PMC

Determination of kinetic parameters of Vc-Mrp in everted membrane vesicles isolated from EP432 transformant cells. Assays were performed at pH 9.0 with varying concentrations of NaCl (circles, a), LiCl (squares, b), or KCl (triangles, c) as described in ‘Experimental Procedures’. ΔQ = Percent of dequenching of the AO fluorescence.

Structure of the mrp operon in V. cholerae. Shown are the genes comprising the operon, their length, names of corresponding loci (according to the TIGR database, //www.tigr.org/tigr-scripts/CMR2/ GenomeTabs.spl?database=gvc#4), together with calculated length and molecular mass of putative products, as well as the number of putative transmembrane segments (TMS) for each protein.

Vc-Mrp protects E. coli EP432 cells against sodium and lithium ions. Cells were transformed with either pVcMrp (closed symbols) or ‘empty’ pBAD24 (open symbols) and grown aerobically as described in ‘Experimental Procedures’ in the presence of 0.5% L-arabinose and indicated concentrations of NaCl or LiCl for 18 h. The growth yield was measured as optical density at 600 nm (OD₆₀₀). Starting OD₆₀₀ in all cases was approximately 0.05. Plotted are averages of at least four independent experiments. Bars show standard deviation.

Probing the stoichiometry of Vc-Mrp. Vesicles were isolated and assayed for Δψ at pH 9.0 in a sorbitol-based medium devoid of K⁺ and Cl-as described in ‘Experimental Procedures’. Diethanolamine at 15 m M was added to the experimental mixture 5 min prior the addition of Oxonol V. Due to the absence of K⁺ in the system, protonophore CCCP was added at the end of each measurement instead of valinomycin for the control. All other conditions as in figure 6b. Representative traces of five independent experiments are shown.

Vc-Mrp confers resistance to cholate in E. coli EP432 transformants. Cells were transformed with either pVcMrp (closed symbols) or ‘empty’ pBAD24 (open symbols) and grown aerobically as described in ‘Experimental Procedures’ in the presence of 0.5% arabinose and varying concentrations of sodium cholate for 16 h. The growth yield was measured as optical density at 600 nm (OD₆₀₀). The OD₆₀₀ at the start of the experiments was approximately 0.05 in all cases. Plotted are averages of at least four independent experiments. Bars show standard deviation.

Kinetic analysis of Vc-Mrp. a A high concentration of intravesicular K⁺ prevents cation-proton exchange by Vc-Mrp. Vesicles were isolated from EP432/pVcMrp cells in a high potassium buffer and assayed in the same buffer at pH 9.0. 5 mM Tris- D -lactate was used to energize respiration-dependent proton pumping, then 10 mM NaCl was added to initiate antiport in the absence (upper trace) or presence of 100 n M valinomycin (lower trace). b Addition of alkali cation (50 mM LiCl) does not perturb the Δψ of the membrane of vesicles. Fluorescence of Δψ-sensitive dye, oxonol V, was monitored instead of AO; all other conditions as in a. Representative of five independent experiments is shown. c Kinetic model of Vc-Mrp based on the binding exchange mechanism. See the text for discussion.

Activity of Vc-Mrp in everted membrane vesicles. a pH profiles of antiport activities mediated by Vc-Mrp. Everted membrane vesicles were isolated from EP432 cells transformed with pVcMrp or ‘empty’ pBAD24 and assayed with 10 mM of specified salt at various pH values as described in ‘Experimental Procedures’. In each case, residual nonspecific activity measured in ‘empty’ vesicles was subtracted from that registered in Vc-Mrpcontaining vesicles and the resulting Vc-Mrp-dependent activity was plotted as a function of pH. Experiments were repeated 3 times for each cation and gave nearly identical results. b High concentrations of external KCl do not prevent Na + /H + antiport mediated by Vc-Mrp. Vesicles were assayed with 10 mM NaCl at pH 9.0 in the standard choline chloride experimental medium supplemented with 140 mM KCl (upper trace) or the same medium containing 280 mM of choline-Cl instead of 140 mM (lower trace) to compensate the difference in osmolarity. Representative data of three independent experiments are shown. c K⁺ ions compete with Na⁺ for the antiporter. Dequenching of AO fluorescence (ΔQ) in response to the addition of varying [NaCl] was registered at pH 9.0 in the standard choline chloride medium supplemented with indicated concentrations of KCl. Concentrations of choline-Cl were varied accordingly to keep the osmolarity constant. Data were plotted in reciprocal coordinates. Obtained apparent K_m values for Na⁺ are: 1.3 mM (at 10 mM KCl), 1.9 mM (at 60 mM KCl), and 2.8 mM (at 140 mM KCl).