Control of derepressed β-galactosidase synthesis inEscherichia coli

1. The synthesis of β-galactosidase in a constitutive mutant ofEscherichia coli (ML 308, i^-z⁺y⁺a⁺) responds to the nutritional environment. Repression can be reversed by cyclic AMP.
2. The greatest degree (%) of repression by metabolisable compounds is obtained when cells utilising glycerol (0%) are given, in addition, pyruvate (67%), serine (57%) which can be converted to pyruvate, or substrates of phosphotransferase systems (20–40%) which liberate pyruvate in their operation. Furthermore, pyruvate represses β-galactosidase synthesis in a phosphoenolpyruvate synthaseless mutant. Pyruvate, however, does not repress in a pyruvate dehydrogenaseless mutant and it follows that pyruvate itself is not the agent of repression.
3. Raffinose, a non-metabolisable galactoside, represses synthesis of β-galactosidase during growth on glycerol. Over a wide range, repression depends on raffinose concentration as does a lowered pool of ATP, rate of oxygen consumption and growth rate. All these parameters are inter-related but, in particular, β-galactosidase synthesis depends on the size of the ATP-pool presumably because this also limits synthesis of cyclic AMP under these conditions.

     

Further observations on the effect of ethionine on the synthesis of β-galactosidase inEscherichia coli: Formation of an immunologically cross-reacting protein

It was found that ethionine partially inhibits the transport of the inducer (TMG) of β-galactosidase into the cells ofEscherichia coli ML-30. The synthesis of β-galactosidase-specific messenger RNA is not inhibited. Ethionine appears to be incorporated into proteins synthesized by the strains used. The incorporation of ethionine into the molecule of β-galactosidase results in the synthesis of an enzymically inactive, immunologically cross-reacting protein.     

The synthesis of β-galactosidase inEscherichia coli in the presence of 7-azatryptophan

The presence of 7-azatryptophan an analogue of tryptophan in the growth medium ofEscherichia coli resulted in a considerable inhibition of the synthesis of active β-galactosidase. No synthesis of an immunologically cross-reacting protein was detected. In addition, the replacement of tryptophan by the analogue rendered the enzyme more susceptible to heat, urea and trypsin as compared with the normal enzyme. The inhibition of growth and enzyme synthesis by 7-azatryptophan was reversed by tryptophan. The analogue did not exhibit any effect on the synthesis and activity of β-galactoside permease.     

Latent β-galactosidase inEscherichia coli

Incubation of washedEscherichia coli cells with crystalline RNase lead to increased β-galactosidase activity. The height of the increase depended on the type of strain and the conditions of cultivation. RNase only raised the level of the β-galactosidase which was bound to the relatively easily sedimenting cellular particles. It had no effect on the activity of β-galactosidase present in soluble form in the supernatant after the disruption of cells or on the activity of purified β-galactosidase in solution. Another basic protein, histone, was found to have a similar effect to that of RNase.     

Effect of cyclic adenosine-3′,5′-monophosphate on the simultaneous synthesis of β-galactosidase and tryptophanase inEscherichia coli

When inducing simultaneously β-galactosidase and tryptophanase in a batch culture either the synthesis of tryptophanase or of both enzymes is decreased due to an insufficient cAMP concentration. The addition of this nucleotide can overcome this decrease. In a continuous culture both enzymes are synthesized at the maximum rate, as the amount of cAMP produced during carbon limitation of growth is probably sufficient for the simultaneous synthesis of both enzymes. In the β-galactosidase hyperproduction mutant cultivated continuously the level of β-galactosidase markedly decreases when tryptophanase is simultaneously induced. Also this decrease is caused by cAMP insufficiency and can be overcome by increasing its concentration. cAMP is thus an important regulatory factor of both enzymes and becomes a limiting factor in their simultaneous synthesis; a competition for this regulatory compound apparently occurs and probably also a different mutual affinity of the regulatory complex with the promoter site of the enzyme opérons is involved.     

Synthesis of β-galactosidase inEscherichia coli in the presence of phenylalanine analogues

The effect of phenylalanine analogues (p-F-phenylalanine, phenylserine and furylalanine) is described on the synthesis of inducible β-galactosidase inEscherichia coli ML-30 and phenylalanine requiring mutant ML-48. The incorporation of these analogues into the enzyme molecule results in the formation of a protein sensitive to a different extent to heat, urea and trypsin. The influence of the analogues on the ability to concentrate inducer inside the cells is also described. The different effect of the analogues on the synthesis and stability of the enzyme is discussed.     

The effect of anaerobiosis on the induced synthesis of β-galactosidase in non-growingEscherichia coli

Depending on conditions of aeration maltose and glucose were found to exhibit different effects on the inducible synthesis of β-galactosidase in aerobically grown cells ofEscherichia coli starving for an exogenous source of nitrogen; both saccharides repressed the synthesis of the enzyme under aerobic conditions, while the above-mentioned saccharides were essential for the enzyme synthesis under anaerobic conditions. The presence of maltose in the medium resulted in the repression of the enzyme synthesis in anaerobically grown cells starving for an exogenous nitrogen source under anaerobic conditions. The synthesis of β-galactosidase-specific messenger RNA was completely blocked and the synthesis of the enzyme proper considerably inhibited in aerobically grown cells incubated anaerobically in a medium without nitrogen and carbon sources.     

Inhibition of β-galactosidase synthesis inEscherichia coli after infection with different DNA and RNA phages

Infection ofEscherichia coli with T1, T2r⁺, T3 and T4 phages leads to an immediate inhibition of β-galactosidase synthesis. Similar results were obtained with the virulent mutant of phage lambda. The degree of inhibition of β-galactosidase synthesis depends on the time delay between the addition of the inducer and the phage particles, and on the amount of phage DNA, which has penetrated into the host cell. RNA phage MS2 exhibited no inhibitory effect on enzyme synthesis.     

Significance of β-galactosidase repression in glucose inhibition of lactose utilization inEscherichia coli

The role of -galactosidase repression in glucose inhibition of lactose utilization was studied inEscherichia coli. Escherichia coli 3300 constitutively produces -galactosidase even in the presence of glucose. When this strain was grown in a mixture of glucose and lactose, lactose utilization did not occur until glucose was depleted. The addition of glucose to a 3300 culture grown in lactose immediately caused a permanent inhibition of lactose utilization and only a mild transient repression of -galactosidase. Exogenous cyclic adenosine monophosphate (AMP) did not overcome the glucose inhibition of lactose utilization but did relieve the transient repression. Thus glucose inhibition of lactose utilization is not related to -galactosidase repression and is independent of cyclic AMP.     

Catabolite repression of β-galactosidase synthesis in Escherichia coli

1. Repression by glucose of β-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i⁻) and permease-less (y⁻) cells as well as in the corresponding i⁺ and y⁺ strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-β-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of β-galactosidase synthesis (e.g. isopropyl thio-β-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-β-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.     

The effect of nucleosides on the induced synthesis of β-galactosidase in non-growing cells ofEscherichia coli

The induced synthesis of β-galactosidase in non-growing cells ofEscherichia coli starving for exogenous carbon and nitrogen sources was stimulated markedly by the addition of any of four nucleosides tested: adenosine, guanosine, cytidine, and uridine. Adenosine was used as a representative of this group of compounds in most experiments. The decrease of ability of the cells to synthesize β-galactosidase, resulting from a prolonged starvation for exogenous carbon and nitrogen, was removed by adenosine. This compound also considerably reduced the inhibitory effect of metabolic poisons on the induced synthesis of β-galactosidase. The blockade of induced β-galactosidase synthesis evoked in aerobically grown cells by anaerobic starvation for exogenous sources of carbon and nitrogen was also significantly reduced by adenosine. The weak transient catabolic repression of induced synthesis of β-galactosidase evoked by glucose in non-growing cells ofEscherichia coli deprived of exogenous carbon and nitrogen sources was prevented by adenosine. The total repression caused by higher glucose concentrations was not influenced by this compound. The results are discussed from the point of view of the role of the energy state ofEscherichia coli cells in the regulation of β-galactosidase synthesis.     

Effects ofin-vitro protein stabilizers on the overproduction of recombinant β-lactamase inEscherichia coli

Effects ofin-vitro protein stabilizers, such as glycerol, fructose and NaCl, were investigated on the overproduction of the secreted recombinant protein, -lactamas, inE. coli. With the addition of glycerol, a five-fold increase in the amount of soluble -lactamase was obtained under optimal conditions. The extent of excretion was dependent on the glycerol concentration. Fructose showed similar effects. The effect of NaCl was dependent on the carbon source used.     

Effect of some pre-treatments on UV-sensitivity and on the course of macromolecular synthesis inEscherichia coli 15 T−, U−, his−

The UV-sensitivity ofEscherichia coli 15 T⁻, U⁻, his⁻ cells after a 45 minutes glucose, thymine uracil, or histidine pre-irradiation starvation, as well as the course of DNA, RNA, and protein synthesis during starvation and during a 60 minute post-treatment in a complete medium were investigated. An increased radioresistance was observed when starvation for some compounds resulted in a consequent inhibition of protein synthesis, as it was observed in the case of glucose, histidine, or uracil starvation. During thymine starvation, which led to a decreased resistance, no inhibition of protein synthesis was recorded. The postirradiation time-course of DNA synthesis did not show any correlation with the increased rate of resistance. The DNA synthesis after U⁻ pre-treatment was greatly delayed, however, after glucose pre-treatment no retardation was observed although both factors increased the rate of surviving cells approximately to the same extent. We assume that the factors which increase the radio-resistance could act by a similar mechanism which would take part in the inhibition of protein synthesis. This mechanism could consist in a decrease of the m-RNA turnover.     

Ethanol-mediated gene-amplification in Escherichia coli enhances derepressed synthesis of β-galactosidase

Summary The presence of ethanol (5 % v/v), in nutrient medium, ehanced DNA synthesis per E. coli cell nearly 2.8-fold compared to that in control cells. At this concentration, the derepressed synthesis of -galactosidase per bacterium also increased about 3-fold. We, therefore, propose that the ethanol-mediated gene-amplification proportionately elevated the induced synthesis of -galactosidase.     

Effects of immobilization on growth,substrate consumption, β-galactosidase induction,and byproduct formation inEscherichia coli

Summary Some metabolic properties of both suspended and immobilized aerobically and anaerobically growingEscherichia coli cells were investigated. Metabolic activity was found to be substantially different whenE. coli cells were immobilized in alginate. Cells grown immobilized in alginate, and then released from the gel, synthesized 1.6 (aerobic growth) and 4.9 (anaerobic growth) times as much -galactosidase per cell in response to induction as did suspended cells. Under both aerobic and anaerobic conditions, the cell yield from glycerol for immobilized cells was half that for suspended cells. At specific growth rates that were not significantly different from those of suspended cells, immobilized cells consumed glycerol at twice the rate of suspended cells. Immobilized cells produced elevated quantities of acetate, pyruvate, and lactate. Interpretation of these findings is discussed in terms of the kinetics of energy metabolism and the regulation of inducible protein synthesis inE. coli.     

Simultaneous and successive induction of synthesis of β-Galactosidase and tryptophanase inEscherichia coli K 12 in the chemostat

β-Galactosidase and tryptophanase were induced either simultaneously or successively during continuous cultivation of the inducible strainEscherichia coli K 12 in the chemostat. Growth was limited by glycerol and the dilution rate was 0.1 h⁻¹. During both the simultaneous and successive induction specific rates of synthesis, as well as maximum enzyme levels, were identical with those obtained after independent induction of individual enzymes. As compared with batch cultivation, β-galactosidase reached the same specific rate of synthesis in the chemostat, whereas the specific rate of synthesis of tryptophanase in the chemostat was up to five times higher.     

Inducible synthesis of β-galactosidase inKluyveromyces fragilis

Lactose,d-galactose, andl-arabinose induce the synthesis of β-galactosidase inKluyveromyces fragilis. Lactose is the best inducer with a maximum effect at 1.4 mm. The induced synthesis of the enzyme in glycerol grown stationary phase cells is triggered within 30 min of inducer addition, the full induction being achieved within subsequent 30–40 min.