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Molecular studies on the mechanism of tetracycline resistance mediated by Tet(O)
E K Manavathu1, C L Fernandez, B S Cooperman
1Department of Medical Microbiology, University of Alberta, Edmonton, Canada.
Abstract:
The mechanism of resistance to tetracycline in Escherichia coli mediated by the Campylobacter jejuni-derived resistance determinant Tet(O) was investigated. The cloned Tet(O) protein had no detectable effect on the intracellular accumulation of tetracycline. The presence of Tet(O) markedly diminished the inhibitory effect of tetracycline on protein synthesis both in vivo and in vitro. Ribosomes prepared from tetracycline-resistant and susceptible E. coli cells bound almost identical amounts of radiolabeled tetracycline. Thus, a reduction in the binding of the antibiotic to its target site on the ribosome is not the primary mechanism of resistance. Poly(U)-directed polyphenylalanine synthesis revealed that an S-100 fraction prepared from tetracycline-resistant cells made the ribosomes prepared from susceptible cells considerably more resistant to the inhibitory action of tetracycline. The N-terminal portion (1 to 150 residues) of Tet(O) is highly homologous to the GTP-binding domain of elongation factor Tu and to elongation factor G, indicating that the Tet(O) protein has the potential to bind GTP. These data suggest that the Tet(O) protein could function either as a tetracycline-resistant analog of this elongation factor(s) or by modifying the target sites on the ribosomes in a catalytic fashion.
Insights
The Tet(O) protein confers tetracycline resistance in Escherichia coli by preventing the antibiotic from inhibiting protein synthesis. This resistance mechanism does not involve reduced tetracycline binding to ribosomes.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Tetracycline resistance is a significant challenge in treating bacterial infections.
- The Tet(O) determinant from Campylobacter jejuni confers resistance to tetracycline in Escherichia coli.
- Understanding resistance mechanisms is crucial for developing effective antimicrobial strategies.
Purpose of the Study:
- To elucidate the mechanism by which the Tet(O) determinant confers tetracycline resistance in Escherichia coli.
- To determine if Tet(O) affects tetracycline accumulation or binding to ribosomes.
- To investigate the functional role of the Tet(O) protein in bacterial resistance.
Main Methods:
- Cloning and expression of the Tet(O) gene in E. coli.
- Measurement of intracellular tetracycline accumulation.
- Assays for tetracycline inhibition of protein synthesis in vivo and in vitro.
- Ribosome binding assays with radiolabeled tetracycline.
- In vitro protein synthesis assays using S-100 fractions and ribosomes.
Main Results:
- Tet(O) expression did not alter intracellular tetracycline levels.
- Tet(O) significantly reduced tetracycline's inhibition of protein synthesis.
- Ribosomes from resistant and susceptible cells bound similar amounts of tetracycline.
- An S-100 fraction from Tet(O)-resistant cells conferred resistance to susceptible ribosomes.
- Sequence homology suggests Tet(O) may bind GTP, similar to elongation factors.
Conclusions:
- Tet(O)-mediated tetracycline resistance in E. coli does not primarily result from reduced antibiotic binding to ribosomes.
- The Tet(O) protein likely interferes with tetracycline's action on protein synthesis through a mechanism involving GTP binding or ribosome modification.
- These findings provide insights into novel tetracycline resistance mechanisms and potential therapeutic targets.