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.     

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.     

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.     

Control of mixed-substrate utilization in continuous cultures of Escherichia coli

The chemostat culture technique was used to study the control mechanisms which operate during utilization of mixtures of glucose and lactose and glucose and l-aspartic acid by populations of Escherichia coli B6. Constitutive mutants were rapidly selected during continuous culture on a mixture of glucose and lactose, and the beta-galactosidase level of the culture increased greatly. After mutant selection, the specific beta-galactosidase level of the culture was a decreasing function of growth rate. In cultures of both the inducible wild type and the constitutive mutant, glucose and lactose were simultaneously utilized at moderate growth rates, whereas only glucose was used in the inducible cultures at high growth rates. Catabolite repression was shown to be the primary mechanism of control of beta-galactosidase level and lactose utilization in continuous culture on mixed substrates. In batch culture, as in the chemostat, catabolite repression acting by itself on the lac enzymes was insufficient to prevent lactose utilization or cause diauxie. Interference with induction of the lac operon, as well as catabolite repression, was necessary to produce diauxic growth. Continuous cultures fed mixtures of glucose and l-aspartic acid utilized both substrates at moderate growth rates, even though the catabolic enzyme aspartase was linearly repressed with increasing growth rate. Although the repression of aspartase paralleled the catabolite repression of beta-galactosidase, l-aspartic acid could be utilized even at very low levels of the catabolic enzyme because of direct anabolic incorporation into protein.     

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.     

Corepressor system for catabolite repression of the lac operon in Escherichia coli

Acetylated amino sugars, normally used in the biosynthesis of cell walls and cell membranes, were found to play a role as corepressors for catabolite repression of the lac operon in Escherichia coli. This conclusion was derived from studies conducted on mutants of E. coli that were able to assimilate an exogenous source of N-acetylglucosamine (AcGN) but were unable to dissimilate or grow on this compound. At concentrations less than 10(-4)m, AcGN caused severe catabolite repression of beta-galactosidase synthesis in cultures grown under either nonrepressed or partially repressed conditions. This repression occurred in the absence of any effect of AcGN on either the carbon and energy metabolism or the growth of the organism. In addition, this repression by AcGN occurred in a mutant strain that is constitutive for beta-galactosidase production, demonstrating that the AcGN effect does not involve the uptake of inducer. This model for the corepressor system of catabolite repression is discussed in relation to the existing theories on repression of the lac operon.     

Regulation of transcription of the Escherichia coli phosphoenolpyruvate carboxykinase locus: studies with pck-lacZ operon fusions

Mutants of Escherichia coli containing genetic fusions of lacZ to the pck (phosphoenolpyruvate carboxykinase) locus were isolated by using Mu d(lacZ Ampr) bacteriophage. Synthesis of beta-galactosidase in these strains is regulated by cyclic AMP and glucose (catabolite repression). Synthesis of beta-galactosidase by pck-lacZ fusions was induced in log-phase cells growing on gluconeogenic media, was repressed by glucose, and was also induced up to 100-fold at the onset of stationary phase in LB medium. This stationary-phase induction required cyclic AMP and some other unknown regulatory signal.     

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.     

Regulation of formation of the intracellular beta-galactosidase activity of Aspergillus nidulans

The regulation of formation of the single intracellular beta-galactosidase activity of Aspergillus nidulans was investigated. beta-Galactosidase was not formed during growth on glucose or glycerol, but was rapidly induced during growth on lactose or D-galactose. L-Arabinose, and -- with lower efficacy -- D-xylose also induced beta-galactosidase activity. Addition of glucose to cultures growing on lactose led to a rapid decrease in beta-galactosidase activity. In contrast, in cultures growing on D-galactose, addition of glucose decreased the activity of beta-galactosidase only slightly. Glucose inhibited the uptake of lactose, but not of D-galactose, and required the carbon catabolite repressor CreA for this. In addition, CreA also repressed the formation of basal levels of beta-galactosidase and partially interfered with the induction of beta-galactosidase by D-galactose, L-arabinose, and D-xylose. D-Galactose phosphorylation was not necessary for beta-galactosidase induction, since induction by D-galactose occurred in an A. nidulans mutant defective in galactose kinase, and by the non-metabolizable D-galactose analogue fucose in the wild-type strain. Interestingly, a mutant in galactose-1-phosphate uridylyl transferase produced beta-galactosidase at a low, constitutive level even on glucose and glycerol and was no longer inducible by D-galactose, whereas it was still inducible by L-arabinose. We conclude that biosynthesis of the intracellular beta-galactosidase of A. nidulans is regulated by CreA, partially repressed by galactose-1-phosphate uridylyl transferase, and induced by D-galactose and L-arabinose in independent ways.     

Catabolite repression of inositol dehydrogenase and gluconate kinase syntheses in Bacillus subtilis

The regulation of induction of inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) was studied in Bacillus subtilis. Inositol dehydrogenase is induced by myo-inositol and gluconate kinase is induced by D-gluconate. Both inductions were strongly repressed by rapidly metabolizable carbohydrates such as D-glucose, D-mannose, D-fructose and glycerol (D-glucose had the strongest repressive effect) but they were weakly repressed by slowly metabolizable carbohydrates. Although each carbohydrate exerted a stronger effect on the induction of inositol dehydrogenase than that of gluconate kinase, it showed a similar tendency with respect to the degree of repression of each induction. This catabolite repression could not be diminished by addition of cyclic AMP to medium. In addition, non-metabolizable D-glucose analogues had no or weak repressive effects. On the assumption that rapidly metabolizable carbohydrates might be metabolized to repress both inductions, it was investigated whether several mutants blocked in the Embden-Meyerhof pathway could produce metabolite(s) (repressor) to repress them. A phosphoglycerate kinase (EC 2.7.2.3) deficient mutant could produce the repressor from D-glucose, D-mannose, D-fructose and glycerol but other mutants could not produce it from carbohydrates unable to be metabolized ineach mutant. Thus, catabolite repression of both enzyme inductions seemed to be under similar regulation. The identification of the possible repressor of the induction of inositol dehydrogenase and gluconate kinase in vivo was discussed.     

Glycerol kinase, the pacemaker for the dissimilation of glycerol in Escherichia coli

The activity of glycerol kinase is rate-limiting in the metabolism of glycerol by cells of Escherichia coli. A mutant strain producing a glycerol kinase resistant to inhibition by fructose-1,6-diphosphate grows faster than its wild-type parent on glycerol as the sole source of carbon and energy. The amount of intracellular fructose-1,6-diphosphate was determined for wild-type cells growing exponentially on glycerol. The water content of such cells was also determined, allowing calculation of the intracellular concentration of fructose-1,6-diphosphate. This value, 1.7 mm, is adequate to exert substantial inhibition on the wild-type glycerol kinase. The desensitization of glycerol kinase to feedback inhibition also enhances the power of glycerol to exert catabolite repression, both on the enzymes of the glycerol system itself and on those of the lactose system. However, desensitization of glycerol kinase alone does not eliminate the phenomenon of diauxic growth in a glucose-glycerol medium. Biphasic growth in such a medium is abolished if the altered enzyme is produced constitutively. The constitutive production of the mutant kinase at high levels, however, renders the cells vulnerable to glycerol. Thus, when the cells have been grown on a carbon source with a low power for catabolite repression, e.g., succinate, sudden exposure to glycerol leads to overconsumption of the nutrient and cell death.     

Three Kinds of Controls Affecting the Expression of the glp Regulon in Escherichia coli

Three kinds of control mechanisms govern the expression of the members of the glp regulon for glycerol and sn-glycerol 3-phosphate (G3P) catabolism in Escherichia coli K-12: specific repression by the product of the glpR gene; catabolite repression; and respiratory repression (the effect exerted by exogenous hydrogen acceptors). The operons of the glp system show different patterns of response to each control. By growing in parallel a mutant strain with temperature-sensitive repressor (glpR(ts)) and an isogenic control with a deletion in the regulator gene at progressively higher temperatures, it was possible to show that the synthesis of aerobic G3P dehydrogenase (glpD product) is far more sensitive to specific repression than that of either glycerol kinase (glpK product) or G3P transport (glpT product). Conversely, in the strain with a deletion in the regulator gene, the syntheses of glycerol kinase and G3P transport are more sensitive to catabolite repression than that of the aerobic G3P dehydrogenase. The levels of the two flavoprotein G3P dehydrogenases vary in opposite directions in response to changes of exogenous hydrogen acceptors. For example, the ratio of the aerobic enzyme to the anaerobic enzyme (specified by glpA) is high when molecular oxygen or nitrate serves as the hydrogen acceptor and low when fumarate plays this role. This trend is not influenced by the addition of cyclic adenosine 3',5'-monophosphate to the growth medium. Thus, respiratory repression most likely involves a third mechanism of control, independent of specific or catabolite repression.     

Effect of Amino Sugars on Catabolite Repression in Escherichia coli

N-acetylglucosamine was found to be a good repressor source for catabolite repression of the beta-galactosidase system in Escherichia coli. It was found capable of increasing the severity of repression by glucose or gluconate when included in the medium with either of these substrates. N-acetylglucosamine was shown to be assimilated under these conditions, but had no effect on culture growth rates. Its influence on catabolite repression was not altered by growth in the presence of inhibiting levels of penicillin. These findings indicated that catabolite repression may be associated with certain reactions of amino sugar metabolism. A working model has been formulated along these lines and will be used to explore this possible relationship further.     

Cyclic 3′,5′-Adenosine Monophosphate and N-Acetyl-glucosamine-6-Phosphate as Regulatory Signals in Catabolite Repression of the lac Operon in Escherichia coli

When an Escherichia coli mutant lacking the enzyme N-acetyl-glucosamine-6-phosphate (AcGN6P) deacetylase is grown in a succinate-mineral salts medium and exposed to an exogenous source of N-acetylglucosamine, approximately 20 to 30 pmoles of AcGN6P per mug of cell dry weight will accumulate in these cells. This accumulation occurs within 2 to 4 min after the addition of N-acetylglucosamine and is coincident with the production of a severe permanent catabolite repression of beta-galactosidase synthesis. This repression does not occur if adenosine 3',5'-cyclic phosphate (cyclic AMP) is added to the cells before AcGN6P accumulates. An immediate derepression occurs when cyclic AMP is added to cells that have already accumulated a large AcGN6P pool. These findings are consistent with the view that low-molecular-weight carbohydrate metabolites and cyclic AMP play key roles in the catabolite repression phenomenon, and that metabolites such as AcGN6P may participate in the represion mechanism by influencing either the formation or degradation of cyclic AMP in E. coli.     

Regulation of tyramine oxidase synthesis in Klebsiella aerogenes.

Tyramine oxidase in Klebsiella aerogenes is highly specific for tyramine, dopamine, octopamine, and norepinephrine, and its synthesis is induced specifically by these compounds. The enzyme is present in a membrane-bound form. The Km value for tyramine is 9 X 10(-4) M. Tyramine oxidase synthesis was subjected to catabolite repression by glucose in the presence of ammonium salts. Addition of cyclic adenosine 3',5'-monophosphate (cAMP) overcame the catabolite repression. A mutant strain, K711, which can produce a high level of beta-galactosidase in the presence of glucose and ammonium chloride, can also synthesize tyramine oxidase and histidase in the presence of inducer in glucose ammonium medium. Catabolite repression of tyramine oxidase synthesis was relieved when the cells were grown under conditions of nitrogen limitation, whereas beta-galactosidase was strongly repressed under these conditions. A cAMP-requiring mutant, MK54, synthesized tyramine oxidase rapidly when tyramine was used as the sole source of nitrogen in the absence of cAMP. However, a glutamine synthetase-constitutive mutant, MK94, failed to synthesize tyramine oxidase in the presence of glucose and ammonium chloride, although it synthesized histidase rapidly under these conditions. These results suggest that catabolite repression of tyramine oxidase synthesis in K. aerogenes is regulated by the intracellular level of cAMP and an unknown cytoplasmic factor that acts independently of cAMP and is formed under conditions of nitrogen limitation.     

The role of the regulator-gene product (repressor) in catabolite repression of β-galactosidase synthesis in Escherichia coli

1. The specific role of the lac repressor (i-gene product) in transient catabolite repression evoked by the introduction of glucose into the medium has been investigated in Escherichia coli by using mutants of the i-gene. 2. A temperature-sensitive mutant (i(TL)) is normally inducible and demonstrates transient repression when grown at 32 degrees . At 42 degrees it is about 20% constitutive and transient catabolite repression is abolished. 3. A strain carrying an amber suppressor-sensitive mutation in the i-gene is phenotypically constitutive and also fails to show transient catabolite repression. 4. Insertion of Flaci(+) into this strain restores both inducibility and transient repression. 5. It is concluded that the i-gene product interacts with the catabolite co-repressor in such a way that its affinity for the operator is increased.