Second-site suppressor mutations for the Asp-66-->Cys mutant of the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli

Biochemistry
A YamaguchiT Sawai

Abstract

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 resi...Continue Reading

Citations

Mar 13, 2019·The Journal of Biological Chemistry·Srinivas AluriI David Goldman
Dec 1, 1996·Microbiological Reviews·I T PaulsenR A Skurray

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