mrp , a Multigene, Multifunctional Locus in Bacillus subtilis with Roles in Resistance to Cholate and to Na + and in pH Homeostasis

Abstract

ABSTRACT A 5.9-kb region of the Bacillus subtilis chromosome is transcribed as a single transcript that is predicted to encode seven membrane-spanning proteins. Homologues of the first gene of this operon, for which the designation mrp (multiple resistance and pH adaptation) is proposed here, have been suggested to encode an Na + /H + antiporter or a K + /H + antiporter. In the present studies of the B. subtilis mrp operon, both polar and nonpolar mutations in mrpA were generated. Growth of these mutants was completely inhibited by concentrations of added Na + as low as 0.3 M at pH 7.0 and 0.03 M at pH 8.3; there was no comparable inhibition by added K + . A null mutant that was constructed by full replacement of the mrp operon was even more Na + sensitive. A double mutant with mutations in both mrpA and the multifunctional antiporter-encoding tetA (L) gene was no more sensitive than the mrpA mutants to Na + , consistent with a major role for mrpA in Na + resistance. Expression of mrpA from an inducible promoter, upon insertion into the amyE locus, restored significant Na + resistance in both the polar and nonpolar mrpA mutants but did not restore resistance in the null mutant. The mrpA disruption also resulted in an impairment of cytoplasmic pH regulation upon a sudden shift in external pH from 7.5 to 8.5 in the presence of Na + and, to some extent, K + in the range from 10 to 25 mM. By contrast, the mrpA tetA (L) double mutant, like the tetA (L) single mutant, completely lost its capacity for both Na + - and K + -dependent cytoplasmic pH regulation upon this kind of shift at cation concentrations ranging from 10 to 100 mM; thus, tetA (L) has a more pronounced involvement than mrpA in pH regulation. Measurements of Na + efflux from the wild-type strain, the nonpolar mrpA mutant, and the complemented mutant indicated that inducible expression of mrpA increased the rate of protonophore- and cyanide-sensitive Na + efflux over that in the wild-type in cells preloaded with 5 mM Na + . The mrpA and null mutants showed no such efflux in that concentration range. This is consistent with MrpA encoding a secondary, proton motive force-energized Na + /H + antiporter. Studies of a polar mutant that leads to loss of mrpFG and its complementation in trans by mrpF or mrpFG support a role for MrpF as an efflux system for Na + and cholate. Part of the Na + efflux capacity of the whole mrp operon products is attributable to mrpF . Neither mrpF nor mrpFG expression in trans enhanced the cholate or Na + resistance of the null mutant. Thus, one or more other mrp gene products must be present, but not at stoichiometric levels, for stability, assembly, or function of both MrpF and MrpA expressed in trans . Also, phenotypic differences among the mrp mutants suggest that functions in addition to Na + and cholate resistance and pH homeostasis will be found among the remaining mrp genes.