Dataset Information

Caffeine and Theophylline Inhibit ?-Galactosidase Activity and Reduce Expression in Escherichia coli.

ABSTRACT: The ?-galactosidase enzyme is a common reporter enzyme that has been used extensively in microbiological and synthetic biology research. Here, we demonstrate that caffeine and theophylline, common natural methylxanthine products found in many foods and pharmaceuticals, negatively impact both the expression and activity of ?-galactosidase in Escherichia coli. The ?-galactosidase activity in E. coli grown with increasing concentrations of caffeine and theophylline was reduced over sixfold in a dose-dependent manner. We also observed decreasing lacZ mRNA transcript levels with increasing methylxanthine concentrations in the growth media. Similarly, caffeine and theophylline inhibit the activity of the purified ?-galactosidase enzyme, with an approximately 1.7-fold increase in K _M toward o-nitrophenyl-?-galactoside and a concomitant decrease in v _max. The authors recommend the use of alternative reporter systems when such methylxanthines are expected to be present.

SUBMITTER: Horne J

PROVIDER: S-EPMC7758883 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Publications

Caffeine and Theophylline Inhibit β-Galactosidase Activity and Reduce Expression in Escherichia coli.

Horne Jesse J Beddingfield Elizabeth E Knapp Madison M Mitchell Stephanie S Crawford Logan L Mills Shelby Brooks SB Wrist Alexandra A Zhang Shuyuan S Summers Ryan M RM

ACS omega 20201214 50

The β-galactosidase enzyme is a common reporter enzyme that has been used extensively in microbiological and synthetic biology research. Here, we demonstrate that caffeine and theophylline, common natural methylxanthine products found in many foods and pharmaceuticals, negatively impact both the expression and activity of β-galactosidase in Escherichia coli. The β-galactosidase activity in E. coli grown with increasing concentrations of caffeine and theophylline was reduced over si ...[more]

PMID: 33376862

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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.

Dataset Information

Caffeine and Theophylline Inhibit ?-Galactosidase Activity and Reduce Expression in Escherichia coli.

Publications

Caffeine and Theophylline Inhibit β-Galactosidase Activity and Reduce Expression in <i>Escherichia coli</i>.

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