No single irreplaceable acidic residues in the Escherichia coli secondary multidrug transporter MdfA

Abstract

The largest family of solute transporters (major facilitator superfamily [MFS]) includes proton-motive-force-driven secondary transporters. Several characterized MFS transporters utilize essential acidic residues that play a critical role in the energy-coupling mechanism during transport. Surprisingly, we show here that no single acidic residue plays an irreplaceable role in the Escherichia coli secondary multidrug transporter MdfA.

FIG. 1.

Neutralization of individual acidic residues in MdfA. (A) Acidic residues are marked on the secondary structure model of MdfA. Circled residues were analyzed in this study. Squares indicate residues that were analyzed previously (2, 4, 6). (B) Membranes were prepared from cells expressing the indicated His₆-tagged mutants and analyzed by Western blotting. WT, wild type; vec, vector.

FIG. 2.

Functional analyses of MdfA mutants. E. coli cells transformed with empty vector (vec) or plasmids encoding wild-type MdfA (WT) or the indicated MdfA mutants were diluted as marked [Dilution (-log)] and spotted onto LB agar plates with or without the test drug, and plates were visualized after ∼20 h at 37°C. LD₅₀ measurements with chloramphenicol (CAM) and EtBr (4) are indicated (in micrograms/milliliter) on the right. (B) Cells expressing the indicated mutants were loaded with EtBr and energized with glucose (left arrow), and the EtBr efflux was monitored continuously in the fluorimeter. As expected, the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) (right arrow) abolished efflux. The experiments were repeated at least three times, and the results shown are representative. a.u., arbitrary units.

FIG. 3.

Characterization of MdfA mutants at positions D77 and D34. (A) Membranes were prepared from cells expressing the indicated His₆-tagged mutants and analyzed by Western blotting. (B) E. coli was transformed with empty vector (vec) or plasmids encoding wild-type MdfA (WT) or the indicated MdfA mutants. Cells were diluted as marked [dilution (-log)] and spotted onto LB agar plates with or without the test drug, and plates were visualized after ∼20 h at 37°C. (C) Uptake of [³H]chloramphenicol by cells expressing the indicated mutants was assayed by rapid filtration (left panel). The experiment was repeated at least three times. The results shown are the average of triplicates. For the right panel, EtBr efflux was assayed as described in the legend of Fig. 2. a.u., arbitrary units.