Second-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 loop2-3 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 (MIC), 50 micrograms/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.
Yamaguchi
A
A
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Inagaki
Y
Y
Sawai
T
T
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001693:Biological Transport, Active; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D015183:Restriction Mapping; D013489:Suppression, Genetic; D013752:Tetracycline / Q000493:pharmacokinetics; D013753:Tetracycline Resistance / Q000235:genetics
D017920:Antiporters; D001426:Bacterial Proteins; D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C079051:tetA protein, Bacteria; D013752:Tetracycline