The Escherichia coli multidrug transporter MdfA catalyzes both electrogenic and electroneutral transport reactions

Abstract

The resistance of cells to many drugs simultaneously (multidrug resistance) often involves the expression of membrane transporters (Mdrs); each recognizes and expels a broad spectrum of chemically unrelated drugs from the cell. The Escherichia coli Mdr transporter MdfA is able to transport differentially charged substrates in exchange for protons. This includes neutral compounds, namely chloramphenicol and thiamphenicol, and lipophilic cations such as tetraphenylphosphonium and ethidium. Here we show that the chloramphenicol and thiamphenicol transport reactions are electrogenic, whereas the transport of several monovalent cationic substrates is electroneutral. Therefore, unlike with positively charged substrates, the transmembrane electrical potential (negative inside) constitutes a major part of the driving force for the transport of electroneutral substrates by MdfA. These results demonstrate an unprecedented ability of a single secondary transporter to catalyze discrete transport reactions that differ in their electrogenicity and are governed by different components of the proton motive force.

Figure 1

Resistance of E. coli UTmdfA∷kan cells toward differentially charged drugs at various pH values. (A) Cells harboring plasmid pT7-5 (upper line, each panel) or pT7-5 MdfA (lower line, each panel) were tested for their ability to grow in the presence of each of several drugs at various pH values. Ten-fold dilutions of cells were plated from right to left. The drugs tested were EtBr at 500 μg/ml, benzalkonium chloride (Bz) at 20 μg/ml, chloramphenicol (Cm) at 8 μg/ml, and thiamphenicol (Tm) at 20 μg/ml. (B) Mdr of control cells (harboring plasmid pT7-5) was tested at the indicated pH values. Ten-fold dilutions of cells were plated from right to left. The drugs tested were EtBr at 20 μg/ml, benzalkonium chloride at 0.8 μg/ml, chloramphenicol at 0.2 μg/ml, and thiamphenicol at 0.3 μg/ml.

Figure 2

Effect of pH on transport of positively charged and neutral substrates. (A) Efflux of EtBr (5 μM) by preloaded cells. EtBr fluorescence is increased after interaction with DNA and RNA, and efflux of EtBr is represented by a decrease in fluorescence. Trace 1, control cells lacking MdfA at pH 7.0. Traces 2–6, EtBr efflux mediated by MdfA-expressing cells at pH 6.5, 7.0, 7.5, 8.0, and 8.5, respectively. Glucose (0.2%) was added at 100 sec to energize the cells. CCCP (10 μM) was added after 300–500 sec to abolish the Δμ̄_H⁺-driven transport. A.U., arbitrary units. (B Left) MdfA-mediated decreased accumulation of [³H]TPP⁺ (50 μM) in energized cells. (C Left) MdfA-mediated decreased accumulation of [³H]chloramphenicol (2 μM) in energized cells. (B Right and C Right) Accumulation of [³H]TPP⁺ (50 μM) or [³H]chloramphenicol (2 μM) by control cells. For direct comparison, the traces representing control cells assayed at pH 7.0 are shown also as dashed traces (B Left and C Left). Filled squares, pH 6.5; filled triangles, pH 7.0; filled circles, pH 7.5; open squares, pH 8.0; and open triangles, pH 8.5. Error bars are indicated unless they are smaller than the icons.

Figure 3

Effect of dissipation of Δψ or ΔpH on the transport of differentially charged substrates by MdfA. (A) Efflux of EtBr (5 μM) from preloaded EDTA/ethanol-treated E. coli UTLmdfA∷kan cells was measured at pH 7.0. Trace 1, control cells lacking MdfA; trace 2, MdfA-expressing cells; trace 3, MdfA-expressing cells in the presence of 5 μM valinomycin; trace 4, MdfA-expressing cells in the presence of 2 μM nigericin. Glucose (0.2%) was added at 80 sec to energize the cells. CCCP (C*) (10 μM) was added, as indicated by the arrows, to abolish Δμ̄_H⁺-driven transport. A.U., arbitrary units. (B) Transport of Hoechst 33342 in inverted membrane vesicles prepared from cells overexpressing MdfA or control cells at pH 7.0. Transport was initiated by the addition of 0.2 mM ATP as indicated. Trace 1, control vesicles [nigericin (2 μM) was added as indicated by an arrow]; trace 2, MdfA-containing vesicles [nigericin (2 μM) was added as indicated by an arrow]; trace 3, MdfA vesicles [valinomycin (2 μM) was added as indicated by an arrow]; trace 4, control vesicles [valinomycin (2 μM) was added as indicated by an arrow]. (C) Accumulation of [³H]chloramphenicol (2 μM) by EDTA/ethanol-treated E. coli UTLmdfA∷kan cells at pH 7.0 (Left) and 6.5 (Right). Open circles, control cells; filled circles, MdfA-expressing cells; filled triangles, MdfA-expressing cells in the presence of 5 μM valinomycin; filled squares, MdfA-expressing cells in the presence of 2 μM nigericin. Error bars are indicated unless they are smaller than the icons.

Figure 4

Changes in the transmembrane electrical potential (inside positive) after additions of differentially charged substrates. Membrane vesicles were energized by the addition of 0.4 mM ATP, and the consequent membrane potential was monitored by oxonol V (0.5 μM) fluorescence. CCCP (C*, 5 μM) was added to completely abolish the membrane potential. The upper-most trace represents control vesicles, and all other traces represent MdfA-containing vesicles. Substrates were added where indicated at the following concentrations: 15 μM chloramphenicol (Cm), 15 μM thiamphenicol (Tm), 2 μM benzalkonium chloride (Bz), and 0.2 μM nigericin (Nig). Vec, vector, no MdfA.

Figure 5

Uptake of [³H]chloramphenicol or [³H]TPP⁺ into MdfA proteoliposomes. (A) Uptake of 2 μM [³H]chloramphenicol: 2 μl of proteoliposomes (1.5 μg of protein per μl) were diluted 100-fold into the appropriate buffer (as detailed in Materials and Methods) to impose the indicated diffusion potentials. (B) Uptake of 1 μM [³H]TPP⁺: 0.5 μl of proteoliposomes (8 μg of protein per μl) were diluted 100-fold into the appropriate buffer (as detailed in Materials and Methods) to impose the indicated diffusion potentials. Net values are shown, after subtraction of the amount of substrate accumulated in liposomes devoid of MdfA, under identical conditions. Error bars are indicated.