Diversity of TETX-Like Proteins

Abstract

<p> The most uncommon form of tetracycline resistance is enzymatic inactivation of the drug. The first protein characterized that was shown to have this ability was TetX, a 44-kDa cytoplasmic protein responsible for inactivating tetracycline in E. coli. The associated gene, tetX, was found on a transposon in the bacterium Bacteroides fragilis, and encodes an NADP-requiring, FAD-dependent monooxygenase. TetX modifies the structure of tetracycline by the addition of a hydroxyl to the C-11a position, altering the β-diketone system of the tetracycline that is responsible for antibiotic activity. This project was designed to search for novel tetracycline inactivators, and to determine the origin of the tetX. Initially, the search for TetX-like proteins in S. coelicolor and C. hutchinsonii was performed using homologous protein sequences found using BLAST searches. Each of the genes encoding the homologous protein sequences was cloned, over-expressed and purified, then analyzed using HPLC and LC/MS methods to determine their tetracycline inactivating ability. Next, the published tetracycline inactivators Tet34 and Tet37 were tested for their ability to inactivate the drug using HPLC and LC/MS methods, after being cloned, over-expressed and purified. Finally, a search of the Actinomycete library belonging to the Wright Laboratory was conducted looking for novel tetracycline inactivators. Bioassays were the first step in a series of experiments done, with HPLC and LC/MS assays eventually being used to determine if an inactivation event was occurring. The homologous sequences from S. coelicolor and C. hutchinsonii did not inactivate tetracycline as determined by HPLC and LC/MS data. The potential inactivators, Tet34 and Tet37, were also found to be void of tetracycline inactivating activity. Finally, one isolate in the actinomycete library was thought to be inactivating the drug, however, upon further inspection via HPLC and LC/MS methods this inactivation event was dismissed. Future research should focus on the search for novel enzymes capable of modifying the structure of tetracycline, as well as the origin of the only known tetracycline inactivator, tetX.</p>