Penicillinamidohydrolase inEscherichia coli

Synthesis of penicillinamidohydrolase (penicillin acylase, EC 3.5.1.11) in Escherichia coli is subjected to the absolute catabolite repression by glucose and partial repression by acetate. Both types of catabolite repression of synthesis of the enzyme in Escherichia coli are substantially influenced by cyclic 3',5'-adenosinemonophosphate (cAMP). Growth diauxie in a mixed medium containing glucose and phenylacetic acid serving as carbon and energy sources is overcome by cAMP. cAMP does not influence the basal rate of the enzyme synthesis (without the inducer). Derepression of synthesis of penicillinamidohydrolase by cAMP in a medium with glucose and inducer (phenylacetic acid) is associated with utilization of the inducer, due probably to derepression of other enzymes responsible for degradation of phenylacetic acid. Lactate can serve as a "catabolically neutral" source of carbon suitable for the maximum production of penicillinamidohydrolase. The gratuitous induction of the enzyme synthesis in a medium with lactate as the carbon and energy source and with phenylacetic acid is not influenced by cAMP; however, cAMP overcomes completely the absolute catabolite repression of the enzyme synthesis by glucose.     

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.     

A lowered concentration of cAMP receptor protein caused by glucose is an important determinant for catabolite repression in Escherichia coli

A decreased intracellular concentration of cAMP is insufficient to account for catabolite repression in Escherichia coli. We show that glucose lowers the amount of cAMP receptor protein (CRP) in cells. A correlation exists between CRP and β-galactosidase levels in cells growing under various conditions. Exogenous cAMP completely eliminates catabolite repression in CRP-overproducing cells, while it does not fully reverse the effect of glucose on β-galactosidase expression in wild-type cells. When the CRP concentration is reduced by manipulating the crp gene, β-galactosidase expression decreases in proportion to the concentration of CRP. These findings indicate that the lowered concentration of CRP caused by glucose is one of the major factors for catabolite repression. We propose that glucose causes catabolite repression by lowering the intracellular levels of both CRP and cAMP.     

Enzymes of arginine utilization and their formation in Aeromonas formicans NCIB 9232

In Aeromonas formicans two inducible catabolic pathways of L-arginine have been characterized. The arginine decarboxylase is induced by arginine which also induces the three enzymes of the arginine deiminase pathway but only in stress conditions such as a shift from aerobic growth conditions to very low oxygen tension. Addition of glucose to medium containing arginine leads to repression of the enzymes involved in the arginine deiminase pathway while exogenous cAMP prevents that repression of enzyme synthesis by glucose. This suggests that the induction of arginine deiminase pathway is regulated by carbon catabolite repression and the energetic state of the cell.     

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.     

Role of the carbon source in regulating chloramphenicol production by Streptomyces venezuelae: studies in batch and continuous cultures

Both carbon- and nitrogen-limited media that supported a biphasic pattern of growth and chloramphenicol biosynthesis were devised for batch cultures of Streptomyces venezuelae. Where onset of the idiophase was associated with nitrogen depletion, a sharp peak of arylamine synthetase activity coincided with the onset of antibiotic production. The specific activity of the enzyme was highest when the carbon source in the medium was also near depletion at the trophophase-idiophase boundary. In media providing a substantial excess of carbon source through the idiophase, the peak specific activity was reduced by 75%, although the timing of enzyme synthesis was unaltered. Moreover, chemostat cultures in which the growth rate was limited by the glucose concentration in the input medium failed to show a decrease in specific production of chloramphenicol as the steady-state intracellular glucose concentration was increased. The results suggest that a form of "carbon catabolite repression" regulates synthesis of chloramphenicol biosynthetic enzymes during a trophophase-idiophase transition induced by nitrogen starvation. However, this regulatory mechanism does not establish the timing of antibiotic biosynthesis and does not function during nitrogen-sufficient growth in the presence of excess glucose.     

Succinate-mediated catabolite repression of enzymes of glucose metabolism in root-nodule bacteria

Enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways were detected in strains ofRhizobium andBradyrhizobium cultured on glucose. The enzymes, except glyceraldehyde-3-phosphate dehydrogenase, were present only in trace amounts in succinategrown cells. The enzymes of the pentose phosphate pathway, being absent inBradyrhizobium, were detected only in glucose-grown cells ofRhizobium. The presence of the glucose-catabolic enzymes in cells only during growth on glucose suggests that they are inducible in nature. Succinate repressed the glucose catabolic enzymes, and the repression appeared to be similar to catabolite repression. Exogenous addition of cAMP caused no change in the activity of these enzymes, demonstrating that the repression was unlikely to be mediated via cAMP.     

Glycolytic enzymes and intermediates in carbon catabolite repression mutants of Saccharomyces cerevisiae

Summary Glycolytic parameters were determined in recessive yeast mutants with partial defects in carbon catabolite repression. Specific activities of pyruvate kinase and pyruvate decarboxylase in glucose grown cells of all mutant and wild type stains were 4–5 times higher than in ethanol grown cells. Mutants of gene HEX1 had a reduced hexose phosphorylating activity on allmedia wheras those of gene HEX2 had elevated levels but only in glucose grown cells. Mutants of gene CAT80 were normal in this respect. All other glycolytic enzymes were normal in all mutants. This was also true for glycolytic intermediates. Only hexlmutants showed a reduced fermentation of repressing sugars. The three genes appear to be involved in catabolite repression of several but not of all repressible enzymes. Even though all three types of mutants show a limited overlap in their effects on certain enzymes, they still are distinctly different in their action spectra. Carbon catabolite repression apparently does not depend on the sole accumulation of glycolytic intermediales. The activity of the products of the three genes HEX1, HEX2 and CAT80 are required directly or indirectly for triggering carbon catabolite repression. Even a small segment of carbon catabolite repression is controlled by several genes with regulatory functions indicating that the entire regulatory circuit is highly complex.     

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.     

Properties of cyclic AMP-independent catabolite gene activator proteins of Escherichia coli

The protein products of two crp alleles encoding mutationally altered catabolite gene activator proteins CAP and CAPc, which are functionally active in vivo in the absence of cAMP, were purified by an immunoaffinity purification procedure. These proteins bind cAMP with the same affinity as does the wild-type catabolite gene activator protein. From their susceptibility to the proteolytic enzyme subtilisin, we conclude that the two mutationally altered proteins adopt structural features adequate for biological activity and similar to the conformation that cAMP elicits or stabilizes in wild-type catabolite gene activator protein. We note, however, that their conformation is not unique and can be modulated by cAMP. The two altered proteins, CAP and CAPc, bind to the lactose promoter, giving rise to specific DNA-protein complexes in the absence of cAMP and promote initiation of specific lac messenger RNA synthesis.     

Catabolite repression during single and multiple induction inEscherichia coli

Intracellular concentration of cAMP regulates the synthesis of enzymes sensitive to catabolite repression. The relationship between the single and multiple induction of beta-galactosidase (EC 3.2.1.23), L-tryptophanase (EC 4.1.99.1), D-serine deaminase (EC 4.2.1.14), L-asparaginase (EC 3.5.1.1) and L-malate dehydrogenase (EC 1.1.1.37) was studied and the effect of cAMP level on the induction in Escherichia coli Crookes (ATCC 8739) was investigated. A varying degree of catabolite repression was observed during induction of individual enzymes induced separately on different energy sources. The synthesis of l-tryptophanase was most sensitive, whereas l-asparaginase was not influenced at all. Exogenous cAMP was found to overcome partially the catabolite repression of beta-galactosidase and D-serine deaminase, both during single induction. The synthesis of l-malate dehydrogenase was negatively influenced by the multiple induction even in the presence of cAMP; on the other hand, the synthesis of l-tryptophanase was stimulated, independently of the level of the exogenous cAMP. Similarly, the activity of L-asparaginase slightly but significantly increased during the multiple induction of all five enzymes; here too the activity increase did not depend on exogenous cAMP.     

Control of the Pathway of γ-Aminobutyrate Breakdown in Escherichia coli K-12

Mutants of Escherichia coli K-12 isolated for their ability to utilize gamma-aminobutyrate (GABA) as the sole source of nitrogen exhibit a concomitant several-fold increase in the activities of gamma-aminobutyrate-alpha-ketoglutarate transaminase (GSST, EC 2.6.1.19) and succinic semialdehyde dehydrogenase (SSDH, EC 1.2.1.16). The increase in rate of enzymatic activity is not accompanied by any changes in the affinities of the mutant enzymes for their respective substrates. The synthesis of the two enzymes is highly coordinate under a great variety of conditions, in spite of the wide range of activities observed. In cultures grown in minimal media with ammonium salts as the source of nitrogen, both GSST and SSDH are severely repressed by glucose. Substitution of ammonia with GABA, glutamate, or aspartate greatly reduces the effect of glucose on the synthesis of the GABA utilization enzymes. This escape from catabolite repression is specific for GSST and SSDH and does not involve other enzymes sensitive to catabolite repression (e.g., beta-galactosidase, EC 3.2.1.23, and aspartase, EC 4.3.1.1).     

Cyclic AMP phosphodiesterase in Salmonella typhimurium: characteristics and physiological function.

The physiological function of cyclic AMP (cAMP) phosphodiesterase in Salmonella typhimurium was investigated with strains which were isogenic except for the cpd locus. In crude broken-cell extracts the properties of the enzyme were found to be similar to those reported for Escherichia coli. The specific activity in the mutant was less than 1% that in the wild type. Rates of cAMP production in the mutant were as much as twice those observed in the wild type. The amount of cAMP accumulated when cells grew overnight with limiting glucose was 4.5-fold greater in the mutant than in the wild type. The intracellular concentration of cAMP in the two strains was measured directly, using four different techniques to wash the cells to remove extracellular cAMP. The cAMP level in the cpd strain was only 25% greater than in the wild type. The functional concentration of the cAMP receptor protein-cAMP complex was estimated indirectly from the specific activity of beta-galactosidase in the two strains after introducing F'lac. When cells were grown with carbon sources permitting synthesis of different levels of cAMP, the specific activity of the enzyme was at most 25% greater in the cpd strain. The cpd strain was more sensitive to the effects of exogenous cAMP. Exogenous cAMP relieved both permanent and transient catabolite repression of the lac operon at lower concentrations in the cpd strain than in the wild type. When cells grew with glucose, glycerol, or ribose, exogenous cAMP inhibited growth of the mutant strain more than the wild type.     

Induction and regulation of alpha-amylase synthesis in a cellulolytic thermophilic fungus Myceliophthora thermophila D14 (ATCC 48104).

The alpha-amylase enzyme synthesis was higher when M. thermophila D-14 (ATCC 48104) was grown in culture medium incorporated with starch or other carbohydrates containing maltose units. Maximum enzyme production was attained with 1% starch followed by a gradual decrease with increasing concentration. Marked decrease in alpha-amylase synthesis occurred with the addition of glucose to the culture medium and this decreasing activity was proportional to the concentration of glucose. The enzyme synthesis was resumed as soon as the glucose concentration fell below a critical level. The addition of cAMP did not eliminate the repressive activity of glucose. The findings suggest that extracellular alpha-amylase synthesis in M. thermophila D-14 was inducible and subject to catabolite repression.