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-cobalt͞H ؉ , 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-cobalt͞H ؉ 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.

Role of the nhaC-encoded Na+/H+ antiporter of alkaliphilic Bacillus firmus OF4

“…NhaB, which can take over in a ⌬nhaA mutant strain, is also electrogenic, albeit with a lower coupling ratio than NhaA, and has an even higher V max than activated NhaA (34,38). A major Na ϩ (K ϩ )/H ϩ antiporter of B. subtilis, the TetA(L) protein, similarly is electrogenic and has a high K m and V max for Na ϩ (8,15). Whereas the current data are most consistent with NhaC being electrogenic, its K m for Na ϩ is likely to be relatively low.…”

“…Enhancement of Na ϩ /H ϩ antiporter activity was observed upon complementation of E. coli EP432. Although this enhancement was modest, it was similar to that observed with other Bacillus antiporters, including one that has been purified and rigorously shown to possess Na ϩ /H ϩ antiporter activity (8,15). Most importantly, studies with B. firmus OF4811M itself, in comparison with the ⌬nhaC strain, N13, provided strong support for NhaC as an Na ϩ /H ϩ antiporter, i.e., in the correlated reductions in N13, relative to the wildtype strain, in electrogenic 22 Na ϩ extrusion and in pH homeostasis upon an upward pH shift.…”

“…Many of these already have obvious homologs in other genera (11,28,29,32,39), and more such homologs are emerging from genome sequencing projects. Three of the antiporter gene products, those of nhaA and nhaB from E. coli (34,38) and tetA(L) from B. subtilis (8), have been rigorously shown to encode Na ϩ /H ϩ antiporters through studies of the purified, reconstituted gene products, and in the last case the protein has been shown to be a multifunctional antiporter that can also catalyze efflux of K ϩ and a tetracycline-metal complex. The activities of gene products for other antiporter genes have been inferred from studies of the enhanced antiport activity or ⌬pH-dependent Na ϩ resistance conferred by the cloned gene in Na ϩ -sensitive mutants of either E. coli or the homologous bacterium.…”

“…Seven structurally distinct genes that putatively encode Na ϩ /H ϩ antiporters have been identified in eubacteria to date, i.e., nhaA (13), nhaB (33), and chaA (19) from Escherichia coli, nhaC from alkaliphilic Bacillus firmus OF4 (17), napA from Enterococcus hirae (41), tetA(L) from Bacillus subtilis (8), and a gene from alkaliphilic Bacillus sp. strain C-125 (16).…”

Importance of the GP Dipeptide of the Antiporter Motif and Other Membrane-Embedded Proline and Glycine Residues in Tetracycline Efflux Protein Tet(L)

“…All the antiport reactions catalyzed by Tet(L) and Tet(K) are electrogenic i.e. more than one H + is translocated inward per Na + , K + or mono-cationic Tc (−1) -Me 2+ complex translocated outward (36)(37)(38)(39) in contrast to the finding with the 12-TMS Tn10 Tet protein (35). Both Tet(L) and Tet(K) can also substitute K + for part of the H + coupling ion complement, especially at high pH, thus catalyzing both net K + and H + uptake under particular conditions of pH and K + status (40)(41)(42).…”