Catalytic properties of Staphylococcus aureus and Bacillus members of the secondary cation/proton antiporter-3 (Mrp) family are revealed by an optimized assay in an Escherichia coli host

Monovalent cation proton antiporter-3 (Mrp) family antiporters are widely distributed and physiologically important in prokaryotes. Unlike other antiporters, they require six or seven hydrophobic gene products for full activity. Standard fluorescence-based assays of Mrp antiport in membrane vesicles from Escherichia coli transformants have not yielded strong enough signals for characterization of antiport kinetics. Here, an optimized assay protocol for vesicles of antiporter-deficient E. coli EP432 transformants produced higher levels of secondary [Na.sup.+] ([Li.sup.+])/[H.sup.+] antiport than previously reported. Assays were conducted on Mrps from alkaliphilic Bacillus pseudofirmus OF4 and Bacillus subtilis and the homologous antiporter of Staphylococcus aureus (Mnh), all of which exhibited [Na.sup.+]([Li.sup.+])/[H.sup.+] antiport. A second paralogue of S. aureus (Mnh2) did not. [K.sup.+], [Ca.sup.2+], and [Mg.sup.2+] did not support significant antiport by any of the test antiporters. All three [Na.sup.+] ([Li.sup.+])/[H.sup.+] Mrp antiporters had alkaline pH optima and apparent [K.sub.m] values for [Na.sup.+] that are among the lowest reported for bacterial [Na.sup.+]/[H.sup.+] antiporters. Using a fluorescent probe of the transmembrane electrical potential ([DELTA][PSI]), Mrp [Na.sup.+]/[H.sup.+] antiport was shown to be [DELTA][PSI] consuming, from which it is inferred to be electrogenic. These assays also showed that membranes from E. coli EP432 expressing Mrp antiporters generated higher [DELTA][PSI] levels than control membranes, as did membranes from E. coli EP432 expressing plasmid-borne NhaA, the well-characterized electrogenic E. coli antiporter. Assays of respiratory chain components in membranes from Mrp and control E. coli transformants led to a hypothesis explaining how activity of secondary, [DELTA][PSI]-consuming antiporters can elicit increased capacity for [DELTA][PSI] generation in a bacterial host.