The influence of chelating agents upon the dissimilatory reduction of Fe(III) by Shewanella putrefaciens

The ability of S. putrefaciens to reduce Fe(III) complexed by a variety of ligands has been investigated. All of the ligands tested caused the cation to be more susceptible to reduction by harvested whole cells than when uncomplexed, although some complexes were more readily reduced than others. Monitoring rates of reduction by a ferrozine assay for Fe(II) formation proved inadequate using Fe(III) ligands giving Fe(II) complexes of low kinetic lability (e.g. EDTA). A more suitable assay for Fe(III) reduction in the presence of such ligands proved to be the observation of associated cytochrome oxidation and re-reduction. Where possible, an assay for Fe(III) reduction based upon the disappearance of Fe(III) complex was also employed. Reduction of all Fe(III) complexes tested was totally inhibited by the presence of O₂, partially inhibited by HQNO and slower in the absence of a physiological electron donor. Upon cell fractionation, Fe(III) reductase activity was detected exclusively in the membranes. Using different physiological electron donors in assays on membranes, relative reduction rates of Fe(III) complexes complemented the data from whole cells. The differences in susceptibility to reduction of the various complexes are discussed, as is evidence for the respiratory nature of the reduction.     

The influence of chelating agents upon the dissimilatory reduction of Fe(III) byShewanella putrefaciens. Part 2. Oxo-and hydroxo-bridged polynuclear Fe(III) complexes

The susceptibility to dissimilatory reduction of polynuclear oxo- and hydroxo-bridged Fe(III) complexes byShewanella putrefaciens intact cells and membranes has been investigated. These complexes were ligated by the potential tetradentates heidi (H₃heidi =N-(2-hydroxyethyl)iminodiacetic acid) or nta (H₃nta = nitrilotriacetic acid), or the potential tridentate ida (H₂ida = iminodiacetic acid). A number of defined small complexes with varied nuclearity and solubility properties were employed, as well as undefined species prepared by mixing different molar ratios of ida or heidi:Fe(III) in solution. The rates of Fe(III) reduction determined by an assay for Fe(II) formation with ferrozine were validated by monitoringc-type cytochrome oxidation and re-reduction associated with electron transport. For the undefined Fe(III) polymeric species, reduction rates in whole cells and membranes were considerably faster in the presence of heidi compared to ida. This is believed to result from generally smaller and more reactive clusters forming with heidi as a consequence of the alkoxo function of this ligand being able to bridge between Fe(III) nuclei, with access to an Fe(III) reductase located at the cytoplasmic membrane being of some importance. The increases in reduction rates of the undefined ida species with Fe(III) using membranes relative to whole cells reinforce such a view. Using soluble synthetic Fe(III) clusters, slow reduction was noted for an oxo-bridged dimer coordinatively saturated with ida and featuring unligated carboxylates. This suggests that sterically hindering the cation can influence enzyme action. A heidi dimer and a heidi multimer (17 or 19 Fe(III) nuclei), which are both of poor solubility, were found to be reduced by whole cells, but dissimilation rates increased markedly using membranes. These data suggest that Fe(III) reductase activity may be located at both the outer membrane and the cytoplasmic membrane ofS. putrefaciens. Slower reduction of the heidi multimer relative to the heidi dimer reflects the presence of a central hydroxo(oxo)-bridged core containing nine Fe(III) nuclei within the former cluster. This unit is a poor substrate for dissimilation, owing to the fact that the Fe(III) is not ligated by aminocarboxylate. The faster reduction noted for the heidi dimer in membranes than for a soluble ida monomer suggests that the presence of ligating water molecules may relieve steric hindrance to enzyme attack. Furthermore, reduction of an insoluble oxo-bridged nta dimer featuring ligating water molecules in intact cells was faster than that of a soluble monomer coordinatively saturated by nta and possessing an unligated carboxylate. This suggests that steric factors may override solubility considerations with respect to the susceptibility to reduction of certain Fe(III) complexes by the bacterium.Previous paper in this series: Dobbin PS, Powell AK, McEwan AG, Richardson DJ. 1995 The influence of chelating agents upon the dissimilatory reduction of Fe(III) byShewanella putefraciens.BioMetals 8, 163–173.     

Reversion by Fe(III) of the inhibition by hydroxamic acids of the cyanide-Insensitive respiration in the yeast Saccharomycopsis lipolytica

The specific inhibitory effect of benzhydroxamic acid on the cyanide-insensitive respiration could be reversed in whole cells of the yeast Saccharomycopsis lipolytica, by addition of Fe(III), in a way suggesting a competition between the added iron and an enzyme-bound metallic ion, both central atoms for the ligand benzhydroxamic acid. The possibility that added metal ions modify the penetration of BHAM into the cells was ruled out. Co(II), Cu(II) and Al(III) could substitute for Fe(III). A linear relation between the concentration in added Fe(III) and the reversed respiration rate was observed. At a given cell concentration. the reversion by added Fe(III) of the inhibitory effect of benzhydroxamic acid on the alternative respiration appeared more related to the degree of inhibition rather than to the concentration in added inhibitor. Increasing cell concentrations required increasing amounts of Fe(III) to reach the same level of reversion. No reversal occurred at concentrations in added Fe(III) lower than 0.1 mM, whatever the benzhydroxamic concentration, the cell concentration or the yeast batch.     

Reduction of Fe(III), Mn(III), and Cu(II) chelates by roots of pea (Pisum sativum L.) or soybean (Glycine max)

Neither the reduction of Fe(III) to Fe(II) by roots nor its induction by Fe-deficiency are unique characteristics of the reductive activities of roots. We show that chelated Mn(III) or chelated Cu(II), as well as chelated Fe(III), may be reduced by Fe-stressed roots of pea (Pisum sativum L.). Deficiency of Fe stimulated the reduction of Fe(III)EDTA about 20-fold, the reduction of Mn(III)CDTA about 11-fold, the reduction of Cu(II)(BPDS)₂ about 5-fold, and the reduction of Fe(III)(CN)₆ by only about 50%. Not only are metals other than Fe reduced as part of the Fe-stress response, but deficiencies of metals other than Fe stimulate the reductive activity of roots. We show that depriving peas or soybeans (Glycine max) of Cu or Zn stimulates the reduction of Fe(III).     

Bacterial siderophores: the solution stoichiometry and coordination of the Fe(III) complexes of pyochelin and related compounds

Pyochelin, its analog 3′′-nor-NH-pyochelin, and the related methyl hydroxamate, 2-(2′-hydroxyphenyl)-4,5-dihydrothiazol-4-carboxylic acid methoxymethyl amide, have been prepared together with their Fe(III) complexes. The solution stoichiometry and the coordination of the three Fe(III) complexes in methanol or buffered (pH∼2) 50:50 (v/v) methanol–water mixtures were determined using various spectroscopic methods: UV–vis absorption, X-ray absorption, extended X-ray absorption fine structure and electron paramagnetic resonance. All three systems showed both a 1:1 and 2:1 ligand–Fe(III) stoichiometry, but presented different coordination properties. Conditional formation constants (pH∼2) were determined for both the 1:1 and 2:1 complexes in all three systems. Computation of the coordination-conformational energies by semiempirical methods indicated that the coordination in the case of the 2:1 complexes of pyochelin–Fe(III) and 3′′-nor-NH-pyochelin–Fe(III) was asymmetrical, with one molecule of pyochelin (or 3′′-nor-NH-pyochelin) tetradentately coordinated (O1, N1, N2 and O3) to the Fe(III), and the second molecule bound bidentately (O1, N1 or N2, O3), to complete the octahedral geometry. In contrast, two molecules of the methyl hydroxamate each provided a set of tridentate ligand atoms in the formation of the 2:1 ligand–Fe(III) complex. These results are consistent with the role of pyochelin in the uptake of iron by the FptA receptor in the outer membrane of Pseudomonas aeruginosa and in several gram-negative bacteria.     

Shewanella oneidensis MR‐1 mutants selected for their inability to produce soluble organic‐Fe(III) complexes are unable to respire Fe(III) as anaerobic electron acceptor

Summary Recent voltammetric analyses indicate that Shewanella putrefaciens strain 200 produces soluble organic‐Fe(III) complexes during anaerobic respiration of sparingly soluble Fe(III) oxides. Results of the present study expand the range of Shewanella species capable of producing soluble organic‐Fe(III) complexes to include Shewanella oneidensis MR‐1. Soluble organic‐Fe(III) was produced by S. oneidensis cultures incubated anaerobically with Fe(III) oxides, or with Fe(III) oxides and the alternate electron acceptor fumarate, but not in the presence of O₂, nitrate or trimethylamine‐N‐oxide. Chemical mutagenesis procedures were combined with a novel MicroElectrode Screening Array (MESA) to identify four (designated Sol) mutants with impaired ability to produce soluble organic‐Fe(III) during anaerobic respiration of Fe(III) oxides. Two of the Sol mutants were deficient in anaerobic growth on both soluble Fe(III)‐citrate and Fe(III) oxide, yet retained the ability to grow on a suite of seven alternate electron acceptors. The rates of soluble organic‐Fe(III) production were proportional to the rates of iron reduction by the S. oneidensis wild‐type and Sol mutant strains, and all four Sol mutants retained wild‐type siderophore production capability. Results of this study indicate that the production of soluble organic‐Fe(III) may be an important intermediate step in the anaerobic respiration of both soluble and sparingly soluble forms of Fe(III) by S. oneidensis.     

异化铁还原细菌Clostridium sp.LQ25分离及其产氢与铁还原特性

[背景] 一些异化铁还原细菌兼具铁还原和发酵产氢能力,可作为发酵型异化铁还原细菌还原机制研究的对象。[目的] 筛选出一株发酵型异化铁还原细菌。在异化铁还原细菌培养体系中,设置不同电子供体并分析电子供体。[方法] 通过三层平板法从海洋沉积物中筛选纯菌株,基于16S rRNA基因序列进行菌株鉴定。通过测定细菌培养液Fe （II）浓度及发酵产氢量分析菌株异化铁还原和产氢性质。[结果] 菌株LQ25与Clostridium butyricum的16S rRNA基因序列相似性达到100%,结合电镜形态观察,菌株命名为Clostridium sp.LQ25。在氢氧化铁为电子受体培养条件下,菌株生长较对照组（未添加氢氧化铁）显著提高。菌株LQ25能够利用丙酮酸钠、葡萄糖和乳酸钠进行生长。丙酮酸钠为电子供体时,菌株LQ25细胞生长和异化铁还原效率最高,菌体蛋白质含量是（78.88±3.40） mg/L,累积产生Fe （II）浓度为（8.27±0.23） mg/L。以葡萄糖为电子供体时,菌株LQ25发酵产氢量最高,达（475.2±14.4） mL/L,相比对照组（未添加氢氧化铁）产氢量提高87.7%。[结论] 筛选到一株具有异化铁还原和发酵产氢能力的菌株Clostridium sp.LQ25,为探究发酵型异化铁还原细菌胞外电子传递机制提供了新的实验材料。     

Potentiometric and P NMR studies on inositol phosphates and their interaction with iron(III) ions

Potentiometric, conductometric and ³¹P NMR titrations have been applied to study interactions between myo-inositol hexakisphosphate (phytic acid), (±)-myo-inositol 1,2,3,5-tetrakisphosphate and (±)-myo-inositol 1,2,3-trisphosphate with iron(III) ions. Potentiometric and conductometric titrations of myo-inositol phosphates show that addition of iron increases acidity and consumption of hydroxide titrant. By increasing the Fe(III)/InsP₆ ratio (from 0.5 to 4) 3 mol of protons are released per 2 mol of iron(III). At first, phytates coordinate iron octahedrally between P2 and P1,3. The second coordination site represents P5 and neighbouring P4,6 phosphate groups. Complexation is accompanied with the deprotonation of P1,3 and P4,6 phosphate oxygens. At higher concentration of iron(III) intermolecular P–O–Fe–O–P bonds trigger formation of a polymeric network and precipitation of the amorphous Fe(III)–InsP₆ aggregates. ³¹P NMR titration data complement the above results and display the largest chemical shift changes at pD values between 5 and 10 in agreement with strong interactions between iron and myo-inositol phosphates. The differences in T₁ relaxation times of phosphorous atoms have shown that phosphate groups at positions 1, 2 and 3 are complexated with iron(III). The interactions between iron(III) ions and inositol phosphates depend significantly on the metal to ligand ratio and an attempt to coordinate more than two irons per InsP₆ molecule results in an unstable heterogeneous system.     

Antimicrobial,spectral, magnetic and thermal studies of Cu(II), Ni(II), Co(II), UO2(VI) and Fe(III) complexes of the Schiff base derived from oxalylhydrazide

The Schiff base ligand, oxalic bis[(2-hydroxybenzylidene)hydrazide], H₂L, and its Cu(II), Ni(II), Co(II), UO₂(VI) and Fe(III) complexes were prepared and tested as antibacterial agents. The Schiff base acts as a dibasic tetra- or hexadentate ligand with metal cations in molar ratio 1:1 or 2:1 (M:L) to yield either mono- or binuclear complexes, respectively. The ligand and its metal complexes were characterized by elemental analyses, IR, ¹H NMR, Mass, and UV-Visible spectra and the magnetic moments and electrical conductance of the complexes were also determined. For binuclear complexes, the magnetic moments are quite low compared to the calculated value for two metal ions complexes and this shows antiferromagnetic interactions between the two adjacent metal ions. The ligand and its metal complexes were tested against a Gram + ve bacteria (Staphylococcus aureus), a Gram -ve bacteria (Escherichia coli), and a fungi (Candida albicans). The tested compounds exhibited high antibacterial activities.     

Fe(III)-enhanced Azo Reduction by <Emphasis Type="Italic">Shewanella decolorationis</Emphasis> S12

Shewanella decolorationis S12 is capable of high rates of azo dye decolorization and dissimilatory Fe(III) reduction. Under anaerobic conditions, when Fe(III) and azo dye were copresent in S12 cultures, dissimilatory Fe(III) reduction and azo dye biodecolorization occurred simultaneously. Furthermore, the dye decolorization was enhanced by the presence of Fe(III). When 1 mM Fe(III) was added, the methyl red decolorizing efficiency was 72.1% after cultivation for 3 h, whereas the decolorizing efficiency was only 60.5% in Fe(III)-free medium. The decolorizing efficiencies increased as the concentration of Fe(III) was increased from 0 to 6 mM. Enzyme activities, which mediate the dye decolorization and Fe(III) reduction, were not affected by preadaption of cells to Fe(III) and azo dye nor by the addition of chloramphenicol. Both the Fe(III) reductase and the azo reductase were membrane associated. The respiratory electron transport chain inhibitors metyrapone, dicumarol, and stigmatellin showed significantly different effects on Fe(III) reduction than on azo dye decolorization.     

Ion‐Exchange and Adsorption of Fe(III) by Sepia Melanin

Sepia eumelanin is associated with many metal ions, yet little is known about its metal binding capacity and the chemical nature of the binding site(s). Herein, the natural concentrations of metal ions are presented and the ability to remove metals by exposure of the melanin granules to EDTA is quantified. The results reveal that the binding constants of melanin at pH 5.8 for Mg(II), Ca(II), Sr(II) and Cu(II) are, respectively, 5, 4, 14 and 34 times greater than the corresponding binding constants of these ions with EDTA. By exposing Sepia eumelanin to aqueous solutions of FeCl₃, the content of bound Fe(III) can be increased from a natural concentration of ～180 ppm to a saturation limit of ～80 000 ppm or 1.43 mmol/g of melanin. Similar saturation limits are found for Mg(II) and Ca(II). Exposure of Sepia melanin granules to aqueous solutions containing Ca(II) results in the stoichiometric replacement of the initially bound Mg(II), arguing that these two ions occupy the same binding site(s) in the pigment. The pH‐dependent binding of Mg(II) and Ca(II) suggests coordination of these ions to carboxylic acid groups in the pigment. Mg(II) and Ca(II) can be added to a Fe(III)‐saturated melanin sample without affecting the amount of Fe(III) pre‐adsorbed, clearly establishing Fe(III) and Mg(II)/Ca(II) occupy different binding sites. Taking recent Raman spectroscopic data into account, the binding of Fe(III) is concluded to involve coordination to o‐dihydroxyl groups. The effects of metal ion content on the surface morphology were analyzed. No significant changes were found over the full range of Fe(III) concentration studied, which is supported by the Brunauer–Emmett–Teller surface area analysis. These observations imply the existence of channels within the melanin granules that can serve to transport metal ions.     

Uptake and degradation of EDTA by Escherichia coli

It was found that Escherichia coli exhibited a growth by utilization of Fe(III)EDTA as a sole nitrogen source. No significant growth was detected when Fe(III)EDTA was replaced by EDTA complexes with other metal ions such as Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, and Zn²⁺. When EDTA uptake was measured in the presence of various ions, it was remarkable only when Fe³⁺ was present. The cell extract of E. coli exhibited a significant degradation of EDTA only in the presence of Fe³⁺. It is likely that the capability of E. coli for the growth by utilization of Fe(III)EDTA results from the Fe³⁺-dependent uptake and degradation of EDTA.     

Alkaline iron(III) reduction by a novel alkaliphilic,halotolerant, <Emphasis Type="Italic">Bacillus</Emphasis> sp. isolated from salt flat sediments of Soap Lake

A halotolerant, alkaliphilic dissimilatory Fe(III)-reducing bacterium, strain SFB, was isolated from salt flat sediments collected from Soap Lake, WA. 16S ribosomal ribonucleic acid gene sequence analysis identified strain SFB as a novel Bacillus sp. most similar to Bacillus agaradhaerens (96.7% similarity). Strain SFB, a fermentative, facultative anaerobe, fermented various hexoses including glucose and fructose. The fructose fermentation products were lactate, acetate, and formate. Under fructose-fermenting conditions in a medium amended with Fe(III), Fe(II) accumulated concomitant with a stoichiometric decrease in lactate and an increase in acetate and CO₂. Strain SFB was also capable of respiratory Fe(III) reduction with some unidentified component(s) of Luria broth as an electron donor. In addition to Fe(III), strain SFB could also utilize nitrate, fumarate, or O₂ as alternative electron acceptors. Optimum growth was observed at 30°C and pH 9. Although the optimal salinity for growth was 0%, strain SFB could grow in a medium with up to 15% NaCl by mass. These studies describe a novel alkaliphilic, halotolerant organism capable of dissimilatory Fe(III) reduction under extreme conditions and demonstrate that Bacillus species can contribute to the microbial reduction of Fe(III) in environments at elevated pH and salinity, such as soda lakes.     

Electron transfer. Part 165: Oxidations of Ti(II)(aq) with ligated iron(III) and ruthenium(III)

Titanium(II) solutions, prepared by dissolving titanium wire in triflic acid + HF, contain equimolar quantities of Ti(IV). Treatment of such solutions with excess Fe(III) or Ru(III) complexes yield Ti(IV), but reactions with Ti(II) in excess give Ti(III). Oxidations by (NH₃)₅Ru(III) complexes, but not by Fe(III) species, are catalyzed by titanium(IV) and by fluoride. Stoichiometry is unchanged. The observed rate law for the Ru(III)-Ti(II)-Ti(IV) reactions in fluoride media points to competing reaction paths differing by a single F⁻, with both routes involving a Ti(II)-Ti(IV) complex which is activated by deprotonation. It is suggested that coordination of Ti(IV) to Ti^II(aq) minimizes the mismatch of Jahn-Teller distortions which would be expected to lower the Ti(II,III) self-exchange rate.     

Redox activity of caffeic acid towards iron(III) complexed in a polygalacturonate network

The transfer of several metal ions from the soil to the plant absorbing cells is mediated principally by organic molecules of low molecular weight with complexing and reducing activity, among which caffeic acid (CAF) is particularly important. Here we report the results of a survey which deals with the oxidation of CAF by the Fe(III) ions bound to a polygalacturonate network (Fe(III)-PGA network). The interaction between Fe(III) and CAF was studied by using Fe(III)-PGA networks equilibrated in the 2.4-7.0 pH range by means of kinetic and spectroscopic methods. The reducing power was found to depend on the nature of the Fe(III)-PGA network complexes: when the ferric ion was complexed only by the PGA carboxylic groups, a high redox activity was observed, whereas the Fe(III) reduction was found to be lower when a hydroxylic group was inserted in the Fe(III) coordination sphere. The iron complexed in the network was protected from hydrolysis reactions, as shown by the high pH values at which its reduction occurred. Two different fractions of Fe(II) produced were identified, one diffusible and another exchangeable with CaCl₂ 6.0 mM. The existence of the exchangeable form was attributed to the electrostatic interaction of the Fe(II) ions with the carboxylate groups of the fibrils and with the degradation products of CAF. The arrangement of the fibrils was altered following the substitution of Ca(II) by Fe(III) ions and was restored following the reduction of Fe (III) by CAF.     

异化铁还原细菌LQ25还原重金属Cr (VI)的特性研究

[背景] 异化铁还原细菌能够在还原Fe （III）的同时将毒性较大的Cr （VI）还原成毒性较小的Cr （III）,解决铬污染的问题。[目的] 基于丁酸梭菌（Clostridium butyricum） LQ25异化铁还原过程制备生物磁铁矿,开展异化铁还原细菌还原Cr （VI）的特性研究。[方法] 构建以氢氧化铁为电子受体和葡萄糖为电子供体的异化铁培养体系。菌株LQ25培养结束时制备生物磁铁矿。设置不同初始Cr （VI）浓度（5、10、15、25和30 mg/L）,分别测定菌株LQ25对Cr （VI）还原效率以及生物磁铁矿对Cr （VI）的还原效率。[结果] 菌株LQ25在设置的Cr （VI）浓度范围内都能良好生长。当Cr （VI）浓度为15 mg/L时,在异化铁培养条件下,菌株LQ25对Cr （VI）的还原率为63.45%±5.13%,生物磁铁矿对Cr （VI）的还原率为87.73%±9.12%,相比菌株还原Cr （VI）的效率提高38%。pH变化能影响生物磁铁矿对Cr （VI）的还原率,当pH 2.0时,生物磁铁矿对Cr （VI）的还原率最高,几乎达到100%。电子显微镜观察发现生物磁铁矿表面有许多孔隙,X-射线衍射图谱显示生物磁铁矿中Fe （II）的存在形式是Fe （OH）₂。[结论] 基于异化铁还原细菌制备生物磁铁矿可用于还原Cr （VI）,这是一种有效去除Cr （VI）的途径。     

Effect of Al(III) on surface properties of Bifidobacterium thermophilum as a function of temperature

The effects of Al(III) on surface properties and lactate accumulation by Bifidobacterium thermophilum were investigated. Bacteria were treated with Al(III) at 37°C and 4°C, then exposed to free radicals or nisin. When exposed to Al(III) at 37°C, the organism exhibited spreading on hydrophobic surfaces and showed high susceptibility to free-radical alteration as indicated by Fe(III) binding, but showed little effect on lactate production in the presence or absence of nisin, even after washing with 2 mM EDTA. At 4°C, there was no increased surface spreading or binding of Fe(III), but protection against nisin action was present. This, however, was abolished after washing with EDTA. It was concluded that membrane fluidity is required to affect membrane lipid rearrangement, resulting in surface spreading and increased susceptibility to peroxidation, whereas only loose binding of Al(III) to membrane surfaces is sufficient to prevent transmembrane channel formation by nisin.     

Suberoylanilide hydroxamic acid, a potent histone deacetylase inhibitor; its X-ray crystal structure and solid state and solution studies of its Zn(II), Ni(II), Cu(II) and Fe(III) complexes

Reaction of the potent hydroxamate-based histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), with hydrated metal salts of Fe(III), Cu(II), Ni(II) and Zn(II) yielded a tris-hydroxamato complex in the case of Fe(III) and bis-hydroxamato complexes in the case of Cu(II), Ni(II) and Zn(II) both in the solid state and in solution. Reaction of the secondary hydroxamic acid, N-Me-SAHA, also yielded a tris-hydroxamato complex in the case of Fe(III) and bis-hydroxamato complexes in the case of Cu(II), Ni(II) and Zn(II) in solution. These metal complexes have the hydroxamato moiety coordinated in an O,O’-bidentate fashion. Stability constants of the metal complexes formed with SAHA and N-Me-SAHA in a DMSO/H₂O 70/30%(v/v) mixture are described. A novel crystal structure of SAHA together with a novel synthesis for N-Me-SAHA are also reported.     

Fe(III) Oxide Reduction by Anaerobic Biofilm Formation-DeficientS-Ribosylhomocysteine Lyase (LuxS) Mutant of Shewanella oneidensis

Shewanella oneidensis respires a variety of terminal electron acceptors, including solid phase Fe(III) oxides. S. oneidensis transfers electrons to Fe(III) oxides via direct (outer membrane- or nanowire-localized c-type cytochromes) and indirect (electron shuttling and Fe(III) solubilization) pathways. In the present study, the influence of anaerobic biofilm formation on Fe(III) oxide reduction by S. oneidensis was determined. The gene encoding the activated methyl cycle (AMC) enzyme S-ribosylhomocysteine lyase (LuxS) was deleted in-frame to generate the corresponding mutant ΔluxS. Conventional biofilm assays and visual inspection via confocal laser scanning microscopy indicated that the wild-type strain formed anaerobic biofilms on Fe(III) oxide-coated silica surfaces, while the ΔluxS mutant was severely impaired in anaerobic biofilm formation on such surfaces. Cell-hematite attachment isotherms demonstrated that the ΔluxS mutant was also severely impaired in attachment to hematite surfaces under anaerobic conditions. The S. oneidensis ΔluxS mutant, however, reduced Fe(III) at wild-type rates during anaerobic incubation with Fe(III) oxide-coated silica surfaces or in batch cultures with Fe(III) oxide or hematite as a terminal electron acceptor. Anaerobic biofilm formation by the ΔluxS mutant was restored to wild-type rates by providing a wild-type copy of luxS in trans or by the addition of AMC or transsulfurylation pathway metabolites involved in organic sulfur metabolism. LuxS is thus required for wild-type anaerobic biofilm formation on Fe(III) oxide surfaces, yet the inability to form wild-type anaerobic biofilms on Fe(III) oxide surfaces does not alter Fe(III) oxide reduction activity.     

Oxidative mutagenesis of doxorubicin-Fe(III) complex

Doxorubicin has a high affinity for inorganic iron, Fe(III), and has potential to form doxorubicin-Fe(III) complexes in biological systems. Indirect involvement of iron has been substantiated in the oxidative mutagenicity of doxorubicin. In this study, however, direct involvement of Fe(III) was evaluated in mutagenicity studies with the doxorubicin-Fe(III) complex. The Salmonella mutagenicity assay with strain TA102 was used with a pre-incubation step. The highest mutagenicity of doxorubicin-Fe(III) complex was observed at the dose of 2.5 nmol/plate of the complex. The S9-mix decreased this highest mutagenicity but increased the number of revertants at a higher dose of 10 nmol/plate of the complex. On the other hand, the mutagenicity of the doxorubicin-Fe(III) complex at the doses of 0.25, 0.5, 1 and 2 nmol/plate was enhanced about twice by the addition of glutathione plus H₂O₂. This enhanced mutagenicity as well as of the complex itself, the complex plus glutathione, and the complex plus H₂O₂ were reduced by the addition of ADR-529, an Fe(III) chelator, and potassium iodide, a hydroxyl radical scavenger. These results indicate that doxorubicin-Fe(III) complex exert the mutagenicity through oxidative DNA damage and that Fe(III) is a required element in the mutagenesis of doxorubicin.