Mutational analysis of quinolone resistance protein QnrB1

Abstract

Alanine substitutions and selected deletions have been used to localize amino acids in QnrB essential for its protective activity. Essential amino acids are found at positions i and i(-2) in the pentapeptide repeat module and in the larger of two loops, where deletion of only a single amino acid compromises activity. Deletion of 10 amino acids at the N terminus is tolerated, but removal of 3 amino acids in the C-terminal dimerization unit destroys activity.

Fig 1

Protection of DNA gyrase from ciprofloxacin inhibition of supercoiling by wild-type QnrB1-His₆ (A), the T106A mutant (B), and the ΔT106 deletion mutant (C). Reaction mixtures of 30 μl were analyzed by agarose gel electrophoresis. Each contained 0.5 μg relaxed pUC19 DNA, reaction buffer, and 1 unit gyrase (lanes 1 to 11), 2.5 μM ciprofloxacin (lanes 2 to 11), and QnrB1-His₆ fusion protein at 7 μM (lane 3), 3.5 μM (lane 4), 1.75 μM (lane 5), 880 nM (lane 6), 440 nM (lane 7), 220 nM (lane 8), 110 nM (lane 9), 55 nM (lane 10), and 27 nM (lane 11).