Ferrous iron uptake byBifidobacterium breve

Bifidobacterium breve transports ferrous iron in preference to the ferric form in a saturable, concentration-dependent manner with an optimum pH of 6. Iron transport is highly temperature sensitive. Two transport systems with apparent Km's of 86 +/- 27 and 35 +/- 20 microM (p greater than 0.01) were distinguished, one operating at high iron concentrations, the other at low iron concentrations. Iron uptake could not be accounted for by surface binding. Uptake of iron was inhibited by iron chelators, a protein ionophore, and ATPase inhibitors, and it was stimulated by potassium ionophores. The presence of a ferri reductase in the insoluble cell fraction of B. breve and its "spent" growth medium was demonstrated. The hypothesis is presented that iron uptake by bifidobacteria is related to the nutritional immunity phenomenon.     

Iron uptake by the microaerophilic anaerobe Bifidobacterium bifidum var. pennsylvanicus

A system was designed to investigate ferrous iron transport into Bifidobacterium bifidum var. pennsylvanicus. It involved the incubation of the organisms with labeled ferrous iron in the Norris medium at pH 5, in which the bacteria had grown. Iron uptakes were similar under aerobic and anaerobic conditions. Ferrous but not ferric iron was taken up by the organisms. Iron uptake showed saturation kinetics and a marked temperature dependence. 2,4-Dinitrophenol and thenoltrifluoroacetate but not azide or trypsin treatment inhibited iron uptake. Zinc inhibited iron uptake competitively. Iron uptake from used medium was much greater than that from fresh medium at the same pH. It is concluded that ferrous iron uptake by the microorganisms is a carrier-mediated active phenomenon, inhibited by zinc, which may involve a substance elaborated into the medium by the organism.     

Transferrin iron interactions with cultured hepatocellular carcinoma cells (PLC/PRF/5)

Hepatocellular carcinoma cells of the PLC/PRF/5 cell line had 1.9 x 10(5) transferrin receptors per tumor cell with a Kd of 1.5 x 10(-8) M. At high concentrations of transferrin the binding was not saturable. Transferrin internalization by hepatoma cells was shown by time and temperature-dependent binding studies and by pronase experiments. Transferrin recycling was confirmed by the demonstration of a progressive increase in the cellular molar ratios of iron to transferrin and by chase experiments. Ammonium chloride interfered with iron unloading. The vinca alkaloid vincristine inhibited iron and transferrin uptake. The hepatocarcinoma cells appeared to lack asialoglycoprotein receptors and therefore internalized partially desialated transferrin by the regular route. Iron uptake from transferrin was markedly inhibited by the hydrophobic ferrous chelator 2,2' bipyridine but was relatively unaffected by the hydrophilic ferric chelator desferroxamine. The implication that ferrous iron was involved in postendocytic transvesicular membrane iron transport was supported by a study in which hepatoma cells were shown to take up large amounts of ferrous iron suspended in 270 mM sucrose at pH 5.5. The interaction at this pH between surface labeled hepatoma cell extracts and ferrous iron on a Sephacryl S-300 column suggested that the postendocytic transvesicular transport of iron through the membrane was in part protein mediated. The endocytosed iron in hepatoma cells was found in association with ferritin (33%), transferrin (31%) and a low molecular weight fraction (21%).     

Ferrous iron uptake by a magnesium transport system is toxic for Escherichia coli and Salmonella typhimurium.

At low magnesium concentrations, Escherichia coli and Salmonella typhimurium LT2 accumulate ferrous iron independent of the ferrous iron transport system feo. Mutant strains with mutations in the magnesium transport gene corA accumulated less ferrous iron than the parent strains. corA+ and corA strains also differed in their sensitivity to ferrous iron under oxic conditions. corA mutants were more resistant to ferrous iron than their parent corA+ strains. Part of the ferrous iron accumulated can be chased by the addition of magnesium. Much less iron was chased when ferric iron was taken up by the siderophore ferrichrome. These results may indicate that the intracellular metabolism of the iron taken up by these systems differs and that it depends on the uptake route of the iron.     

Separate pathways for cellular uptake of ferric and ferrous iron

Separate pathways for transport of nontransferrin ferric and ferrous iron into tissue cultured cells were demonstrated. Neither the ferric nor ferrous pathway was shared with either zinc or copper. Manganese shared the ferrous pathway but had no effect on cellular uptake of ferric iron. We postulate that ferric iron was transported into cells via beta(3)-integrin and mobilferrin (IMP), whereas ferrous iron uptake was facilitated by divalent metal transporter-1 (DMT-1; Nramp-2). These conclusions were documented by competitive inhibition studies, utilization of a beta(3)-integrin antibody that blocked uptake of ferric but not ferrous iron, development of an anti-DMT-1 antibody that blocked ferrous iron and manganese uptake but not ferric iron, transfection of DMT-1 DNA into tissue culture cells that showed enhanced uptake of ferrous iron and manganese but neither ferric iron nor zinc, hepatic metal concentrations in mk mice showing decreased iron and manganese but not zinc or copper, and data showing that the addition of reducing agents to tissue culture media altered iron binding to proteins of the IMP and DMT-1 pathways. Although these experiments show ferric and ferrous iron can enter cells via different pathways, they do not indicate which pathway is dominant in humans.     

Uptake of ionic 55Fe by mouse peritoneal macrophages in vitro.

Mouse peritoneal macrophages were maintained in vitro up to 3 days and exposed to radiolabelled ⁵⁵Fe in the form of ferrous citrate, ferrous sulfate, and ferric chloride in concentrations of 3–5 γ Fe/ml. The divalent iron compounds were taken up 10–40 times more extensively per weight of iron than the trivalent iron compounds. The net uptake of ferrous citrate was linear during the first day and thereafter increased at a slower rate. Macrophages in culture for 1 week showed one-third the average uptake of freshly cultured cells during comparable periods of exposure to ferrous citrate. The iron taken up was used in the synthesis of mouse ferritin. Uptake of ferrous citrate was influenced by serum concentration in the tissue culture medium, temperature, pinocytosis and phagocytosis of both latex particles and heated rat erythrocytes. Uptake of ferrous citrate was enhanced by exposure to either sodium fluoride (5×10^?3 M), or 2,4-dinitrophenol (1×10^?5 M), but was not affected by cyanide, azide, or cycloheximide. The effect of sodium fluoride was not demonstrated when ferrous sulfate was substituted for ferrous citrate. The results reported here suggest that the ability of macrophages to take up ferrous citrate is good in freshly explanted cultures, is a temperature-dependent process, is suppressed by pinocytosis and phagocytosis, and paradoxically enhanced by certain metabolic inhibitors.     

Uptake of ionic Fe by mouse peritoneal macrophages in vitro

Mouse peritoneal macrophages were maintained in vitro up to 3 days and exposed to radiolabelled ⁵⁵Fe in the form of ferrous citrate, ferrous sulfate, and ferric chloride in concentrations of 3–5 γ Fe/ml. The divalent iron compounds were taken up 10–40 times more extensively per weight of iron than the trivalent iron compounds. The net uptake of ferrous citrate was linear during the first day and thereafter increased at a slower rate. Macrophages in culture for 1 week showed one-third the average uptake of freshly cultured cells during comparable periods of exposure to ferrous citrate. The iron taken up was used in the synthesis of mouse ferritin. Uptake of ferrous citrate was influenced by serum concentration in the tissue culture medium, temperature, pinocytosis and phagocytosis of both latex particles and heated rat erythrocytes. Uptake of ferrous citrate was enhanced by exposure to either sodium fluoride (5×10⁻³ M), or 2,4-dinitrophenol (1×10⁻⁵ M), but was not affected by cyanide, azide, or cycloheximide. The effect of sodium fluoride was not demonstrated when ferrous sulfate was substituted for ferrous citrate. The results reported here suggest that the ability of macrophages to take up ferrous citrate is good in freshly explanted cultures, is a temperature-dependent process, is suppressed by pinocytosis and phagocytosis, and paradoxically enhanced by certain metabolic inhibitors.     

An in vitro examination of intestinal iron absorption in a freshwater teleost, rainbow trout (Oncorhynchus mykiss)

This study investigated the physiological characteristics of intestinal iron absorption in a freshwater teleost, rainbow trout (Oncorhynchus mykiss). Using an in vitro gastro-intestinal sac technique, we evaluated the spatial pattern and concentration dependent profile of iron uptake, and also the influence of luminal chemistry (pH and chelation) on iron absorption. We demonstrated that the iron uptake rate in the anterior intestine is significantly higher than that in the mid and posterior intestine. Interestingly, absorption of iron in the anterior intestine occurs likely via simple diffusion, whereas a carrier-mediated pathway is apparent in the mid and posterior intestine. The uptake of ferric and ferrous iron appeared to be linear over the entire range of iron concentration tested (0–20 μM), however the uptake of ferrous iron was significantly higher than that of ferric iron at high iron concentrations (>15 μM). An increase in mucosal pH from 7.4 to 8.2 significantly reduced iron absorption in both mid and posterior intestine, implying the involvement of a Fe²⁺/H⁺ symporter. Iron chelators (nitrilotriacetic acid and desferrioxamine mesylate) had no effects on iron absorption, which suggests that fish are able to acquire chelated iron via intestine.     

Humic acid and iron uptake by plants

Summary The behaviour of ferric EDTA and ferric citrate in nutrient solution and their interaction with humic acid was investigated at various hydrogen ion concentrations using the technique of membrane ultrafiltration to separate small iron species from high molecular weight products of hydrolysis and to estimate the binding of iron by humic acid. Ferric EDTA was found to be of small molecular size at all pH values between 5.0 and 7.0 whilst ferric citrate solutions contained an increasing proportion of high molecular weight material as pH was increased from 5.0 to 7.0. Some iron present in solutions of both ferric EDTA and ferric citrate was bound by humic acid at all pH values from 5.0 to 7.0. Studies were also made of the uptake of iron by wheat roots from nutrient solutions containing either ferric EDTA or ferric citrate and of the effect of humic acid on uptake. More iron was absorbed from ferric EDTA than from ferric citrate at all pH values. Increasing pH between 5.0 and 7.0 resulted in a progressive decrease in the uptake of iron in both cases. The presence of humic acid depressed iron absorption from both solutions at all pH values.     

Effect of ferric sulfate on activity of moderately thermophilic acidophilic iron-oxidizing microorganisms

The effect of high ferric sulfate concentrations on the organisms predominating in biohydrometallurgical processes (bacteria of genus Sulfobaсillus and archaea of the genus Acidiplasma) was studied. Ability of the studied strains to grow and oxidize ferrous iron in the media with 125 to 500 mM ferric sulfate was determined. High concentrations of ferric sulfate significantly inhibited the oxidative activity and growth of the studied microorganisms. Bacteria of the genus Sulfobaсillus were found to be incapable of active iron oxidation in the presence of ferric iron sulfate at concentrations exceeding 250 mM. Archaea of the genus Acidiplasma oxidized ferrous iron completely in the presence of 500 mM Fe³⁺. Microbial growth was suppressed by relatively low ferric sulfate concentrations. Almost no growth occurred at ferric sulfate concentrations exceeding 199 mM, while lysis of the cells of all studied strains was observed at higher Fe³⁺ concentrations. Archaea (genus Acidiplasma, family Ferroplasmaceae) were shown to be more tolerant to high ferric sulfate concentrations than bacteria of the genus Sulfobaсillus. The results obtained may be used for improvement of biohydrometallurgical technologies and are also important for the understanding of the patterns of formation of microbial communities carrying out the technological processes.     

Genetics and environmental regulation of Shigella iron transport systems

Shigella spp. have transport systems for both ferric and ferrous iron. The iron can be taken up as free iron or complexed to a variety of carriers. All Shigella species have both the Feo and Sit systems for acquisition of ferrous iron, and all have at least one siderophore-mediated system for transport of ferric iron. Several of the transport systems, including Sit, Iuc/IutA (aerobactin synthesis and transport), Fec (ferric di-citrate uptake), and Shu (heme transport) are encoded within pathogenicity islands. The presence and the genomic locations of these islands vary considerably among the Shigella species, and even between isolates of the same species. The expression of the iron transport systems is influenced by the concentration of iron and by environmental conditions including the level of oxygen. ArcA and FNR regulate iron transport gene expression as a function of oxygen tension, with the sit and iuc promoters being highly expressed in aerobic conditions, while the feo ferrous iron transporter promoter is most active under anaerobic conditions. The effects of oxygen are also seen in infection of cultured cells by Shigella flexneri; the Sit and Iuc systems support plaque formation under aerobic conditions, whereas Feo allows plaque formation anaerobically.     

Ferrous iron is transported across the peribacteroid membrane of soybean nodules

Symbiosomes and bacteroids isolated from soybean (Glycine max Merr.) nodules are able to take up ferrous iron. This uptake activity was completely abolished in the presence of ferrous-iron chelators. The kinetics of uptake were characterized by initially high rates of iron internalization, but no saturation was observed with increasing iron concentration. This process does not appear to involve the ferric reductase of the peribacteroid membrane. The transport of ferrous iron was inhibited by other transition metals, particularly copper. Ferrous iron was taken up by symbiosomes more efficiently than the ferric form. This indicates that the iron transport from the plant host cell to the microsymbiont in vivo may occur mainly as the ferrous form. Received: 11 February 1998 / Accepted: 29 May 1998     

Anaerobic and aerobic oxidation of ferrous iron at neutral pH by chemoheterotrophic nitrate-reducing bacteria

Nine out of ten anaerobic enrichment cultures inoculated with sediment samples from various freshwater, brackish-water, and marine sediments exhibited ferrous iron oxidation in mineral media with nitrate and an organic cosubstrate at pH 7.2 and 30° C. Anaerobic nitrate-dependent ferrous iron oxidation was a biological process. One strain isolated from brackish-water sediment (strain HidR2, a motile, nonsporeforming, gram-negative rod) was chosen for further investigation of ferrous iron oxidation in the presence of acetate as cosubstrate. Strain HidR2 oxidized between 0.7 and 4.9 mM ferrous iron aerobically and anaerobically at pH 7.2 and 30° C in the presence of small amounts of acetate (between 0.2 and 1.1 mM). The strain gained energy for growth from anaerobic ferrous iron oxidation with nitrate, and the ratio of iron oxidized to acetate provided was constant at limiting acetate supply. The ability to oxidize ferrous iron anaerobically with nitrate at approximately pH 7 appears to be a widespread capacity among mesophilic denitrifying bacteria. Since nitrate-dependent iron oxidation closes the iron cycle within the anoxic zone of sediments and aerobic iron oxidation enhances the reoxidation of ferrous to ferric iron in the oxic zone, both processes increase the importance of iron as a transient electron carrier in the turnover of organic matter in natural sediments. Received: 24 April 1997 / Accepted: 22 September 1997     

革兰氏阴性菌亚铁离子转运系统的组成及作用机制

几乎所有细菌的生长都离不开铁元素。在有氧的环境中,三价铁离子几乎无法被细菌直接利用。但是在宿主胃肠道中,铁元素主要以可溶性的亚铁离子形式存在,它们可通过革兰氏阴性菌外膜直接进入胞周质,在周质通过亚铁离子转运系统,将铁离子转运至胞浆供细菌利用。绝大多数阴性菌主要是通过Feo转运系统利用亚铁离子,大肠杆菌的Feo转运系统由feoA、feoB和feoC3个基因组成。除Feo转运系统外,还发现Yfe转运系统、Efe转运系统、Sit转运系统等。本文重点介绍革兰氏阴性菌Feo转运系统的组成及作用机制,以期为进一步研究细菌亚铁离子的转运机制提供参考。     

Uptake of iron by apoferritin from a ferric dihydrolipoate complex

A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin-bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin. Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin-bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron. Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25-40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50-60 mol iron/mol ferritin after a 48-h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.     

Different iron sources to study the physiology and biochemistry of iron metabolism in marine micro-algae

We compared ferric EDTA, ferric citrate and ferrous ascorbate as iron sources to study iron metabolism in Ostreococcus tauri, Phaeodactlylum tricornutum and Emiliania huxleyi. Ferric EDTA was a better iron source than ferric citrate for growth and chlorophyll levels. Direct and indirect experiments showed that iron was much more available to the cells when provided as ferric citrate as compared to ferric EDTA. As a consequence, growth media with iron concentration in the range 1–100 nM were rapidly iron-depleted when ferric citrate—but not ferric EDTA was the iron source. When cultured together, P. tricornutum cells overgrew the two other species in iron-sufficient conditions, but E. huxleyi was able to compete other species in iron-deficient conditions, and when iron was provided as ferric citrate instead of ferric EDTA, which points out the critical influence of the chemical form of iron on the blooms of some phytoplankton species. The use of ferric citrate and ferrous ascorbate allowed us to unravel a kind of regulation of iron uptake that was dependent on the day/night cycles and to evidence independent uptake systems for ferrous and ferric iron, which can be regulated independently and be copper-dependent or independent. The same iron sources also allowed one to identify molecular components involved in iron uptake and storage in marine micro-algae. Characterizing the mechanisms of iron metabolism in the phytoplankton constitutes a big challenge; we show here that the use of iron sources more readily available to the cells than ferric EDTA is critical for this task.     

Iron transport mechanisms in reticulocytes and mature erythrocytes

The mechanism of iron transport into erythroid cells was investigated using rabbit reticulocytes and mature erythrocytes incubated with ⁵⁹Fe-labelled Fe(II) in isotonic sucrose or in solutions in which the sucrose was replaced with varying amounts of isotonic NaCl or KCl. Iron uptake was inhibited at all concentrations of NaCl, in a concentration-dependent manner, but with KCl inhibition occurred only at concentrations up to 10 mM. Higher KCl concentrations stimulated iron uptake to the cytosol of the cells, but inhibited its incorporation into heme. This effect became more marked as the iron concentration was raised. It was found that KCl inhibits iron incorporation into heme and stimulates iron uptake by mature erythrocytes, as well as by reticulocytes. It is concluded that erythroid cells can take up nontransferrin-bound Fe(II) by two mechanisms. One is a high-affinity mechanism that is limited to reticulocytes, saturates at a low iron concentration, and is inhibited by metabolic inhibitors. The other is a low-affinity process that is found in both reticulocytes and erythrocytes, becomes more prominent at higher iron concentrations, and is stimulated by KCl, as well as RbCl, LiCl, CsCl, and choline Cl. The KCl stimulation is inhibited by amiloride, but not by metabolic inhibitors, and its operation is not dependent on changes in cell volume or membrane potential, but it does require the presence of a permeant extracellular anion. Iron uptake by this process appears to occur by facilitated transport and is possibly assoicated with exchange of Na⁺. A further aspect of this study was a comparison of iron uptake by reticulocytes from Fe(II)-sucrose and Fe(II)-ascorbate using a variety of incubation conditions. No major differences were observed. © 1995 Wiley-Liss, Inc.     

The ferrireductase paraferritin contains divalent metal transporter as well as mobilferrin

Inorganic iron can be transported into cells in the absence of transferrin. Ferric iron enters cells utilizing an integrin-mobilferrin-paraferritin pathway, whereas ferrous iron uptake is facilitated by divalent metal transporter-1 (DMT-1). Immunoprecipitation studies using antimobilferrin antibody precipitated the previously described large-molecular-weight protein complex named paraferritin. It was previously shown that paraferritin functions as an intracellular ferrireductase, reducing ferric iron to ferrous iron utilizing NADPH as the energy source. It functions in the pathway for the cellular uptake of ferric iron. This multipeptide protein contains a number of active peptides, including the ferric iron binding protein mobilferrin and a flavin monooxygenase. The immunoprecipitates and purified preparations of paraferritin also contained DMT-1. This identifies DMT-1 as one of the peptides constituting the paraferritin complex. Since paraferritin functions to reduce newly transported ferric iron to ferrous iron and DMT-1 can transport ferrous iron, these findings suggest a role for DMT-1 in conveyance of iron from paraferritin to ferrochelatase, the enzyme utilizing ferrous iron for the synthesis of heme in the mitochondrion.     

Iron and Pathogenesis of Shigella: Iron Acquisition in the Intracellular Environment

Shigella species are able to grow in a variety of environments, including intracellularly in host epithelial cells. Shigella have a number of different iron transport systems that contribute to their ability to grow in these diverse environments. Siderophore iron uptake systems, heme transporters, and ferric and ferrous iron transport systems are present in these bacteria, and the genes encoding some of these systems appear to have spread among the Shigella species by horizontal transmission. Iron is not only essential for growth of Shigella but also plays an important role in regulation of metabolic processes and virulence determinants in Shigella. This regulation is mediated by the repressor protein Fur and the small RNA RyhB.     

The effects of pH and the iron redox state on iron uptake in the intestine of a marine teleost fish, gulf toadfish (Opsanus beta)

In the marine teleost intestine the secretion of bicarbonate increases pH of the lumen (pH 8.4 -9.0) and importantly reduces Ca2+ and Mg2+ concentrations by the formation of insoluble divalent ion carbonates. The alkaline intestinal environment could potentially also cause essential metal carbonate formation reducing bioavailability. Iron accumulation was assessed in the Gulf toadfish (Opsanus beta) gut by mounting intestine segments in modified Ussing chambers fitted to a pH-stat titration system. This system titrates to maintain lumen pH constant and in the process prevents bicarbonate accumulation. The luminal saline pH was clamped to pH 5.5 or 7.0 to investigate the effect of proton concentrations on iron uptake. In addition, redox state was altered (gassing with N2, addition of dithiothreitol (DTT) and ascorbate) to evaluate Fe3+ versus Fe2+ uptake, enabling us to compare a marine teleost intestine model for iron uptake to the mammalian system for non-haem bound iron uptake that occurs via a ferrous/proton (Fe2+/H+) symporter called Divalent Metal Transporter 1 (DMT1). None of the redox altering strategies affected iron (Fe3+ or Fe2+) binding to mucus, but the addition of ascorbate resulted in a 4.6-fold increase in epithelium iron accumulation. This indicates that mucus iron binding is irrespective of valency and suggests that ferrous iron is preferentially transported across the apical surface. Altering luminal saline pH from 7.0 to 5.5 did not affect ferric or ferrous iron uptake, suggesting that if iron is entering via DMT1 in marine fish intestine this transporter works efficiently under circumneutral conditions.