Roles of arginine and canavanine in the synthesis and repression of ornithine transcarbamylase by Escherichia coli

Conditions were found under which the processes of repression and derepression of ornithine transcarbamylase were separated from the process of enzyme synthesis. After 10 min of arginine deprivation followed by the addition of 2 to 200 mug of l-arginine per ml, a number of strains of Escherichia coli exhibited a significant burst of ornithine transcarbamylase synthesis which lasted 3 to 4 min before the onset of repression. The rapid increase of enzyme activity was shown to require protein synthesis, and was not due to a slow uptake of arginine or induction of an arginine-inducible ornithine transcarbamylase. The capacity of E. coli to synthesize the burst of ornithine transcarbamylase reached a maximum after 10 min of arginine deprivation and then remained constant. The observed increase in enzyme synthesis may reflect the level of unstable messenger ribonucleic acid (RNA) for ornithine transcarbamylase present in the cell at the time protein synthesis was reinitiated. After the addition of arginine in the absence of protein synthesis, the burst of ornithine transcarbamylase decayed with a half-life of about 3 min. The data implied that arginine prevents synthesis of new messenger RNA that can translate this enzyme. Repression of ornithine transcarbamylase by l-canavanine (100 to 200 mug/ml) was observed, and no active enzyme was formed in the presence of this analogue. The action of canavanine as a repressor was distinguished from the inhibitory effect of this compound on protein synthesis.     

The effects of bisulfite on growth and macromolecular synthesis in Escherichia coli

Bisulfite reversibly inhibits the growth of a variety of microorganisms and has been used as a preservative in foods and beverages for that reason. We have now measured macromolecule synthesis in Escherichia coli K12 after bisulfite treatment. RNA synthesis, the synthesis of total protein, and of an inducible enzyme, beta-galactosidase, stopped almost immediately upon addition of 2 mM (or higher concentrations) of bisulfite. These functions resumed after a lag whose duration depended on the concentration of bisulfite added. The synthesis of DNA was slowed upon bisulfite addition, but did not stop entirely. The inhibition of RNA synthesis by bisulfite took place in both stringent and relaxed strains of E. coli and was not relieved upon addition of chloramphenicol. Stringent control was therefore not involved in this effect. No effect on protein synthesis was observed in the cell-free system of E. coli (using poly(U) or MS2 RNA as messenger) at bisulfite concentrations up to 10 mM. Protein synthesis inhibition in vivo was apparently not due to a reaction of bisulfite with a component of this system. In additional experiments, RNA polymerase was not impaired by bisulfite, and the growth inhibition effect was shown to proceed in the presence of inhibitors of free radical chain reactions.     

Dual regulation by arginine of the expression of the Escherichia coli argECBH operon.

The correlation between the level of messenger ribonucleic acid (mRNA) specific for the argECBH gene cluster (argECBH mRNA) measured by ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization and the rates of synthesis of N-acetylornithine deacetylase (argE enzyme) and of argininosuccinate lyase (argH enzyme) of Escherichia coli strain K-12 were determined for steady-state growth with and without added L-arginine and during the transition periods between these two states. During the transient period after arginine removal (transient derepression), the synthesis of enzymes argE and argH was initially three to five times greater than the steady-state derepressed rate finally reached 50 min later. The level of argECHB mRNA correlated well both quantitatively and temporally with the rates of enzyme synthesis during this transition. The level of in vivo charged arginyl-transfer RNA (tRNAarg), monitored simultaneously, was initially only 5 to 10% and gradually increased to a final level of 80% after 45 min. During the transient period after arginine addition (transient repression), the rates of synthesis of enzymes argE and argH decreased to almost zero and gradually reached steady-state repressed rates after about 180 min. The argECBH mRNA level remained constant at the steady-state repressed level throughout transient repression, revealing a discontinuity between the level of this mRNA and rates of enzyme synthesis. A similar discrepancy was noted during the transition after ornithine addition. In vivo charged tRNAarg remained constant at 80% during this transition. After removal of arginine, the zero-level transient enzyme synthesis developed after only 7.5 min of arginine deprivation and was maximum after 30 min. The results suggest an accumulation of a molecule regulated by arginine that plays a role in transient repression. Our data indicate that arginyl-tRNA synthetase is not this molecule since its synthesis was unaffected by arginine. The ratios of steady-state argE and argH enzyme synthesis without arginine to that with arginine were 12 and 20, respectively, whereas the similar ratio for argECBH mRNA was 2 to 3. The repressed level of argECBH mRNA was not affected by attempts to repress or derepress the ppc+ gene (carried on the DNA used for hybridization), and the repressed level of argECBH mRNA was lowered about 50% in cells carrying an internal argBH deletion. These data taken together indicate the presence of an excess of untranslated argECBH mRNA during both transient and steady-state repression by arginine. Thus, a second regulatory mechanism, not yet defined, appears to play an important role in arginine regulation of enzyme synthesis.     

Transient repression of catabolite-sensitive enzyme synthesis elicited by 2,4-dinitrophenol.

Transient inhibition of catabolic enzyme synthesis in Escherichia coli occurred when a low concentration of 2,4-dinitrophenol (DNP) was simultaneously added with inducer. Using mutant strains defective for gamma-gene product or constitutive for lac enzymes, it was found that the inhibition is not due to the exclusion of inducer by uncoupling. The addition of cyclic adenosine 3',5'-monophosphate overcame repression. The components of the lac operon coordinately responded to DNP inhibition. From deoxyribonucleic acid-ribonucleic acid hybridization experiments, it was found that the inhibition of beta-galactosidase induction occurred at the level of messenger ribonucleic acid synthesis specific for the lac operon. It seems probable that DNP represses induction in a similar manner to that of transient repression observed upon the addition of glucose. Furthermore, it was found that transient repression disappeared if cells were preincubated with DNP before induction. This indicates that new contact of cells with DNP is obligatory for transient repression. From these results, it is suggested that the cell membrane may be responsible for regulation of catabolite-sensitive enzyme synthesis.     

Induction of Galactokinase in Saccharomyces cerevisiae: Kinetics of Induction and Glucose Effects

The induced synthesis of galactokinase and the repressing effects of glucose on this synthesis have been investigated in whole yeast cells rendered permeable by treatment with dimethyl sulfoxide. It was found that the induction response of uninduced cells to galactose is clearly dependent on the nature of the carbon source upon which the culture was grown prior to exposure to galactose. Glucose-grown cells exhibited a long lag before induction, whereas lactate-grown cells exhibited induced synthesis within 8 min. A concentration of 0.5% galactose was found to be optimal for induction. The addition of glucose to yeast cultures growing on galactose resulted in a severe transient repression of synthesis which was followed by a resumed rate of synthesis characteristic of a weaker permanent catabolite repression. Neither 2-deoxygalactose nor fucose acted as gratuitous inducers of the pathway, nor did they serve as a substrates for galactokinase.     

Release of the β-Galactosidase-Synthesizing System from Ultraviolet Catabolite Repression by Cyclic 3′,5′-Adenosine Monophosphate, Dark Repair, Photoreactivation, and Cold Treatment

Recovery from the inhibitory effect of ultraviolet irradiation on the induced synthesis of beta-galactosidase was studied in Escherichia coli B/r. When irradiated cells (520 ergs/mm(2) at 254 nm) were induced and incubated in minimal medium supplemented with Casamino Acids (conditions of catabolite repression), the ability to form enzyme was greatly reduced for about 100 min and then recovery began. The inhibition observed immediately after ultraviolet irradiation was partially reversed by cyclic 3',5'-adenosine monophosphate (cyclic AMP) or by photoreactivation treatment. Inhibition was reduced if the cells were given cold treatment (5 C) before or during irradiation; the kinetics of induced enzyme formation in each case were similar to those of irradiated cells receiving cyclic AMP. These kinetics suggest that the cold treatments, like cyclic AMP, cause the release of the beta-galactosidase-synthesizing system from catabolite repression. When irradiated cells were incubated for various times before cyclic AMP or photoreactivation treatment, some reversal of the inhibition of induced enzyme formation was obtained, but by 100 min the treatments were ineffective. Because 100 min was also the time at which dark recovery of enzyme formation began, the recovery process was interpreted to be the result of completion of DNA repair, which, in turn, released the beta-galactosidase-synthesizing system from catabolite repression.     

Control of synthesis of mRNA''s for T4 bacteriophage-specific dihydrofolate reductase and deoxycytidylate hydroxymethylase.

A 30 degrees C, functional messengers for dCMP hydroxymethylase first appeared 3 to 6 min postinfection and reached their maximum levels at 12 min. Chloramphenicol, added before the phage, reduced the rate of mRNA accumulation. When the antibiotic was added 6 min postinfection, mRNA levels increased at their normal rate but there was no obvious repression of messenger accumulation. Delaying the addition of drug until 8 or 12 min had progressively less effect on the pattern of hydroxymethylase mRNA metabolism. When chloramphenicol was present from preinfection times or from 6 min postinfection, all hydroxymethylase mRNA's synthesized were stable; at later times, however, the ability of the drug to stabilize mRNA decreased with its ability to delay the turnoff of mRNA production. An overaccumulation of hydroxymethylase mRNA was also seen when phage-specific DNA synthesis was inhibited either by mutational lesion in an essential viral gene or by 5-fluorodeoxyuridine. By min 20 of a DNA-negative program, hydroxymethylase mRNA synthesis was repressed to the point where it no longer compensated for decay. However, a finite level of hydroxymethylase mRNA synthesis was maintained at later times of a DNA-negative infection. Such results indicate that replication of the phage chromosome is necessary but not sufficient for a complete turnoff of hydroxymethylase mRNA production. Functions controlled by the maturation-defective proteins (the products of genes 55 and 33) played only a minor role in the regulation of hydroxymethylase mRNA, metabolism. Thus, we favor the hypothesis that a complete turnoff of hydroxymethylase messenger production requires one or more new proteins as well as an interval of DNA replication. The absence of DNA synthesis had no particular effect upon dihydrofolate reductase messenger production. The preinfection addition of chloramphenicol likewise had little effect on dihydrofolate reductase messenger metabolism. These latter data imply that prior synthesis of a phage-coded protein synthesis may not be required for the turnoff of reductase messenger production.     

Lifetime of bacterial messenger ribonucleic acid

Moses, V. (University of California, Berkeley), and M. Calvin. Lifetime of bacterial messenger ribonucleic acid. J. Bacteriol. 90:1205-1217. 1965.-When cells from a stationary culture of Escherichia coli were placed in fresh medium containing inducer for beta-galactosidase, growth, as represented by increase in turbidity and by total protein synthesis, started within 30 sec. By contrast, beta-galactosidase synthesis was greatly delayed compared with induction during exponential growth. Two other inducible enzymes (d-serine deaminase and l-tryptophanase) and one repressible enzyme (alkaline phosphatase) showed similar lags. The lags were not due to catabolite repression. They could not be reduced by pretreatment of the culture with inducer, or by supplementing the fresh medium with amino acids or nucleotides. The lag was also demonstrated by an i(-) mutant constitutive for beta-galactosidase synthesis. An inhibitor of ribonucleic acid (RNA) synthesis, 6-azauracil, preferentially inhibited beta-galactosidase synthesis compared with growth in both inducible and constitutive strains. Puromycin, an inhibitor of protein synthesis, acted as an inhibitor at additional sites during the induction of beta-galactosidase synthesis. No inhibition of the reactions proceeding during the first 20 sec of induction was observed, but puromycin seemed to prevent the accumulation of messenger RNA during the period between 20 sec and the first appearance of enzyme activity after 3 min. It is suggested that these observations, together with many reports in the literature that inducible enzyme synthesis is more sensitive than total growth to some inhibitors and adverse growth conditions, can be explained by supposing that messenger RNA for normally inducible enzymes is biologically more labile than that for some normally constitutive proteins. The possible implications of this hypothesis for the achievement of cell differentiation by genetic regulation of enzyme synthesis are briefly discussed.     

Stimulation of thiamine diphosphatase activity by ascorbic acid in rat brain microsomes.

The effect of ascorbic acid on microsomal thiamine diphosphatase activity in rat brain was examined. Ascorbic acid at 0.02--0.1 mM increased the thiamine diphosphatase activity by 20--600% and produced a significant amount of lipid peroxide, which was measured with thiobarbiturate under the same conditions as the enzyme. A lag period of about 10 min was observed in the process of stimulation of enzyme activity by ascorbic acid. The stimulation of enzyme activity and the lipid peroxidation induced by ascorbic acid were blocked by metal-binding compounds (EDTA, alpha,alpha'-dipyridyl, o-phenanthroline) and an antioxidant (N,N'-diphenyl p-phenylenediamine). GSH significantly enhanced the stimulation of enzyme activity and formation of lipid peroxide by 0.02--0.05 mM ascorbic acid. The effect of GSH was due in part to maintenance of the concentration of ascorbic acid in the medium, since GSH could convert dehydroascorbic acid, an oxidized form of ascorbic acid, to ascorbic acid.     

Kinetics of Penicillinase Induction and Variation of Penicillinase Translation in Staphylococcus aureus

At neutral pH, the rate of penicillinase synthesis by staphylococci declines gradually after removal of free inducer, while at pH 5.4 enzyme formation is generally linear for an extended period. Linear synthesis of penicillinase was observed at neutral pH in nonsaturating concentrations (1 μg/ml) of actinomycin D. The rate of enzyme synthesis, corrected for inhibition of growth caused by the antibiotic, was relatively independent of the time of actinomycin addition. The lag preceding linear enzyme formation increased with the interval between induction and the addition of actinomycin. The findings are consistent with the concept that, at neutral pH, “operons” activated by induction are rapidly repressed, while at pH 5.4, this process is delayed.

At a concentration of 4 μg/ml, actinomycin D blocked penicillinase messenger synthesis and also elicited a short-lived acceleration of the increase of penicillinase activity in uninduced and, late after induction, in induced cultures. This effect did not require a functional genomic repressor mechanism since it occurred also in a penicillinase-constitutive strain. It required protein synthesis and could not be attributed to a greater enzyme stability in the presence of actinomycin. The results suggest enhanced penicillinase translation after addition of actinomycin D.

     

Human fat cell adenylate cyclase. Enzyme characterization and guanine nucleotide effects on epinephrine responsiveness in cell membranes.

Human adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) has been studied in preparations of fat cell membranes ("ghosts"). As reported earlier, under ordinary assay conditions (1.0 mM ATP, 5 mM Mg2+, 30 degrees C, 10 min incubation) the enzyme was activated 6-fold by epinephrine in the presence of the GTP analog, 5'-guanylyl-imidodiphosphate [GMP-P(NH)P] (Cooper, B. et al. (1975) J. Clin. Invest. 56, 1350-1353). Basal activity was highest during the first 2 min of incubation then slowed and was linear for at least the next 18 min. Epinephrine, added alone, was often without effect. but sometimes maintained the initial high rate of basal activity. GMP-P(NH)P alone produced inhibition ("lag") of basal enzyme early in the incubation periods. Augmentation of epinephrine effect by GMP-P(NH)P, which also proceeded after a brief (2 min) lag period, was noted over a wide range of substrate (ATP) concentrations. GTP inhibited basal levels of the enzyme by about 50%. GTP also allowed expression of an epinephrine effect, but only in the sense that the hormone abolished the inhibition by GTP. Occasionally a slight stimulatory effect on epinephrine action was seen with GTP. At high Mg2+ concentration (greater than 10 mM) or elevated temperatures (greater than 30 degrees C) GMP-P(NH)P alone activated the enzyme. Maximal activity of human fat cell adenylate cyclase was seen at 50 mM Mg2+, 1.0 mM ATP, pH 8.2, and 37 degrees C in the presence of 10(-4) M GMP-P(NH)P; under these conditions addition of epinephrine did not further enhance activity. Human fat cell adenylate cyclase of adults was insensitive to ACTH and glucagon even in the presence of GMP-P(NH)P.     

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.     

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.     

Stimulation of thiamine diphosphate activity by ascorbic acid in rat brain microsomes

The effect of ascorbic acid on microsomal thiamine diphosphate activity in rat brain was examined. Ascorbic acid at 0.02–0.1 mM increased the thiamine diphosphate activity by 20–600% and produced a significant amount of lipid peroxide, which was measured with thiobarbiturate under the same conditions as the enzyme. A lag period of about 10 min was observed in the process of stimulation of enzyme activity by ascorbic acid. The stimulation of enzyme activity and the lipid peroxidation induced by ascorbic acid were blocked by metal-binding compounds (EDTA, α,α′-dipyridyl, o-phenanthroline) and an antioxidant (N,N′-diphenyl p-phenylenediamine). GSH significantly enhanced the stimulation of enzyme activity and formation of lipid peroxide by 0.02–0.05 mM ascorbic acid. The effect of GSH was due in part to maintenance of the concentration of ascorbic acid in the medium, since GSH could convert dehydroascorbic acid, an oxidized form of ascorbic acid, to ascorbic acid.     

The utilisation of d-galactonate and d-2-oxo-3-deoxygalactonate by Escherichia coli K-12

1. Escherichia coli K-12 mutants unable to grow on d-galactonate have been isolated and found to be defective in either galactonate dehydratase, 2-oxo-3-deoxygalactonate 6-phosphate aldolase or devoid of both of these enzymes and of 2-oxo-3-deoxygalactonate kinase.
2. 2-Oxo-3-deoxygalactonate kinase and 2-oxo-3-deoxygalactonate 6-phosphate aldolase are still induced by galactonate in mutants lacking galactonate dehydratase, suggesting that galactonate rather than a catabolic product of galactonate is the inducer of the galactonate catabolic enzymes. Synthesis of the enzymes is subject to glucose catabolite repression.
3. Mutants defective in 2-oxo-3-deoxygalactonate 6-phosphate aldolase accumulate 2-oxo-3-deoxygalactonate 6-phosphate when exposed to galactonate and this compound causes general growth inhibition.
4. Secondary mutants that no longer show this inhibition fail to make 2-oxo-3-deoxygalactonate 6-phosphate due to additional defects in galactonate transport, galactonate dehydratase, 2-oxo-3-deoxygalactonate kinase or a putative promoter mutation that prevents formation of these enzymes.
5. A spontaneous mutant capable of growth on 2-oxo-3-deoxygalactonate has been isolated. It has two genetically distinct mutations. One permits constitutive formation of the galactonate catabolic enzymes and the other allows the uptake of 2-oxo-3-deoxygalactonate. Neither mutation on its own permitted growth on 2-oxo-3-deoxygalactonate.
6. Genes specifying the various galactonate catabolic enzymes have been located at min 81.7 on the E. coli K-12 linkage map and probably constitute an operon. The gene sequence in this region was shown to by: pyrE uhp dgo dnaA.

     

Effects of Ca2+ and Mg2+ on the activity of pyruvate dehydrogenase phosphate phosphatase within toluene-permeabilized mitochondria.

Mitochondria from rat epididymal white adipose tissue were made permeable to small molecules by toluene treatment and were used to investigate the effects of Mg2+ and Ca2+ on the re-activation of pyruvate dehydrogenase phosphate by endogenous phosphatase. Re-activation of fully phosphorylated enzyme after addition of 0.18 mM-Mg2+ showed a marked lag of 5-10 min before a maximum rate of reactivation was achieved. Increasing the Mg2+ concentration to 1.8 mM (near saturating) or the addition of 100 microM-Ca2+ resulted in loss of the lag phase, which was also greatly diminished if pyruvate dehydrogenase was not fully phosphorylated. It is concluded that, within intact mitochondria, phosphatase activity is highly sensitive to the degree of phosphorylation of pyruvate dehydrogenase and that the major effect of Ca2+ may be to overcome the inhibitory effects of sites 2 and 3 on the dephosphorylation of site 1. Apparent K0.5 values for Mg2+ and Ca2+ were determined from the increases in pyruvate dehydrogenase activity observed after 5 min. The K0.5 for Mg2+ was diminished from 0.60 mM at less than 1 nM-Ca2+ to 0.32 mM at 100 microM-Ca2+; at 0.18 mM-Mg2+, the K0.5 for Ca2+ was 0.40 microM. Ca2+ had little or no effect at saturating Mg2+ concentrations. Since effects of Ca2+ are readily observed in intact coupled mitochondria, it follows that Mg2+ concentrations within mitochondria are sub-saturating for pyruvate dehydrogenase phosphate phosphatase and hence less than 0.5 mM.     

Comparative Study of the Events Associated with Colicin Induction

Colicinogenic factors ColI and ColV, which have been shown to behave as sex factors, could not be induced with mitomycin C. In contrast, the ColE(1), ColE(2), and ColE(3) factors, which do not exhibit any fertility factor characteristics, are inducible by this agent. The induced production of colicins E(1), E(2), and E(3) was accompanied by a loss in viability at a concentration of mitomycin C which was bacteriostatic to noncolicinogenic cells or to cells carrying the ColV or ColI factors. The loss in viability accompanying the mitomycin C induction of the ColE(1), ColE(2), or ColE(3) factors also occurred when colicin synthesis was blocked by chloramphenicol or amino acid starvation. However, chloramphenicol was able to block the loss of viability of a recipient cell after mitomycin C induction of a newly acquired Col factor if the antibiotic was present throughout the mating period. No detectable internal colicin or colicin precursor could be demonstrated during the lag period prior to the appearance of colicin outside the cell 20 to 30 min after the addition of mitomycin C. If chloramphenicol was present during the lag period following the addition of mitomycin C, colicin synthesis began immediately after the removal of these antibiotics. The synthesis of tryptophan synthetase and induced beta-galactosidase proceeded normally throughout the lag period and well into the period of colicin production. Regulation of beta-galactosidase synthesis did not seem to be profoundly affected during the lag period subsequent to mitomycin C addition. Induced colicin synthesis, like bacterial or induced prophage protein synthesis, was subject to inhibition by virulent phage infection.