The only approach to the control of bacterial infections is the use of antibacterial drugs. However, antibiotic resistance can compromise treatment. One of the most frequently used antibiotics is tetracycline. The Tet M resistance determinant, which we discovered during the course of this project, has proven to have an extremely broad host range among bacterial pathogens. We have shown that Tet(M) protein protects the protein biosynthetic machinery of the cell from the inhibitory action of antibiotic at the level of the ribosome. The goal of this project is to understand the molecular basis for this protection mechanism with the hope that such information may be useful in design of new tetracycline derivatives which may be useful in treating infections with a possible secondary outcome being a better understand the mechanism of tetracycline action. To this end we plan to study the certain steps during protein synthesis for their susceptibility to tetracycline and the consequence of the presence of Tet(M) protein. In particular preliminary experiments suggest that Tet(M) protein may function as a homolog of the normal elongation factor G (which functions during ribosome translocation). some extent the approaches outlined below will be guided by this observation. Two general approaches will be employed: (1) A biochemical approach will be used to evaluate the physical interaction of Tet(M) with protein biosynthetic components. For example, we have shown that the protein has high affinity for the ribosome, although it may also interact with elongation factors. The nature of such interactions will be examined by use of protein-protein and protein-nucleic acid crosslinking reagents to deduce interaction of Tet(M) with these components, and by application of alkylation protection methods to assess rRNA interactions with tetracycline in the presence and absence Tet(M). (2) These biochemical experiments will be complemented by genetic analysis of Escherichia coli chromosomal mutations that were isolated during the previous grant period which confer tetracycline sensitivity on the cell even in the presence of functional Tet(M). Our current hypothesis is that such mutations alter the gene(s) that encode tetracycline binding activities and/or components which interact directly with Tet(M). Preliminary mapping of these mutations place them within the major cluster of ribosomal protein genes (73 min). We hope to clone and identify the corresponding genes and characterize the nature of the mutations conferring this phenotype. In a related approach, we plan attempted isolation of mutations in the rRNA coding sequences to see if we can identify alterations in these molecules that result in tetracycline resistance.
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