Role of the iron-dependent NrdAB homologue, NrdEFThe genome of Escherichia coli encodes two class I ribonucleotide reductases.The first, NrdAB, is a well-studied iron-dependent enzyme that is essential for aerobic growth.The second, NrdEF, is not functional under routine conditions, and its role is obscure.Recent studies demonstrated that NrdEF can be activated in vitro by manganese as well as iron.Since iron enzymes are potential targets for hydrogen peroxide (H2O2), and since the nrdHIEF operon is induced during H2O2 stress, we hypothesized that H2O2 might inactivate NrdAB and that NrdEF might be induced to compensate.This idea was tested using E. coli mutants that are chronically stressed by H2O2.Contrary to expectation, NrdAB remained active.Its resistance to H2O2 depended upon YfaE, which helps to activate NrdB.The induction of NrdEF during H2O2 stress was mediated by the inactivation of Fur, an iron-dependent repressor.This regulatory arrangement implied that NrdEF has a physiological role during periods of iron starvation.Indeed, NrdEF supported cell replication in iron-depleted cells.Iron bound to NrdF when it was expressed in iron-rich cells, but NrdEF was functional only in cells that were both iron-depleted and manganese-rich.Thus NrdEF supports DNA replication when iron is unavailable to activate the housekeeping NrdAB enzyme.Role of mntS in manganese metabolism Iron is involved in many cellular processes and is essential for almost all organisms.However, it is sometimes scarce in the contemporary aerobic world due to oxidation.When Escherichia coli is deficient in iron or stressed by hydrogen peroxide, it uses manganese rather than iron to populate mononuclear enzymes.When iron becomes available, excess manganese is exported out of the cell through the efflux pump, MntP.It is unclear how these cells traffic intracellular manganese, since cells maintain manganese at concentrations below those of most other metals. Recent identification and characterization of the novel manganese homeostasis gene, mntS, sparked our interest in this problem.The mntS RNA encodes an extremely small protein of 42 amino acids that is repressed by the transcriptional regulator, MntR, in response to high intracellular manganese levels. Furthermore, hydrogen peroxide-stressed cells need manganese to sustain enzyme activity, and we confirmed that mntS allowed cells to cope with H2O2-stress. We hypothesized that MntS makes manganese more available to proteins when manganese is scarce.We showed that mntS assists the activation of two manganese-dependent enzymes:manganese-superoxide dismutase and manganese-ribonucleotide reductase.Titration experiments demonstrated that mntS is needed only when manganese is scarce.Conversely, MntS overexpression made cells more vulnerable to high concentrations of manganese, via activation of Fur, the ferric uptake regulator.These cells exhibited profound repression of the Fur regulon, which resulted in a significant reduction in intracellular free iron (<2 µM compared to ~90 µM under routine aerobiosis) and accumulation of 3-fold more manganese.This ultimately led to the failure of heme synthesis.Furthermore, a similar effect was observed in mntP mutants with respect to manganese toxicity.Taken together, this study reveals that the small protein MntS makes manganese more available to proteins possibly by inhibiting MntP function.
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