Mutations in the Tn10 tet repressor that interfere with induction. Location of the tetracycline-binding domain

Abstract

Tetracycline induces transcription of the Tn10 tetracycline resistance gene (tetA) by binding to the tet repressor, thereby reducing the repressor's affinity for two operator sites that overlap the tet promoters. We characterized mutations in the tet repressor (tetRs mutations) that interfere with induction of tetA expression. The mutations were isolated on multicopy Tn10 tet plasmids by selecting for resistance to the inducer 5a,6-anhydrotetracycline. Under these conditions, maximal induction of tetA expression inhibits the growth of Escherichia coli K-12. DNA sequence analysis of 25 spontaneous tetRs mutations identified amino acid changes at 13 different positions clustered near the middle of the 207 amino acid residue sequence of tet repressor. This region (residues 64 to 107) is distinct from the bihelical DNA-binding motif of tet repressor (residues 26 to 47). The capacity of tetRs repressors to bind tet operator DNA and to respond to inducer was examined in vivo in tetA-lacZ fusion strains. In three cases, the capacity of tetRs repressors to bind tetracycline was examined in vitro using cell extracts enriched in repressor. Mutations 64Y (His64----Tyr) and 82H (Asn82----His) reduce the repressor's affinity for tetracycline more than 1000-fold and more than 100-fold, respectively, suggesting that His64 and Asn82 may be part of the inducer-binding site or directly involved in maintaining its conformation. Mutation 103I (Thr103----Ile) reduces the repressor's affinity for tetracycline less than tenfold, yet it interferes with induction to a greater extent than either 64Y or 82H, suggesting that 103I may also reduce the repressor's capacity to undergo a conformational change required for induction. The properties of tetRs mutants suggest that the region of amino acid residues 64 to 107 is involved in inducer binding and in signalling between the inducer-binding and operator-binding domains of the repressor.