Active efflux of doxorubicin (A) and rhodamine 6G (B) from E. coli KAM3 and KAM3ΔyhiUV cells overproducing EvgA. Active efflux of doxorubicin and rhodamine 6G from E. coli cells was measured as previously described (16). In order to obtain maximal preloading of the fluorophore, the cells were preincubated with 11.5 μM doxorubicin (A) or 1 μM rhodamine 6G (B) in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (40 μM) at 37°C for 1 h. The cells were then centrifuged and resuspended in the same medium containing 25 mM glucose without fluorescent drugs and carbonyl cyanide m-chlorophenylhydrazone, followed by fluorescence measurement. Doxorubicin transport was measured with excitation at 478 nm and emission at 591 nm. Rhodamine 6G transport was measured with excitation at 529 nm and emission at 553 nm.

Gel mobility shift profile of EvgA-responsive DNA fragments. DNA fragments of 338 bp including the mac, emr, and yhi promoter regions were prepared by PCR. The binding reaction was done in a total volume of 10 μl of binding buffer (20 mM Tris-HCl [pH 8.0], 50 mM NaCl, 1 mM EDTA, 1 mM β-mercaptoethanol, and 10% glycerol). Each reaction mixture contained 5 μg of His₆-EvgA (+) or bovine serum albumin as a negative control (−) as well as 1 μg of DNA. After they were incubated at 37°C for 30 min, the mixtures were put onto a 4% NuSieve 3:1 (BioWhittaker Molecular Applications) agarose gel, and electrophoresis was done. The gel was stained with ethidium bromide and photographed under UV illumination.

Northern blot analysis of mRNA from E. coli cells. KAM3 cells harboring pUC119 or pUCA were grown in Luria-Bertani medium to an optical density at 600 nm of 0.8 at 37°C, and the RNA was isolated by using the SV total RNA isolation system (Promega). The membranes (Hybond-N; Amersham Pharmacia Biotech) were hybridized at high stringency (68°C) with the probe for yhiU labeled with [α-³²P]dCTP by random priming. The arrow indicates the main transcript. Each lane contains 15 μg of total RNA.