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。     

Iron-limited growth and kinetics of iron uptake in Magnetospirillum gryphiswaldense

Growth and magnetite formation in Magnetospirillum gryphiswaldense MSR-1 were found close to the maximum at an extracellular iron concentration of 15–20 μM. Ferrous iron was incorporated by a slow, diffusion-like process. Several iron chelators including various microbial siderophores were unable to promote transport of iron into the cells. In contrast, spent culture fluids stimulated the uptake of ferric iron in iron-depleted cells at a high rate, whereas fresh medium and transport buffer were unable to promote iron uptake. However, no siderophore-like compound could be detected in spent culture fluids by the Chrome Azurol S assay. Ferric iron uptake followed Michaelis-Menten kinetics with a K _m of 3 μM and a V _max of 0.86 nmol min^–1 (mg dry weight)^–1, suggesting a comparatively low-affinity, but high-velocity transport system. Iron incorporation was sensitive to 2,4-dinitrophenol and carbonylcyanide-m-chlorophenylhydrazone, indicating an energy-dependent transport process. Received: 21 May 1996 / Accepted: 7 August 1996     

Chlorobium ferrooxidans sp. nov., a phototrophic green sulfur bacterium that oxidizes ferrous iron in coculture with a “Geospirillum” sp. strain

A green phototrophic bacterium was enriched with ferrous iron as sole electron donor and was isolated in defined coculture with a spirilloid chemoheterotrophic bacterium. The coculture oxidized ferrous iron to ferric iron with stoichiometric formation of cell mass from carbon dioxide. Sulfide, thiosulfate, or elemental sulfur was not used as electron donor in the light. Hydrogen or acetate in the presence of ferrous iron increased the cell yield of the phototrophic partner, and hydrogen could also be used as sole electron source. Complexed ferric iron was slowly reduced to ferrous iron in the dark, with hydrogen as electron source. Similar to Chlorobium limicola, the phototrophic bacterium contained bacteriochlorophyll c and chlorobactene as photosynthetic pigments, and also resembled representatives of this species morphologically. On the basis of 16S rRNA sequence comparisons, this organism clusters with Chlorobium, Prosthecochloris, and Pelodictyon species within the green sulfur bacteria phylum. Since the phototrophic partner in the coculture KoFox is only moderately related to the other members of the cluster, it is proposed as a new species, Chlorobium ferrooxidans. The chemoheterotrophic partner bacterium, strain KoFum, was isolated in pure culture with fumarate as sole substrate. The strain was identified as a member of the ɛ-subclass of the Proteobacteria closely related to “Geospirillum arsenophilum” on the basis of physiological properties and 16S rRNA sequence comparison. The “Geospirillum” strain was present in the coculture only in low numbers. It fermented fumarate, aspartate, malate, or pyruvate to acetate, succinate, and carbon dioxide, and could reduce nitrate to dinitrogen gas. It was not involved in ferrous iron oxidation but possibly provided a thus far unidentified growth factor to the phototrophic partner. Received: 17 November 1998 / Accepted: 26 April 1999     

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.     

The Bradyrhizobium japonicum fsrB gene is essential for utilization of structurally diverse ferric siderophores to fulfill its nutritional iron requirement

In Bradyrhizobium japonicum, iron uptake from ferric siderophores involves selective outer membrane proteins and non-selective periplasmic and cytoplasmic membrane components that accommodate numerous structurally diverse siderophores. Free iron traverses the cytoplasmic membrane through the ferrous (Fe²⁺) transporter system FeoAB, but the other non-selective components have not been described. Here, we identify fsrB as an iron-regulated gene required for growth on iron chelates of catecholate- and hydroxymate-type siderophores, but not on inorganic iron. Utilization of the non-physiological iron chelator EDDHA as an iron source was also dependent on fsrB. Uptake activities of ⁵⁵Fe³⁺ bound to ferrioxamine B, ferrichrome or enterobactin were severely diminished in the fsrB mutant compared with the wild type. Growth of the fsrB or feoB strains on ferrichrome were rescued with plasmid-borne E. coli fhuCDB ferrichrome transport genes, suggesting that FsrB activity occurs in the periplasm rather than the cytoplasm. Whole cells of an fsrB mutant are defective in ferric reductase activity. Both whole cells and spheroplasts catalyzed the demetallation of ferric siderophores that were defective in an fsrB mutant. Collectively, the data support a model whereby FsrB is required for reduction of iron and its dissociation from the siderophore in the periplasm, followed by transport of the ferrous ion into the cytoplasm by FeoAB.     

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.     

Mechanisms of ferric and ferrous iron uptake by Bifidobacterium bifidum var. pennsylvanicus

Iron uptake studies in Bifidobacterium bifidum var. pennsylvanicus were carried out using ferric citrate at iron concentrations above 0.01 mM and pH 7, ferrous iron at concentrations less than 0.01 mM at pH 5. Two ferric iron transport systems were distinguished: the temperature-insensitive polymer, and the temperature-sensitive monomer uptake. Both showed a saturation phenomenon. The transport of ferrous iron at concentrations below 0.01 mM was temperature-dependent, and its affinity for iron was higher than that of a system operating at iron concentrations higher than 0.01 mM. The use of various metabolic inhibitors indicated that ferrous iron transport at pH 5 at both high and low iron concentrations was mediated by transport-type ATPase. Proton gradient dissipators abolished ferrous iron uptakes as well as the ferric monomer uptake. Uptake of the ferric polymer was insensitive to metabolic inhibitors. The functional significance of the various types of iron transport systems may be related to the nutritional immunity phenomenon.     

Inhibition effect of ferric iron on the kinetics of ferrous iron

Ferric iron acted as a non-competitive inhibitor for the biological oxidation of ferrous iron and decreased the inhibitory effects of high concentrations of ferrous iron as well as the auto-inhibitive effect the bacterial cells. A previously developed kinetic model for this reaction was modified to incorporate the inhibition effects of ferric iron. © Rapid Science Ltd. 1998     

Iron and cadmium uptake by duodenum of hypotransferrinaemic mice

Absorption from food is an important route for entry of the toxic metal, cadmium, into the body. Both cadmium and iron are believed to be taken up by duodenal enterocytes via the iron regulated, proton-coupled transporter, DMT1. This means that cadmium uptake could be enhanced in conditions where iron absorption is increased. We measured pH dependent uptake of ¹⁰⁹Cd and ⁵⁹Fe by duodenum from mice with an in vitro method. Mice with experimental (hypoxia, iron deficiency) or hereditary (hypotransferrinaemia) increased iron absorption were studied. All three groups of mice showed increased ⁵⁹Fe uptake (p<0.05) compared to their respective controls. Hypotransferrinaemic and iron deficient mice exhibited an increase in ¹⁰⁹Cd uptake (p<0.05). Cadmium uptake was not, however, increased by lowering the medium pH from 7.4 to 6. In contrast, ⁵⁹Fe uptake (from ⁵⁹FeNTA₂) and ferric reductase activity was increased by lowering medium pH in control and iron deficient mice (p<0.05). The data show that duodenal cadmium uptake can be increased by hereditary iron overload conditions. The uptake is not, however, altered by lowering medium pH suggesting that DMT1-independent uptake pathways may operate.     

Nitrite inhibition of aerobic bacteria

Nitrite was shown to inhibit active transport, oxygen uptake, and oxidative phosphorylation byPseudomonas aeruginosa. The evidence strongly suggested that nitrite exerted its inhibitory effect by oxidizing ferrous iron of an electron carrier(s), such as cytochrome oxidase, to ferric iron. The inhibitory effect of nitrite was readily reversible by washing the cells. Glucose transport byStreptococcus faecalis andS. lactis was not inhibited by nitrite, presumably because these species lack cytochromes and because glucose is transported by the phosphoenolpyruvate: phosphotransferase system rather than by active transport.     

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     

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 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.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line off-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the specific growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the specific growth rate, μ, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum specific growth rate, μ _max, measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q _O2,max, of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly differ from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-specific rates are up to 1.3 times higher in continuous cultures. Received: 8 February 1999 / Accepted: 17 February 1999     

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.     

Two pathways of iron uptake in bovine spleen apoferritin dependent on iron concentration

Iron incorporation by bovine spleen apoferritin either with ferrous ammonium sulfate in different buffers or with ferrous ammonium sulfate and phosphate was studied. Iron uptake and iron autoxidation were recorded spectrophotomerically. The buffers [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid (Hepes) and tris(hydroxymethyl)aminoethane (Tris) exhibited pH-dependent iron autoxidation, with Tris showing less iron autoxidation than Hepes. An Eadie-Scatchard plot (v/[s] versus v) of the iron uptake rate in Hepes was a curved rather than a straight line, suggesting that there are two iron uptake pathways. On the other hand, the Eadie-Scatchard plots of Tris and of Hepes after the addition of phosphate showed a straight line. Phosphate accelerated the iron uptake rate. The iron loading kinetics of apoferritin in Hepes was dependent on apoferritin concentration. The K_m value obtained from iron uptake kinetics was 4.5 M, corresponding to the physiological iron concentration. These results demonstrate that iron loading of apoferritin was accomplished at physiological iron concentrations, which is essential for iron uptake, via two uptake pathways of dependent on iron concentration.     

Fatty acids alter monolayer integrity, paracellular transport, and iron uptake and transport in Caco-2 cells

The Caco-2 cell line was used as a model to determine if the type of fatty acid, unsaturated versus saturated, differentially altered the uptake and transport of iron in the human intestine and if the changes were the result of alterations in monolayer integrity and paracellular transport. Cells were cultured in either a lower-iron or normal-iron medium and incubated with a bovine serum albumin control, linoleate, oleate, palmatate, or stearate. Oleate, palmatate, and stearate enhanced (p<0.05) iron uptake in cells grown in lower iron. The fatty acid effect on iron uptake by cells grown in normal iron was not as pronounced. Iron transport was not affected (p>0.05) by an interaction between the type of medium (iron concentration) and the type of fatty acid. Iron transport was enhanced (p<0.05) with palmatate and stearate. Various indicators of monolayer integrity and paracellular transport were also affected by the fatty acids, thus impacting iron uptake and transport. These results indicate that oleate, palmatate, and stearic can enhance iron uptake and transport; however, this enhancement may be the result of alterations in the integrity of the intestine. A portion of the data was presented at Experimental Biology 96 as a poster session. E. A. Droke, L. K. Johnson, and H. C. Lukaski. Fatty acids affect iron uptake and transport in Caco-2 cells. FASEB J. 10, 1431 (1996).     

Regulated redox processes at the plasmalemma of plant root cells and their function in iron uptake

Plants take up iron as ferric chelates or, after reduction, as ferrous ions. Ferric reduction takes place at the plasma membrane of the root epidermis cells by a transmembrane redox system, which can be activated when iron is getting short. It is proposed that this inducible system, with NADPH as electron donor, is separate from a system, presumably present in all plant cells, which transports electrons from NADH or NADPH to ferricyanide, or,in vivo, oygen.     

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.     

The determination of ferric iron in plants by HPLC using the microbial iron chelator desferrioxamine E

Common methods for plant iron determination are based on atomic absorption spectroscopy, radioactive measurements or extraction with subsequent spectrophotometry. However, accuracy is often a problem due to background, contamination and interfering compounds. We here describe a novel method for the easy determination of ferric iron in plants by chelation with a highly effective microbial siderophore and separation by high performance liquid chromatography (HPLC). After addition of colourless desferrioxamine E (DFE) to plant fluids, the soluble iron is trapped as a brown-red ferrioxamine E (FoxE) complex which is subsequently separated by HPLC on a reversed phase column. The formed FoxE complex can be identified due to its ligand-to-metal charge transfer band at 435 nm. Alternatively, elution of both, DFE and FoxE can be followed as separate peaks at 220 nm wavelength with characteristic retention times. The extraordinarily high stability constant of DFE with ferric iron of K=10³² enables extraction of iron from a variety of ferrous and ferric iron compounds and allows quantitation after separation by HPLC without interference by coloured by-products. Thus, iron bound to protein, amino acids, citrate and other organic acid ligands and even insoluble ferric hydroxides and phosphates can be solubilized in the presence desferrioxamine E. The “Ferrioxamine E method” can be applied to all kinds of plant fluids (apoplasmic, xylem, phloem, intracellular) either at physiological pH or even at acid pH values. The FoxE complex is stable down to pH 1 allowing protein removal by perchloric acid treatment and HPLC separation in the presence of trifluoroacetic acid containing eluents. Published online December 2004