M. Ito et al., mrp, a multigene, multifunctional locus in Bacillus subtilis with roles inresistance to cholate and to NA(+) and in pH homeostasis, J BACT, 181(8), 1999, pp. 2394-2402
A 5.9-kb region of the Bacillus subtilis chromosome is transcribed as a sin
gle transcript that is predicted to encode seven membrane-spanning proteins
, Homologues of the first gene of this operon, for which the designation mr
p (multiple resistance and pH adaptation) is proposed here, have been sugge
sted to encode an Na+/H+ antiporter or a K+/H+ antiporter, In the present s
tudies of the B. subtilis mrp operon, both polar and nonpolar mutations in
mrpA were generated, Growth of these mutants was completely inhibited by co
ncentrations of added Na+ as low as 0.3 M at pH 7.0 and 0.03 M at pH 8.3; t
here was no comparable inhibition by added K+. A null mutant that was const
ructed by full replacement of the mrp operon was even more Nai sensitive, A
double mutant with mutations in both mrpA and the multifunctional antiport
er-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 mr
pA 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 re
sulted 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 cat
ion concentrations ranging from 10 to 100 mM; thus, tetA(L) has a more pron
ounced involvement than mrpA in pH regulation. Measurements of Na+ efflux f
rom the wild-type strain, the nonpolar mrpA mutant, and the complemented mu
tant indicated that inducible expression of mrpA increased the rate of prot
onophore- and cyanide-sensitive Na+ efflux over that in the wild-type in ce
lls preloaded with 5 mM Na+. The mrpA and null mutants showed no such efflu
x in that concentration range. This is consistent with MrpA encoding a seco
ndary, 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 attribu
table to mrpF. Neither mrpF nor mrpFG expression in trans enhanced the chol
ate 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, pheno
typic differences among the mrp mutants suggest that functions in addition
to Na+ and cholate resistance and pH homeostasis will be found among the re
maining mrp genes.