The mntH gene encodes the major Mn2+ 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 Mn²⁺ transport and for maintenance of cellular manganese homeostasis. Transport activity was induced under manganese deficiency, and Fe²⁺ did not compete with ⁵⁴Mn²⁺ 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 Mn²⁺ 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. Mn²⁺ 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 Mn²⁺ transporter in B. japonicum , and show that Fur is a manganese-responsive regulator in that organism. Furthermore, Fe²⁺ is neither a substrate for MntH nor a regulator of mntH expression in vivo .     

New insights into the metal specificity of the Pseudomonas aeruginosa pyoverdine–iron uptake pathway

Pyoverdine (PvdI) is the major siderophore secreted by Pseudomonas aeruginosa PAOI in order to get access to iron. After being loaded with iron in the extracellular medium, PvdI is transported across the bacterial outer membrane by the transporter, FpvAI. We used the spectral properties of PvdI to show that in addition to Fe³⁺, this siderophore also chelates, but with lower efficiencies, all the 16 metals used in our screening. Afterwards, FpvAI at the cell surface binds Ag⁺, Al³⁺, Cd²⁺, Co²⁺, Cu²⁺, Fe³⁺, Ga³⁺, Hg²⁺, Mn²⁺, Ni²⁺ or Zn²⁺ in complex with PvdI. We used Inductively Coupled Plasma-Atomic Emission Spectrometry to monitor metal uptake in P. aeruginosa : TonB-dependent uptake, in the presence of PvdI, was only efficient for Fe³⁺. Cu²⁺, Ga³⁺, Mn²⁺ and Ni²⁺ were also transported into the cell but with lower uptake rates. The presence of Al³⁺, Cu²⁺, Ga³⁺, Mn²⁺, Ni²⁺ and Zn²⁺ in the extracellular medium induced PvdI production in P. aeruginosa . All these data allow a better understanding of the behaviour of the PvdI uptake pathway in the presence of metals other than iron: FpvAI at the cell surface has broad metal specificity at the binding stage and it is highly selective for Fe³⁺ only during the uptake process.     

Indispensability of Iron for the Growth of Cultured Chick Cells

In order to clarify the role of iron in the growth promoting effect of transferrin (Tf), the effects of the following substances were examined in cultured chick skeletal myogenic cells: transition metal ions (Fe²⁺, Fe³⁺, Cr³⁺, Cu²⁺, Mn²⁺, Co²⁺, Cd²⁺, Zn²⁺ and Ni²⁺), Tf complexes with these metals and metal-free apoTf.
The cells did not grow well when incubated in a culture medium composed of Eagle's minimum essential medium and horse serum. But they grew well in the presence of Fe²⁺ or Fe³⁺ (10–100 μM) or iron-bound Tf (10–500 nM) in the medium. None of the transition metal ions other than iron was effective. Neither apoTf nor Tf complexes with these metals showed the growth promoting effect. The generality of the requirement of iron for cell growth was ascertained in the primary culture of other types of chick embryonic cells: fibroblasts, cardiac myocytes, retinal pigment cells and spinal nerve cells.
The results show that iron is one of the indispensable substances for cell growth and suggest that Tf protein plays a role in facilitating the transport of iron into the cells.     

Plasma membrane-bound H+-ATPase and reductase activities in Fe-deficient cucumber roots

Physiological and biochemical modifications induced by Fe-deficiency have been studied in cucumber ( Cucumis sativus L. cv. Marketer) roots, a Strategy I plant that initiates a rapid acidification of the medium and an increase in the electric potential difference when grown under Fe-deficiency. Using the aqueous two-phase partitioning method, a membrane fraction which has the plasmalemma characteristics was purified from roots of plants grown in the absence and in the presence of iron. The plasma membrane vesicles prepared from Fe-deficient plants showed an H⁺-ATPase activity (EC 3.6.1.35) that is twice that of the non-deficient control. Furthermore, membranes from Fe-deficient plants showed a higher capacity to reduce Fe³⁺-chelates. The difference observed in the reductase activity was small with ferricyanide (only 30%) but was much greater with Fe³-EDTA and Fe³-citrate (210 and 250%, respectively). NADH was the preferred electron donor for the reduction of Fe³⁺ compounds. Fe³⁺ reduction in plasma membrane from cucumber roots seems to occur with utilisation of superoxide anion, since addition of superoxide dismutase (SOD; EC 1.15.1.1) "in vitro" decreased Fe³⁺ reduction by 60%.
The response and the difference induced by iron starvation on these two plasma membrane activities together with a possible involvement of O₂ in controlling the Fe³⁺/Fe²⁺ ratio in the rhizosphere are discussed.     

Identification of a tyrosine-based motif (YGSI) in the amino terminus of Nramp1 (Slc11a1) that is important for lysosomal targeting

In macrophages, Nramp1 (Slc11a1) is expressed in lysosomes and restricts replication of intracellular pathogens by removing divalent metals (Mn2+ and Fe2+) from the phagolysosome. Nramp2 (DMT1, Slc11a2) is expressed both at the duodenal brush border where it mediates uptake of dietary iron and ubiquitously at the plasma membrane/recycling endosomes of many cell types where it transports transferrin-associated iron across the endosomal membrane. In Nramp2, a carboxyl-terminal cytoplasmic motif ((555)YLLNT(559)) is critical for internalization and recycling of the transporter from the plasma membrane. Here we studied the subcellular trafficking properties of Nramp1 and investigated the cis-acting sequences responsible for targeting to lysosomes. For this, we constructed and studied Nramp1/Nramp2 chimeric proteins where homologous domains of each protein were exchanged. Chimeras exchanging the amino-(upstream TM1) and carboxyl-terminal (downstream TM12) cytoplasmic segments of both transporters were stably expressed in porcine LLC-PK1 kidney cells and were studied with respect to expression, maturation, stability, cell surface targeting, transport activity, and subcellular localization. An Nramp2 isoform II chimera bearing the amino terminus of Nramp1 was not expressed at the cell surface but was targeted to lysosomes. This lysosomal targeting was abolished by single alanine substitutions at Tyr15 and Ile18 of a (15)YGSI(18) motif present in the amino terminus of Nramp1. These results identify YGSI as a tyrosine-based sorting signal responsible for lysosomal targeting of Nramp1.     

Nramp1 expression by dendritic cells modulates inflammatory responses during Salmonella Typhimurium infection

Host resistance against Salmonella enterica serovar Typhimurium ( S . Typhimurium) is mediated by natural resistance-associated macrophage protein 1 (Nramp1/Slc11a1). Nramp1 is critical to host defence, as mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S . Typhimurium. Despite this crucial role, the mechanisms underlying Nramp1's protective effects are unclear. Dendritic cells (DCs) that sample the intestinal lumen are among the first cells encountered by S. Typhimurium following oral infection and act as a conduit for S. Typhimurium to cross the intestinal epithelial barrier. We report that DCs, including intestinal, splenic and bone marrow-derived DCs (BMDCs), express Nramp1 protein. In the small intestine, Nramp1 expression is greater in a subset of DCs (CD11c⁺CD103^-) characterized by the elevated expression of pro-inflammatory cytokines in response to bacterial products. While Nramp1 expression did not affect S. Typhimurium replication in BMDCs, infected Nramp1+/+ BMDCs and intestinal CD11c⁺CD103^- DCs secreted more inflammatory cytokines (IL-6, IL-12 and TNF-α) than Nramp1−/−, suggesting that Nramp1 expression may promote a more rapid inflammatory response following infection. Collectively, these findings reveal a new role for DCs and Nramp1 in modulating the host inflammatory response to S. Typhimurium.     

Loss of Zhf and the tightly regulated zinc-uptake system SpZrt1 in Schizosaccharomyces pombe reveals the delicacy of cellular zinc balance

Zinc is an essential micronutrient, and yet it can be toxic when present in excess. Zinc acquisition and distribution are dependent on tightly controlled transport of Zn²⁺ ions. Schizosaccharomyces pombe represents a second eukaryotic model to study cellular metal homeostasis. In several ways its micronutrient metabolism is fundamentally different from Saccharomyces cerevisiae . We identified the first Zn²⁺-uptake system in S. pombe and named it SpZrt1. Knock-out strains for all three ZIP (Zrt, Irt-like protein) transporters in fission yeast were constructed. Only zrt1 Δ cells were unable to grow at low Zn²⁺ and showed reduced ⁶⁵Zn²⁺ uptake. Elemental profiles revealed a strong decrease in zinc accumulation. Cd²⁺ ions inhibited uptake but Fe²⁺ or Mn²⁺ did not. Both mRNA abundance and protein amount are tightly regulated. Zrt1 activity is rapidly shut down upon transfer of zinc-deficient cells to zinc-replete conditions. In cells lacking Zhf, a transporter mediating endoplasmic reticulum storage of zinc, this response is about 100-fold more sensitive. Thus, removal of excess of zinc from the cytosol is largely Zhf dependent. Moreover, cells deficient for both transporters are no longer able to adjust to changing external Zn²⁺ concentrations. Optimal growth is restricted to a narrow range of Zn²⁺ concentrations, illustrating the fine balance between micronutrient deficiency and toxicity.     

OXYGEN CONSUMPTION ASSOCIATED WITH FERRIC REDUCTASE ACTIVITY AND IRON UPTAKE BY IRON-LIMITED CELLS OF CHLORELLA KESSLERI (CHLOROPHYCEAE)

Plasma membrane ferric reductase activity was enhanced 5-fold under iron limitation in the unicellular green alga Chlorella kessleri Fott et Nováková. Furthermore, ferric reductase activity in iron-limited cells was approximately 50% higher in the light than in the dark. In contrast, iron uptake rates of iron-limited cells were unaffected by light versus dark treatments. Rates of iron uptake were much lower than rates of ferric reduction, averaging approximately 2% of the dark ferric reduction rate. Ferric reduction was associated with an increased rate of O₂ consumption in both light and dark, the increase in the light being approximately 1.5 times as large as in the dark. The increased rate of O₂ consumption could be decreased by half by the addition of catalase, indicating that H₂O₂ is the product of the O₂ consumption and that the increased O₂ consumption is nonrespiratory. The stimulation of O₂ consumption was almost completely abolished by the addition of bathophenanthroline disulfonate, a strong chelator of Fe^{2 +} . Anaerobic conditions or the presence of exogenous superoxide dismutase affected neither ferric reduction nor iron uptake. We suggest that the O₂ consumption associated with ferric reductase activity resulted from superoxide formation from the aerobic oxidation of Fe^{2 +} , which is the product of ferric reductase activity. At saturating concentrations of Fe^{3 +} chelates, ferric reductase activity is much greater than the iron uptake rate, leading to rapid oxidation of Fe^{2 +} and superoxide generation. Therefore, O₂ consumption is not an integral part of the iron assimilation process.     

Nramp1 modifies the fusion of Salmonella typhimurium-containing vacuoles with cellular endomembranes in macrophages.

Salmonella survive and replicate within mammalian cells by becoming secluded within specialized membrane-bound vacuoles inaccessible to the host defense mechanisms. Delayed acidification of the vacuole and its incomplete fusion with lysosomes have been implicated in intracellular Salmonella survival. Nramp1 confers to macrophages resistance to a variety of intracellular pathogens, including Salmonella, but its precise mode of action is not understood. We investigated whether Nramp1 affects the maturation and acidification of Salmonella-containing vacuoles (SCV). A mouse-derived macrophage line (RAW/Nramp1(-)) devoid of Nramp1 and therefore susceptible to infection was compared with isogenic clones stably transfected with Nramp1 (RAW/Nramp1(+)). Intravacuolar pH, measured in situ, was similar in Nramp1-expressing and -deficient cells. SCV acquired LAMP1 and fused with preloaded fluid-phase markers in both cell types. In contrast, although few vacuoles in RAW/Nramp1(-) acquired mannose 6-phosphate receptor, many more contained M6PR in RAW/Nramp1(+) cells. Shortly after closure, SCV in RAW/Nramp1(-) became inaccessible to extracellular markers, suggesting inability to fuse with newly formed endosomes. Expression of Nramp1 markedly increased the access to extracellularly added markers. We propose that Nramp1 counteracts the ability of Salmonella to become secluded in a compartment that limits access of bactericidal agents, allowing the normal degradative pathway of the macrophage to proceed.     

Mechanism of iron uptake by plants

Abstract. Green plants require a continuous supply of Fe as they grow, because Fe does not not move from the older to the newer leaves. Soils do not lack Fe per se , but it may not be available to plants grown in alkaline soils. Plants are classed 'Fe-efficient' if they respond to Fe-deficiency stress by inducing biochemical reactions that make Fe available in a useful form, and 'Fe-inefficienT' if they do not. Iron uptake induced in response to Fe stress involves release of hydrogen ions and reductants by the root. The lowered pH and presence of reductant at the root zone, along with reduction of Fe³⁺ to Fe²⁺ at the root surface, enables Fe²⁺ to be taken up primarily through the young lateral roots. Ferrous iron is present throughout the protozylem and may or may not have entered the root by a carrier. The root-absorbed Fe²⁺ is oxidized to Fe³⁺ at the junction of the protoxylem and the metaxylem, chelated by citrate, and then transported in the metaxylem to the plant top. In the plant, the chemical reactions injuced by Fe-deficiency stress may affect nitrate reductase activity, use of Fe from Fe³⁺ phosphate and chelating agents, and tolerance to heavy metals. An efficient mechanism for Fe uptake in roots appears to be important for the efficient use of Fe in plant tops.     

Apoplastic pH and FeIII reduction in young sunflower (Helianthus annuus) roots

The relationship between the apoplastic pH in young sunflower roots ( Helianthus annuus L.) and the plasmalemma ferric chelate reductase (FC-R; EC 1.16.1.7) activity in roots was investigated. The hypothesis was tested that a high apoplastic pH depresses FC-R activity, thereby restricting the uptake of Fe²⁺ into the cytosol. Until recently, little has been known about this relationship, because pH and redox reaction measurements are difficult to perform within the confines of the root apoplast. We recorded the apoplastic pH by means of the fluorescence ratio in conjunction with video microscopy by covalently tagging fluorescein boronic acid to OH groups of the root cell wall. Fe^III reduction was measured using a similar approach by tagging ferrozine diboronic acid with OH groups of the cell wall. Ferrozine forms an Fe²⁺ complex, thus indicating the reduction of ferric iron. In roots bathing in buffered outer solutions of different pH, a high pH sensitivity of apoplastic Fe^III reduction was found, with the highest ferric iron reduction rates at an apoplastic pH of 4.9; above an apoplastic pH of 5.3, no reduction was observed. Nitrate in the bathing solution increased the apoplastic pH and hence depressed the Fe^III reduction; ammonium had the reverse effect. Nitrate together with HCO₃^–, a combination which is typical of calcareous soils, had the strongest depressing effect. From the results, it can be concluded that the main reason for the frequently occurring iron deficiency chlorosis of plants grown on calcareous soils is the inhibition of Fe^III reduction in the apoplast, and hence Fe²⁺ uptake into the cytosol.     

Effect of chelating agents on bud induction and accumulation of iron and copper by the moss Bartramidula bartramioides

The chelating agents, EDDHA, its iron salt, EDTA, and salicylic acid enhance bud formation in Bartramidula bartramioides (Griff.) Wijk & Marg. Salicylic acid elicits optimal response at 10^–4 M , whereas the other substances do so at 10^–7 M . Increased concentration of ferric citrate and cupric sulphate also stimulate bud induction. The accumulation of Fe³⁺ and Cu²⁺ is facilitated by chelators. The endogenous iron content is maximum at 10^–7 M EDDHA or EDTA, which is also the concentration optimal for bud induction.     

Nramp1 drives an accelerated inflammatory response during Salmonella-induced colitis in mice

A recently developed model for enterocolitis in mice involves pre-treatment with the antibiotic streptomycin prior to infection with Salmonella enterica serovar Typhimurium ( S.  Typhimurium). The contribution of Nramp1/Slc11a1 protein, a critical host defence mechanism against S.  Typhimurium, to the development of inflammation in this model has not been studied. Here, we analysed the impact of Nramp1 expression on the early development of colitis using isogenic Nramp1^+/+ and Nramp1^−/− mice. We hypothesized that Nramp1 acts by rapidly inducing an inflammatory response in the gut mucosa creating an antibacterial environment and limiting spread of S.  Typhimurium to systemic sites. We observed that Nramp1^+/+ mice showed lower numbers of S.  Typhimurium in the caecum compared with Nramp1^−/− mice at all times analysed. Acute inflammation was much more pronounced in Nramp1^+/+ mice 1 day after infection. The effect of Nramp1 on development of colitis was characterized by higher secretion of the pro-inflammatory cytokines IFN-γ, TNF-α and MIP-1α and a massive infiltration of neutrophils and macrophages, compared with Nramp1^−/− animals. These data show that an early and rapid inflammatory response results in protection against pathological effects of S.  Typhimurium infection in Nramp1^+/+ mice.     

Escherichia coli K-12 ferrous iron uptake mutants are impaired in their ability to colonize the mouse intestine

Abstract The streptomycin-treated mouse colonization model was used to investigate the role of the Fe²⁺ uptake system (Feo) of Escherichia coli K12 in the colonization of the mouse intestine. Mutants impaired in the uptake of Fe²⁺ ions were shown to be deficient also in their colonization ability. Both enterochelin-producing and enterochelin-nonproducing Escherichia coli feo mutants were unable to colonize the mouse intestine. These results demonstrated that Fe(II) is an essential source of iron for E. coli grown in the intestine.     

The requirement for Mn2+ and Ca2+ in nitrogen fixation by the photosynthetic bacterium Rhodospirillum rubrum

Abstract: The role of Ca²⁺ and Mn²⁺ in Rhodospirillum rubrum grown under different conditions with respect to nitrogen source has been studied. The results show that this phototroph does not have an absolute requirement for these cations. In vitro studies of one of the enzymes operative in the metabolic regulation of nitrogenase in Rsp. rubrum have shown that Mn²⁺ or Fe²⁺ is required for activity. This investigation indicates that Mn²⁺ is not required in vivo for the function of this enzyme, suggesting that either Fe²⁺ is functional or that the enzyme has other properties when active in the cell.     

Prevention of phosphate inhibition of cephalosporin synthetases by ferrous ion

Abstract Phosphate interference in the production of cephalosporins by Streptomyces clavuligerus had been associated with repression of expandase (desacetoxycephalosporin C synthetase) and inhibition of both expandase and cyclase (isopenicillin N synthetase). The present work shows that inhibition of enzyme action could be prevented by increasing the Fe²⁺ added to the cell-free reactions or to resting cells incubated with chloramphenicol. Since excess Fe²⁺ could not reverse phosphate interference of antibiotic synthesis in complete fermentations, it is clear that the major cause of the phosphate effect in fermentations is phosphate repression, rather than phosphate inhibition caused by Fe²⁺ deprivation.