The incorporation of β-2-thienylalanine into the β-galactosidase of Escherichia coli

The incorporation of the phenylalanine analog, β-2-thienylalanine, into the β-galactosidase (EC 3.2.1.23) of Escherichia coli is described. The catalytic properties of β-galactosidase in which at least 95% of the phenylalanine residues have been replaced by the analog were apparently unchanged. Phenylalanine replacement, however, rendered the β-galactosidase more labile than the normal enzyme to heat, urea, toluene, and trypsin (EC 3.4.4.4). Bacteria grown in the presence of β-2-thienylalanine had lost the ability to accumulate galactosides against a concentration gradient.     

The effect of histone on the inducible β-galactosidase in Escherichia coli

In E, coli ML 39 grown logarithmically histone inhibited, while in the stationary-phase cells histone stimulated the inducible synthesis of β-galactosidase. In the constitative strain ML 308 enzyme synthesis was not influenced. The effect of histone was antagonized by Mg⁺⁺ and Ca⁺⁺ lons and by spermine.     

The relationships in biosynthesis of the β-galactosidase- and Pz-proteins in Escherichia coli

E. coli cells possess a protein, Pz, which appears to be synthesized under all conditions of growth. In the presence of a galactoside, the cells also synthesize another protein, Gz, endowed with β-galactosidase properties, and very closely similar to Pz in antigenic structure as well as in solubility properties. The synthesis of Gz interferes very significantly with the net rate of Pz synthesis. Only those species of Enterobacteriaceae which possess the Pz protein are competent to synthesize β-galactosidase.     

Extracellular β-lactamase from Escherichia coli

Summary Escherichia coli strain O 127: K63: (B8): H—was grown in nutrient broth (Difco). Penicillinase activity was found in the culture supernatant after only five hours of incubation, i.e. during the exponential phase of growth. At this phase the levels of typical intracellular markers, did not indicate cell lysis or gross cell damage. The bioautographic revelation of penicillin-splitting enzymes on electropherograms of cell-free liquids confirmed the presence of one basic broad-spectrum -lactamase. This possibly extracellular -lactamase seems to be also present in the cellular extracts where it coexists with several other cell-bound penicillinases.     

The effects of selenite on β-galactosidase induction in Escherichia coli

Toxicity of selenite to Escherichia coli ML-30 is enhanced if the organism is inoculated into a selenite medium which contains lactose as the only carbon source. In media of this type, the cells must synthesize β-galactosidase before growth can take place, and a lag period is produced which is proportional to the selenite concentration. This phenomenon is not observed when another carbon source such as succinate is present, or, if the cells are fully induced for β-galactosidase.Isotope uptake studies demonstrate that the above phenomenon is not the result of a permeability barrier produced by selenite. The data suggest that the opposite is true and permeability to the inducer, lactose, is increased by selenite.The rate of β-galactosidase biosynthesis is increased in selenite supplemented media using lactose, or melibiose as the inducers, and succinate, maltose, glycerol, or lactic acid as carbon sources. By use of differential rate plots, the biosynthesis rate constants were evaluated and found to be greater than the controls which contain no selenite.The inhibition of β-galactosidase by selenite was studied and found to occur only at concentrations higher than those employed in the studies described above.Two explanations are offered for the results described herein.It is postulated that selenite uncouples growth from cell division. This would allow an increase in bacterial protein without cell division and give the net effect of an increased enzyme biosynthesis rate.Another possibility is that β-galactosidase is synthesized at a faster rate since it contains more methionine than the other cell protein. Thus, it is a potential depository for selenomethionine which is the product of selenite detoxification.     

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 establishment of β-galactosidase repression in the mating system of Escherichia coli K12

Experiments designed to explain the one-hour delay (latent period) in the establishment of repression of β-galactosidase synthesis following injection of F′i⁺ genes into F⁻i⁻, Escherichia coli K12 have shown the following:

• 1.(1) During the latent period, isopropyl-β-d-thiogalactoside doubled the rate of enzyme synthesis, indicating the existence of a low degree of repression.
• 2.(2) Complete repression was established rapidly at the end of the latent period.
• 3.(3) Puromycin or dl-4-methyltryptophan present from the beginning of mating blocked the establishment of repression. After the inhibitor was removed, a latent period of normal duration (60 minutes) was required to establish repression. Puromycin blocked protein synthesis, and 4-methyltryptophan was incorporated into protein. Therefore, synthesis of functional protein appears to be required to establish repression.
• 4.(4) During the first hour, addition of the same inhibitors for a brief period caused a subsequent partial repression. The degree of repression was proportional to the time interval between mating and addition of inhibitor. A new full-length latent period was subsequently required for the complete establishment of repression.
• 5.(5) De-repression upon removal of puromycin was never observed. This suggests that the repressor is stable under these conditions.

The data are interpreted in the context of a repressor made of protein precursors, called here pro-repressor. Pro-repressor is synthesized and accumulates following mating. It can partly repress enzyme formation and can interact with isopropyl-β-d-thiogalactoside. When a sufficiently high concentration of prorepressor accumulates, aggregation occurs forming active repressor. As suggested by Sadler & Novick (1965), the lag in the establishment of repression might be explained by a high power dependence of the rate of formation of repressor on the concentration of pro-repressor.     

Improved extraction procedure for the isolation of β-D-galactosidase from Escherichia coli

Summary A fast 4-step isolation procedure for -D-galactosidase from E. coli has been developed: cell disruption, two-stage aqueous two-phase extraction and ultrafiltration. A 60-fold purification of the enzyme with a total yield of 75% was achieved.     

The reconstituted Escherichia coli Bam complex catalyzes multiple rounds of β-barrel assembly

β-Barrel proteins are folded and inserted into the outer membranes of Escherichia coli by the Bam complex. The Bam complex has been purified and functionally reconstituted in vitro. We report conditions for reconstitution that increase the folding yield 10-fold and allow us to monitor the time course of folding directly. We use these conditions to analyze the effect of a mutation in the Bam complex and to demonstrate the ability of the reconstituted complex to catalyze more than one round of substrate assembly.     

Interaction of the lacZ β-galactosidase of Escherichia coli with some β-d-galactopyranoside competitive inhibitors

1. The location of the bivalent metal cation with respect to bound competitive inhibitors in Escherichia coli (lacZ) beta-galactosidase was investigated by proton magnetic resonance. 2. Replacement of Mg(2+) by Mn(2+) enhances both longitudinal and transverse relaxation of the methyl groups of the beta-d-galactopyranosyltrimethylammonium ion, and of methyl 1-thio-beta-d-galactopyranoside; linewidths are narrowed by increasing temperature. 3. The Mn(2+) ion is located 8-9A (0.8-0.9nm) from the centroid of the trimethylammonium group and 9A (0.9nm) from the average position of the methylthio protons. 4. The effective charge at the active site was probed by measurement of competitive inhibition constants (K(i) (o) and K(i) (+) respectively) for the isosteric ligands, beta-d-galactopyranosylbenzene and the beta-d-galactopyranosylpyridinium ion. 5. The ratio of inhibition constants (Q=K(i) (+)/K(i) (o)) obtained with 2-(beta-d-galactopyranosyl)-naphthalene and the beta-d-galactopyranosylisoquinolinium ion at pH7 with Mg(2+)-enzyme was identical, within experimental error, with that obtained with the monocyclic compounds. 6. The variation of Q for Mg(2+)-enzyme can be described by Q=0.1(1+[H(+)]/4.17x10(-10))/1+[H(+)]/10(-8)). 7. This, in the theoretical form for a single ionizable group, is ascribed to the ionization of the phenolic hydroxy group of tyrosine-501. 8. The variation of Q for Mg(2+)-free enzyme is complex, probably because of deprotonation of the groups normally attached to Mg(2+) as well as tyrosine-501.     

Escherichia coli processivity clamp β from DNA polymerase III is dynamic in solution

Escherichia coli DNA polymerase III is a highly processive replicase because of the presence of the β clamp protein that tethers DNA polymerases to DNA. The β clamp is a head-to-tail ring-shaped homodimer, in which each protomer contains three structurally similar domains. Although multiple studies have probed the functions of the β clamp, a detailed understanding of the conformational dynamics of the β clamp in solution is lacking. Here we used hydrogen exchange mass spectrometry to characterize the conformation and dynamics of the intact dimer β clamp and a variant form (I272A/L273A) with a weakened ability to dimerize in solution. Our data indicate that the β clamp is not a static closed ring but rather is dynamic in solution. The three domains exhibited different dynamics, though they share a highly similar tertiary structure. Domain I, which controls the opening of the clamp by dissociating from domain III, contained several highly flexible peptides that underwent partial cooperative unfolding (EX1 kinetics) with a half-life of ~4 h. The comparison between the β monomer variant and the wild-type β clamp showed that the β monomer was more dynamic. In the monomer, partial unfolding was much faster and additional regions of domain III also underwent partial unfolding with a half-life of ~1 h. Our results suggest that the δ subunit of the clamp loader may function as a "ring holder" to stabilize the transient opening of the β clamp, rather than as a "ring opener".     

The isolation and properties of β-galactosidase from Escherichia coli grown on sodium selenate

β-Galactosidase (EC 3.21.23) was isolated from Escherichia coli grown on 0.01 M selenate and compared with the enzyme from cells grown on sulfate. Neutron activation analyses indicated that only the β-galactosidase from selenate cells contained selenium. Amino acid analyses showed that about 80 of the 150 methionine residues had been substituted by selenomethionine while none of the cystine residues were replaced. The catalytic parameters, K_m and v_max, were not changed; however, the stability of the selenium β-galactosidase to heat and urea was decreased. It was noted that the urea-denatured enzyme containing selenium, renatured more rapidly and to a greater extent than did the enzyme with the normal sulfur content. A protein sedimenting with a sedimentation coefficient of 4 S was present during most of the isolation steps of both sulfur and selenium enzymes. The relative amount present in the selenium preparation was, however, much greater. Amino acid analysis suggested that this 4-S protein may be a subunit of the active β-galactosidase. A greater number of the methionines of the selenium 4-S protein were replaced by selenomethionine than in the active enzyme. The cystine contents were, however, nearly the same in both preparations.     

Over-production of β-carotene from metabolically engineered Escherichia coli

To produce recombinant β-carotene in vitro, synthetic operons encoding genes governing its biosynthesis were engineered into Escherichia coli. Constructs harboring these operons were introduced into either a high-copy or low-copy cloning vector. β-Carotene production from these recombinant E. coli cells was either constitutive or inducible depending upon plasmid copy number. The most efficient β-carotene production was with the low-copy based vector. The process was increased incrementally from a 5 l to a 50 l fermentor and finally into a 300 l fermentor. The maximal β-carotene yields achieved using the 50 l and 300 l fermentor were 390 mg l⁻¹ and 240 mg l⁻¹, respectively, with overall productivities of 7.8 mg l⁻¹ h⁻¹ and 4.8 mg l⁻¹ h⁻¹.