Metal-specific control of gene expression mediated by Bradyrhizobium japonicum Mur and Escherichia coli Fur is determined by the cellular context.

Bradyrhizobium japonicum Mur and Escherichia coli Fur are manganese- and iron-responsive transcriptional regulators, respectively, that belong to the same protein family. Here, we show that neither Mur nor Fur discriminate between Fe(2+) and Mn(2+) in vitro nor is there a metal preference for conferral of DNA-binding activity on the purified proteins. When expressed in E. coli, B. japonicum Mur responded to iron, but not manganese, as determined by in vivo promoter occupancy and transcriptional repression activity. Moreover, E. coli Fur activity was manganese-dependent in B. japonicum. Total and chelatable iron levels were higher in E. coli than in B. japonicum under identical growth conditions, and Mur responded to iron in a B. japonicum iron export mutant that accumulated high levels of the metal. However, elevated manganese content in E. coli did not confer activity on Fur or Mur, suggesting a regulatory pool of manganese in B. japonicum that is absent in E. coli. We conclude that the metal selectivity of Mur and Fur depends on the cellular context in which they function, not on intrinsic properties of the proteins. Also, the novel iron sensing mechanism found in the rhizobia may be an evolutionary adaptation to the cellular manganese status.

Magnesium-dependent processes are targets of bacterial manganese toxicity.

A Bradyrhizobium japonicum mutant defective in the gene encoding the high-affinity Mn(2+) transporter MntH has a severe growth phenotype under manganese limitation. Here, we isolated suppressor mutants of an mntH strain that grew under manganese limitation, and activities of high-affinity Mn(2+) transport and Mn(2+) -dependent enzymes were partially rescued. The suppressor strains harbour gain-of-function mutations in the gene encoding the Mg(2+) channel MgtE. The MgtE variants likely allow Mn(2+) entry via loss of a gating mechanism that normally holds the transporter in the closed state when cellular Mg(2+) levels are high. Both MgtE-dependent and MgtE-independent suppressor phenotypes were recapitulated by magnesium-limited growth of the mntH strain. Growth studies of wild-type cells suggest that manganese is toxic to cells when environmental magnesium is low. Moreover, extracellular manganese and magnesium levels were manipulated to inhibit growth without substantially altering the intracellular content of either metal, implying that manganese toxicity depends on its cellular distribution rather than the absolute concentration. Mg(2+) -dependent enzyme activities were found to be inhibited or stimulated by Mn(2+) . We conclude that Mn(2+) can occupy Mg(2+) binding sites in cells, and suggest that Mg(2+) -dependent processes are targets of manganese toxicity.

Manganese is required for oxidative metabolism in unstressed Bradyrhizobium japonicum cells.

Recent studies of Mn(2+) transport mutants indicate that manganese is essential for unstressed growth in some bacterial species, but is required primarily for induced stress responses in others. A Bradyrhizobium japonicum mutant defective in the high-affinity Mn(2+) transporter gene mntH has a severe growth phenotype under manganese limitation, suggesting a requirement for the metal under unstressed growth. Here, we found that activities of superoxide dismutase and the glycolytic enzyme pyruvate kinase were deficient in an mntH strain grown under manganese limitation. We identified pykM as the only pyruvate kinase-encoding gene based on deficiency in activity of a pykM mutant, rescue of the growth phenotype with pyruvate, and pyruvate kinase activity of purified recombinant PykM. PykM is unusual in that it required Mn(2+) rather than Mg(2+) for high activity, and that neither fructose-1,6-bisphosphate nor AMP was a positive allosteric effector. The mntH-dependent superoxide dismutase is encoded by sodM, the only expressed superoxide dismutase-encoding gene under unstressed growth conditions. An mntH mutant grew more slowly on pyruvate under manganese-limited conditions than did a pykM sodM double mutant, implying additional manganese-dependent processes. The findings implicate roles for manganese in key steps in unstressed oxidative metabolism in B. japonicum.

Bacterial outer membrane channel for divalent metal ion acquisition.

The prevailing model of bacterial membrane function predicts that the outer membrane is permeable to most small solutes because of pores with limited selectivity based primarily on size. Here, we identified mnoP in the Gram-negative bacterium Bradyrhizobium japonicum as a gene coregulated with the inner membrane Mn(2+) transporter gene mntH. MnoP is an outer membrane protein expressed specifically under manganese limitation. MnoP acts as a channel to facilitate the tranlocation of Mn(2+), but not Co(2+) or Cu(2+), into reconstituted proteoliposomes. An mnoP mutant is defective in high-affinity Mn(2+) transport into cells and has a severe growth phenotype under manganese limitation. We suggest that the outer membrane is a barrier to divalent metal ions that requires a selective channel to meet the nutritional needs of the cell.

Transcriptional control of the Bradyrhizobium japonicum irr gene requires repression by fur and Antirepression by Irr.

Bradyrhizobium japonicum Fur mediates manganese-responsive transcriptional control of the mntH gene independently of iron, but it also has been implicated in iron-dependent regulation of the irr gene. Thus, we sought to address the apparent discrepancy in Fur responsiveness to metals. Irr is a transcriptional regulator found in iron-limited cells. Here, we show that irr gene mRNA was regulated by both iron and manganese, and repression occurred only in the presence of both metals. Under these conditions, Fur occupied the irr promoter in vivo in the parent strain, and irr mRNA expression was derepressed in a fur mutant. Under low iron conditions, the irr promoter was occupied by Irr, but not by Fur, and control by manganese was lost. Fur occupancy of the irr promoter was dependent on manganese, but not iron, in an irr mutant, suggesting that Irr normally interferes with Fur binding. Correspondingly, regulation of irr mRNA was dependent only on manganese in the irr strain. The Irr binding site within the irr promoter partially overlaps the Fur binding site. DNase I footprinting analysis showed that Irr interfered with Fur binding in vitro. In addition, Fur repression of transcription from the irr promoter in vitro was relieved by Irr. We conclude that Fur mediates manganese-dependent repression of irr transcription and that Irr acts as an antirepressor under iron limitation by preventing Fur binding to the promoter.

Control of bacterial iron homeostasis by manganese.

Perception and response to nutritional iron availability by bacteria are essential to control cellular iron homeostasis. The Irr protein from Bradyrhizobium japonicum senses iron through the status of heme biosynthesis to globally regulate iron-dependent gene expression. Heme binds directly to Irr to trigger its degradation. Here, we show that severe manganese limitation created by growth of a Mn(2+) transport mutant in manganese-limited media resulted in a cellular iron deficiency. In wild-type cells, Irr levels were attenuated under manganese limitation, resulting in reduced promoter occupancy of target genes and altered iron-dependent gene expression. Irr levels were high regardless of manganese availability in a heme-deficient mutant, indicating that manganese normally affects heme-dependent degradation of Irr. Manganese altered the secondary structure of Irr in vitro and inhibited binding of heme to the protein. We propose that manganese limitation destabilizes Irr under low-iron conditions by lowering the threshold of heme that can trigger Irr degradation. The findings implicate a mechanism for the control of iron homeostasis by manganese in a bacterium.

The mntH gene encodes the major Mn(2+) transporter in Bradyrhizobium japonicum and is regulated by manganese via the Fur protein.

The bacterial Nramp family protein MntH is a divalent metal transporter, but mntH mutants have little or no phenotype in organisms where it has been studied. Here, we identify the mntH homologue of Bradyrhizobium japonicum, and demonstrate that it is essential for Mn(2+) transport and for maintenance of cellular manganese homeostasis. Transport activity was induced under manganese deficiency, and Fe(2+) did not compete with (54)Mn(2+) for uptake by cells. The steady-state level of mntH mRNA was negatively regulated by manganese, but was unaffected by iron. Control of mntH expression and Mn(2+) transport by manganese was lost in a fur strain, resulting in constitutively high activity. Fur protected a 35 bp region of the mntH promoter in DNase I footprinting analysis that includes three imperfect direct repeat hexamers that are needed for full occupancy. Mn(2+) increased the affinity of Fur for the mntH promoter by over 50-fold, with a K(d) value of 2.2 nM in the presence of metal. The findings identify MntH as the major Mn(2+) transporter in B. japonicum, and show that Fur is a manganese-responsive regulator in that organism. Furthermore, Fe(2+) is neither a substrate for MntH nor a regulator of mntH expression in vivo.