Effects of nonpolar mutations in each of the seven Bacillus subtilis mrp genes suggest complex interactions among the gene products in support of Na⁺ and alkali but not cholate resistance

The Bacillus subtitis mrp (multiple resistance and pH) operon supports Na⁺ and alkali resistance via an Na⁺/H⁺ antiport, as well as cholate efflux and resistance. Among the individual mutants with nonpolar mutations in each of the seven mrp genes, only the mrpF mutant exhibited cholate sensitivity and a cholate efflux defect that were complemented by expression of the deleted gene in trans. Expression of mrpF in the mrp null (VKN1) strain also restored cholate transport and increased Na⁺ efflux, indicating that MrpF does not require even low levels of other mrp gene expression for its own function. In contrast to MrpF, MrpA function had earlier seemed to depend upon at least modest expression of other mrp genes, i.e., mrpA restored Na⁺ resistance and efflux to strain VK6 (a polar mrpA mutant which expresses low levels of mrpB to -G) but not to the null strain VKN1. In a wild-type background, each nonpolar mutation in individual mrp genes caused profound Na⁺ sensitivity at both pH 7.0 and 8.3. The mrpA and mrpD mutants were particularly sensitive to alkaline pH even without added Na⁺. While transport assays in membrane vesicles from selected strains indicated that MrpA-dependent antiport can occur by a secondary, proton motive force-dependent mechanism, the requirement for multiple mrp gene products suggests that there are features of energization, function, or stabilization that differ from typical secondary membrane transporters. Northern analyses indicated regulatory relationships among mrp genes as well. All the mrp mutants, especially the mrpA, -B, -D, -E, and -G mutants, had elevated levels of mrp RNA relative to the wild type. Expression of an upstream gene, maeN, that encodes an Na⁺/malate symporter, was coordinately regulated with mrp, although it is not part of the operon.