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 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 Escherichia coli, 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 microg tetracycline ml(-1), compared to 30 microg tetracycline ml(-1) 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 E. coli. It is concluded that these regions are of functional significance in the TetA(P)-mediated efflux of tetracycline from the bacterial cell.
Bannam
T L
TL
Department of Microbiology, Monash University, Clayton, Victoria, Australia.
Rood
J I
JI
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D003016:Clostridium perfringens / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics*; D017124:Conserved Sequence; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013753:Tetracycline Resistance / Q000235:genetics / Q000502:physiology; D013754:Tetracyclines
D000596:Amino Acids; D000900:Anti-Bacterial Agents; D017920:Antiporters; D001426:Bacterial Proteins; D008565:Membrane Proteins; D013754:Tetracyclines; C079051:tetA protein, Bacteria