Ferric Iron Reduction by Acidophilic Heterotrophic Bacteria

Fifty mesophilic and five moderately thermophilic strains of acidophilic heterotrophic bacteria were tested for the ability to reduce ferric iron in liquid and solid media under aerobic conditions; about 40% of the mesophiles (but none of the moderate thermophiles) displayed at least some capacity to reduce iron. Both rates and extents of ferric iron reduction were highly strain dependent. No acidophilic heterotroph reduced nitrate or sulfate, and (limited) reduction of manganese(IV) was noted in only one strain (Acidiphilium facilis), an acidophile which did not reduce iron. Insoluble forms of ferric iron, both amorphous and crystalline, were reduced, as well as soluble iron. There was evidence that, in at least some acidophilic heterotrophs, iron reduction was enzymically mediated and that ferric iron could act as a terminal electron acceptor. In anaerobically incubated cultures, bacterial biomass increased with increasing concentrations of ferric but not ferrous iron. Mixed cultures of Thiobacillus ferrooxidans or Leptospirillum ferrooxidans and an acidophilic heterotroph (SJH) produced sequences of iron cycling in ferrous iron-glucose media.     

Reduction of ferric iron by L-lactate and DL-glycerol-3-phosphate in membrane preparations from Staphylococcus aureus and interactions with the nitrate reductase system.

Membrane fractions with L-lactate dehydrogenase, sn-glycerol-3-phosphate (G3P) dehydrogenase, and nitrate reductase activities were prepared from Staphylococcus aureus wild-type and hem mutant strains. These preparations reduced ferric to ferrous iron with L-lactate or G3P as the source of reductant, using ferrozine to trap the ferrous iron. Reduction of ferric iron was insensitive to 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) with either L-lactate or G3P as reductant, but oxalate and dicumarol inhibited reduction with L-lactate as substrate. The membranes had L-lactate- and G3P-nitrate reductase activities, which were inhibited by azide and by HQNO. Reduction of ferric iron under anaerobic conditions was inhibited by nitrate with preparations from the wild-type strain. This effect of nitrate was abolished by blocking electron transport to the nitrate reductase system with azide or HQNO. Nitrate did not inhibit reduction of ferric iron in heme-depleted membranes from the hem mutant unless hemin was added to restore L-lactate- and G3P-nitrate reductase activity. We conclude that reduced components of the electron transport chain that precede cytochrome b serve as the source of reductant for ferric iron and that these components are oxidized preferentially by a functional nitrate reductase system.     

Reduction of ferric citrate catalyzed by NADH:nitrate reductase

We show that NADH:nitrate reductase from squash cotyledons can catalyze the reduction of ferric citrate. When nitrate reductase was purified to homogeneity using a two-step affinity chromatography procedure, an NADH:Fe(III)-citrate reductase activity copurified with it and had identical electrophoretic mobility to it. The iron reductase activity was optimum near pH 6.3, had an apparent Km for Fe(III)-citrate of 0.02 mM, and was inhibited by monospecific anti-nitrate reductase rabbit sera. Differential inhibition of the enzyme's activities indicated iron and nitrate were reduced at different sites. In addition to its role in nitrogen assimilation, nitrate reductase catalyzes ferric citrate reduction and could have a role in iron assimilation.     

NADH-ferric reductase activity associated with dihydropteridine reductase

In mammals dietary ferric iron is reduced to ferrous iron for more efficient absorption by the intestine. Analysis of a pig duodenal membrane fraction revealed two NADH-dependent ferric reductase activities, one associated with a b-type cytochrome and the other not. Purification and characterization of the non-cytochrome ferric reductase identified a 31 kDa protein. MALDI-MS analysis and amino acid sequencing identified the ferric reductase as being related to the 26 kDa liver NADH-dependent quinoid dihydropteridine reductase (DHPR). The NADH-dependent DHPR ferric reductase activity was found to be pteridine-independent since exhaustive dialysis did not reduce activity and heat-inactivation destroyed activity. In intestinal Caco-2 cells, DHPR mRNA levels were found to be regulated by iron. Thus, DHPR appears to be a dual function enzyme, a NADH-dependent dihydopteridine reductase and an iron-regulated, NADH-dependent, pteridine-independent ferric reductase.     

Siderophore reduction catalyzed by higher plant NADH:nitrate reductase

Squash cotyledon NADH:nitrate reductase catalyzes the reduction of the siderophore ferrioxamine B. The enzyme also reduced ferric ion in a buffer system containing the chelators oxalate and maleate. Ferrioxamine B reduction was maximal at pH 4; ferric ion reduction was maximal at pH 8. The present study indicates that iron assimilation by higher plants may occur with microbial siderophores serving as ferric ion sources and nitrate reductase functioning as the siderophore reductase.     

Metabolic characterization of lactic acid bacterium Lactococcus garvieae sk11, capable of reducing ferric iron, nitrate, and fumarate

A lactic acid bacterium capable of anaerobic respiration was isolated from soil with ferric iron-containing glucose basal medium and identified as L. garvieae by using 16S rDNA sequence homology. The isolate reduced ferric iron, nitrate, and fumarate to ferrous iron, nitrite, and succinate, respectively, under anaerobic N2 atmosphere. Growth of the isolate was increased about 30-39% in glucose basal medium containing nitrate and fumarate, but not in the medium containing ferric iron. Specifically, metabolic reduction of nitrate and fumarate is thought to be controlled by the specific genes fnr, encoding FNR-like protein, and nir, regulating fumarate-nitrate reductase. Reduction activity of ferric iron by the isolate was estimated physiologically, enzymologically, and electrochemically. The results obtained led us to propose that the isolate metabolized nitrate and fumarate as an electron acceptor and has specific enzymes capable of reducing ferric iron in coupling with anaerobic respiration.     

Nitrate reductase from Rhodopseudomonas sphaeroides.

The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed.     

Acidocella aromatica sp. nov.: an acidophilic heterotrophic alphaproteobacterium with unusual phenotypic traits

Three obligately heterotrophic bacterial isolates were identified as strains of a proposed novel species of extremely acidophilic, mesophilic Alphaproteobacteria, Acidocella aromatica. They utilized a restricted range of organic substrates, which included fructose (but none of the other monosaccharides tested), acetate and several aromatic compounds (benzoate, benzyl alcohol and phenol). No growth was obtained on complex organic substrates, such as yeast extract and tryptone. Tolerance of the proposed type strain of the species (PFBC) to acetic acid was much greater than that typically reported for acidophiles. The bacteria grew aerobically, and catalyzed the dissimilatory reductive dissolution of the ferric iron mineral schwertmannite under both micro-aerobic and anaerobic conditions. Strain PFBC did not grow anaerobically via ferric iron respiration, though it has been reported to grow in co-culture with acid-tolerant sulfidogenic bacteria under strictly anoxic conditions. Tolerance of strains of Acidocella aromatica to nickel were about two orders of magnitude greater than those of other Acidocella spp., though similar levels of tolerance to other metals tested was observed. The use of this novel acidophile in solid media designed to promote the isolation and growth of other (aerobic and anaerobic) acidophilic heterotrophs is discussed.     

Overexpression of AtFRO6 in transgenic tobacco enhances ferric chelate reductase activity in leaves and increases tolerance to iron-deficiency chlorosis

The Arabidopsis gene FRO6(AtFRO6) encodes ferric chelate reductase and highly expressed in green tissues of plants. We have expressed the gene AtFRO6 under the control of a 35S promoter in transgenic tobacco plants. High-level expression of AtFRO6 in transgenic plants was confirmed by northern blot analysis. Ferric reductase activity in leaves of transgenic plants grown under iron-sufficient or iron-deficient conditions is 2.13 and 1.26 fold higher than in control plants respectively. The enhanced ferric reductase activity led to increased concentrations of ferrous iron and chlorophyll, and reduced the iron deficiency chlorosis in the transgenic plants, compared to the control plants. In roots, the concentration of ferrous iron and ferric reductase activity were not significantly different in the transgenic plants compared to the control plants. These results suggest that FRO6 functions as a ferric chelate reductase for iron uptake by leaf cells, and overexpression of AtFRO6 in transgenic plants can reduce iron deficiency chlorosis.     

The siderophore-interacting protein YqjH acts as a ferric reductase in different iron assimilation pathways of Escherichia coli

Siderophore-interacting proteins (SIPs), such as YqjH from Escherichia coli, are widespread among bacteria and commonly associated with iron-dependent induction and siderophore utilization. In this study, we show by detailed biochemical and genetic analyses the reaction mechanism by which the YqjH protein is able to catalyze the release of iron from a variety of iron chelators, including ferric triscatecholates and ferric dicitrate, displaying the highest efficiency for the hydrolyzed ferric enterobactin complex ferric (2,3-dihydroxybenzoylserine)(3). Site-directed mutagenesis revealed that residues K55 and R130 of YqjH are crucial for both substrate binding and reductase activity. The NADPH-dependent iron reduction was found to proceed via single-electron transfer in a double-displacement-type reaction through formation of a transient flavosemiquinone. The capacity to reduce substrates with extremely negative redox potentials, though at low catalytic rates, was studied by displacing the native FAD cofactor with 5-deaza-5-carba-FAD, which is restricted to a two-electron transfer. In the presence of the reconstituted noncatalytic protein, the ferric enterobactin midpoint potential increased remarkably and partially overlapped with the effective E(1) redox range. Concurrently, the observed molar ratios of generated Fe(II) versus NADPH were found to be ~1.5-fold higher for hydrolyzed ferric triscatecholates and ferric dicitrate than for ferric enterobactin. Further, combination of a chromosomal yqjH deletion with entC single- and entC fes double-deletion backgrounds showed the impact of yqjH on growth during supplementation with ferric siderophore substrates. Thus, YqjH enhances siderophore utilization in different iron acquisition pathways, including assimilation of low-potential ferric substrates that are not reduced by common cellular cofactors.     

Humic Acid Reduction by Propionibacterium freudenreichii and Other Fermenting Bacteria

Iron-reducing bacteria have been reported to reduce humic acids and low-molecular-weight quinones with electrons from acetate or hydrogen oxidation. Due to the rapid chemical reaction of amorphous ferric iron with the reduced reaction products, humic acids and low-molecular-weight redox mediators may play an important role in biological iron reduction. Since many anaerobic bacteria that are not able to reduce amorphous ferric iron directly are known to transfer electrons to other external acceptors, such as ferricyanide, 2,6-anthraquinone disulfonate (AQDS), or molecular oxygen, we tested several physiologically different species of fermenting bacteria to determine their abilities to reduce humic acids. Propionibacterium freudenreichii, Lactococcus lactis, and Enterococcus cecorum all shifted their fermentation patterns towards more oxidized products when humic acids were present; P. freudenreichii even oxidized propionate to acetate under these conditions. When amorphous ferric iron was added to reoxidize the electron acceptor, humic acids were found to be equally effective when they were added in substoichiometric amounts. These findings indicate that in addition to iron-reducing bacteria, fermenting bacteria are also capable of channeling electrons from anaerobic oxidations via humic acids towards iron reduction. This information needs to be considered in future studies of electron flow in soils and sediments.     

Iron reductases from Pseudomonas aeruginosa

Cell-free extracts of Pseudomonas aeruginosa contain enzyme activities which reduce Fe(III) to Fe(II) when iron is provided in certain chelates, but not when the iron is uncomplexed. Iron reductase activities for two substrates, ferripyochelin and ferric citrate, appear to be separate enzymes because of differences in heat stabilities, in locations in fractions of cell-free extracts, in reductant specificity, and in apparent sizes during gel filtration chromatography. Ferric citrate iron reductase is an extremely labile activity found in the cytoplasmic fraction, and ferripyochelin iron reductase is a more stable activity found in the periplasmic as well as cytoplasmic fraction of extracts. A small amount of activity detectable in the membrane fraction seemed to be loosely associated with the membranes. Although both enzymes have highest activity reduced nicotinamide adenine dinucleotide, reduced glutathione also worked with ferripyochelin iron reductase. In addition, oxygen caused an irreversible loss of a percentage of the ferripyochelin iron reductase following sparge of reaction mixtures, whereas the reductase for ferric citrate was not appreciably affected by oxygen.     

Enzymatic reduction of iron oxide by fungi

The occurrence of the iron-reducing phenomenon among some common fungi was studied. Results indicated that (i) the reduction of ferric iron to the ferrous state by fungi seems to be restricted to nitrate reductase-inducible strains such as Actinomucor repens, Alternaria tenuis, Fusarium oxysporum, and F. solani and (ii) the amount of dissolved ferrous iron may be reduced progressively by increasing the amount of nitrate added to the medium. Compared with a complex medium (Sabouraud medium), less iron became reduced if NO₃^- was the only nitrogen source (Czapek Dox medium). These data strongly support the view that ferric iron is acting as an hydrogen acceptor in respiration, competing with nitrate for electrons that are mediated by the enzyme nitrate reductase. The significance of this property from an ecological viewpoint is discussed.     

Effect of ammonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium species

BACKGROUND AND AIMS: Most Vaccinium species have strict soil requirements for optimal growth, requiring low pH, high iron availability and nitrogen primarily in the ammonium form. These soils are limited and are often located near wetlands. Vaccinium arboreum is a wild species adapted to a wide range of soils, including high pH, low iron, and nitrate-containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake compared with the cultivated Vaccinium species. METHODS: Nitrate, ammonium and iron uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in two Vaccinium species grown hydroponically in either nitrate or ammonia, with or without iron. The species studied were the wild V. arboreum and the cultivated V. corymbosum interspecific hybrid, which exhibits the strict soil requirements of most Vaccinium species. RESULTS: Ammonium uptake was significantly greater than nitrate uptake in both species, while nitrate uptake was greater in the wild species, V. arboreum, compared with the cultivated species, V. corymbosum. The increased nitrate uptake in V. arboreum was correlated with increased root NR activity compared with V. corymbosum. The lower nitrate uptake in V. corymbosum was reflected in decreased plant dry weight in this species compared with V. arboreum. Root FCR activity increased significantly in V. corymbosum grown under iron-deficient conditions, compared with the same species grown under iron-sufficient conditions or with V. arboreum grown under either iron condition. CONCLUSIONS: V. arboreum appears to be more efficient in acquiring nitrate compared with V. corymbosum, possibly due to increased NR activity and this may partially explain the wider soil adaptation of V. arboreum.     

Anaerobic respiratory growth of Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi with trimethylamine N-oxide, nitrate and fumarate: ecological implications

Two symbiotic species, Photobacterium leiognathi and Vibrio fischeri, and one non-symbiotic species, Vibrio harveyi, of the Vibrionaceae were tested for their ability to grow by anaerobic respiration on various electron acceptors, including trimethylamine N-oxide (TMAO) and dimethylsulphoxide (DMSO), compounds common in the marine environment. Each species was able to grow anaerobically with TMAO, nitrate or fumarate, but not with DMSO, as an electron acceptor. Cell growth under microaerophilic growth conditions resulted in elevated levels of TMAO reductase, nitrate reductase and fumarate reductase activity in each strain, whereas growth in the presence of the respective substrate for each enzyme further elevated enzyme activity. TMAO reductase specific activity was the highest of all the reductases. Interestingly, the bacteria-colonized light organs from the two squids, Euprymna scolopes and Euprymna morsei, and the light organ of the ponyfish, Leiognathus equus, also had high levels of TMAO reductase enzyme activity, in contrast to non-symbiotic tissues. The ability of these bacterial symbionts to support cell growth by respiration with TMAO may conceivably eliminate the competition for oxygen needed for both bioluminescence and metabolism.     

Aerobic ferrisiderophore reductase assay and activity stain for native polyacrylamide gels

The reduction of ferric iron from microbial iron-binding compounds (siderophores) releases the iron from the siderophore so that it may be utilized by the microorganism. A method to detect aerobic ferrisiderophore reductase activity using ferrozine as a ferrous iron trap is shown to be applicable to cytoplasmic fractions from Rhodopseudomonas sphaeroides and four other different species of bacteria. The ferrisiderophore reductase uses reduced nicotinamide cofactors as reducing agents, and activity is stimulated by flavins. This assay has been adapted as a staining method to locate ferrisiderophore reductase activity in native polyacrylamide gels.     

Phenazines and other redox-active antibiotics promote microbial mineral reduction

Natural products with important therapeutic properties are known to be produced by a variety of soil bacteria, yet the ecological function of these compounds is not well understood. Here we show that phenazines and other redox-active antibiotics can promote microbial mineral reduction. Pseudomonas chlororaphis PCL1391, a root isolate that produces phenazine-1-carboxamide (PCN), is able to reductively dissolve poorly crystalline iron and manganese oxides, whereas a strain carrying a mutation in one of the phenazine-biosynthetic genes (phzB) is not; the addition of purified PCN restores this ability to the mutant strain. The small amount of PCN produced relative to the large amount of ferric iron reduced in cultures of P. chlororaphis implies that PCN is recycled multiple times; moreover, poorly crystalline iron (hydr)oxide can be reduced abiotically by reduced PCN. This ability suggests that PCN functions as an electron shuttle rather than an iron chelator, a finding that is consistent with the observation that dissolved ferric iron is undetectable in culture fluids. Multiple phenazines and the glycopeptidic antibiotic bleomycin can also stimulate mineral reduction by the dissimilatory iron-reducing bacterium Shewanella oneidensis MR1. Because diverse bacterial strains that cannot grow on iron can reduce phenazines, and because thermodynamic calculations suggest that phenazines have lower redox potentials than those of poorly crystalline iron (hydr)oxides in a range of relevant environmental pH (5 to 9), we suggest that natural products like phenazines may promote microbial mineral reduction in the environment.