PHYSIOLOGICAL CHARACTERIZATION OF A SYNECHOCOCCUS SP. (CYANOPHYCEAE) STRAIN PCC 7942 IRON‐DEPENDENT BIOREPORTER FOR FRESHWATER ENVIRONMENTS1

The complex chemical speciation of Fe in aquatic systems and the uncertainties associated with biological assimilation of Fe species make it difficult to assess the bioavailability of Fe to phytoplankton in relation to total dissolved Fe concentrations in natural waters. We developed a cyanobacterial Fe‐responsive bioreporter constructed in Synechococcus sp. strain PCC 7942 by fusing the Fe‐responsive isiAB promoter to Vibrio harveyi luxAB reporter genes. A comprehensive physiological characterization of the bioreporter has been made in defined Fraquil medium at free ferric ion concentrations ranging from pFe 21.6 to pFe 19.5. Whereas growth and physiological parameters are largely constrained over this range of Fe bioavailability, the bioreporter elicits a luminescent signal that varies in response to Fe deficiency. A dose‐response characterization of bioreporter luminescence made over this range of Fe^{3 +} bioavailability demonstrates a sigmoidal response with a dynamic linear range extending between pFe 21.1 and pFe 20.6. The applicability of using this Fe bioreporter to assess Fe availability in the natural environment has been tested using water samples from Lake Huron (Laurentian Great Lakes). Parallel assessment of dissolved Fe and bioreporter response from these samples reinforces the idea that measures of dissolved Fe should not be considered alone when assessing Fe availability to phytoplankton communities.     

Arsenic precipitation in the bioleaching of enargite by Sulfolobus BC at 70 °C

Enargite (Cu₃AsS₄) was leached at 70°C by Sulfolobus BC in shake-flasks. The highest copper dissolution (52% after 550 h of leaching) was obtained with bacteria and 1 g l^–1 ferric ion. In the absence of ferric ion, Sulfolobus BC catalyzes the bioleaching of enargite through a direct mechanism after adhesion onto the mineral surface. In ferric bioleaching, arsenic precipitated as ferric arsenate and arsenic remained associated to the solid residues, preventing the presence of a high dissolved arsenic concentration in the leaching solution. About 90% inhibition of bacterial growth rate and activity was observed for dissolved arsenic concentrations above 600 mg l^–1 for As(III) and above 1000 mg l^–1 for As(V). Arsenic-bearing copper ores and concentrates could be leached by Sulfolobus BC in the presence of ferric iron due to the favourable precipitation of arsenic ion as ferric arsenate, avoiding significant bacterial inhibition.     

A high sensitivity iron-dependent bioreporter used to measure iron bioavailability in freshwaters

A Nostoc sp. PCC 7120 iron bioreporter containing iron-regulated schizokinen transporter gene alr0397 promoter fused to the luxAB genes was examined to optimize its response to bioavailable iron. Dose-response relationships between luciferase activity and free ferric ion (Fe(3+) ) concentrations pFe (-lg [Fe(3+) ]) were generated by measuring luciferase activities of the bioreporter in trace metal-buffered Fraquil medium with various incubation times. The results were best demonstrated by sigmoidal curves (pFe 18.8-21.7, Fe(3+) =?10(-18.8) -10(-21.7) M) with the linear range extending from pFe 19.6-21.5 (Fe(3+) =?10(-19.6) -10(-21.5) M) after a 12-h incubation time. Optimal conditions for the use of this bioreporter to sense the iron bioavailability were determined to be: a 12-h exposure time, initial cell density of OD(730?nm) =?0.06, high nitrate (100?μM), high phosphate (10?μM), moderate Co(2+) (0.1-22.5?nM), Zn(2+) (0.16-12?nM), Cu(2+) (0.04-50?nM), and wide range of Mn(2+) concentration (0.92-2300?nM). The applicability of using this iron bioreporter to assess iron availability in the natural environment has been tested using water samples from eutrophic Taihu, Donghu, and Chaohu lakes. It is indicated that the bioreporter is a useful tool to assess bioavailable iron in various water quality samples, especially in eutrophic lakes with high bioavailable iron.     

Ferric chelate reduction by suspension culture cells and roots of soybean: A kinetic comparison

The abilities of suspension cultures and intact roots of soybean (Glycine max L. cv. Hawkeye) to reduce ferric chelate were compared. Ferric chelate was supplied as ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) and reduction was measured spectrophotometrically using bathophenan-throlinedisulfonic acid (BPDS) as the ferrous scavenger. Ferric chelate reduction by cell suspension cultures showed typical saturation kinetics; however, no difference was observed between cells that had been continuously grown with Fe (+Fe) and those that had been grown for four days without added Fe (–Fe). Values for K_m and V_max, determined from a Lineweaver-Burk plot, were 57 M and nmoles mg^-1 dry weight for the +Fe cells and 50 M and 22 nmoles mg^-1 dry weight for the -Fe cells, respectively. Ferric chelate reduction by Fe-deficient roots also exhibited saturation kinetics, while roots grown with adequate Fe did not reduce ferric chelate. The K_m and V_max values for Fe-deficient roots were 45 M and 20 nmoles mg^-1 dry weight, respectively, and did not differ from values obtained for cells in culture. This study offers strong evidence that the mechanism responsible for the reduction of ferric chelate is the same for cultured cells and roots and that the process is controlled at the cellular level. We propose that suspension cultures can be used as an alternative to intact roots in the study of ferric chelate reduction.     

Toxicity and bioaccumulation of iron in soil microalgae

Microalgae are extensively used in the remediation of heavy metals like iron. However, factors like toxicity, bioavailability and iron speciation play a major role in its removal by microalgae. Thus, in this study, toxicity of three different iron salts (FeSO₄, FeCl₃ and Fe(NO₃)₃) was evaluated towards three soil microalgal isolates, Chlorella sp. MM3, Chlamydomonas sp. MM7 and Chlorococcum sp. MM11. Interestingly, all the three iron salts gave different EC50 concentrations; however, ferric nitrate was found to be significantly more toxic followed by ferrous sulphate and ferric chloride. The EC₅₀ analysis revealed that Chlorella sp. was significantly resistant to iron compared to other microalgae. However, almost 900 μg g^?1 iron was accumulated by Chlamydomonas sp. grown with 12 mg L^?1 ferric nitrate as an iron source when compared to other algae and iron salts. The time-course bioaccumulation confirmed that all the three microalgae adsorb the ferric salts such as ferric nitrate and ferric chloride more rapidly than ferrous salt, whereas intracellular accumulation was found to be rapid for ferrous salts. However, the amount of iron accumulated or adsorbed by algae, irrespective of species, from ferrous sulphate medium is comparatively lower than ferric chloride and ferric nitrate medium. The Fourier transform infrared spectroscopy (FTIR) analysis shows that the oxygen atom and P?=?O group of polysaccharides present in the cell wall of algae played a major role in the bioaccumulation of iron ions by algae.     

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.     

Inhibition of Sulfur Use by Sulfite Ion in Thiobacillus ferrooxidans

In Thiobacillus ferrooxidans AP19-3, elemental sulfur is oxidized by the cooperation of three enzymes, namely, hydrogen sulfide: ferric ion oxidoreductase (SFORase), sulfite: ferric ion oxidoreductase, and iron oxidase. Sulfite ions are one of the products when elemental sulfur is oxidized by SFORase. Under the conditions in which sulfite ions are accumulated in the cells, use of sulfur as an energy source by this strain was strongly inhibited. So the mechanism of inhibition by sulfite ions in T. ferrooxidans AP19-3 was studied. The activities of SFORase and iron oxidase were completely inhibited by 0.8 mm and 1.5 mm NaHSO₃, respectively. ¹⁴CO₂ uptake into washed intact cells was also completely inhibited by 1mm NaHSO₃ when ferrous ion or elemental sulfur was used as an energy source. However, the activities of ribulose-1,5-bisphosphate carboxylase, phosphoribulokinase, and ribosephosphate isomerase measured with a cell-free extract were not inhibited by NaHSO₃ at 1 mm, indicating that sulfite ions didn’t inhibit key enzymes of the Calvin cycle. Since the activity of CO₂ uptake into washed intact cells was absolutely dependent on Fe^{2 +} - or S0-oxidation, mechanism of inhibition of sulfur use by sulfite ions is proposed as follows: sulfite ions inhibit SFORase and iron oxidase, as a result T. ferrooxidans AP19-3 can not obtain a carbon source for CO₂ fixation and stops cell growth on sulfur-salts medium.     

Ferrous iron dependent nitric oxide production in nitrate reducing cultures of Escherichia coli

l-Lactate-driven ferric and nitrate reduction was studied in Escherichia coli E4. Ferric iron reduction activity in E. coli E4 was found to be constitutive. Contrary to nitrate, ferric iron could not be used as electron acceptor for growth. Ferric iron reductase activity of 9 nmol Fe²⁺ mg^-1 protein min^-1 could not be inhibited by inhibitors for the respiratory chain, like Rotenone, quinacrine, Actinomycin A, or potassium cyanide. Active cells and l-lactate-driven nitrate respiration in E. coli E4 leading to the production of nitrite, was reduced to about 20% of its maximum activity with 5 mM ferric iron, or to about 50% in presence of 5 mM ferrous iron. The inhibition was caused by nitric oxide formed by a purely chemical reduction of nitrite by ferrous iron. Nitric oxide was further chemically reduced by ferrous iron to nitrous oxide. With electron paramagnetic resonance spectroscopy, the presence of a free [Fe²⁺-NO] complex was shown. In presence of ferrous or ferric iron and l-lactate, nitrate was anaerobically converted to nitric oxide and nitrous oxide by the combined action of E. coli E4 and chemical reduction reactions (chemodenitrification).     

Redox control of proton transfers in membrane b-type cytochromes: an absorption and resonance Raman study on bis(imidazole) and bis(imidazolate) model complexes of iron-protoporphyrin

Optical absorption spectra and resonance Raman (RR) spectra, obtained with Soret excitation, are reported for bis(imidazole) and bis(imidazolate) complexes of iron(II)- and iron(III)-protoporphyrin IX, prepared in aqueous conditions. Perdeuteration experiments on the axial ligands permitted the assignment of the symmetric Fe-(ligand)₂ stretching mode of Fe[x]PP(L)₂ to RR bands at 203 (x = II; L = ImH), 212 (x = II; L = Im^–), 201 (x = III; L = ImH) and 226 cm^–1 (x = III; L = Im^–). These frequency differences indicate a strengthening of the axial bonds when the imidazole deprotonations occur. The larger difference observed for the ferric derivatives reflects the stronger -donor capability of the Im^– anion for iron(III) over iron(II). For the ferrous derivatives, the frequencies of several skeletal porphyrin modes (₄, ₁₀, ₁₁ and ₃₈) are downshifted by 2–10 cm^–1 upon deprotonation of the ligands. This effect corresponds to an increased back-bonding from the metal atom to the porphyrin ring when the axial ligand decreases its -acid strength. Bringing further support to this interpretation, an inverse linear relationship is established between the frequencies of (Fe(Il)-L₂) and ₁₁. This correlation is expected to monitor the overall H-bonding state of histidine ligands of reduced cytochromes b. On the other hand, absorption measurements have characterized large pK_a differences for the sequential imidazole ionizations of Fe[x]PP(ImH)₂ in aqueous cetyltrimethylammonium bromide (9.0 and 10.8 for x = 111; 13.0 and 14.1 for x = II). These titrations show that Fe(II)PP(Im^–)₂ and Fe(III)PP(ImH)₂ are good proton-acceptor and proton-donor, respectively, and suggest a model by which heme, located in a favorable environment inside a cytochrome, could couple a cycle of electron transfer with a proton transfer. Based on sequence data and structural models, it is further proposed that, in several membrane cytochromes b (b, b ₆, b ₅₅₉), a positively charged amino acid residue and an imidazolate ligand of the ferriheme could form an ion pair involved in a redox control of proton transfer.Abbreviations RR resonance Raman - EPR electron paramagnetic resonance - PP protoporphyrin IX - ImH imidazole - Im^– imidazolate - Im* imidazole or imidazolate - 1MeIm 1-methylimidazole - HisH histidine - His^– histidinate - CTABr cetyltrimethylammonium bromide - NaDS sodium dodecylsulphate - VLP very low potential - LP low potential - HP high potential     

Nicotianamine and the distribution of iron into the apoplasm and symplasm of tomato (Lycopersicon esculentum Mill.)

The apoplasmic and symplasmic iron pools were determined in roots and leaves of Lycopersicon esculentum Mill. cv. Bonner Beste and its mutant chloronerva. The mutant is auxotrophic for the ubiquitous plant constituent nicotianamine (NA) and exhibits an impaired iron metabolism. Formation of apoplasmic iron pools in roots was dependent on the iron source in the nutrient solution. With Fe-ethylenediaminedi-(2-hydroxyphenylacetate) (FeEDDHA) only a very small apoplasmic iron pool was formed in the roots of both genotypes. Plants grown with FeEDTA increased their apoplasmic iron pool with increasing exogenous iron concentrations in the nutrient solution. The size of the apoplasmic pools in roots did not differ between the wild-type and the mutant (about 85 mol Fe · g^–1 DW). By contrast, the symplasmic iron concentrations in roots and leaves of the mutant were significantly higher when compared to the wild-type. An exogenous NA supply to the leaves of the mutant reduced the high symplasmic iron concentrations to the level of the wild-type. Mutant leaves exhibited a gradient of symplasmic iron concentrations depending on the developmental age of the leaves. The oldest leaves contained considerably more symplasmic iron than the youngest. The results demonstrate that the apparent iron deficiency of the mutant is not the consequence of an impaired iron transport from the apoplasm to the symplasm. Therefore, it is concluded that NA is not required for the transport of Fe(II) through the plasmalemma into the cell.Abbreviations BPDS bathophenanthroline disulfonic acid, Na₂ salt - FeEDDHA ferric N-N-ethylenediaminedi-(2-hydroxy-phenylacetate) - NA nicotianamine Part 40 in the series The normalizing factor for the tomato mutant chloronerva. For part 39 see Pich et al. (1991)The valuable technical assistance of Mrs. Christa Kallas and Mr. Günter Faupel is gratefully acknowledged.     

A comparative study of the effects of natural and synthetic ligands on iron uptake by plants

Summary The uptake of iron by wheat seedlings was investigated using half-strength Hoagland's nutrient solution containing 2.0 M ferric chloride labelled with⁵⁹Fe. The iron content of root tissue, which includes adsorbed iron, was depressed by the presence in the solution of the synthetic ligands EDTA and polymaleic acid (PMA) and by the natural ligands, humate, fulvate and a water-extractable soil polycarboxylate. The patterns of change in iron content of the shoots were in all cases different from those of the roots and were of two types. EDTA and humate increased the iron content of the shoots to maximum values, at ligand concentrations of 5.0 M and 2.5 mg l^–1 respectively, and decreased it at higher concentrations. Fulvate, water-extractable soil polycarboxylate and PMA increased the iron content of the shoots up to the maximum ligand concentrations tested (25 mg l^–1). These results are discussed in the light of the likely solution chemistry of iron and the various ligands.     

Growth and nutrition of small Betula pendula plants at different relative addition rates of iron

Summary Small birch plants (Betula pendula Roth.) were cultivated in a hydroponic spray solution where the relative addition rate of iron (R_Fe; g g^–1 day^–1), was the growth-controlling variable. All other elements were added in free access. An additional treatment was performed where all nutrients, including iron, were in free access (FA). The plants showed deficiency symptoms at steady-state growth and severe limitation of iron, R_Fe 0.05 and 0.10 day^–1. There were few symptoms at R_Fe of 0.15 or above. Plant relative growth rate (R_G; g g^–1 day^–1), equalled the relative rate of increase in iron supply, R_Fe. Internal iron concentration of the plants ranged from 40 to 70 g g^–1 dry weight (DW) over the range for which iron supply was limiting growth. At FA, the internal concentration was approximately 200 g g^–1 DW without further increase in R_G, demonstrating that iron may be taken up in excess without affecting growth. Internal concentrations of macronutrients were stable at the different R_Fe, except for Ca and Mg in shoots which were higher at low iron supply. Uptake rates of iron, calculated per root growth rate (mol g^–1 root DW), were approximately twice as high at R_Fe 0.20 as at 0.05 day^–1. The effect of iron limitation on dry matter allocation to leaves was small, with increases in the root fraction being largely at the expense of the stem. Leaf area ratio was constant regardless of R_Fe and the specific leaf area tended to increase with increasing iron limitation. Net assimilation rate decreased by a factor of 6 from free access to severe iron limitation, largely accounting for the differences in plant R_G.     

利用蓝藻生物报告体检测水体中可利用铁的研究

鱼腥藻PCC 7120 中的alr2581 基因编码的蛋白质在缺铁胁迫时显著上调。将该基因的启动子Palr2581和费氏弧菌的luxAB 基因融合, 通过同源单交换, 整合到鱼腥藻PCC 7120 的基因组上, 构建了可以感知环境中铁的生物报告体Palr2581-luxAB。该藻株在不含铁的BG11 中培养时, 启动子Palr2581 的转录活性增强, LuxAB酶活显著升高。通过测定Palr2581-luxAB 藻株在不同铁浓度Fraquil 培养基中的LuxAB 酶活, 得到了铁浓度pFe(-lgFe3+)与 LuxAB 酶活的剂量反应曲线。结果显示, 12h 时, LuxAB 酶活随培养基中Fe3+浓度增加呈S形递减关系, 其中在pFe=20.7—21.2 范围内有很好的线性关系。根据这一特性, 我们利用Palr2581-luxAB 作为铁的生物报告体, 测定了武汉市东湖水体中可利用的铁浓度为10-20.56 mol/L。研究显示, 通过这一方法可以较方便地监测各种淡水中可利用的铁浓度。       

Effects of metal speciation on growth of phytoplankton with special reference to iron

This paper is a compilation of studies concerning the effects of metal speciation on the growth of phytoplankton. Special attention is paid to the speciation and availability of iron in lake Tjeukemeer, The Netherlands. Under laboratory conditions the free ionic metal species generally appear to be most effective in determining metal availability and toxicity. A variety of factors controlling solubility, ion-exchange, complexation or chelation, sorption and electrostatical attraction of metal ions affect the metal speciation, mostly resulting in reduced availabilities. However, some organic metal chelates such as citrates, nitrillotriacetates and the specifically iron chelating siderophores, are sometimes more available than the corresponding free ions. The presence of other metals also influences the availability of a given metal by competing for the same binding sites on the algal cell. This competition leads to antagonism betweene.g. iron nutrition and cadmium toxicity in marine diatoms. In the eutrophic, alkaline, hard and humic lake Tjeukemeer, the free Fe³⁺ concentration is below measurable levels and does not match the iron requirement of the phytoplankton. So most of the algal iron must have been provided by the predominant inorganic iron colloids and particles bye.g. dissolution or photo-degradation (reduction). If the provision rates of available iron are slow in relation to that of iron uptake, the growth of some phytoplankton species may become iron-limited. Continuous culture work indicated that the iron fraction <0.2 m from Tjeukemeer,i.e., the soluble fraction, is about one third as much available as iron from NH₄Fe(SO₄)₂.12H₂O. Different phytoplankton species vary widely in their metal requirements and tolerances. Therefore, metal speciation and availability may affect species composition and succession within phytoplankton communities. So far the assessment of metal availability in natural waters has been complicated by the complex metal chemistry and by methodological limits.     

Iron-efficient and iron-inefficient oats and corn respond differently to iron-deficiency stress

Iron-efficient (WF9 corn and Coker 227 oat) and Fe-inefficient (ys₁ corn and TAM 0–312 oat) cultivars were comparatively tested for their response to Fe-deficiency stress induced by the use of either ferrous or ferric chelators. Corn and oats were grown in 20 M Fe with 0, 60, and 120 M BPDS and 40 M Fe with 0, 120, and 240 M BPDS and 20 M Fe with 0 and 40 M EDDHA. All four cultivars tested, both Fe-efficient and Fe-inefficient, continuously reduced Fe³⁺ to Fe²⁺ at a low level as evidenced by the production of Fe²⁺ (BPDS)₃ in test nutrient solutions over time. Severity of chlorosis increased as more BPDS was added to the nutrient solutions for both WF9 and ys₁ corn, but unlike corn, Coker 227 and TAM 0-312 oats were both able to obtain Fe from the Fe²⁺ (BPDS)₃ complex and were less chlorotic as a result. In short-term (4-hour) in vivo measurements, iron-stressed WF9 (Fe-efficient) corn reduced more Fe³⁺ to Fe²⁺ than similarly stressed ys₁ corn, Coker 227 oat or TAM 0-312 oat. Thus, at the same time that Fe-efficient WF9 corn reduces more Fe than the other cultivars, it is also unable to compete with BPDS for that Fe in the nutrient solution. These differences coupled with the observation that only Coker 227 oat produced measureable iron solubilizing substances (phytosiderophores) suggest that these two species differ in their mechanisms for obtaining Fe during Fe-deficiency stress.     

Phytosiderophore release in relation to micronutrient metal deficiencies in barley

Phytosiderophore release occurs under both iron and zinc deficiencies in representative Poaceae and has been speculated to be a general adaptive response to enhance the acquisition of micronutrient metals. To test this hypothesis, phytosiderophore (PS) release rates from barley (Hordeum vulgare cv. CM72) subjected to deficiencies of Fe, Zn, Mn, and Cu were compared using chelator-buffered nutrient solutions. PS release rates were determined at two day intervals during onset and development of deficiency symptoms. Plant dry matter yields and nutrient concentrations, measured at three time points were used to construct growth curves for calculation of PS release per unit root mass and estimation of critical internal nutrient levels associated with PS release. In comparison to trace metal-sufficient control plants, dry matter production was markedly reduced in the Zn, Mn, and Cu deficiency treatments, with final relative yields of 49, 61, and 34%, respectively. Relative yields for Fe-deficient plants grown at three suboptimal Fe levels ranged from 95 to 33% of control, and provided a basis for comparison of PS release rates by Zn-, Mn-, and Cu-deficient plants at similar levels of growth inhibition. Under Fe deficiency, PS release increased with severity of the deficiency as measured by foliar Fe concentration, yield reduction, and chlorosis. Changes in PS release rates over time suggested a cyclical pattern that may be regulated by Fe concentration in the plant shoot. The highest rate of PS release (35 mol g^–1 root dw 2 h^–1) was measured after 10 days of growth at pFe 19, whereas control plants adapted for growth at pFe 17 released only 2 to 3 mol g^–1 root dw 2 h^–1. In a second experiment, maximum PS release rates for barley subjected to Zn, Mn, and Cu deficiencies were only 2.6, 2.5 and 1 mol g^–1 2 h^–1, respectively and were only slightly elevated over those of the control plants (ca. 1 mol g^–1 root dw 2 h^–1) grown at pFe 16.5. Moreover, enhanced PS release under Zn deficiency occurred much later, after the deficiency had already caused severely reduced growth. The results suggest that phytosiderophore release in this barley cultivar is a specific response to Fe deficiency and is not significantly induced in response to deficiencies of other trace metals.     

The gills as an important uptake route for the essential nutrient iron in freshwater rainbow trout Oncorhynchus mykiss

Short-term (3h) acquisition of iron (16 nmol ⁵⁹FeCl₃ l⁻¹) from oxic, alkaline fresh water was assessed in rainbow trout Oncorhynchus mykiss in the presence or absence of a range of iron chelators, all of which had differing binding affinities for ferric iron [100 μmol l⁻¹ of desferrioxamine (DFO), Log₁₀K₁ 32·5; citric acid Log₁₀K₁ 11·9; nitrilotriacetic acid (NTA) Log₁₀K₁ 15·9, CP20 and CP94 (Log₁₀K₁ > 30), as well as humic acid (HA), Log₁₀K₁ 5·04, 5 mg l⁻¹]. In the absence of chelators (control conditions) O. mykiss acquired iron from the water under laboratory lights (wavelength range of the lights 440–650 nm, peak intensity 548–626 nm) via the gill. In these conditions iron uptake onto the gill had a maximum transport capacity (J_max) of 11·2 pmol Fe g⁻¹ h⁻¹ (gill organ mass) and a K_m of 21·3 nmol Fe l⁻¹ h⁻¹. Furthermore, there were two components to iron accumulation into the carcass of these fish, a slow rate of aqueous iron uptake at low concentrations (6–24 nmol Fe l⁻¹), followed by a faster rate of uptake at higher iron concentrations (48–96 nmol Fe l⁻¹), suggesting that the rate-limiting step of iron uptake at low iron concentrations is the apical entry step. O. mykiss also acquired iron in the presence of HA, although the majority of the other chelators prevented iron uptake. Ultraviolet light (354 nm) treatment of Fe-DFO increased iron bioavailability. Results suggest that rainbow trout are able to access either the predicted very low concentrations (picomolar) of ferrous iron present in fresh water or the ferric oxide complexes present in oxic environments. The iron uptake rate measured (0·75 pmol g⁻¹ h⁻¹) would be sufficient to provide a substantial proportion (c. 85%) of the daily iron requirements of growing salmonid fry.     

Kinetic properties of a micronutrient transporter from<Emphasis Type="Italic"> Pisum sativum</Emphasis> indicate a primary function in Fe uptake from the soil

Fe uptake in dicotyledonous plants is mediated by a root plasma membrane-bound ferric reductase that reduces extracellular Fe(III)-chelates, releasing Fe²⁺ ions, which are then absorbed via a metal ion transporter. We previously showed that Fe deficiency induces an increased capacity to absorb Fe and other micronutrient and heavy metals such as Zn²⁺ and Cd²⁺ into pea (Pisum sativum L.) roots [Cohen et al. (1998) Plant Physiol 116:1063–1072). To investigate the molecular basis for this phenomenon, an Fe-regulated transporter that is a homologue of the Arabidopsis IRT1 micronutrient transporter was isolated from pea seedlings. This cDNA clone, designated RIT1 for root iron transporter, encodes a 348 amino acid polypeptide with eight putative membrane-spanning domains that is induced under Fe deficiency and can functionally complement yeast mutants defective in high- and low-affinity Fe transport. Chelate buffer techniques were used to control Fe²⁺ in the uptake solution at nanomolar activities representative of those found in the rhizosphere, and radiotracer methodologies were employed to show that RIT1 is a very high-affinity ⁵⁹Fe²⁺ uptake system (K _m =54–93 nM). Additionally, radiotracer (⁶⁵Zn, ¹⁰⁹Cd) flux techniques were used to show that RIT can also mediate a lower affinity Zn and Cd influx (K _m of 4 and 100 M, for Zn²⁺ and Cd²⁺, respectively). These findings suggest that, in typical agricultural soils, RIT1 functions primarily as a high-affinity Fe²⁺ transporter that mediates root Fe acquisition. This is consistent with recent findings with Arabidopsis IRT1 knockout mutants that strongly suggest that this transporter plays a key role in root Fe uptake and nutrition. However, the ability of RIT1 to facilitate Zn and Cd uptake when these metals are present at elevated concentrations suggests that RIT1 may be one pathway for the entry of toxic metals into the food chain. Furthermore, the finding that plant Fe deficiency status may promote heavy metal uptake via increased expression of this transporter could have implications both for human nutrition and also for phytoremediation, the use of terrestrial plants to sequester toxic metals from contaminated soil.     

Ferredoxin-NADP+ Reductase from Pseudomonas putida Functions as a Ferric Reductase

Pseudomonas putida harbors two ferredoxin-NADP⁺ reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally contained within the Fpr superfamily. Interestingly, ferric reductase is not annotated on the chromosome of P. putida. In an effort to elucidate the function of the Fpr as a ferric reductase, we used a variety of biochemical and physiological methods using the wild-type and mutant strains. In both the ferric reductase and flavin reductase assays, FprA and FprB preferentially used NADPH and NADH as electron donors, respectively. Two Fprs prefer a native ferric chelator to a synthetic ferric chelator and utilize free flavin mononucleotide (FMN) as an electron carrier. FprB has a higher k_cat/K_m value for reducing the ferric complex with free FMN. The growth rate of the fprB mutant was reduced more profoundly than that of the fprA mutant, the growth rate of which is also lower than the wild type in ferric iron-containing minimal media. Flavin reductase activity was diminished completely when the cell extracts of the fprB mutant plus NADH were utilized, but not the fprA mutant with NADPH. This indicates that other NADPH-dependent flavin reductases may exist. Interestingly, the structure of the NAD(P) region of FprB, but not of FprA, resembled the ferric reductase (Fre) of Escherichia coli in the homology modeling. This study demonstrates, for the first time, the functions of Fprs in P. putida as flavin and ferric reductases. Furthermore, our results indicated that FprB may perform a crucial role as a NADH-dependent ferric/flavin reductase under iron stress conditions.Commonly, Fprs are ubiquitous, monomeric, reversible flavin enzymes. Fprs evidence a profound preference for NADP(H) over NAD(H) (3). They harbor a prosthetic flavin cofactor (FAD) and catalyze the reversible electron exchange between NADPH and either ferredoxin (Fd) or flavodoxin (Fld) (4, 5). In oxygenic photosynthesis, the Fd is reduced by the photosystem and subsequently passes electrons on to NADP⁺ via the Fpr. This reaction provides the cellular NADPH pool required for CO₂ assimilation and other biosynthetic processes (4, 5). In heterotrophic organisms such as bacteria, reduced ferredoxin, owing to the reverse enzymatic activity of the Fpr, can donate an electron to several Fd-dependent enzymes, such as nitrite reductase, sulfite reductase, glutamate synthase, and Fd-thioredoxin reductase, allowing ferredoxin to function in a variety of systems, including oxidative stress (1, 4, 5).Iron is the fourth most abundant element in the natural environment and exists primarily as an oxidized form, Fe(III), which has very low solubility under neutral pH conditions (9, 34) and thus presents problems in terms of bioavailability. However, ferrous iron, of Fe(II), is soluble and available at neutral pH in bacterial cytosol (34). Most bacteria secrete siderophores, which are natural chelators of ferric iron. After they bind to ferric iron, that complex enters the bacteria and releases ferric iron into the cytosol in ferric or ferrous form (9). In the bacterial cytosol, ferric iron must be reduced to ferrous form, and thus ferric reductase is essential to bacterial iron utilization.Commonly, prokaryotic ferric reductases are divided into two groups—namely, the bacterial and archaeal types (34). The typical bacterial type ferric reductase is Escherichia coli Fre, which also functions as a flavin reductase. In other words, the ferric reductase can reduce free flavin as flavin reductase, rather than having the flavin cofactor as a prosthetic group in E. coli (38). The archaeal ferric reductase harbors a flavin cofactor in the enzyme and thus does not require a flavin carrier for ferric reduction (26, 34). E. coli Fre includes a Rosmann folding structure at the NAD(P) binding region, whereas the archaeal ferric reductase (FeR) of Archaeoglobus fulgidus does not evidence that folding structure (6, 34). Many bacterial ferric reductases utilize free flavins, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and riboflavin, as electron carrier and, NADH (NAD) or NADP as electron donors to ferric reductase (14, 34). However, reduced ferric iron by reduced free flavin gives rise to the Fenton reaction, which generates the hydroxyl radical within the cell (20, 38). The Fenton reaction is known to generate hydroxyl radicals from ferrous iron and hydrogen peroxide (20). The hydroxyl radical is the most reactive radical and can damage DNA, proteins, and membrane lipids (16, 20, 34, 38). Therefore, the fine-tuning of ferric reduction regulation is required for the survival of bacterial cells.Many Pseudomonas strains, including Pseudomonas putida, a gram-negative soil model bacteria, and Pseudomonas aeruginosa, a human pathogen bacteria, do not harbor annotated ferric reductase within their genome sequences. Commonly, the pathogens compete with the host for available iron, whichis crucial for their survival within the host. Thus, studies of P. aeruginosa regarding iron utilization, siderophores, and ferric reduction are considered to be essential for a better understanding of human infections (9, 19). Studying the physiology and ecology of P. putida also provides us with a new framework for elucidating the basis of the metabolic versatility and environmental stress response of soil microorganisms. Thus, the study of ferric reductase in strains of Pseudomonas at the molecular level is certainly required. From the structural perspective, ferric reductases are generally considered to be contained within the structurally diverse ferredoxin-NADP⁺ reductase (Fprs; EC 1.18.1.2) superfamily, which is frequently involved in the transfer of electrons between Fd/Fld and NADP(H) (2, 15, 34). Thus, we tested the role of the Fpr as a ferric reductase using free flavin (FMN or FAD), NADH, or NADPH as electron donors, and ferric-citrate or ferric-EDTA as terminal electron acceptors (37). We determined that FprA could efficiently utilize NADPH in ferric reduction. Rather, FprB could use NADH as an electron donor and may perform a crucial role as a NADH-dependent ferric reductase under iron stress conditions.     

Plasma membrane ferric reductase activity of iron-limited algal cells is inhibited by ferric chelators

Iron-limited cells of the green alga Chlorella kesslerii use a reductive mechanism to acquire Fe(III) from the extracellular environment, in which a plasma membrane ferric reductase reduces Fe(III)-chelates to Fe(II), which is subsequently taken up by the cell. Previous work has demonstrated that synthetic chelators both support ferric reductase activity (when supplied as Fe(III)-chelates) and inhibit ferric reductase. In the present set of experiments we extend these observations to naturally-occurring chelators and their analogues (desferrioxamine B mesylate, schizokinen, two forms of dihydroxybenzoic acid) and also two formulations of the commonly-used herbicide N-(phoshonomethyl)glycine (glyphosate). The ferric forms of the larger siderophores (desferrioxamine B mesylate, schizokinen) and Fe(III)-N-(phoshonomethyl)glycine (as the isopropylamine salt) all supported rapid rates of ferric reductase activity, while the iron-free forms inhibited reductase activity. The smaller siderophores/siderophore precursors, 2,3- and 3,4-dihydroxybenzoic acids, did not support high rates of reductase in the ferric form but did inhibit reductase activity in the iron-free form. Bioassays indicated that Fe(III)-chelates that supported high rates of ferric reductase activity also supported a large stimulation in the growth of iron-limited cells, and that an excess of iron-free chelator decreased the growth rate. With respect to N-(phosphonomethyl)glycine, there were differences between the pure compound (free acid form) and the most common commercial formulation (which also contains isopropylamine) in terms of supporting and inhibiting ferric reductase activity and growth. Overall, these results suggest that photosynthetic organisms that use a reductive strategy for iron acquisition both require, and are potentially simultaneously inhibited by, ferric chelators. Furthermore, these results also may provide an explanation for the frequently contradictory results of N-(phosphonomethyl)glycine application to crops: we suggest that low concentrations of this molecule likely solubilize Fe(III), making it available for plant growth, but that higher (but sub-lethal) concentrations decrease iron acquisition by inhibiting ferric reductase activity.