Iron homeostasis in Bacillus subtilis relies on three differentially expressed efflux systems

In Bacillus subtilis, iron homeostasis is maintained by the ferric uptake regulator (Fur) and manganese homeostasis relies on the manganese transport regulator (MntR). Both Fur and MntR function as bi-functional metalloregulators that repress import and activate metal ion efflux systems. The ferrous iron efflux ATPase, PfeT, is derepressed by hydrogen peroxide (H₂O₂) as sensed by PerR and induced by iron as sensed by Fur. Mutants lacking PfeT are sensitive to iron intoxication. Here, we show that mntR mutants are also iron-sensitive, largely due to decreased expression of the MntR-activated MneP and MneS cation diffusion facilitator (CDF) proteins previously defined for their role in Mn²⁺ export. The ability of MneP and MneS to export iron is apparent even when their expression is not induced by Mn²⁺. Our results demonstrate that PfeT, MneP and MneS each contribute to iron homeostasis, and a triple mutant lacking all three is more iron-sensitive than any single mutant. We further show that sensitivity to H₂O₂ does not correlate with iron sensitivity. For example, an mntR mutant is H₂O₂-sensitive due to elevated Mn(II) that increases PerR-mediated repression of peroxide resistance genes, and this repression is antagonized by elevated Fe²⁺ in an mntR pfeT mutant. Thus, H₂O₂-sensitivity reflects the relative levels of Mn²⁺ and Fe²⁺ as sensed by the PerR regulatory protein. These results underscore the complex interplay between manganese, iron and oxidative stress in B. subtilis.