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.


Article metrics loading...

Loading full text...

Full text loading...



  1. Abraham, L. J., Berryman, D. I. & Rood, J. I. (1988). Hybridization analysis of the class P tetracycline resistance determinant from the Clostridiumperfringens R-plasmid, pCW3. Plasmid 19, 113-120.[CrossRef] [Google Scholar]
  2. Allard, J. D. & Bertrand, K. P. (1992). Membrane topology of the pBR322 tetracycline resistance protein. TetA-PhoA gene fusions and implications for the mechanism of TetA membrane insertion. J Biol Chem 267, 17809-17819. [Google Scholar]
  3. Boyd, D., Traxler, B. & Beckwith, J. (1993). Analysis of the topology of a membrane protein by using a minimum number of alkaline phosphatase fusions. J Bacteriol 175, 553-556. [Google Scholar]
  4. Claros, M. G. & von Heijne, G. (1994). TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10, 685-686. [Google Scholar]
  5. Eckert, B. & Beck, C. F. (1989). Topology of the transposon Tn10-encoded tetracycline resistance protein within the inner membrane of Escherichia coli. J Biol Chem 264, 11663-11670. [Google Scholar]
  6. Fraser, C. M., Casjens, S., Huang, W. M. & 35 other authors (1997). Genomic sequence of a lyme disease spirochaete, Borrelia burgdorferi. Nature 390, 580–586.[CrossRef] [Google Scholar]
  7. Fujihira, E., Kimura, T., Shiina, Y. & Yamaguchi, A. (1996). Transmembrane glutamic acid residues play essential roles in the metal-tetracycline/H+ antiporter of Staphylococcus aureus. FEBS Lett 391, 243-246.[CrossRef] [Google Scholar]
  8. Fujihira, E., Kimura, T. & Yamaguchi, A. (1997). Roles of acidic residues in the hydrophilic loop regions of metal-tetracycline/H+ antiporter Tet(K) of Staphylococcus aureus. FEBS Lett 419, 211-214.[CrossRef] [Google Scholar]
  9. Ginn, S. L., Brown, M. H. & Skurray, R. A. (1997). Membrane topology of the metal-tetracycline/H+ antiporter TetA(K) from Staphylococcus aureus. J Bacteriol 179, 3786-3789. [Google Scholar]
  10. von Heijne, G. (1992). Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol 225, 487-494.[CrossRef] [Google Scholar]
  11. Henderson, P. J. (1990). Proton-linked sugar transport systems in bacteria. J Bioenerg Biomembr 22, 525-569.[CrossRef] [Google Scholar]
  12. Jessen-Marshall, A. E., Paul, N. J. & Brooker, R. J. (1995). The conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), in hydrophilic loop 2/3 of the lactose permease. J Biol Chem 270, 16251-16257.[CrossRef] [Google Scholar]
  13. Kennan, R. M., McMurry, L. M., Levy, S. B. & Rood, J. I. (1997). Glutamate residues located within putative transmembrane helices are essential for TetA(P)-mediated tetracycline efflux. J Bacteriol 179, 7011-7015. [Google Scholar]
  14. Kimura, T. & Yamaguchi, A. (1996). Asp-285 of the metal-tetracycline/H+ antiporter of Escherichia coli is essential for substrate binding. FEBS Lett 388, 50-52.[CrossRef] [Google Scholar]
  15. Kimura, T., Ohnuma, M., Sawai, T. & Yamaguchi, A. (1997). Membrane topology of the transposon 10-encoded metal-tetracycline/H+ antiporter as studied by site-directed chemical labeling. J Biol Chem 272, 580-585.[CrossRef] [Google Scholar]
  16. Kimura, T., Nakatani, M., Kawabe, T. & Yamaguchi, A. (1998). Roles of conserved arginine residues in the metal-tetracycline/H+ antiporter of Escherichia coli. Biochemistry 37, 5475-5480.[CrossRef] [Google Scholar]
  17. Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105-132.[CrossRef] [Google Scholar]
  18. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[CrossRef] [Google Scholar]
  19. Levy, S. B. (1992). Active efflux mechanisms for antimicrobial resistance. Antimicrob Agents Chemother 36, 695-703.[CrossRef] [Google Scholar]
  20. McMurry, L. M., Park, B. H., Burdett, V. & Levy, S. B. (1987). Energy-dependent efflux mediated by class L (tetL) tetracycline resistance determinant from streptococci. Antimicrob Agents Chemother 31, 1648-1650.[CrossRef] [Google Scholar]
  21. McNicholas, P., Chopra, I. & Rothstein, D. M. (1992). Genetic analysis of the tetA(C) gene on plasmid pBR322. J Bacteriol 174, 7926-7933. [Google Scholar]
  22. Miller, J. H. (1972).Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  23. Mojumdar, M. & Khan, S. A. (1988). Characterization of the tetracycline resistance gene of plasmid pT181 of Staphylococcus aureus. J Bacteriol 170, 5522-5528. [Google Scholar]
  24. Pao, S. S., Paulsen, I. T. & Saier, M. H.Jr (1998). Major facilitator superfamily. Microbiol Mol Biol Rev 62, 1-34. [Google Scholar]
  25. Paulsen, I. T., Brown, M. H., Dunstan, S. J. & Skurray, R. A. (1995). Molecular characterization of the staphylococcal multidrug resistance export protein QacC. J Bacteriol 177, 2827-2833. [Google Scholar]
  26. Paulsen, I. T., Brown, M. H. & Skurray, R. A. (1996). Proton-dependent multidrug efflux systems. Microbiol Rev 60, 575-608. [Google Scholar]
  27. Roberts, M. C. (1996). Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility, and distribution. FEMS Microbiol Rev 19, 1-24. [Google Scholar]
  28. Roy, C. R. & Isberg, R. R. (1997). Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages. Infect Immun 65, 571-578. [Google Scholar]
  29. Schnappinger, D. & Hillen, W. (1996). Tetracyclines: antibiotic action, uptake, and resistance mechanisms. Arch Microbiol 165, 359-369.[CrossRef] [Google Scholar]
  30. Seol, W. & Shatkin, A. J. (1993). Membrane topology model of Escherichia coli alpha-ketoglutarate permease by PhoA fusion analysis. J Bacteriol 175, 565-567. [Google Scholar]
  31. Sloan, J., McMurry, L. M., Lyras, D., Levy, S. B. & Rood, J. I. (1994). The Clostridium perfringens Tet P determinant comprises two overlapping genes: tetA(P), which mediates active tetracycline efflux, and tetB(P), which is related to the ribosomal protection family of tetracycline-resistance determinants. Mol Microbiol 11, 403-415.[CrossRef] [Google Scholar]
  32. Speer, B. S., Shoemaker, N. B. & Salyers, A. A. (1992). Bacterial resistance to tetracycline: mechanisms, transfer, and clinical significance. Clin Microbiol Rev 5, 387-399. [Google Scholar]
  33. Tomb, J. F., White, O., Kerlavage, A. R. & 39 other authors (1997). The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539–547.[CrossRef] [Google Scholar]
  34. Yamaguchi, A., Ono, N., Akasaka, T., Noumi, T. & Sawai, T. (1990a). Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10. The role of the conserved dipeptide, Ser65-Asp66, in tetracycline transport. J Biol Chem 265, 15525-15530. [Google Scholar]
  35. Yamaguchi, A., Udagawa, T. & Sawai, T. (1990b). Transport of divalent cations with tetracycline as mediated by the transposon Tn10-encoded tetracycline resistance protein. J Biol Chem 265, 4809-4813. [Google Scholar]
  36. Yamaguchi, A., Akasaka, T., Ono, N., Someya, Y., Nakatani, M. & Sawai, T. (1992a). Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. Roles of the aspartyl residues located in the putative transmembrane helices. J Biol Chem 267, 7490-7498. [Google Scholar]
  37. Yamaguchi, A., Ono, N., Akasaka, T. & Sawai, T. (1992b). Serine residues responsible for tetracycline transport are on a vertical stripe including Asp-84 on one side of transmembrane helix 3 in transposon Tn10-encoded tetracycline/H+ antiporter of Escherichia coli. FEBS Lett 307, 229-232.[CrossRef] [Google Scholar]
  38. Yamaguchi, A., Nakatani, M. & Sawai, T. (1992c). Aspartic acid-66 is the only essential negatively charged residue in the putative hydrophilic loop region of the metal-tetracycline/H+ antiporter encoded by transposon Tn10 of Escherichia coli. Biochemistry 31, 8344-8348.[CrossRef] [Google Scholar]
  39. Yamaguchi, A., Someya, Y. & Sawai, T. (1992d). Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. The role of a conserved sequence motif, GXXXXRXGRR, in a putative cytoplasmic loop between helices 2 and 3. J Biol Chem 267, 19155-19162. [Google Scholar]
  40. Yamaguchi, A., Akasaka, T., Kimura, T., Sakai, T., Adachi, Y. & Sawai, T. (1993a). Role of the conserved quartets of residues located in the N- and C-terminal halves of the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli. Biochemistry 32, 5698-5704.[CrossRef] [Google Scholar]
  41. Yamaguchi, A., Someya, Y. & Sawai, T. (1993b). The in vivo assembly and function of the N- and C-terminal halves of the Tn10-encoded TetA protein in Escherichia coli. FEBS Lett 324, 131-135.[CrossRef] [Google Scholar]
  42. Yamaguchi, A., Samejima, T., Kimura, T. & Sawai, T. (1996). His257 is a uniquely important histidine residue for tetracycline H+ antiport function but not mandatory for full activity of the transposon Tn10-encoded metal-tetracycline H+ antiporter. Biochemistry 35, 4359-4364.[CrossRef] [Google Scholar]
  43. Yoshida, K., Seki, S., Fujimura, M., Miwa, Y. & Fujita, Y. (1995). Cloning and sequencing of a 36-kb region of the Bacillus subtilis genome between the gnt and iol operons. DNA Res 2, 61-69.[CrossRef] [Google Scholar]

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error