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.     

Regulation of galactose operon expression: glucose effects and role of cyclic adenosine 3',5'-monophosphate.

We studied the following two aspects of the glucose effect on galactose operon expression in Escherichia coli K-12: catabolite repression and inducer exclusion. Using both inducible and constitutive strains and measuring the rate of promoter-proximal enzyme synthesis, we found that the galactose operon did not seem to exhibit catabolite repression. The only glucose effect on galactose operon expression which we observed was inducer exclusion, as shown by the existence of diauxic growth in the presence of glucose and galactose. This diauxie was not relieved by cyclic adenosine 3',5'-monophosphate. Cyclic adenosine 3',5'-monophosphate did not seem to be an antagonist of any glucose effect on galactose operon expression; its only effect was to stimulate promoter-distal gene expression.     

Cyclic adenosine 3',5'-monophosphate levels in Pseudomonas putida and Pseudomonas aeruginosa during induction and carbon catabolite repression of histidase synthesis.

Inducibility of histidase (histidine ammonia-lyase, EC 4.3.1.3) in Pseudomonas putida and Pseudomonas aeruginosa was observed to be strongly affected by succinate-provoked catabolite repression, but this did not occur as a consequence of reduced intracellular cyclic adenosine 3',5'-monophosphate levels, and repression could not be alleviated by exogenously added cyclic adenosine 3,'5'-monophosphate. Milder repression of histidase by lactate was also not reversed by the addition of cyclic adenosine 3',5'-monophosphate. These results, along with data showing intracellular cyclic adenosine 3',5'-monophosphate levels remained essentially constant during growth on such diverse carbon sources as histidine, acetamide, glucose, and succinate, indicated that catabolite repression of histidase synthesis by efficient carbon sources was not mediated through variations in internal cyclic adenosine 3,'5'-monophosphate.     

Catabolite and transient repression in Escherichia coli do not require enzyme I of the phosphotransferase system.

Transient and catabolite repression with changes in intracellular concentrations of cyclic adenosine 3',5-monophosphate is produced by glycerol and by glucose-6-phosphate in a strain with a partial deletion of the structural gene for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.     

Preparation of Mutants Resistant to Catabolite Repression of Trichoderma reesei

The development of agar plate screening techniques has allowed the isolation of mutants of Trichoderma reesei capable of synthesizing cellulase under the conditions of a high concentration of glucose. Mutants resistant to catabolite repression by glycerol or glucose were isolated on Walseth’s cellulose (WC) agar plates containing 5% glycerol or 5% glucose, respectively. Mutants resistant to catabolite repression by glycerol were not derepressed enough for the production of cellulase on WC agar plates containing 5% glucose or in flask cultures with a mixture of 1% Avicel and 3% glucose. On the contrary, two mutant strains resistant to catabolite repression by glucose (KDD-10 and DGD-16) produced large clearing zones on WC agar plates containing 5% glucose. Both strains could begin to produce CMCase even in the presence of residual glucose and finally produced 1.5 times the CMCase activity, in flask cultures on 1% Avicel and 3% glucose, than that with 1% Avicel alone. These results suggest that KDD-10 and DGD-16 are comparatively derepressed by glucose for cellulase production.     

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.     

Adenosine 3′:5′-cyclic monophosphate and catabolite repression in Escherichia coli

1. Both permanent and transient catabolite repression of beta-galactosidase synthesis in Escherichia coli are abolished by 5mm-3':5'-cyclic-AMP when elicited by glucose, but not when caused by a mixture of glucose, glucose 6-phosphate, gluconate and casein hydrolysate (casamino acids). 2. Glucose uptake is slightly increased by 3':5'-cyclic-AMP. 3. No significant effects of the nucleotide were found on the synthesis of protein and RNA, either in exponential growth on one substrate, or during a growth shift from glycerol to glycerol plus glucose. 4. Marked changes in the soluble-protein profiles of cells growing in glycerol and glucose were caused by the presence of 3':5'-cyclic-AMP. 5. Measurements of (14)CO(2) release from specifically-labelled glucose showed that 3':5'-cyclic-AMP greatly stimulated glycolytic activity while having a minor depressing effect on the metabolic flow through the pentose phosphate cycle. 6. The concentrations of several metabolic intermediates, particularly fructose 1,6-diphosphate, were greatly affected by the presence of 3':5'-cyclic-AMP. 7. Several metabolites partially relieved glucose repression of beta-galactosidase synthesis in EDTA-treated cells; three out of five of these metabolites reversed the effect more effectively than did 3':5'-cyclic-AMP. 8. The evidence for and against a direct role for 3':5'-cyclic-AMP is discussed. It is concluded that the evidence for indirect action is at least as strong as that for direct action.     

Regulation of arginine and proline catabolism in Bacillus licheniformis

The enzymes in the arginine breakdown pathway (arginase, ornithine-delta-transaminase, and Delta'-pyrroline-5-carboxylate dehydrogenase) were found to be present in Bacillus licheniformis cells during exponential growth on glutamate. These enzymes could be coincidentally induced by arginine or ornithine to a very high level and their synthesis could be repressed by the addition of glucose, clearly demonstrating catabolite repression control of the arginine degradative pathway. The strongest catabolite repression control of arginase occurred when cells were grown on glucose and this control decreased when cells were grown on glycerol, acetate, pyruvate, or glutamate. The proline catabolite pathway was present in B. licheniformis during exponential growth on glutamate. The proline oxidation and the Delta'-pyrroline-5-carboxylate dehydrogenase in this breakdown pathway were induced by l-proline to a high level. The Delta'-pyrroline-5-carboxylate dehydrogenase was found to be under catabolite repression control. Arginase could be induced by proline and arginine addition induced proline oxidation, suggesting a common in vivo inducer for these convergent pathways.     

Isolation and characterization of catabolite-resistant mutants in the D-serine deaminase system of Escherichia coli K-12.

Two classes of D-serine deaminase (Dsdase)-specific secondary mutants of Escherichia coli K-12 were isolated from a Dsdase low constitutive nonhyperinducible mutant as types which could grow in the presence of both D-serine and glucose. These strains contain cis dominant, nonsuppressible mutations in the dsdO (operator-initiator) region. In the first class of mutants (e.g., FB4010), Dsdase synthesis is completely insensitive to catabolite repression, and synthesis occurs at a high constitutive rate in the absence of cyclic adenosine 5'-monophosphate. In the second class (e.g., FB4005), Dsdase synthesis is partially insensitive to catabolite repression, and catabolite repression is reversed by the addition of cyclic adenosine 5'-monophosphate. Dsdase synthesis in strain FB4005 is partially independent of the cyclic adenosine 5'-monophosphate binding protein, as constitutive synthesis is reduced only 65% (relative to the cap+ strain) in strains unable to synthesize the cyclic adenosine 5'-monophosphate binding protein. Surprisingly, the constitutive rate of Dsdase synthesis is fourfold higher in all mutants of both classes than in the parent, indicating a close interrelationship between the sites of response to induction and catabolite repression.     

Fracture Faces in the Cell Envelope of Escherichia coli

Freeze-fracturing of Escherichia coli cells in the presence of 30% (v/v) glycerol resulted in a double cleavage of the cell envelope exposing two convex and two concave fracture faces ([Formula: see text], [Formula: see text] and [Formula: see text], [Formula: see text]) with characteristic patterns. Complementary replicas revealed the relationship of the fracture faces to their corresponding fracture planes. The inner fracture plane splits the plasma membrane at one particular level. Apparently the outer fracture plane was located in the outer part of the wall, as it was separated by a layer ([Formula: see text]) from the fractured profile (CW1) presumably corresponding to the murein layer. The outer fracture plane did alternate toward the cell periphery, exposing complementary smooth areas ([Formula: see text] and [Formula: see text]). When cells were freeze-fractured in the absence of glycerol, the outer cell surface appeared as an etching face rather than a fracture face. A schematic representation of the relative location of the different fracture faces in the E. coli cell envelope is given.     

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.     

Kinetics of the onset of catabolite repression in Escherichia coli as determined by lac messenger ribonucleic acid initiations and intracellular cyclic adenosine 3'',5''-monophosphate levels.

The rates of synthesis of beta-galactosidase (EC 3.2.1.23) and the intracellular levels of cyclic 3',5'-adenosine monophosphate (cAMP) soon after the addition of glucose or glycerol to exponentially growing cultures of Escherichia coli have been determined. Within 10 s of its addition, glucose, but not glycerol, lowered the apparent initiation frequency of lac messenger ribonucleic acid. The glucose-generated reduction in initiations is identified as catabolite repression by its reversibility with cAMP. The intracellular cAMP levels respond virtually identically to glucose and glycerol additions. Thus, no correlation was observed between the rate of messenger ribonucleic acid initiation and the level of cAMP.     

Characterization of Escherichia coli Flagellar Mutants That are Insensitive to Catabolite Repression

In Escherichia coli, the synthesis of the flagellar organelle is sensitive to catabolite repression. Synthesis requires the presence of the cyclic adenosine monophosphate receptor protein (Crp) and 3',5'-cyclic adenosine monophosphate (cAMP); i.e., mutants that lack Crp or adenylcyclase (Cya) synthesize no flagella. We isolated and characterized a series of mutants (cfs) that restored flagella-forming ability in a Crp strain of E. coli. The mutations in these strains were transferred onto episomes and they were then introduced into a variety of other strains. The presence of the mutation resulted in flagella synthesis in Cya and Crp strains as well as in the wild type grown under conditions of catabolite repression. Deletion analysis and other genetic studies indicated that: (i) the cfs mutations had a dominant effect when they were in the transconfiguration in merodiploids: (ii) they occurred in or very close to the flaI gene: and (iii) their expression required the presence of an intact flaI gene adjacent to the cfs mutation. Biochemical studies showed that the synthesis of at least two flagellar polypeptides, the hook subunit and an amber fragment of flagellin, were absent in strains that carried a cya mutation. Their synthesis was depressed in strains grown under conditions of catabolite repression. The presence of the cfs mutation restored the specific synthesis of these two polypeptides. We suggest that the formation of the flaI gene product is the step in flagellar synthesis that is catabolite sensitive and requires cAMP. We propose a regulatory function for the product of the flaI gene.     

Arylsulfatase in Salmonella typhimurium: detection and influence of carbon source and tyramine on its synthesis.

Arylsulfatase synthesis was shown to occur in Salmonella typhimurium LT2. The enzyme had a molecular weight of approximately 50,000 and was separated into five forms by isoelectrofocusing. The optimal pH for substrate hydrolysis was pH 6.7, with Michaelis constants for nitrocatechol sulfate and nitrophenyl sulfate being 4.1 and 7.9 mM, respectively. Enzyme synthesis was strongly influenced by the presence of tyramine in the growth medium. The uptake of [14C]tyramine and arylsulfatase synthesis were initiated during the second phase of a diauxie growth response, when the organism was cultured with different carbon sources. Adenosine 3',5'-cyclic monophosphoric acid enhanced the uptake of tyramine and the levels of arylsulfatase synthesized. However, the addition of glucose and glycerol to organisms actively transporting tyramine and synthesizing enzyme caused a rapid inhibition of both of these processes. This inhibition was not reversed by adding adenosine 3',5'-cyclic monophosphoric acid. The results suggest that the effect of the carbon source on tyramine transport and arylsulfatase synthesis may be explained in terms of inducer exclusion.     

Induction of Nonpigmented Variants of Erwinia herbicola by Incubation at Supraoptimal Temperatures

As with other inducible enzymes, the induced synthesis of l-arabinose isomerase (l-arabinose ketol isomerase, EC 5.3.1.4) in Salmonella typhimurium is subject to catabolite repression. Of the three catabolite repressors tested, glucose produces maximum repression. Analogues of catabolite repressors like 2-deoxy-d-glucose and d-fucose also inhibit the synthesis of the enzyme. The catabolite repression is completely reversed in the presence of 1.5 x 10(-3)m cyclic 3',5'-adenosine monophosphate (AMP). The maximum repression is produced in glucose-grown cells in glucose-containing induction medium. Cyclic 3',5-AMP reverses this repression provided that the cells are treated with ethylenediaminetetraacetic acid (EDTA). In normal cells, cyclic 3',5'-AMP has no effect on the induction but in EDTA-treated cells the cyclic nucleotide enhances synthesis of the enzyme. The inhibition produced by d-fucose cannot be reversed by cyclic 3',5'-AMP. d-Fucose competes with the inducer l-arabinose in some step(s) involved in the process of induction.     

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.     

Effect of glucose on glutamate dehydrogenase and acid phosphatase and its reversal by cyclic adenosine 3':5'-monophosphate in single cell cultures of Asparagus officinalis

Cell cultures of Asparagus officinalis L. ev. "Argenteuil" were used to study the effect of glucose on the synthesis of glutamate dehydrogenase and acid phosphatase. Both the enzymes are progressively repressed by increasing the glucose concentration in the culture medium. Furthermore, synthesis of glutamatc dehydrogenase and acid phosphatase in cells grown in the presence of high glucose concentrations is derepressed by the addition of the cyclic adenosine 3':5'-monophosphate to the culture medium. These data support the hypothesis that also in cells from higher plants a regulatory mechanism exists with some similarities to the "catabolite repression" mechanism operating in several prokaryotes and lower eukaryotes.     

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.