GLUTAMATE RESIDUES LOCATED WITHIN PUTATIVE TRANSMEMBRANE HELICES ARE ESSENTIAL FOR TETA(P)-MEDIATED TETRACYCLINE EFFLUX

The tetA (P) gene from Clostridium perfringens encodes a unique membra ne protein that is responsible for the active efflux of tetracycline f rom resistant cells, The novel TetA(P) protein has neither the typical structure nor the conserved motifs that are found in tetracycline eff lux proteins from classes A through H or classes K and L. Site-directe d mutagenesis of selected residues within TetA(P) was performed to elu cidate their role in tetracycline efflux. Glutamate residues 52 and 59 , negatively charged residues located within putative transmembrane he lix 2, could not be replaced by either glutamine or aspartate and so w ere essential for tetracycline efflux. Replacement of Glu89, which was located at the end of helix 3, by aspartate but not by glutamine allo wed TetA(P) function, indicating the importance of a carboxyl group at this position, After mutation of the Asp67 residue, located within cy toplasmic loop 1, no immunoreactive protein was detected, It is conclu ded that negatively charged residues that appear to be located within or near the membrane are important for the function of TetA(P).