Identification of structural and functional domains of the tetracycline efflux protein TetA(P) from Clostridium perfringens

The Clostridium perfringens tetracycline-resistance protein, TetA(P), is an integral inner-membrane protein that mediates the active efflux of tetracy cline from the cell. TetA(P) acts as an antiporter, presumably transporting a divalent cation-tetracycline complex in exchange for a proton, and is pr edicted to have 12 transmembrane domains (TMDs). Two glutamate residues tha t are located in predicted TMD 2 were previously shown to be required for t he active efflux of tetracycline by TetA(P). To identify additional residue s that are required for the structure or function of TetA(P), a random muta genesis approach was used. Of the 61 tetracycline-susceptible mutants that were obtained in Escherichia coli, 31 different derivatives were shown to c ontain a single amino acid change that resulted in reduced tetracycline res istance. The stability of the mutant TetA(P) proteins was examined by immun oblotting and 19 of these strains were found to produce a detectable TetA(P ) protein. The MIC of these derivatives ranged from 2 to 15 mu g tetracycli ne ml(-1), compared to 30 mu g tetracycline ml(-1) for the wild-type. The m ajority of these mutants clustered into three potential loop regions of the TetA(P) protein, namely the cytoplasmic loops 2-3 and 4-5, and loop 7-8, w hich is predicted to be located in the periplasm in E. coli. It is conclude d that these regions are of functional significance in the TetA(P)-mediated efflux of tetracycline from the bacterial cell.