Interactions between metabolic intermediates and beta-galactosidase from Escherichia coli.
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ABSTRACT:
SUBMITTER: Moses V
PROVIDER: S-EPMC1179217 | biostudies-other | 1970 Jul
REPOSITORIES: biostudies-other
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Project description:1. The intermediary metabolism of two strains of Escherichia coli has been examined. One strain (Q22) exhibits acute transient repression of beta-galactosidase synthesis when glucose is supplied to cells growing on glycerol; the other strain (W3110) does not. The two strains do not differ genetically in their lac operons. 2. Strain Q22 uses about twice as much glucose as strain W3110 per unit of cell mass produced. 3. Pentose phosphate-cycle activity in the presence of glucose is much stronger in strain Q22 than in strain W3110. 4. In strain Q22 the pool sizes of glucose 6-phosphate, 6-phosphogluconate, fructose 1,6-diphosphate and NADPH increase when glucose is added to cells growing on glycerol, and beta-galactosidase synthesis is severely inhibited. After about 1hr. the synthesis of beta-galactosidase is partly resumed, and the pool sizes of the four compounds fall. ATP, NADH and several other phosphorylated compounds show no concentration changes. 5. These concentration changes do not occur in strain W3110, in which beta-galactosidase synthesis is only rather weakly repressed by glucose. 6. It is suggested that repression of enzyme synthesis by glucose requires the rapid operation of the pentose phosphate cycle, and is mediated by one of the four substances whose concentration rises and later falls in strain Q22. A definite choice of effector from among these four possibilities cannot at present be made.
| S-EPMC1270415 | biostudies-other
Project description:The rate of differential synthesis of beta-galactosidase (alphalac) was measured in maximally induced cultures of Escherichia coli B/r with 0.01 M-inducer and 0.01 M-cyclic AMP. The value of alphalac decreases with growth rate (60% between 0.67 and 2.1 doublings/h) and after a nutritional shift-up. This decrease is presumed to reflect a decrease in the intracellular concentration of free active RNA polymerase after a shift-up, which implies that the increase in ribosome synthesis after a shift-up is due to an active induction of the ribosomal components.
| S-EPMC1165696 | biostudies-other
Project description:1. Repression by glucose of beta-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-beta-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of beta-galactosidase synthesis (e.g. isopropyl thio-beta-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-beta-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.
| S-EPMC1265142 | biostudies-other
Project description:Carboxymethylated beta-galactosidase from Escherichia coli was dissociated at 100 degrees C to form carboxymethylated fragments A and B. The mol.wts. of carboxymethylated fragments A and B were determined by gel filtration to be 64300 and 22400 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of carboxymethylated fragments A and B that had been pretreated with 2-mercaptoethanol and sodium dodecyl sulphate yielded mol.wts. of 64000 and 22100 respectively. Carboxymethylated fragments A and B had arginine as their C-terminal amino acid. When a crude extract of E. coli M15 was filtered through a column of Sepharose 6B, it was found that carboxymethylated fragment B could restore beta-galactosidase activity when added to fractions having mol.wts. estimated to be 123000, 262000 and 506000. These fractions are referred to as ;complementable fractions'. Similarly, it was found that carboxymethylated fragment A could restore enzyme activity to tractions having mol.wts. estimated to be 63000, 253000 and 506000. Estimates of the molecular weights of the beta-galactosidase activity obtained by restoration with carboxymethylated fragments A and B were made by filtering the active enzyme through another column of Sepharose 6B. The enzyme obtained by complementation with carboxymethylated fragment B, i.e. the complemented enzyme, had mol.wt. 525000, and that obtained with carboxymethylated fragment A had mol.wts. of 525000, 646000 and 2000000. The latter finding suggests that multiple forms of complemented beta-galactosidase can exist.
| S-EPMC1164922 | biostudies-other
Project description:BETA-Galactosidase (EC 3.2.1.23), prepared from strains ML 308 and K12 3300 of Escherichia coli, dissociated into an inactive monomer in the presence of Ag+. When such a monomer preparation is treated with excess of thiol an enzymically active dimer is formed in addition to an active tetramer. It is suggested that Ag+ may be of value in studies on other multimeric proteins as a mild dissociating agent.
| S-EPMC1172374 | biostudies-other
Project description:Understanding interactions between antibiotics used in combination is an important theme in microbiology. Using the interactions between the antifolate drug trimethoprim and the ribosome-targeting antibiotic erythromycin in Escherichia coli as a model, we applied a transcriptomic approach for dissecting interactions between two antibiotics with different modes of action. When trimethoprim and erythromycin were combined, the transcriptional response of genes from the sulfate reduction pathway deviated from the dominant effect of trimethoprim on the transcriptome. We successfully altered the drug interaction from additivity to suppression by increasing the sulfate level in the growth environment and identified sulfate reduction as an important metabolic determinant that shapes the interaction between the two drugs. Our work highlights the potential of using prioritization of gene expression patterns as a tool for identifying key metabolic determinants that shape drug-drug interactions. We further demonstrated that the sigma factor-binding protein gene crl shapes the interactions between the two antibiotics, which provides a rare example of how naturally occurring variations between strains of the same bacterial species can sometimes generate very different drug interactions.
| S-EPMC8564399 | biostudies-literature
Project description:The tolQRAB-pal operon is conserved in Gram-negative genomes. The TolQRA proteins of Escherichia coli form an inner membrane complex in which TolQR uses the proton-motive force to regulate TolA conformation and the in vivo interaction of TolA C-terminal region with the outer membrane Pal lipoprotein. The stoichiometry of the TolQ, TolR, and TolA has been estimated and suggests that 4-6 TolQ molecules are associated in the complex, thus involving interactions between the transmembrane helices (TMHs) of TolQ, TolR, and TolA. It has been proposed that an ion channel forms at the interface between two TolQ and one TolR TMHs involving the TolR-Asp(23), TolQ-Thr(145), and TolQ-Thr(178) residues. To define the organization of the three TMHs of TolQ, we constructed epitope-tagged versions of TolQ. Immunodetection of in vivo and in vitro chemically cross-linked TolQ proteins showed that TolQ exists as multimers in the complex. To understand how TolQ multimerizes, we initiated a cysteine-scanning study. Results of single and tandem cysteine substitution suggest a dynamic model of helix interactions in which the hairpin formed by the two last TMHs of TolQ change conformation, whereas the first TMH of TolQ forms intramolecular interactions.
| S-EPMC3064227 | biostudies-literature
Project description:Genetically encoded reporter proteins are important and widely used tools for the identification and capture of a promoter, tracking the dynamic behavior of transcription, and the quantification of promoter activity. The sensitivity of the reporter gene is a critical factor for an ideal reporter system because weak transcriptional signal has usually failed to be detected using classical reporters. In this study, we present a novel reporter system for improved monitoring of transcription in E. coli based on β-galactosidase α-complementation. In this reporter system, the β-galactosidase activity resulting from the assembly of a reporter lacZα and an existing α-acceptor in advance serves as a measure of transcriptional activity in vivo. To validate the potential of the lacZα-derived reporter system, a series of artificial operons were constructed, and the moderately strong lac promoter, ara promoter, and weak pbr promoter were chosen as the detection promoters. The response profiles of lacZα was similar to that of wild type lacZ in artificial lac operons. Due to its small size and efficient expression profile, the detection sensitivity of a lacZα-derived reporter system was significantly higher than that of the traditional full-length β-galactosidase and the fluorescent protein mCherry reporter system in artificial ara operons. As expected, the response sensitivity of the lacZα-derived reporter system was also demonstrated to be significantly higher than that of the β-galactosidase and mCherry reporter systems in lead-sensitive artificial pbr operons. The lacZα-derived reporter system may prove to be a valuable tool for detecting promoter activity, especially low-level transcription in vivo.
| S-EPMC6607957 | biostudies-literature
Project description:The molecular weight of Escherichia coli beta-galactosidase was determined in 6m- and 8m-guanidine hydrochloride by meniscus-depletion sedimentation equilibrium, sedimentation velocity and viscosity. Sedimentation equilibrium revealed heterogeneity with the smallest component having a molecular weight of about 50000. At lower speeds, the apparent weight-average molecular weight is about 80000. By use of a calculation based on an empirical correlation for proteins that are random coils in 6m-guanidine hydrochloride, sedimentation velocity gave a molecular weight of 91000, and the intrinsic viscosity indicated a viscosity-average molecular weight of 84000. Heating in 6m-guanidine hydrochloride lowered the viscosity of beta-galactosidase in a variable manner.
| S-EPMC1179595 | biostudies-other
Project description: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.
| S-EPMC1186350 | biostudies-other