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.

Effects of Efflux Transporter Genes on Susceptibility of Escherichia coli to Tigecycline (GAR-936)

“…Cells were grown in 1 liter of medium A (22) Fluorescence measurement of ethidium bromide efflux mediated by AcrAB in E. coli KAM3/pAc8 (acrAB) whole cells. Ethidium bromide transport in intact E. coli cells was measured by a modification of the method of Lewinson et al (10). E. coli KAM3 or E. coli KAM3/pAc8 (acrAB) (7) was grown in 10 ml of 2ϫ yeast extract-tryptone medium (22) at 37°C.…”

“…An identical concentration of ethidium bromide was added to cells without CCCP; and after the fluorescence had reached equilibrium, glucose was added to start the efflux reaction; i.e., the efflux of ethidium bromide started, as indicated by the decrease in fluorescence due to the dissociation of ethidium bromide from DNA or RNA (Fig. 2c, bottom trace line, no drug) (10). The fluorescence returned to a plateau when CCCP was added to dissipate the proton motive force.…”

Determinants of Substrate Recognition by the Escherichia coli Multidrug Transporter MdfA Identified on Both Sides of the Membrane

“…Inverted vesicles prepared from cells expressing either of these mutants generated a stable electrochemical gradient (inside positive and acid) in the presence of ascorbate and phenazine methosulfate (30), as demonstrated by fluorescence spectroscopy using ⌬ -and ⌬pH-sensitive dyes (data not shown). Under these conditions chloramphenicol is actively transported into the vesicles (15). The vesicles were preincubated with or without chloramphenicol, energized to generate ⌬˜Hϩ, and then labeled by [ 14 C]NEM.…”

“…A recent study demonstrates that MdfA is able to bind chloramphenicol and TPP ϩ simultaneously and that the two binding sites are closely related (16). Transport experiments show that MdfA is driven by the proton electrochemical gradient and functions as a drug/proton antiporter (17,15). As predicted from the hydropathy plot of the protein and gene fusion analysis, the putative 12 transmembrane domains (TMs) of the protein contain at least one membrane-embedded charged amino acid residue, Glu-26, in the middle of the TM1 (18,19) (see Fig.…”

“…Recently, MdfA orthologues were identified in several pathogenic bacteria: Shigella flexneri (99% identity) (11), Salmonella enterica serovar Typhi (90% identity) (12), and Yersinia pestis (73% identity) (13). Cells expressing MdfA from a multicopy plasmid exhibit multidrug resistance to a diverse group of toxic compounds and also export other substrates including (i) lipophilic cations such as ethidium bromide (EtBr), tetraphenylphosphonium (TPP ϩ ), and benzalkonium; (ii) zwitterionic drugs such as ciprofloxacin, and (iii) neutral compounds such as chloramphenicol, thiamphenicol, and the sugar isopropyl-1-thio-␤-D-galactopyranoside (8,14,15). A recent study demonstrates that MdfA is able to bind chloramphenicol and TPP ϩ simultaneously and that the two binding sites are closely related (16).…”