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 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.     

Catabolite repression and derepression of arylsulfatase synthesis in Klebsiella aerogenes

When a mutant (Mao(-)) of Klebsiella aerogenes lacking an enzyme for tyramine degradation (monoamine oxidase) was grown with d-xylose as a carbon source, arylsulfatase was repressed by inorganic sulfate and repression was relieved by tyramine. When the cells were grown on glucose, tyramine failed to derepress the arylsulfatase synthesis. When grown with methionine as the sole sulfur source, the enzyme was synthesized irrespective of the carbon source used. Addition of cyclic adenosine monophosphate overcame the catabolite repression of synthesis of the derepressed enzyme caused by tyramine. Uptake of tyramine was not affected by the carbon source. We isolated a mutant strain in which derepression of arylsulfatase synthesis by tyramine occurred even in the presence of glucose and inorganic sulfate. This strain also produced beta-galactosidase in the presence of an inducer and glucose. These results, and those on other mutant strains in which tyramine cannot derepress enzyme synthesis, strongly suggest that a protein factor regulated by catabolite repression is involved in the derepression of arylsulfatase synthesis by tyramine.     

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.     

Loss of protein kinase-catalyzed phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system, by mutation of the ptsH gene confers catabolite repression resistance to several catabolic genes of Bacillus subtilis.

In gram-positive bacteria, HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), is phosphorylated by an ATP-dependent, metabolite-activated protein kinase on seryl residue 46. In a Bacillus subtilis mutant strain in which Ser-46 of HPr was replaced with a nonphosphorylatable alanyl residue (ptsH1 mutation), synthesis of gluconate kinase, glucitol dehydrogenase, mannitol-1-P dehydrogenase and the mannitol-specific PTS permease was completely relieved from repression by glucose, fructose, or mannitol, whereas synthesis of inositol dehydrogenase was partially relieved from catabolite repression and synthesis of alpha-glucosidase and glycerol kinase was still subject to catabolite repression. When the S46A mutation in HPr was reverted to give S46 wild-type HPr, expression of gluconate kinase and glucitol dehydrogenase regained full sensitivity to repression by PTS sugars. These results suggest that phosphorylation of HPr at Ser-46 is directly or indirectly involved in catabolite repression. A strain deleted for the ptsGHI genes was transformed with plasmids expressing either the wild-type ptsH gene or various S46 mutant ptsH genes (S46A or S46D). Expression of the gene encoding S46D HPr, having a structure similar to that of P-ser-HPr according to nuclear magnetic resonance data, caused significant reduction of gluconate kinase activity, whereas expression of the genes encoding wild-type or S46A HPr had no effect on this enzyme activity. When the promoterless lacZ gene was put under the control of the gnt promoter and was subsequently incorporated into the amyE gene on the B. subtilis chromosome, expression of beta-galactosidase was inducible by gluconate and repressed by glucose. However, we observed no repression of beta-galactosidase activity in a strain carrying the ptsH1 mutation. Additionally, we investigated a ccpA mutant strain and observed that all of the enzymes which we found to be relieved from carbon catabolite repression in the ptsH1 mutant strain were also insensitive to catabolite repression in the ccpA mutant. Enzymes that were repressed in the ptsH1 mutant were also repressed in the ccpA mutant.     

ADHII in Aspergillus nidulans is induced by carbon starvation stress

In Aspergillus nidulans there are three NAD(+)-dependent alcohol dehydrogenases (ADHs) that are capable of utilizing ethanol as a substrate. ADHI is the physiological enzyme of ethanol catabolism and ADHIII is induced under conditions of anaerobiosis. The physiological role of ADHII (structural gene alcB) is unknown. We have measured beta-galactosidase in a transformant with an alcB::lacZ fusion and have shown that alcB is maximally expressed under conditions of carbon starvation. The behavior of the alcB::lacZ transformant suggests a hierarchy of repressing carbon sources characteristic of repression by the general carbon catabolite repressor protein, CreA, but in a creA(d)30 background the transformant shows only partial derepression of beta-galactosidase on 1% glucose compared to the creA+ strain. Our results suggest that, in addition to carbon catabolite repression acting via CreA, a CreA-independent mechanism is involved in induction of alcB on carbon starvation.     

In situ assay for 5-aminolevulinate dehydratase and application to the study of a catabolite repression-resistant Saccharomyces cerevisiae mutant.

To facilitate the study of the effects of carbon catabolite repression and mutations on 5-aminolevulinate dehydratase (EC 4.2.1.24) from Saccharomyces cerevisiae, a sensitive in situ assay was developed, using cells permeabilized by five cycles of freezing and thawing. Enzymatic activity was measured by colorimetric determination of porphobilinogen with a modified Ehrlich reagent. For normal strains, porphobilinogen production was linear for 15 min, and the reaction rate was directly proportional to the permeabilized cell concentration up to 20 mg (dry weight) per ml. The reaction exhibited Michaelis-Menten-type kinetics, and an apparent Km of 2.6 mM was obtained for 5-aminolevulinic acid. This value is only slightly higher than the value of 1.8 mM obtained for the enzyme assayed in cell extracts. The in situ assay was used to assess catabolite repression-dependent changes in 5-aminolevulinate dehydratase during batch culture on glucose medium. In normal S. cerevisiae cells, the enzyme is strongly repressed as long as glucose is present in the medium. In contrast, a strain bearing the hex2-3 mutation exhibits derepressed levels of enzyme activity during growth on glucose. Synthesis of cytochromes by this strain is also resistant to catabolite repression. Similar studies employing a strain containing the glc1 mutation, which enhances porphyrin accumulation, did not reveal any significant phenotypic change in catabolite regulation of 5-aminolevulinate dehydratase.     

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.     

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.     

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

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

Production and Catabolite Repression of the Constitutive Polygalacturonic Acid trans-Eliminase of Aeromonas liquefaciens

Production of polygalacturonic acid (PGA) trans-eliminase was greatly stimulated under conditions of restricted growth of Aeromonas liquefaciens. This was accomplished either by substrate restriction in a continuous-feeding culture or by restricting divalent cations in a batch culture, with the use of PGA as the sole source of carbon in a chemically defined medium containing inorganic nitrogen. Slow feeding of glucose, glycerol, or PGA to carbon-limited cultures allowed PGA trans-eliminase to be formed at a maximum differential rate 500 times greater than in batch cultures with excess substrate present. The differential rate of enzyme formation obtained by slow feeding of these three substrances or of a mixture of PGA plus glucose was observed to be the same. Therefore, PGA trans-eliminase produced by A. liquefaciens, contrary to the current view, appears to be constitutive. These observations also indicate that production of PGA trans-eliminase is subject to catabolite repression and that limiting the substrate reverses this repression. It was also found that, under conditions of unrestricted growth, any compound which the bacteria can use as a source of carbon and energy repressed constitutive PGA trans-eliminase production. The heritable reversal of catabolite repression of PGA trans-eliminase synthesis was demonstrated by isolation of mutant strain Gc-6 which can readily synthesize the constitutive catabolic enzyme PGA trans-eliminase while growing in the presence of excess substrate.     

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.     

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.