1887

Abstract

The tetracycline-resistance protein, TetA(P), is an integral inner-membrane protein that mediates the active efflux of tetracycline from the cell. TetA(P) acts as an antiporter, presumably transporting a divalent cation–tetracycline complex in exchange for a proton, and is predicted to have 12 transmembrane domains (TMDs). Two glutamate residues that are located in predicted TMD 2 were previously shown to be required for the active efflux of tetracycline by TetA(P). To identify additional residues that are required for the structure or function of TetA(P), a random mutagenesis approach was used. Of the 61 tetracycline-susceptible mutants that were obtained in , 31 different derivatives were shown to contain a single amino acid change that resulted in reduced tetracycline resistance. The stability of the mutant TetA(P) proteins was examined by immunoblotting and 19 of these strains were found to produce a detectable TetA(P) protein. The MIC of these derivatives ranged from 2 to 15 μg tetracycline ml, compared to 30 μg tetracycline ml for the wild-type. The majority 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, which is predicted to be located in the periplasm in . It is concluded that these regions are of functional significance in the TetA(P)-mediated efflux of tetracycline from the bacterial cell.

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1999-10-01
2020-01-19
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