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.     

Effect of ethionine on the synthesis of β-galactosidase inEscherichia coli

Ethionine at concentrations of 10⁻³M, 5×10⁻³M and 10⁻²M inhibits growth, both of β-galactosidase inducible ML-30 and constitutive ML-308Escherichia coli strains. The protein synthesis (measured by the incorporation of l-leucine-¹⁴C and l-aspartic-¹⁴C acid into proteins) of these strains is inhibited to the same extent as their growth. The synthesis of inducible and constitutive β-galactosidase produced by the strains ML-30 and ML-308, respectively, is considerably inhibited by ethionine.     

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.     

Activity of different forms of initiation factor 2 in the vitro synthesis of beta-galactosidase.

Two forms of initiation factor 2, (IF-2α, M_r, 118,000 and IF-2β, M_r 90,000) have been isolated from Escherichia coli extracts and tested for their ability to support β-galactosidase synthesis in a phage DNA-directed in vitro protein synthesis system. Although both forms are equally active in supporting the binding of fMet-tRNA to ribosomes only IF-2α functions in β-galactosidase synthesis.     

Analytical limits of four β-glucuronidase and β-galactosidase-based commercial culture methods used to detect Escherichia coli and total coliforms

Colilert^® (Colilert), Readycult^® Coliforms 100 (Readycult), Chromocult^® Coliform agar ES (Chromocult), and MI agar (MI) are β-galactosidase and β-glucuronidase-based commercial culture methods used to assess water quality. Their analytical performance, in terms of their respective ability to detect different strains of Escherichia coli and total coliforms, had never been systematically compared with pure cultures. Here, their ability to detect β-glucuronidase production from E. coli isolates was evaluated by using 74 E. coli strains of different geographic origins and serotypes encountered in fecal and environmental settings. Their ability to detect β-galactosidase production was studied by testing the 74 E. coli strains as well as 33 reference and environmental non-E. coli total coliform strains. Chromocult, MI, Readycult, and Colilert detected β-glucuronidase production from respectively 79.9, 79.9, 81.1, and 51.4% of the 74 E. coli strains tested. These 4 methods detected β-galactosidase production from respectively 85.1, 73.8, 84.1, and 84.1% of the total coliform strains tested. The results of the present study suggest that Colilert is the weakest method tested to detect β-glucuronidase production and MI the weakest to detect β-galactosidase production. Furthermore, the high level of false-negative results for E. coli recognition obtained by all four methods suggests that they may not be appropriate for identification of presumptive E. coli strains.     

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.     

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.

     

Production of the hybrid protein staphylococcal protein A/Escherichia coli β-galactosidase with E. coli

We have investigated the cultivation of an Escherichia coli strain producing the hybrid protein SpA-βgal. The hybrid protein consists of protein A from Staphylococcus aureus and β-galactosidase from E. coli with retained biological activity of both protein A and β-galactosidase. The expression was controlled by the temperature regulated P_R promoter from phage lambda. By late induction of the product synthesis it was possible to circumvent the problem with plasmid instability. The amount of produced SpA-βgal corresponded to approximately 1256 of the cell dry weight. In shake flask cultures most of the hybrid protein was found in an insoluble form and typical inclusion bodies were observed. However, the major part of the protein could be produced in a soluble and biological active form under controlled conditions in a reactor.     

Characterization of burden on growth due to the nutritional state of media and pre-induced gene expression

Studies have shown that the production of unnecessary proteins burdens the cellular growth mainly due to allocation of cellular resources to unnecessary protein synthesis, thereby limiting the resources available for growth. In the current study, we focus on the effect of pre-induction and nutritional status of the medium on the burden imposed on growth due to the synthesis of unnecessary protein. Escherichia coli cells with different history were grown in a glycerol media with and without IPTG to characterize the burden imposed due to the synthesis of β-galactosidase. Effect of pre-induced lac operon on growth and β-galactosidase expression on lactose milieu was also investigated. The study demonstrates that pre-induction has a strong influence on the extent of burden and is sustained in several generations. Further, the burden was much lower in a rich media relative to that observed in a minimal media.     

G6P-capturing molecules in the periplasm of Escherichia coli accelerate the shikimate pathway

Escherichia coli, the most studied prokaryote, is an excellent host for producing valuable chemicals from renewable resources as it is easy to manipulate genetically. Since the periplasmic environment can be easily controlled externally, elucidating how the localization of specific proteins or small molecules in the periplasm affects metabolism may lead to bioproduction development using E. coli. We investigated metabolic changes and its mechanisms occurring when specific proteins are localized to the E. coli periplasm. We found that the periplasmic localization of β-glucosidase promoted the shikimate pathway involved in the synthesis of aromatic chemicals. The periplasmic localization of other proteins with an affinity for glucose-6-phosphate (G6P), such as inactivated mutants of Pgi, Zwf, and PhoA, similarly accelerated the shikimate pathway. Our results indicate that G6P is transported from the cytoplasm to the periplasm by the glucose transporter protein EIICB^Glc, and then captured by β-glucosidase.     

Quaternary structure,Mg2+ interactions,and some kinetic properties of the (β-galactosidase fromThermoanaerobacterium thermosulfurigenes EM1

Theβ-galactosidase fromThermoanaerobacterium thermosulfurigenes EM1 was found to be a dimer with a monomer molecular weight of about 85,000. It lacks theα-peptide and an importantα-helix that are both needed for dimer-dimer interaction and there is no homology in other important dimer-dimer interaction areas. These differences in structure probably account for the dimeric (rather than tetrameric) structure. Only 0.19 Mg²⁺ bound per monomer and Mg²⁺ had only small effects on the activity and heat stability. The absence of residues equivalent to Glu-416 and His-418 (two of the three ligands to Mg²⁺ in theβ-galactosidase fromEscherichia coli) probably accounts for the low level of Mg²⁺ binding and the consequent lack of response to Mg²⁺. Both Na⁺ and K⁺ also had no effect on the activity. The enzyme activity witho-nitrophenyl-β-D-galactopyanoside (ONPG) was very similar to that withp-nitrophenyl-β-D-β-D-galactopyranoside (PNPG) and the ONPG pH profile was very similar to the PNPG pH profile. These differences are in contrast to theE. coli β-galactosidase, which dramatically discriminates between these two substrates. The lack of discrimination by theT. thermosulfurigenes β-galactosidase could be due to the absence of the sequence equivalent to residues 910-1023 of theE. coli β-galactosidase. Trp-999 is probably of the most importance. Trp-999 of theE. coli β-galactosidase is important for aglycone binding and ONPG and PNPG differ only in their aglycones. The suggestion that the aglycone site of theT. thermosulfurigenes β-galactosidase is different was strengthened by competitive inhibition studies. Compared toE. coli β-galactosidase, D-galactonolactone was a very good inhibitor of theT. thermosulfurigenes enzyme, while L-ribose inhibited poorly. These are transition-state analogs and the results indicate thatT. thermosulfurigenes β-galactosidase binds the transition state differently than doesE. coli β-galactosidase. Methanol and glucose were good acceptors of galactose, and allolactose was formed when glucose was the acceptor. Allolactose could not, however, be detected by TLC when lactose was the substrate. The differences noted may be due to the thermophilic nature ofT. thermosulfurigenes.     

An autoradiographic study of the effect of the plant hormone abscisic acid on nucleic acid and protein metabolism

Summary Abscisic acid maintains embryos in a state of dormancy and inhibits the incorporation of H³uridine and H³thymidine but not the incorporation of H³leucine. Ribosomes present in imbibed but dormant embryos do not become associated into polysomes until actual germination of the embryos. Protein synthesis still occurs in embryos when RNA synthesis is inhibited and therefore stable m-RNA must be present in dormant embryos. It is concluded that abscisic acid maintains dormancy by inhibiting the production of specific types of m-RNA, and therefore the formation of specific proteins. The activity of abscisic acid is antagonistic to the effect of gibberellic acid in dormancy.     

The α-galactosidase from Escherichia coli K12

Growth of Escherichia coli on melibiose requires the induced synthesis of α-galactoside permease and α-galactosidase. Hydrolysis of the chromogenic substrate p-nitrophenyl-σ-galactoside by whole bacteria is dependent on intact oxidative metabolism. The α-galactosidase from E. coli was isolated for the first time as a soluble enzyme. In cell-free extracts p-nitrophenyl-α-galactoside hydrolisis was observed only at high protein concentrations and the activity decreased exponentially with the square of the dilution. The reason for this behaviour was shown to be that, unlike other known α-galactosidases, the enzyme of E. coli requires NAD. For optimal activity the enzyme also requires Mn²⁺, a high concentration of 2-mercaptoethanol, and a pH of 8.1. The approximate molecular weight of the active from of α-galactosidase as determined by sedimentation in a sucrose gradient is 200 000. Due to the instability of the enzyme, its purification has not been achieved.     

The effect of some ions on the activity of β-galactosidase and the inhibitory effect of tris (hydroxymethyl) aminomethane

The stimulating effect of phosphate and the inhibitory effect of tris-HCl on the activity of β-galactosidase inEscherichia coli was studied. The phosphate anion antagonizes the inhibitory effect of chloride. Since a similar effect is displayed by sulphate and arsenate no specific “stimulating” effect of phosphate can take place. The tris cation has also an inhibitory effect which is antagonized by univalent cations (K⁺). The resulting β-galactosidase activity reflects the antagonisms between cations and anions present in the reaction medium.