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Second-site suppressor mutations for the serine 202 to phenylalanine substitution within the interdomain loop of the tetracycline efflux protein Tet(C)

Publication Date
  • Tetracycline
  • Efflux
  • Pump
  • Resistance
  • Interdomain Loop
  • Life Sciences :: Microbiology [F11]
  • Sciences Du Vivant :: Microbiologie [F11]
  • Biology


Second-site Suppressor Mutations for the Serine 202 to Phenylalanine Substitution within the Interdomain Loop of the Tetracycline Efflux Protein Tet(C)* Received for publication, March 14, 2003, and in revised form, May 22, 2003 Published, JBC Papers in Press, May 23, 2003, DOI 10.1074/jbc.M302658200 Frederic M. Sapunaric and Stuart B. Levy‡ From the Center for Adaptation Genetics and Drug Resistance and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111 The serine 202 to phenylalanine substitution within the cytoplasmic interdomain loop of Tet(C) greatly re- duces tetracycline resistance and efflux activity (Sara- ceni-Richards, C. A., and Levy, S. B. (2000) J. Biol. Chem. 275, 6101–6106). Second-site suppressor mutations were identified following hydroxylamine and nitrosoguani- dine mutagenesis. Three mutations, L11F in transmem- brane 1 (TM1), A213T in the central interdomain loop, and A270V in cytoplasmic loop 8–9, restored a wild type level of resistance and an active efflux activity in Esch- erichia coli cells bearing the mutant tet(C) gene. The Tet S202F protein with the additional A270V mutation was expressed in amounts comparable with the original mu- tant, whereas L11F and A213T Tet(C) protein mutants were overexpressed. Introduction of each single muta- tion into the wild type tet(C) gene by site-directed mu- tagenesis did not alter tetracycline resistance or efflux activity. These secondary mutations may restore resist- ance by promoting a conformational change in the pro- tein to accommodate the S202F mutation. The data dem- onstrate an interaction of the interdomain loop with other distant regions of the protein and support a role of the interdomain loop in mediating tetracycline resistance. One of the major bacterial protections against the growth inhibitory action of tetracycline is exporting the drug out of the cytoplasm, thus preventing its reaching the ribosome target (2–4). Tet(B) and Tet

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