Fig 7.

Fluorimetric efflux assay. Accumulation studies were performed using EtBr (A) and ciprofloxacin (B) with K. pneumoniae and the ΔkpnEF mutant. The efflux of EtBr in mutant and WT cells was monitored continuously by measuring fluorescence emission at 600 nm upon excitation at 530 nm. After 5 min in a fluorimeter, cells loaded with EtBr were energized by the addition of glucose, and the efflux of EtBr was monitored. After 10 min, 100 μM CCCP was added as indicated to abolish active transport, and fluorescence emission was monitored further. The fluorescence was measured using a spectrofluorimeter (Hitachi). For ciprofloxacin, excitation was set at 275 nm and emission was set at 440 nm. Each data point represents the mean plus the standard deviation of three independent experiments.