2ND-SITE SUPPRESSOR MUTATIONS FOR THE ASP-66-]CYS MUTANT OF THE TRANSPOSON TN10-ENCODED METAL-TETRACYCLINE/H+ ANTIPORTER OF ESCHERICHIA-COLI

Asp66 is the only essential acidic residue in the putative hydrophilic loop(2-3) region of the transposon Tn10-encoded metal-tetracycline/H+ antiporter [TetA(B)] [Yamaguchi, A., Nakatani, M., & Sawai, T. (1992a) Biochemistry 31, 8344-8348]. Escherichia coli cells producing a D66C mutant of TetA(B) showed no tetracycline resistance. A spontaneous second-site revertant was isolated from the cells carrying the D66C mutant gene, which showed moderate resistance to tetracycline [minimum inhibitory concentration (MIG), 50 mu g/mL]. The entire sequencing of the revertant genes revealed two secondary mutations, i.e., the codon 40 of GCT (Ala) --> GAT (Asp) and T --> G at 17 bases upstream from the initiation codon of the tetA gene. There was a T --> G mutation at position -17, which was a mutation in the tet promoter/operator region, which caused a decrease in TetA(B) production. The full expression of the A40D/D66C double and the A40D single mutants, which were constructed by site-directed mutagenesis, was deleterious for cell growth. The -17T --> G mutation mitigated the deleterious effect of these mutants through reduction of expression. The -17T --> G single mutation introduced into the wild-type tet gene did not affect the level of resistance, although the expression was significantly reduced. Intact cells carrying the A40D/D66C and -17T --> G/A40D/D66C mutant plasmids showed a reduced level of tetracycline accumulation due to active efflux, whereas no significant tetracycline uptake was observed in inverted membrane vesicles prepared from these mutant-producing cells. The A40D mutation is located at opposite side of the membrane to the D66C mutation in the putative secondary structure of TetA(B). The second-site mutation might mediate its effects through a structural perturbation propagated along the polypeptide backbone.