Marine Siderophores and Microbial Iron Mobilization

Iron is essential for the growth of nearly all microorganisms yet iron is only sparingly soluble near the neutral pH, aerobic conditions in which many microorganisms grow. The pH of ocean water is even higher, thereby further lowering the concentration of dissolved ferric ion. To compound the problem of availability, the total iron concentration is surprisingly low in surface ocean water, yet nevertheless, marine microorganisms still require iron for growth. Like terrestrial bacterial, bacteria isolated from open ocean water often produce siderophores, which are low molecular weight chelating ligands that facilitate the microbial acquisition of iron. The present review summarizes the structures of siderophores produced by marine bacteria and the emerging characteristics that distinguish marine siderophores.     

Iron Acquisition from Natural Organic Matter by an Aerobic Pseudomonas mendocina Bacterium: Siderophores and Cellular Iron Status

This research investigated the potential role of siderophores in aerobic microbial Fe acquisition from natural organic matter (NOM; XAD-8 isolate and reverse osmosis concentrate pre- and post-Chelex® treatment) through the use of a siderophore-producing Pseudomonas mendocina wild type (WT) bacterium and an engineered mutant (Mt) that was incapable of siderophore production. NOM had complex effects on microbial growth under Fe-limited conditions as measured by optical density, most likely because of the presence of other toxic (trace) metals such as Al, NOM binding interference with additional trace metal nutrients, and/or biofilm development. However, a bioassay for cellular Fe status showed that both WT and Mt readily acquired Fe naturally associated with NOM. Thus, while siderophores may be useful for Fe acquisition from NOM by P. mendocina, they do not appear to be essential for this process.     

假单胞菌荧光与非荧光铁载体对铁离子的应答差异

假单胞菌既能产荧光铁载体也能产非荧光铁载体.通过对假单胞菌在不同铁离子浓度下,在通用CAS(Chrome azroul S)检测平板、改进的蔗糖-天冬氨酸(SA)平板(MSA)上以及通用液体CAS培养基和MSA培养基内的铁载体产生情况的比较,发现在通用CAS的液体培养基上产生的主要为非荧光铁载体(pyochelin),而在改进的MSA培养基上产生的主要为荧光铁载体(pyoverdine);在铁离子的应答方面,pyoverdine较pyochelin灵敏,较低的铁离子浓度即可抑制荧光铁载体的产生,但是不能抑制非荧光铁载体.     

The Role of Ligand Exchange in the Uptake of Iron from Microbial Siderophores by Gramineous Plants

The siderophore rhizoferrin, produced by the fungus Rhizopus arrhizus, was previously found to be as an efficient Fe source as Fe-ethylenediamine-di(o-hydroxphenylacetic acid) to strategy I plants. The role of this microbial siderophore in Fe uptake by strategy II plants is the focus of this research. Fe-rhizoferrin was found to be an efficient Fe source for barley (Hordeum vulgare L.) and corn (Zea mays L.). The mechanisms by which these Gramineae utilize Fe from Fe-rhizoferrin and from other chelators were studied. Fe uptake from 59Fe-rhizoferrin, 59Fe-ferrioxamine B, 59Fe-ethylenediaminetetraacetic acid, and 59Fe-2[prime]-deoxymugineic acid by barley plants grown in nutrient solution at pH 6.0 was examined during periods of high (morning) and low (evening) phytosiderophore release. Uptake and translocation rates from Fe chelates paralleled the diurnal rhythm of phytosiderophore release. In corn, however, similar uptake and translocation rates were observed both in the morning and in the evening. A constant rate of the phytosiderophore's release during 14 h of light was found in the corn cv Alice. The results presented support the hypothesis that Fe from Fe-rhizoferrin is taken up by strategy II plants via an indirect mechanism that involves ligand exchange between the ferrated microbial siderophore and phytosiderophores, which are then taken up by the plant. This hypothesis was verified by in vitro ligand-exchange experiments.     

Iron release from ferritin by alloxan radical

Alloxan in the presence of reduced glutathione released iron from ferritin which is the major intracellular iron storage protein. Superoxide dismutase inhibited by only about 30% the alloxan-dependent iron release from ferritin but completely inhibited the iron release from ferritin induced by hypoxanthine-xanthine oxidase. Under anaerobic conditions, the ESR spectrum of alloxan radical was obtained and interaction with ferritin resulted in a marked diminution of the alloxan radical signal. These results indicate that alloxan radical rapidly releases iron from ferritin.     

Iron release from ferritin by flavin nucleotides

Background

Extensive in-vitro studies have focused on elucidating the mechanism of iron uptake and mineral core formation in ferritin. However, despite a plethora of studies attempting to characterize iron release under different experimental conditions, the in-vivo mobilization of iron from ferritin remains poorly understood.Several iron-reductive mobilization pathways have been proposed including, among others, flavin mononucleotides, ascorbate, glutathione, dithionite, and polyphenols. Here, we investigate the kinetics of iron release from ferritin by reduced flavin nucleotide, FMNH2, and discuss the physiological significance of this process in-vivo.

Methods

Iron release from horse spleen ferritin and recombinant human heteropolymer ferritin was followed by the change in optical density of the Fe(II)–bipyridine complex using a Cary 50 Bio UV–Vis spectrophotometer. Oxygen consumption curves were followed on a MI 730 Clark oxygen microelectrode.

Results

The reductive mobilization of iron from ferritin by the nonenzymatic FMN/NAD(P)H system is extremely slow in the presence of oxygen and might involve superoxide radicals, but not FMNH2. Under anaerobic conditions, a very rapid phase of iron mobilization by FMNH2 was observed.

Conclusions

Under normoxic conditions, FMNH2 alone might not be a physiologically significant contributor to iron release from ferritin.

General significance

There is no consensus on which iron release pathway is predominantly responsible for iron mobilization from ferritin under cellular conditions. While reduced flavin mononucleotide (FMNH2) is one likely candidate for in-vivo ferritin iron removal, its significance is confounded by the rapid oxidation of the latter by molecular oxygen.     

Mechanism of Ferrous Iron Binding and Oxidation by Ferritin from a Pennate Diatom

A novel ferritin was recently found in Pseudo-nitzschia multiseries (PmFTN), a marine pennate diatom that plays a major role in global primary production and carbon sequestration into the deep ocean. Crystals of recombinant PmFTN were soaked in iron and zinc solutions, and the structures were solved to 1.65–2.2-Å resolution. Three distinct iron binding sites were identified as determined from anomalous dispersion data from aerobically grown ferrous soaked crystals. Sites A and B comprise the conserved ferroxidase active site, and site C forms a pathway leading toward the central cavity where iron storage occurs. In contrast, crystal structures derived from anaerobically grown and ferrous soaked crystals revealed only one ferrous iron in the active site occupying site A. In the presence of dioxygen, zinc is observed bound to all three sites. Iron oxidation experiments using stopped-flow absorbance spectroscopy revealed an extremely rapid phase corresponding to Fe(II) oxidation at the ferroxidase site, which is saturated after adding 48 ferrous iron to apo-PmFTN (two ferrous iron per subunit), and a much slower phase due to iron core formation. These results suggest an ordered stepwise binding of ferrous iron and dioxygen to the ferroxidase site in preparation for catalysis and a partial mobilization of iron from the site following oxidation.     

Release of Iron from Ferritin by 6-Hydroxydopamine Under Aerobic and Anaerobic Conditions

6-hydroxydopamine (6-OHDA) proved to be a very effective agent for iron release from ferritin. Iron release was enhanced in the presence of SOD, catalase and under anaerobic conditions. Ascorbic acid, a well known agent able to release iron from ferritin, increased the amount of released iron in more than an additive manner when used in combination with 6-OHDA. Similar to 6-OHDA, 6-hydroxydopa (Topa) and 1,2,4-benzenetriol were also able to release iron in large amounts; in contrast, catecholamines and other benzenediols were comparatively ineffective.     

Release of Iron from Ferritin by 6-Hydroxydopamine Under Aerobic and Anaerobic Conditions

6-hydroxydopamine (6-OHDA) proved to be a very effective agent for iron release from ferritin. Iron release was enhanced in the presence of SOD, catalase and under anaerobic conditions. Ascorbic acid, a well known agent able to release iron from ferritin, increased the amount of released iron in more than an additive manner when used in combination with 6-OHDA. Similar to 6-OHDA, 6-hydroxydopa (Topa) and 1,2,4-benzenetriol were also able to release iron in large amounts; in contrast, catecholamines and other benzenediols were comparatively ineffective.     

Ascorbate-mediated Iron Release from Ferritin in the Presence of Alloxan

Release of iron from ferritin requires reduction of ferric to ferrous iron. The iron can participate in the diabetogenic action of alloxan. We investigated the ability of ascorbate to catalyze the release of iron from ferritin in the presence of alloxan. Incubation of ferritin with ascorbate alone elicited iron release (33 nmol/10 min) and the generation of ascorbate free radical, suggesting a direct role for ascorbate in iron reduction. Iron release by ascorbate significantly increased in the presence of alloxan, but alloxan alone was unable to release measurable amounts of iron from ferritin. Superoxide dismutase significantly inhibited ascorbate-mediated iron release in the presence of alloxan, whereas catalase did not. The amount of alloxan radical (A·⁻) generated in reaction systems containing both ascorbate and alloxan decreased significantly upon addition of ferritin, suggesting that A·⁻ is directly involved in iron reduction. Although release of iron from ferritin and generation of A·⁻ were also observed in reactions containing GSH and alloxan, the amount of iron released in these reactions was not totally dependent on the amount of A·⁻ present, suggesting that other reductants in addition to A·⁻ (such as dialuric acid) may be involved in iron release mediated by GSH and alloxan. These results suggest that A·⁻ is the main reductant involved in ascorbate-mediated iron release from ferritin in the presence of alloxan and that both dialuric acid and A·⁻ contribute to GSH/alloxan-mediated iron release.     

铁核结构对马脾铁蛋白释放铁动力学的影响

建立Ｈ^% 参与马脾铁蛋白释放铁的动力方程,Ｈ^ 以１／２级反应方式参与铁蛋白释放铁核表层的铁。在酸性介质（ＰＨ６．５）中,铁蛋白释放铁的总平均速率（３３２Ｆe^3 /HSF.min)比在碱性介质（Ｐ8Ｈ８．０）中放铁的总平均速率（７３Ｆe^3 /HSF.min)高４．６倍,铁蛋白的铁核结构和外加的磷酸盐均能影响该蛋白释放的速率,但并不改变其反应级数。     

Iron Fortification of Banana by the Expression of Soybean Ferritin

Iron deficiency anemia is one of the serious ailments related to nutrition in the developing countries. Fruit and vegetable crops favor the bioavailability of iron. Banana is consumed as a staple food in the tropics. Iron-fortified bananas provide an effective means of controlling the iron deficiency. Embryogenic cells of banana cv. Rasthali (AAB) were transformed with soybean ferritin cDNA using two different expression cassettes pSF and pEFE-SF to express ferritin. Transgenic nature of the regenerated plants was confirmed by PCR. Transgenic plants were regenerated and analyzed through PCR and PCR-Southern analysis. The expression of ferritin was confirmed by RT-PCR. Iron and zinc levels in the transgenic and control plants were estimated by atomic absorption spectroscopy. A 6.32-fold increase in iron accumulation and a 4.58-fold increase in the zinc levels were noted in the leaves of transgenic plants. Thus, iron- and zinc-fortified bananas could be developed as a functional food to overcome the malnutrition-related iron deficiency. This is the first report on the iron and zinc fortification of banana.     

Iron release analyses from ferritin by visible light irradiation

We investigated the iron release from ferritin by irradiation from a white fluorescent light in the absence or presence of ADP. Irradiation of a ferritin solution at 17,000 lx in the absence of ADP slightly induces iron release from ferritin but only at acidic pH conditions (pH 5.0 or pH 6.0). Irradiation in the presence of ADP markedly enhances iron release from ferritin under the same conditions. In the absence of irradiation, the iron release from ferritin was low even in the presence of ADP. The induction of the iron release by irradiation in the presence of ADP was also affected by various factors such as irradiation dose and acidity, but not temperature (4-47°C), oxygen concentration, or free radical generations during the irradiation. The iron release during the irradiation ceased to increase by turning off the light and was found to increase again after additional irradiation. These results suggest that visible light directly induces iron release from ferritin via the photoreduction of iron stored inside ferritin.     

Effects of Ceruloplasmin on Superoxide-Dependent Iron Release from Ferritin and Lipid Peroxidation

Ceruloplasmin (CP) effectively inhibited superoxide and ferritin-dependent peroxidation of phospholipid liposomes, using xanthine oxidase or gamma irradiation of water as sources of superoxide. In addition, CP inhibited superoxide-dependent mobilization of iron from ferritin. suggesting that CP inhibited lipid peroxidation by decreasing the availability of iron from ferritin. CP also exhibited some superoxide scavenging activity as evidenced by its inhibition of superoxide-dependent cytochrome c reduction. However, superoxide scavenging by CP did not quantitatively account for its inhibitory effects on iron release. The effects of CP on iron-catalyzed lipid peroxidation in systems containing exogenously added ferrous iron was also investigated. CP exhibited prooxidant and antioxidant effects; CP stimulated at lower concentrations, reached a maximum. and inhibited at higher concentrations. However. the addition of apoferritin inhibited CP and Fe(II)-catalyzed lipid peroxidation at all concentrations of CP. In addition, CP catalyzed the incorporation of Fe(II) into apoferritin. Collectively these data suggest that CP inhibits superoxide and ferritin-dependent lipid peroxidation via its ability to incorporate reductively-mobilized iron into ferritin.     

Roles of Siderophores,Oxalate, and Ascorbate in Mobilization of Iron from Hematite by the Aerobic Bacterium Pseudomonas mendocina

In aerobic, circumneutral environments, the essential element Fe occurs primarily in scarcely soluble mineral forms. We examined the independent and combined effects of a siderophore, a reductant (ascorbate), and a low-molecular-weight carboxylic acid (oxalate) on acquisition of Fe from the mineral hematite (α-Fe₂O₃) by the obligate aerobe Pseudomonas mendocina ymp. A site-directed ΔpmhA mutant that was not capable of producing functional siderophores (i.e., siderophore⁻ phenotype) did not grow on hematite as the only Fe source. The concentration of an added exogenous siderophore (1 μM desferrioxamine B [DFO-B]) needed to restore wild-type (WT)-like growth kinetics to the siderophore⁻ strain was ∼50-fold less than the concentration of the siderophore secreted by the WT organism grown under the same conditions. The roles of a reductant (ascorbate) and a simple carboxylic acid (oxalate) in the Fe acquisition process were examined in the presence and absence of the siderophore. Addition of ascorbate (50 μM) alone restored the growth of the siderophore⁻ culture to the WT levels. A higher concentration of oxalate (100 μM) had little effect on the growth of a siderophore⁻ culture; however, addition of 0.1 μM DFO-B and 100 μM oxalate restored the growth of the mutant to WT levels when the oxalate was prereacted with the hematite, demonstrating that a metabolizing culture benefits from a synergistic effect of DFO-B and oxalate.Iron (Fe) is essential for almost all life. However, in aerobic, circumneutral environments, Fe is bound primarily in scarcely soluble minerals and amorphous solids [e.g., the solubility product (K_SP) for amorphous Fe(OH)₃ is 10⁻³⁸] (53) and is therefore poorly bioavailable. Aerobic microorganisms directly transform mineral-bound Fe(III) into soluble, highly bioavailable forms (1), overcoming significant kinetic and thermodynamic barriers to mineral dissolution and serving as primary transporters of Fe from the geosphere into global biogeochemical cycles.A primary means by which aerobic microorganisms enhance Fe mobility and bioavailability is by secreting siderophores, which are structurally diverse, low-molecular-weight chelating agents with extremely high affinities for Fe(III) (12, 27, 37, 40). Fe(III)-siderophore stability constants can be as high as 10⁵² (1, 40), which is many orders of magnitude higher than the stability constants for low-molecular-weight organic acids, such as oxalic acid [for Fe(III) + 3 oxalate ⇆ Fe(oxalate)₃, K = 10^18.6] (45). While their high affinity for Fe(III) is clearly important for helping siderophores mobilize Fe from Fe(III) (hydr)oxides in the aqueous phase, the mechanisms of Fe mobilization appear to be complex and are the subject of much recent study (14, 17, 18, 26, 28, 49). In particular, the role of siderophores in ligand-promoted dissolution mechanisms has undergone careful evaluation in vitro. The model is described simply here as follows for amorphous Fe(OH)₃ and has been described in detail by Kraemer (26): Fe(OH)₃ + 3H⁺ ⇆ Fe(III) + 3H₂O (K_SP) (equation 1); Fe(III) + H₃L ⇆ FeL + 3H⁺ (K_FeL) (equation 2); and Fe(OH)₃ + H₃L ⇆ FeL + 3H₂O (K_eq = K_SPK_FeL = [FeL]/[H₃L]) (equation 3). The concentration of the solubilized FeL complex, according to equation 3, is determined as follows: [FeL] = [H₃L]K_{SP ×} K_FeL. The estimated concentration of siderophores in carbonic soil (∼10⁻⁸ to 10⁻⁷ M), combined with their strong affinities for Fe(III) (39), suggests that [FeL] could in principle easily be micromolar or higher and could support vigorous bacterial growth. However, the trishydroxamate siderophores that have been studied most to date adsorb only weakly to Fe(III) (hydr)oxide minerals, likely due to steric constraints, although charge repulsion may also play a role for positively charged siderophores, such as desferrioxamine B (DFO-B) (6, 26, 41, 42). Therefore, it has been proposed that siderophores act primarily in conjunction with other molecules, such as simple plant-derived carboxylic acids or reductants, which interact more strongly with mineral surfaces and release Fe directly through ligand-promoted and/or reductive mechanisms (52). This proposed “synergistic effect,” in which the combined effect of various elements is greater than the sum of the individual effects, suggests that an interaction of biogenic molecules may overcome kinetic and thermodynamic barriers to the release of Fe from minerals in the presence of siderophores. The role of the siderophore in such a synergistic system is not a direct role in surface processes; rather, the siderophore maintains a low concentration of aqueous Fe in equilibrium with the mineral (an Fe sink), thus driving the reaction toward more dissolution (26, 41). Only a low concentration of a siderophore relative to the concentrations of surface-reacting organic species is required to promote efficient dissolution (26).The synergistic effect has been observed directly in in vitro, abiotic experiments using combinations of microbe-derived siderophores and simple organic acids. A combination of environmentally relevant concentrations of oxalate (1 to 80 μM) and DFO-B (40 μM), for example, doubled the rate of Fe(III) hydroxide mineral (goethite) dissolution compared with the rate when only oxalate or DFO-B was present in a recent in vitro study (6). Actively metabolizing aerobic bacteria, which can move Fe from solution into cells and recycle or release new siderophores back into the medium, might be expected to promote the synergistic siderophore-carboxylic acid interaction even further in a batch system. Likewise, it has been suggested that organic reductants may work synergistically with siderophores. In particular, a recent study showed that exogenously added reductants significantly enhance the bioavailability of Fe to an aerobic siderophore-producing bacterium, Pseudomonas mendocina ymp (15), isolated from the Nevada Test Site and used in the work described here.As an obligate aerobe, P. mendocina ymp does not have dissimilatory reduction pathways, so that its use of iron (hydr)oxide minerals is only for acquisition of nutritional Fe and not for cellular respiration. In contrast to dissimilatory Fe-reducing bacteria, which require millimolar concentrations of Fe (2, 29-31, 36, 43, 50), P. mendocina ymp requires micromolar concentrations (19, 20, 24, 32-34). Previously, this strain''s ability to dissolve and use various mineral forms of Fe was quantified in a series of microbial growth studies (23, 24, 32-34). P. mendocina ymp is known to produce hydroxamate-containing siderophores that increase the rate of dissolution of the Fe oxide mineral hematite. A recent study demonstrated that reductants significantly enhanced the bioavailability of Fe-(hydr)oxide minerals to P. mendocina (15). The ymp strain was also shown to have endogenous Fe(III)-reducing activity, which Hersman et al. suggested could be involved in solubilizing ferric minerals (24). Likewise, closely related strains of Pseudomonas stutzeri have been shown to produce pyridine-2,6-bis(thiocarboxylic acid) (PDTC), which they can use in the reduction, transport, and detoxification of metals and metalloids (11, 16, 51). However, control experiments showed that P. mendocina did not secrete molecules that exhibited a significant amount of reducing activity under the conditions used in this study (see the supplemental material). Notably, this species does not appear to contain a set of PDTC biosynthesis genes.In this work we used the wild-type (WT) strain P. mendocina ymp along with a mutant with a site-directed markerless mutation that was not capable of producing siderophores (ΔpmhA mutant with the siderophore⁻ phenotype) (3) in a series of experiments examining siderophore use and potential synergistic effects with either a simple carboxylic acid (oxalate) or an exogenous reductant (ascorbate). Both ascorbate and oxalate are plant products that are frequently found in the shallow subsurface; their effects on in vitro Fe (hydr)oxide dissolution have been well described (6).     

Rapid and Efficient Purification of Haemolymph Ferritin from Cricket, Gryllus bimaculatus

The Gryllus bimaculatus ferritin was purified from the haemolymph by a consecutive four‐step procedures consisting of 50% ammonium sulfate fractionation, anion exchange column chromatography using HiTrap^TM Q column (1.6 x 4 cm, Amersham Bioscience), 70°C heat treatment for 10 min, acid treatment of 0.1 M sodium acetate buffer (pH 6.0), and gel filtration column chromatography using G4000SW column (0.75 x 60 cm, Tosoh, Japan) connected on FPLC system. The purified ferritin was found to have two major subunits of 32 and 30 kDa and three minor subunits of 28, 27, and 25 kDa by 2D electrophoresis analysis. Amino acid composition analysis showed that there are high contents of Asp, Glu, Met, Leu, and Lys residues in ferritin while low contents of Cys, Tyr, and Trp residues in the protein. G. bimaculatus haemolymph ferritin could be classified as a methionine‐rich protein.     

Iron Content and Ferritin in Leaves of Iron Treated Xanthium pensylvanicum Plants

Iron administration to iron-starved cocklebur (Xanthium pensylvanicum) plants causes an increase in the iron content of ferritin fractions extracted from mature leaves. Xanthium plants grown under long days (vegetative stage) have more iron and ferritin than similarly iron-treated plants induced to flower under short day regimes. This first demonstration of ferritin in cocklebur (Compositae) leaves suggests that a substantial portion of iron that enters the iron-starved plant appears as this protein-iron macromolecule.     

A Diatom Ferritin Optimized for Iron Oxidation but Not Iron Storage

Ferritin from the marine pennate diatom Pseudo-nitzschia multiseries (PmFTN) plays a key role in sustaining growth in iron-limited ocean environments. The di-iron catalytic ferroxidase center of PmFTN (sites A and B) has a nearby third iron site (site C) in an arrangement typically observed in prokaryotic ferritins. Here we demonstrate that Glu-44, a site C ligand, and Glu-130, a residue that bridges iron bound at sites B and C, limit the rate of post-oxidation reorganization of iron coordination and the rate at which Fe³⁺ exits the ferroxidase center for storage within the mineral core. The latter, in particular, severely limits the overall rate of iron mineralization. Thus, the diatom ferritin is optimized for initial Fe²⁺ oxidation but not for mineralization, pointing to a role for this protein in buffering iron availability and facilitating iron-sparing rather than only long-term iron storage.     

In Vitro Studies of Ferritin Iron Release and Neurotoxicity

Abstract: The increase in brain iron associated with several neurodegenerative diseases may lead to an increased production of free radicals via the Fenton reaction. Intracellular iron is usually tightly regulated, being bound by ferritin in an insoluble ferrihydrite core. The neurotoxin 6-hydroxydopamine (6-OHDA) releases iron from the ferritin core by reducing it to the ferrous form. Iron release induced by 6-OHDA and structurally related compounds and two other dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium iodide (MPP⁺) and 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo), were compared, to identify the structural characteristics important for such release. 1,2,4-Trihydroxybenzene (THB) was most effective in releasing ferritin-bound iron, followed by 6-OHDA, dopamine, catechol, and hydroquinone. Resorcinol, MPP⁺, and TaClo were ineffective. The ability to release iron was associated with a low oxidation potential. It is proposed that a low oxidation potential and an ortho -dihydroxyphenyl structure are important in the mechanism by which ferritin iron is mobilized. In the presence of ferritin, both 6-OHDA and THB strongly stimulated lipid peroxidation, an effect abolished by the addition of the iron chelator deferoxamine. These results suggest that ferritin iron release contributes to free radical-induced cell damage in vivo.