VmrA, a member of a novel class of Na(+)-coupled multidrug efflux pumps from Vibrio parahaemolyticus

Abstract

Gene vmrA, cloned from Vibrio parahaemolyticus, made Escherichia coli resistant to 4',6-diamino-2-phenylindol, tetraphenylphosphonium chloride, acriflavine, and ethidium bromide. VmrA belongs to the DinF branch of MATE family efflux transporters. VmrA catalyzed acriflavine efflux and showed Na(+)/drug transporter activity because the addition of tetraphenylphosphonium to Na(+)-loaded cells caused Na(+) efflux.

FIG. 1.

Plasmids and restriction maps of cloned V. parahaemolyticus DNA containing the vmrA gene. Physical maps of the DNA inserts derived from the V. parahaemolyticus chromosome in pVCJ6 and its derivatives are shown. Restriction sites determined in pVCJ6 are shown. The growth capabilities of E. coli KAM32 cells harboring each plasmid in L medium containing 10 μg of ethidium bromide/ml are shown on the right. +, Cells grew; −, cells did not grow. The position and direction of the vmrA gene revealed by sequencing are shown at the bottom.

FIG. 2.

Dendrogram for three subfamilies of the MATE family. The dendrogram was obtained by using CLUSTAL W and Decypher of Stanford University computer system (http://dna.stanford.edu/projects.html). Classification of the MATE family into three subfamilies (clusters) is from Brown et al. (4). NorM bolongs to the subfamily 1, ERC1 belongs to the subfamily 2, and DinF belongs to the subfamily 3 (DinF subfamily).

FIG. 3.

Effect of DNA concentration on the fluorescence intensity of acriflavine. The assay mixture contained modified Tanaka medium (25) (Na⁺ salts were replaced with K⁺ salts) and 1.25 μg of acriflavine/ml. DNA was added to the assay mixture at the indicated concentrations, and the fluorescence of acriflavine was measured at the excitation wavelength of 468 nm and the emission wavelength of 499 nm.

FIG. 4.

Acriflavine efflux-accumulation assay. Cells of E. coli KAM32 or KAM32/pVCJ6 were grown in L medium (8) to the late exponential phase of growth under aerobic conditions at 37°C. The cells were then harvested, washed with the modified Tanaka medium (25) supplemented with 2 mM MgSO₄, and suspended in the same medium supplemented with 15 mM NaCl. After preincubation at 25°C for 4 min, acriflavine (1.25 μg/ml) was added to initiate the assay, and the fluorescence intensity was monitored as indicated in the legend for Fig. 2. A H⁺ conductor CCCP (30 μM) was added at time point indicated by a downward arrow to deenergize the membrane. The downward deflection in the pattern indicates accumulation of acriflavine in the cells.

FIG. 5.

Na⁺ efflux from Na⁺-loaded cells elicited by an inwardly directed TPP⁺ gradient. Cells of E. coli KAM32/pSTV28 or E. coli KAM32/pVCJ7 were grown in the modified Tanaka medium supplemented with 1% tryptone, 10 μg of chloramphenicol/ml, and 10 mM melibiose at 30°C under aerobic conditions. It is important to grow cells at this temperature because MelB of E. coli K-12 strains is labile at 37°C (17, 27). Cells were harvested at the late exponential phase of growth, washed twice with 0.1 M morpholinepropanesulfonic acid-tetramethylammonium hydroxide (TMAH; pH 7.0), and resuspended in the same buffer. A portion of this suspension (0.5 ml) was diluted with 2.5 ml of 0.1 M Tricine-TMAH (pH 8.0; ca. 20 mg of protein/ml). NaCl was added to give a final concentration of 33 μM. Cells were incubated at 30°C in a plastic vessel with rapid stirring, and water-saturated N₂ gas was introduced continuously to maintain anaerobic conditions. An Na⁺-electrode (Radiometer, Copenhagen, Denmark) and a reference electrode were put into the vessel (28). The first arrow indicates when an anaerobic solution of MβGal (final concentration, 5 mM) was added to the cell suspension to elicit Na⁺ uptake into cells. The second arrow indicates when an anaerobic solution of TPPCl (final concentration, 0.5 mM) was added to the assay mixture. Upward deflection represents the uptake of Na⁺, and downward deflection represents the efflux of Na⁺. Calibration was carried out by the addition of known amounts of NaCl.