The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline-cobalt/H+ and Na+(K+)/H+ exchange

Abstract

Recent work has suggested that the chromosomally encoded TetA(L) transporter of Bacillus subtilis, for which no physiological function had been shown earlier, not only confers resistance to low concentrations of tetracycline but is also a multifunctional antiporter protein that has dominant roles in both Na(+)- and K(+)-dependent pH homeostasis and in Na+ resistance during growth at alkaline pH. To rigorously test this hypothesis, TetA(L) has been purified with a hexahistidine tag at its C terminus and reconstituted into proteoliposomes. The TetA(L)-hexahistidine proteoliposomes exhibit high activities of tetracycline-cobalt/H+, Na+/ H+, and K+/H+ antiport in an assay in which an outwardly directed proton gradient is artificially imposed and solute uptake is monitored. Tetracycline uptake depends on the presence of cobalt and vice versa, with the cosubstrates being transported in a 1:1 ratio. Evidence for the electrogenicity of both tetracycline-cobalt/H+ and Na+/H+ antiports is presented. K+ and Li+ inhibit Na+ uptake, but there is little cross-inhibition between Na+ and tetracycline-cobalt uptake activities. The results strongly support the conclusion that TetA(L) is a multifunctional antiporter. They expand the roster of such porters to encompass one with a complex organic substrate and monovalent cation substrates that may have distinct binding domains, and provide the first functional reconstitution of a member of the 14-transmembrane segment transporter family.

Figure 1

Electrophoretic characterization of TetA(L)–H₆ purified from E. coli expressing a modified B. subtilis tetA(L) gene on a multicopy plasmid. Purified TetA(L)–H₆ (1 μg) next to empty lanes containing no protein. After electrophoresis, the sample was silver-stained (Right) or transferred and analyzed for cross-reaction with antibody raised against a synthetic peptide corresponding to the N-terminal 14 amino acids of TetA(L) (Left); although not shown, preimmune rabbit serum showed no cross-reaction. The arrowhead indicates the position of TetA(L)–H₆. A higher set of bands found in both lanes of the silver-stained gel is most likely a staining artifact.

Figure 2

Uptake of Tc and Na⁺ into TetA(L)–H₆ proteoliposomes upon imposition of an outwardly directed proton gradient. The proteoliposomes were loaded at pH 7.0 with 100 mM NH₄Cl and then diluted into the following: (Left) ammonium-free buffer at pH 7.5 containing 50 μM [³H]Tc and 100 μM CoCl₂ (▪) alone or also containing either 10 mM NaCl (•) or 100 mM NH₄Cl (□); (Right) ammonium-free buffer at pH 8.5 containing 1 mM ²²Na⁺ alone (▪) or also containing either 50 μM Tc plus 100 μM CoCl₂ (•) or 100 mM NH₄Cl (□). Samples were filtered at intervals and analyzed as described in Materials and Methods.

Figure 3

Uptake of Tc and cobalt into TetA(L)–H₆ proteoliposomes upon imposition of an outwardly directed proton gradient. The proteoliposomes were loaded with NH₄Cl at pH 7.0 as described in Materials and Methods and were diluted into ammonium-free buffer at pH 7.5 containing 50 μM [³H]Tc and 100 μM nonradioactive CoCl₂ (•); 50 μM nonradioactive Tc and 100 μM ⁶⁰CoCl₂ (▪); 50 μM [³H]Tc alone (○); or 100 μM ⁶⁰CoCl₂ alone (□). Samples were filtered at intervals and processed as described in the text. The numbers at the top of the figure indicate the Tc/Co ratios calculated from total accumulation at the points shown by the lines.

Figure 4

Efflux of ⁶⁰Co²⁺ in the presence of Tc or of ²²Na⁺ from preloaded TetA(L)–H₆ proteoliposomes upon imposition of either an inwardly directed proton gradient or a diffusion potential, outside positive. Proteoliposomes were loaded with 100 mM potassium acetate and either 100 μM ⁶⁰Co²⁺ plus 50 μM nonradioactive Tc (A) or 1 mM ²²Na⁺ (B). During the loading, 2.5 μM valinomycin was also present in all but the control sample with no valinomycin (□). The experiment was initiated by the dilution of the appropriately loaded proteoliposomes into buffers at pH 7.0 and containing 5 μM valinomycin and 100 mM potassium acetate (▪), potassium gluconate (▴), or choline acetate (•). The control with no valinomycin was diluted into buffer lacking valinomycin and containing 100 mM potassium acetate.