Messenger RNA potential and the delay before exponential decay of messages

Potential is a parameter of messenger RNA decay that measures the ability of a message population to continue to initiate complete translations. It can be measured from the start of induction using a specific inhibitor of translation-initiation. With very short inductions it can be seen that the potential of the β-galactosidase message is not lost at a constant exponential rate until a time (delay time) approximately equal to the time required to synthesize the β-galactosidase message. The capacity to make enzyme declines only after the induction lag and its subsequent decay shows a somewhat longer delay; unlike potential, capacity is also affected by the variable rate of ribosome movement. The delays in loss of potential and capacity are consistent with either of two mechanisms: (1) the message must be completed before attack at its starting end, or (2) two or more separate events (or hits) with specific time constants are needed to inactivate. Even at very low growth temperatures, functional decay kinetics are consistent with either mechanism, as is the mass decay of β-galactosidase message at 37 °C. Messages for anthranilate synthetase and galactoside acetyl-transferase do not require two hits to inactivate, but the data cannot determine if there is a delay equal to their synthesis time. Either β-galactosidase message is exceptional and, as opposed to other messages, requires two or more hits to be inactivated, or Escherichia coli messages generally do not commence to decay until they are completed.     

Translation and mRNA decay

Summary Degradation of messenger RNA from the lactose operon (lac mRNA) was measured during the inhibition of protein synthesis by chloramphenicol (CM) or of translation-initiation by kasugamycin (KAS). With increasing CM concentration mRNA decay becomes slower, but there is no direct proportionality between rates of chemical decay and polypeptide synthesis. During exponential growth lac mRNA is cleaved endonucleolytically (Blundell and Kennell, 1974). At a CM concentration which completely inhibits all polypeptide synthesis this cleavage is blocked. In contrast, if only the initiation of translation is blocked by addition of KAS, the cleavage rate as well as the rate of chemical decay are increased significantly without delay. These faster rates do not result from immediate degradation of the lengthening stretch of ribosome-free proximal message, since the full-length size is present and the same discrete message sizes are generated during inhibition.These results suggest that neither ribosomes nor translation play an active role in the degradative process. Rather, targets can be protected by the proximity of a ribosome, and without nearby ribosomes the probability of cleavage becomes very high. During normal growth there is a certain probability that any message is in such a vulnerable state, and the fraction of vulnerable molecules determines the inactivation rate of that species.     

Catabolite repression of the lac operon. Separate repression of two enzymes

1. Catabolite repression of β-galactosidase and of thiogalactoside transacetylase was studied in several strains of Escherichia coli K 12, in an attempt to show whether a single site within the structural genes of the lac operon co-ordinately controls translational repression for the two enzymes. In all experiments the rate of synthesis of the enzymes was compared in glycerol–minimal medium and in glucose–minimal medium. 2. In a wild-type strain, glucose repressed the synthesis of the two enzymes equally. 3. The possibility that repression was co-ordinate was investigated by studies of mutant strains that carry deletions in the genes for β-galactosidase or galactoside permease or both. In all of the strains with deletions, the repression of thiogalactoside transacetylase persisted, and it is concluded that there is no part of the structural gene for β-galactosidase that is essential for catabolite repression of thiogalactoside transacetylase. 4. Subculture of one strain through several transfers in rich medium greatly increased its susceptibility to catabolite repression by glucose. It is concluded that unknown features of the genotype can markedly affect sensitivity to catabolite repression. 5. These results make it clear that one cannot draw valid conclusions about the effect of known genotypic differences on catabolite repression from a comparison of two separate strains; to study the effect of a particular genetic change in a lac operon it is necessary to construct a partially diploid strain so that catabolite repression suffered by one lac operon can be compared with that suffered by another. 6. Four such partial diploids were constructed. In all of them catabolite repression of β-galactosidase synthesized by one operon was equal in extent to catabolite repression of thiogalactoside transacetylase synthesized by the other. 7. Taken together, these results suggest that catabolite repression of β-galactosidase and thiogalactoside transacetylase is separate but equal.     

Pressure-induced inactivation of E. coli β-galactosidase: influence of pH and temperatur

In order to assess the feasibility of a high-pressure immunodesorption process using a β-galactosidase-anti-/3-galactosidase complex as a model, the influence of high hydrostatic pressure on the inactivation of E. coli /3-galactosidase has been investigated. The irreversible activity loss of β-galactosidase was studied as a function of pH and temperature for pressures comprised between atmospheric pressure and 500 megapascal (MPa; 1 MPa = 10 bar). This enabled us to establish a practical pressure-temperature diagram of stability for this enzyme. The stability domains determined thus appeared to be strongly dependent on the pH under atmospheric pressure of the phosphate buffer employed for pressurisation. Therefore, to interpret meaningfully this result, the influence of pressure on the pH-activity curve of β-galactosidase was investigated by using a high-pressure stopped-flow device. It appeared that the pH-activity curve of this enzyme was also reversibly affected by pressures lower than 150 MPa. An interpretation of these results in relation to the high-pressure induced changes of ionisation constants is proposed. For our practical purpose, the implications for the elaboration of a high-pressure immunodesorption process using /3-galactosidase as a tag, are discussed.     

Patterns of growth and β-galactosidase production by Bifidobacteria

We investigated the patterns of growth and β-galactosidase production in the strains Bifidobacterium adolescentis GO-13, MS-42, 91-BIM, and 94-BIM and b. bifidum No.1, LVA-3, 791 on media with various carbon sources. The synthesis of β-galactosidase was shown to be associated with exponential growth of the cultures involved. The maximum specific rate of β-galactosidase synthesis of 0.20 U mg^?1 h^?1 was observed in B. bifidum LVA-3 after 3–6 h of cultivation. This value for B. adolescentis 91-BIM and 94-BIM was lower and amounted to 0.03–0.08 U mg^?1h^?1. On the medium with lactose, the highest specific growth rates for B. bifidum LVA-3 and B. bifidum No.1 were 0.38 and 0.60 h^?1, respectively, after 3–6 h of cultivation. For B. adolescentis 91-BIM and 94-BIM, this parameter peaked at 12–15 h of cultivation at 0.13 and 0.22 h^?1, respectively. The hydrolytic activity of β-galactosidase in the growth medium decreased during the stationary growth phase of the tested cultures.     

The effect of high pressure homogenization on the activity of a commercial β-galactosidase

High pressure homogenization (HPH) has been proposed as a promising method for changing the activity and stability of enzymes. Therefore, this research studied the activity of β-galactosidase before and after HPH. The enzyme solution at pH values of 6.4, 7.0, and 8.0 was processed at pressures of up to 150?MPa, and the effects of HPH were determined from the residual enzyme activity measured at 5, 30, and 45?°C immediately after homogenization and after 1?day of refrigerated storage. The results indicated that at neutral pH the enzyme remained active at 30?°C (optimum temperature) even after homogenization at pressures of up to 150?MPa. On the contrary, when the β-galactosidase was homogenized at pH 6.4 and 8.0, a gradual loss of activity was observed, reaching a minimum activity (around 30?%) after HPH at 150?MPa and pH 8.0. After storage, only β-galactosidase that underwent HPH at pH 7.0 retained similar activity to the native sample. Thus, HPH did not affect the activity and stability of β-galactosidase only when the process was carried out at neutral pH; for the other conditions, HPH resulted in partial inactivation of the enzyme. Considering the use of β-galactosidase to produce low lactose milk, it was concluded that HPH can be applied with no deleterious effects on enzyme activity.     

Purification and characterization of β-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis

β-galactosidase is a commercially important enzyme that was purified from probiotic Pediococcus acidilactici. The enzyme was extracted from cells using sonication and subsequently purified using ammonium sulphate fractionation and successive chromatographies on Sephadex G-100 and Q-Sepharose. The enzyme was purified 3.06-fold up to electrophoretic homogeneity with specific activity of 0.883 U/mg and yield of 28.26%. Molecular mass of β-galactosidase as estimated by SDS-PAGE and MALDI-TOF was 39.07 kDa. The enzyme is a heterodimer with subunit mass of 15.55 and 19.58 kDa. The purified enzyme was optimally active at pH 6.0 and stable in a pH range of 5.8–7.0 with more than 97% activity. Purified β-galactosidase was optimally active at 50 °C. Kinetic parameters K_m and V_max for purified enzyme were 400 µM and 1.22 × 10⁻¹ U respectively. Its inactivation by PMSF confirmed the presence of serine at the active site. The metal ions had different effects on enzyme. Ca²⁺, Mg²⁺ and Mn²⁺ slightly activated the enzyme whereas NH₄⁺, Co²⁺ and Fe³⁺ slightly decreased the enzyme activity. Thermodynamic parameters were calculated that suggested that β-galactosidase is less stable at higher temperature (60 °C). Purified enzyme effectively hydrolysed milk lactose with lactose hydrolysing rate of 0.047 min⁻¹ and t_1/2 of 14.74 min. This is better than other studied β-galactosidases. Both sonicated Pediococcus acidilactici cells and purified β-galactosidase synthesized galactooligosaccharides (GOSs) as studied by TLC at 30% and 50% of lactose concentration at 47.5 °C. These findings indicate the use of β-galactosidase from probiotic bacteria for producing delactosed milk for lactose intolerant population and prebiotic synthesis. pH and temperature optima and its activation by Ca²⁺ shows that it is suitable for milk processing.     

Aggregation of sponge cells: a novel mechanism of controlled intercerllular adhesion

From the marine sponge Geodia cydonium a series of macromolecules have been isolated and characterized which are involved in the control of aggregation and separation of sponge cells; these include aggregation factor, aggregation receptor, anti-aggregation receptor, β-glucuronidase, β-glucuronosyltransferase, β-galactosyltransferase, β-galactosidase and a lectin. These components might be linked in the following sequence: (a) activation of the aggregation receptor by its enzymic glucuronylaion; (b) adhesive recognition of the cells, mediated by the aggregation factor and the glucuronylated aggregation receptor; (c) inactivation of the aggregation receptor by its deglucuronylation with the membrane-associated β-glucuronidase; (d) cell separation due either to the loss of the recognition site (glucuronic acid) of the aggregation receptor for the aggregation factor or to an inactivation of the aggregation factor by the anti-aggregation receptor. The activity of the anti-aggregation receptor is probably controlled by the Geodia lectin.     

The specific inhibition of the expression of the lactose operon by D,L-threo-phenylserine inEscherichia coli

Phenylserine, one of the phenylalanine analogues, is incorporated into proteins ofEscherichia coli and replaces the natural amino acid. The incorporation results in the inhibition of the synthesis of both inducible and constitutive β-galactosidase. The rate of the synthesis of β-galactosidase specific m-RNA is only slightly influenced by phenylserine, the steady-state level being decreased by about 40%. The m-RNA formed in the present of the analogue functions normally and its translation after the removal of the inhibitor results in the formation of normal β-galactosidase. The character of the inhibition of the enzyme synthesis by phenylserine is similar to that caused by chloramphenicol. However, phenylserine specifically inhibits only the synthesis of β-galactosidase, whereas other cell proteins are synthesized. No protein immunologically cross-reacting with the antiserum against normal β-galactosidase is formed by inducible ánd constitutiveEscherichia coli strains. The active transport is completely inhibited as the cells induced in the presence of phenylserine do not accumulate¹⁴C-TMG. It follows from the results that phenylserine inhibits both the formation of TMG-specific permease and the synthesis of the active molecule of β-galactosidase inEscherichia coli.     

Proteolytic degradation of fused protein A-β-galactosidase in Escherichia coli

The stability of the fusion protein staphylococcal protein A-E. coli β-galactosidase (SpA-βgal) produced in E. coli has been studied both in cell disintegrate and in purified preparations. SpA-βgal was degraded by a proteolytic cleavage between the two functional parts of the molecule, resulting in one β-galactosidase tetramer and four protein A molecules. Intermediates were detected, namely β-galactosidase containing three, two and one protein A. The β-galactosidase was stable with respect to enzyme activity and molecular weight, while protein A was further degraded. In cell disintegrate the half-life of SpA-βgal was found to be 6 h at 20°C and 1.5 h at 37°C. The protease responsible for initial proteolytic cleavage of SpA-βgal was shown to be cell debris associated.     

The effect of oxygen transfer on the activity of encapsulated whole cell β-galactosidase

The effect of oxygen transfer on the production of immobilized whole cell β-galactosidase has been evaluated. The encapsulated whole cell β-galactosidase was prepared by combining cell encapsulation and culture into one-step. Escherichia coli was encapsulated and cultured in the growth and production media to accumulate β-galactosidase in itself. Sunflower seed oil was coimmobilized to increase the oxygen transfer rate through the capsule membrane. The oxygen transfer rate increased 63 percent and the activity of β-galactosidase increased by 10 percent. The activity of encapsulated β-galactosidase obtained in the concentric air lift reactor was 86 percent higher than that in the shaking incubator. In the concentric air lift reactor, the accumulation of encapsulated whole cell β-galactosidase was primarily dependent on the capsule velocity. While the accumulation of specific β-galactosidase in the capsule increased with volumetric oxygen transfer coefficient, the cell biomass accumulated in the capsule decreased.     

Differences in the Average Single Molecule Activities of E. coli β-Galactosidase: Effect of Source, Enzyme Molecule Age and Temperature of Induction

Using a capillary electrophoresis–based method, single enzyme molecule assays were performed on E. coli β-galactosidase from three different sets of samples. The first set consisted of lysates of induced cells from five different strains of the bacteria, as well as two different commercial preparations of the enzyme. These samples were found to have substantially different distributions of single molecule activities. For the second set of samples, β-galactosidase expression was induced for 1.5 hr, followed by further incubation where expression was repressed. Assays were performed on the lysates of the preinduction and on the lysates from aliquots taken set times postinduction. The recently induced enzyme had a 25% higher average single molecule activity than the basally expressed enzyme. This average activity returned to the basal value 3.5 hr postinduction and remained unchanged thereafter. Finally, β-galactosidase was induced at 26 and 42°C. The enzyme was assayed before and after partial thermal denaturation. The samples were found to be indistinguishable with respect to their average single molecule activities.     

Increased Inactivation of Messengers in <Emphasis Type="Italic">Escherichia coli ts</Emphasis> Mutant

WE recently described some of the properties of a temperature sensitive mutant of Escherichia coli (refs. 1–3 and unpublished work) in which RNAase II activity is increased on transfer to the non-permissive temperature^1,2, while the functional half-life of β-galactosidase mRNA¹ and the chemical half-life of the lac Operon mRNA³ are decreased. Questions raised by these studies were (a) can the strain be considered a general messenger RNAase mutant and (b) what is the direction of messenger inactivation in this strain? The latter question is particularly interesting since the increased RNAase activity in this strain is that of RNAase II (unpublished work) which degrades RNA molecules in the 3′ to 5′ direction⁴, while mRNA is known to be degraded in the 5′ to 3′ direction^5,6.     

Multiple β-galactosidase activities in petunia hybrida: Separation and characterization

Using isoelectrofocusing (IEF), multiple forms of Petunia β-galactosidase activity could be detected. The β-galactosidase pattern showed only minor tissue-specific differences. There were, however, species-specific differences. Zea mays, for instance, showed two bands which differed from the zones obtained with Petunia preparations. Petunia and corn leaves were mixed and extracted commonly. The species-specific activity patterns remained unchanged.Petunia preparations were inactivated by 8 Murea. Following dialysis, enzymatic activity and the Petunia-specific pattern were restored. The same holds true for a mixture of Petunia and E. coli β-galactosidase preparations. On refocusing isolated Petunia zones, untreated or inactivated by 8 M urea and reactivated by dialysis, the original mobilities were shown. Therefore, it seems highly improbable that the β-galactosidase pattern was due to artefacts. Using a Petunia line which was ‘pure’, also in respect to its β-galactosidase pattern, the four main bands were preparatively separated by IEF and characterized. They showed the same pH optimum (4.3), the same temperature optimum (55°), the same inactivation kinetics by urea, the same sensitivity against Cl^?, and closely related K_m. values. In sucrose gradient centrifugation they invariably showed S values of 8–10. The multiple activities could not be separated by zone electrophoresis using various carrier systems, or by gel filtration. It seems possible that they represent forms which differ only in isoelectric points, not in MW.     

The expression of β-galactosidase by Escherichia coli during continuous culture

We have used the technique of continuous culture to study the expression of β-galactosidase in Escherichia coli. In these experiments the cultures were grown on carbon-limited media in which half of the available carbon was supplied as glycerol, glucose, or glucose 6-phosphate, and the other half as lactose. Lactose itself provided the sole source of inducer for the lac operon. The steady-state specific activity of the enzyme passed through a maximal value as a function of dilution rate. Moreover, the rate at which activity was maximal (0.40 h^?1) and the observed specific activity of the enzyme at a given growth rate were found to be identical in each of the three media tested. This result was unexpected, since the steady-state specific activity can be shown to be equal to the differential rate of enzyme synthesis, and since it is known that glycerol, glucose, and glucose-6-P-cause different degrees of catabolite repression in batch culture. The differential rate of β-galactosidase synthesis was an apparently linear function of the rate of lactose utilization per milligram protein regardless of the composition of the input medium. That is, it is independent of the rate of metabolism of substrates other than lactose which are concurrently being utilized and the enzyme level appears to be matched to the metabolic requirement for it. If this relationship is taken to indicate the existence of a fundamental control mechanism, it may represent a form of attenuation of the rate of β-galactosidase synthesis which is independent of cyclic AMP levels.     

The effect of controlled feeding of glycerol on beta-galactosidase production by Escherichia coli in batch culture

A mutant of E. coli constitutive for β-galactosidase has been grown in batch culture with the carbon source, glycerol, fed at various fixed rates to the culture. High feeding rates where growth was only slightly restricted gave final enzyme levels similar to those obtained in cultures where all the glycerol was added initially. Low feeding rates resulted in breakdown of the β-galactosidase formed and gave reduced final levels of the enzyme.