MdfA confers alkaline pH tolerance. (A) Growth on agar–LB plates. Growth of E. coli UTΔmdfA::kan transformed with plasmid pT7-5 or pT7-5-mdfA-6His on solid LB media at pH 7, 8.75, or 9, as indicated. We plated 10-fold dilutions of cells (from left to right). (B) Growth in flasks. E. coli UTΔmdfA::kan transformed with plasmid pT7-5 (•) or pT7-5-mdfA-6His (▪) was grown at pH 7, 9.5, or 10, as indicated. Growth was conducted in LB broth in a 37°C shaker. (C) Growth in a microplate reader. E. coli UTΔmdfA::kan transformed with plasmid pT7-5 (trace 1), pT7-5-LacY (trace 2) or pT7–5-mdfA-6His (trace 3) were grown at pH 7 (Left) or 9.75 (Right). Cells were grown in LB broth at 37°C with intermittent agitation. OD at 600 nm was constantly measured. The results shown are an average of three independent repeats; SEs are <1% of the measured signal and, thus, not shown. (D) Growth in a microplate reader of E. coli UTmdfA::kan expressing various MdfA mutants. At pH 7 (Left), the growth of cells expressing no MdfA, wild-type MdfA (fully active in multidrug resistance), or mutants E26Q, E26D (partially active), E26C, R112C, R112H, or R112K (not active) is indistinguishable. At pH 9.5 (Right), only cells expressing mutants E26Q (trace 1) and E26D (trace 2) exhibit partial growth when compared with those expressing wild-type MdfA (trace 3). All other mutants (E26C, R112C, R112H, and R112K) fail to grow and are indistinguishable from one another.

Effect of the chromosomal deletion of the mdfA gene on bacterial growth under various pH conditions. E. coli UT5600 (□) or E. coli UTΔmdfA::kan (○) were grown in 96-well plates at the indicated pH (pH 7–9.5). Three independent growth experiment repeats are shown. Growth of E. coli UT5600 cells at pH 7 was defined as maximal growth (100%). All other growth curves were quantified as a percentage of this maximal curve.

Growth of E. coli UTΔmdfA::kan transformed with plasmid pT7–5-mdfA-6His under various conditions. (A–F) Growth was monitored continuously at pH 9.5. Cells were grown in the presence of the indicated concentrations of sodium chloride (A); potassium chloride (B); 20, 40, 80, and 100 mM sucrose (C) (growth curves are indistinguishable); 20, 40, and 86 mM choline chloride (D) (growth curves are indistinguishable); sodium gluconate (E); and potassium gluconate (F). (G) Growth of E. coli UTΔmdfA::kan transformed with plasmid pT7–5orpT7–5-mdfA-6His on solid media at pH 7, 8.75, 9, or 9.25 in the presence of 40 mM sodium chloride or potassium chloride, as indicated. We plated 10-fold dilutions of cells (from left to right).

EtdBr efflux from preloaded cells. E. coli UTΔmdfa::kan transformed with plasmid pT7–5orpT7–5mdfA were loaded with 10 μM EtdBr and energized by addition of 0.2% glucose after 90 sec. NaCl was added to both the loading buffer and the reaction mixture. Curves 1–4 show MdfA expressing cells in the presence of 0, 20, 40, and 80 mM NaCl, respectively. Curve 5 shows control cells without NaCl. CCCP was added after 250–300 sec.

In vivo internal pH measurements. Internal pH (pH_in) of cells transformed with plain vector (•) or an MdfA encoding plasmid (▪) was measured under various external alkaline pH (pH_out) conditions by the distribution of [¹⁴C]methylamine.

Cation-driven proton translocation by MdfA. (A and B) The ΔpH measurements in inverted membrane vesicles prepared from E. coli strain UTLΔmdfA::kan (A) or E. coli strain EP432 (B). Vesicles were diluted to 0.6 mg/ml membrane proteins in 1 mM Tris·HCl, pH 7/10 mM magnesium sulfate/1 μM ACMA. ATP·Tris (0.4 mM) was added to impose a ΔpH (internal acidic). In A and B Left, the first arrow indicates the time of addition of ATP, and the second arrow indicates addition of 100 mM sodium or potassium gluconate (as indicated). In B Right, a third arrow indicates the time of addition of 2.5 μM CCCP. Traces marked as control refer to vesicles prepared from cells not expressing MdfA. R112C stands for vesicles prepared from cells expressing the nonfunctional mutant R112C. WT refers to vesicles prepared from cells expressing wild-type MdfA. (C and D) ΔpH measurements in proteoliposomes using acridine orange as a fluorescent probe. A ΔpH (inside acidic) was imposed by means of an ammonium chloride chemical gradient across the membranes of liposomes devoid of protein (Control) or containing the nonfunctional mutant R112C, or wild-type MdfA (WT). The left arrow indicates time of addition of liposomes or proteoliposomes, the middle arrow indicates time of addition of 100 mM sodium gluconate (C) or 100 mM potassium gluconate (D), and the right arrow indicates time of addition of 20 μM CCCP.