose-grown culture, the transport of galactose continued at a reduced but significate rate, whereas further synthesis of beta-galactosidase was halted. Adenosine 3',5'-cyclic monophosphate (camp) or guanosine 3',5'-cyclic monophosphate (Cgmp) did not relieve the glucose inhibition of beta-galactosidase synthesis in the preinduced culture. A method which gave a reproducible assay of c[32P]AMP in Escherichia coli did not detect cAMP or cGMP in a B. megaterium culture undergoing beta-galactosidase induction, but revealed the extracellular accumulation of two unknown phosphorylated compounds. Cell-free extracts prepared from galactose-grown cells did not catalyze the degradation of cAMP or cGMP.     

Galactose repression of beta-galactosidase induction in Escherichia coli

Beggs, William H. (University of Minnesota, Minneapolis), and Palmer Rogers. Galactose repression of beta-galactosidase induction in Escherichia coli. J. Bacteriol. 91:1869-1874. 1966.-Galactose repression of beta-galactosidase induction in Escherichia coli was investigated to determine whether the galactose molecule itself is the catabolite repressor of this enzyme system. Without exception, beta-galactosidase induction by cells grown in a synthetic salts medium with lactate or glycerol as the carbon source was more strongly repressed by glucose than by galactose. This relationship existed even when the organism was previously grown in the synthetic medium containing galactose as the source of carbon. Two observations suggested that the ability of galactose to repress beta-galactosidase formation by Escherichia coli depends directly upon the cells' capacity to catabolize galactose. First, galactose repression of beta-galactosidase synthesis was markedly enhanced in bacteria tested subsequent to gratuitous induction of the galactose-degrading enzymes with d-fucose. Second, galactose failed to exert a repressive effect on beta-galactosidase in a galactose-negative mutant lacking the first two enzymes involved in galactose catabolism. Glucose completely repressed enzyme formation in this mutant. This same mutant, into which the genes for inducible galactose utilization had been introduced previously by transduction, again exhibited galactose repression. Pyruvate was found to be at least as effective as galactose in repressing beta-galactosidase induction by cells grown in synthetic salts medium plus glycerol. It is concluded that the galactose molecule itself is not the catabolite repressor of beta-galactosidase, but that repression is exerted through some intermediate in galactose catabolism.     

Aspartase-hyperproducing mutants of Escherichia coli B

When a wild-type strain of Escherichia coli B was cultured on a medium containing L-aspartic acid as the sole carbon source (Asp-C medium), aspartase formation was higher than that observed in minimal medium. Addition of glucose to Asp-C medium decreased aspartase formation. When also cultured in a medium containing L-aspartic acid as the sole nitrogen source (Asp-N medium), E. coli B showed a low level of aspartase formation and an elongated doubling time. To obtain aspartase-hyperproducing strains, we enriched cells growing faster than cells of the wild-type strain in Asp-N medium by continuous cultivation of mutagenized cells. After plate selection, the doubling times of these mutants were measured. Thereafter, fast-growing mutants were tested for aspartase formation. One of these mutants, strain EAPc7, had a higher level of aspartase formation than did the wild-type strain in medium containing L-aspartic acid as the carbon source, however; addition of glucose to this medium decreased aspartase formation. The other mutant, strain EAPc244, had a higher level of aspartase activity than did the wild-type strain in both media. Therefore, aspartase formation in mutant EAPc244 was released from catabolite repression. In strain EAPc244 the other catabolite-repressible enzymes, beta-galactosidase, tryptophanase, and the three tricarboxylic acid cycle enzymes, were also released from catabolite repression. Both mutants had sevenfold the aspartase formation of the wild-type strain in a medium which contained fumaric acid as the main carbon source and which has been used for industrial production of E. coli B aspartase. However, strain EAPc244 had 2.5-fold the fumarase activity of strain EAPc7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aspartase-hyperproducing mutants of Escherichia coli B.

When a wild-type strain of Escherichia coli B was cultured on a medium containing L-aspartic acid as the sole carbon source (Asp-C medium), aspartase formation was higher than that observed in minimal medium. Addition of glucose to Asp-C medium decreased aspartase formation. When also cultured in a medium containing L-aspartic acid as the sole nitrogen source (Asp-N medium), E. coli B showed a low level of aspartase formation and an elongated doubling time. To obtain aspartase-hyperproducing strains, we enriched cells growing faster than cells of the wild-type strain in Asp-N medium by continuous cultivation of mutagenized cells. After plate selection, the doubling times of these mutants were measured. Thereafter, fast-growing mutants were tested for aspartase formation. One of these mutants, strain EAPc7, had a higher level of aspartase formation than did the wild-type strain in medium containing L-aspartic acid as the carbon source, however; addition of glucose to this medium decreased aspartase formation. The other mutant, strain EAPc244, had a higher level of aspartase activity than did the wild-type strain in both media. Therefore, aspartase formation in mutant EAPc244 was released from catabolite repression. In strain EAPc244 the other catabolite-repressible enzymes, beta-galactosidase, tryptophanase, and the three tricarboxylic acid cycle enzymes, were also released from catabolite repression. Both mutants had sevenfold the aspartase formation of the wild-type strain in a medium which contained fumaric acid as the main carbon source and which has been used for industrial production of E. coli B aspartase. However, strain EAPc244 had 2.5-fold the fumarase activity of strain EAPc7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-induction of beta-galactosidase and the lactose-P-enolpyruvate phosphotransferase system in Streptococcus salivarius and Streptococcus mutans.

The addition of lactose, galactose, or isopropyl-beta-D-thiogalactoside (IPTG) to glucose-grown cells of Streptococcus salivarius 25975 resulted in the co-induction of both the lactose-P-enolpyruvate phosphotransferase system (lactose-PTS) and beta-galactosidase, with the latter the predominant metabolic system. With various strains of Streptococcus mutans and Streptococcus sanguis 10556, on the other hand, the lactose-PTS was the major metabolic pathway with beta-galactosidase induced either to low or negligible levels. In all cases, induction of the lactose-PTS resulted in the concomitant induction of 6-P-beta-galactosidase. The induction by lactose of both the lactose-PTS and beta-galactosidase in all strains was repressed by glucose and other catabolites, notably, fructose. Induction of beta-galactosidase in S. salivarius 25975 by IPTG was, however, relatively resistant to glucose repression. Induction experiments with IPTG and lactose suggested that a cellular metabolite of lactose metabolism was a repressor of enzyme activity. Exogenous cAMP was shown to reverse the transient repression by glucose of beta-galactosidase induction in cells of S. salivarius 25975 receiving lactose, provided the cells were grown with small amounts of toluene to overcome the permeability barrier to this nucleotide, cAMP, was however, unable to overcome the permanent repression of beta-galactosidase activity to a significant extent under these conditions.     

Isolation and characterization of catabolite repression-resistant mutants of Escherichia coli.

A method has been developed for the isolation of Escherichia coli mutants which are resistant to catabolic repression. The method is based on the fact that a mixture of glucose and gluconate inhibits the development of chemotactic motility in the wild type, but not in the mutants. A motile E. coli strain was mutagenized and grown in glucose and gluconate. Mutants which were able to swim into a tube containing a chemotactic attractant (aspartic acid) were isolated. Most of these mutants were able to produce beta-galactosidase in the presence of glucose and gluconate and were normal in their ability to degrade adenosine 3',5-cyclic monophosphate. Some of these mutants were defective in the glucose phosphotransferase system.     

Physiological studies of beta-galactosidase induction in Kluyveromyces lactis

We examined the kinetics of beta-galactosidase (EC 3.2.1.23) induction in the yeast Kluyveromyces lactis. Enzyme activity began to increase 10 to 15 min, about 1/10 of a cell generation, after the addition of inducer and continued to increase linearly for from 7 to 9 cell generations before reaching a maximum, some 125- to 150-fold above the basal level of uninduced cells. Thereafter, as long as logarithmic growth was maintained, enzyme levels remained high, but enzyme levels dropped to a value only 5- to 10-fold above the basal level if cells entered stationary phase. Enzyme induction required the constant presence of inducer, since removal of inducer caused a reduction in enzyme level. Three nongratuitous inducers of beta-galactosidase activity, lactose, galactose, and lactobionic acid, were identified. Several inducers of the lac operon of Escherichia coli, including methyl-, isopropyl- and phenyl-1-thio-beta-d-galactoside, and thioallolactose did not induce beta-galactosidase in K. lactis even though they entered the cell. The maximum rate of enzyme induction was only achieved with lactose concentrations of greater than 1 to 2 mM. The initial differential rate of beta-galactosidase appearance after induction was reduced in medium containing glucose, indicating transient carbon catabolite repression. However, glucose did not exclude lactose from K. lactis, it did not cause permanent carbon catabolite repression of beta-galactosidase synthesis, and it did not prevent lactose utilization. These three results are in direct contrast to those observed for lactose utilization in E. coli. Furthermore, these results, along with our observation that K. lactis grew slightly faster on lactose than on glucose, indicate that this organism has evolved an efficient system for utilizing lactose.     

Inducer exclusion by glucose 6-phosphate in Escherichia coli

The main mechanism causing catabolite repression by glucose and other carbon sources transported by the phosphotransferase system (PTS) in Escherichia coli involves dephosphorylation of enzyme IIA^Glc as a result of transport and phosphorylation of PTS carbohydrates. Dephosphorylation of enzyme IIA^Glc leads to 'inducer exclusion': inhibition of transport of a number of non-PTS carbon sources (e.g. lactose, glycerol), and reduced adenylate cyclase activity. In this paper, we show that the non-PTS carbon source glucose 6-phosphate can also cause inducer exclusion. Glucose 6-phosphate was shown to cause inhibition of transport of lactose and the non-metabolizable lactose analogue methyl-β- D -thiogalactoside (TMG). Inhibition was absent in mutants that lacked enzyme IIA^Glc or were insensitive to inducer exclusion because enzyme IIA^Glc could not bind to the lactose carrier. Furthermore, we showed that glucose 6-phosphate caused dephosphorylation of enzyme IIA^Glc. In a mutant insensitive to enzyme IIA^Glc-mediated inducer exclusion, catabolite repression by glucose 6-phosphate in lactose-induced cells was much weaker than that in the wild-type strain, showing that inducer exclusion is the most important mechanism contributing to catabolite repression in lactose-induced cells. We discuss an expanded model of enzyme IIA^Glc-mediated catabolite repression which embodies repression by non- PTS carbon sources.     

Glucose-induced hyperaccumulation of cyclic AMP and defective glucose repression in yeast strains with reduced activity of cyclic AMP-dependent protein kinase.

Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-mediated cyclic AMP (cAMP) signal that induces a protein phosphorylation cascade. In yeast mutants (tpk1w1, tpk2w1, and tpk3w1) containing reduced activity of cAMP-dependent protein kinase, fermentable sugars, as opposed to nonfermentable carbon sources, induced a permanent hyperaccumulation of cAMP. This finding confirms previous conclusions that fermentable sugars are specific stimulators of cAMP synthesis in yeast cells. Despite the huge cAMP levels present in these mutants, deletion of the gene (BCY1) coding for the regulatory subunit of cAMP-dependent protein kinase severely reduced hyperaccumulation of cAMP. Glucose-induced hyperaccumulation of cAMP was also observed in exponential-phase glucose-grown cells of the tpklw1 and tpk2w1 strains but not the tpk3w1 strain even though addition of glucose to glucose-repressed wild-type cells did not induce a cAMP signal. Investigation of mitochondrial respiration by in vivo 31P nuclear magnetic resonance spectroscopy showed the tpk1w1 and tpk2w1 strains, to be defective in glucose repression. These results are consistent with the idea that the signal transmission pathway from glucose to adenyl cyclase contains a glucose-repressible protein. They also show that a certain level of cAMP-dependent protein phosphorylation is required for glucose repression. Investigation of the glucose-induced cAMP signal and glucose-induced activation of trehalase in derepressed cells of strains containing only one of the wild-type TPK genes indicates that the transient nature of the cAMP signal is due to feedback inhibition by cAMP-dependent protein kinase.