Influence of Tropolone on Poria placenta Wood Degradation

Fenton reactions are believed to play important roles in wood degradation by brown rot fungi. In this context, the effect of tropolone (2-hydroxycyclohepta-2,4,6-trienone), a metal chelator, on wood degradation by Poria placenta was investigated. Tropolone (50 μM) strongly inhibits fungal growth on malt agar, but this inhibition could be relieved by adding iron salts. With an experimental system containing two separate parts, one supplemented with tropolone (100 μM) and the other not, it was shown that the fungus is able to reallocate essential minerals from the area where they are available and also to grow in these conditions on malt-agar in the presence of tropolone. Nevertheless, even in the presence of an external source of metals, P. placenta is not able to attack pine blocks impregnated with tropolone (5 mM). This wood degradation inhibition is related to the presence of the tropolone hydroxyl group, as shown by the use of analogs (cyclohepta-2,4,6-trienone and 2-methoxycyclohepta-2,4,6-trienone). Furthermore, tropolone possesses both weak antioxidative and weak radical-scavenging properties and a strong affinity for ferric ion and is able to inhibit ferric iron reduction by catecholates, lowering the redox potential of the iron couple. These data are consistent with the hypothesis that tropolone inhibits wood degradation by P. placenta by chelating iron present in wood, thus avoiding initiation of the Fenton reaction. This study demonstrates that iron chelators such as tropolone could be also involved in novel and more environmentally benign preservative systems.     

Effect of pH and oxalate on hydroquinone-derived hydroxyl radical formation during brown rot wood degradation

The redox cycle of 2,5-dimethoxybenzoquinone (2,5-DMBQ) is proposed as a source of reducing equivalent for the regeneration of Fe2+ and H2O2 in brown rot fungal decay of wood. Oxalate has also been proposed to be the physiological iron reductant. We characterized the effect of pH and oxalate on the 2,5-DMBQ-driven Fenton chemistry and on Fe3+ reduction and oxidation. Hydroxyl radical formation was assessed by lipid peroxidation. We found that hydroquinone (2,5-DMHQ) is very stable in the absence of iron at pH 2 to 4, the pH of degraded wood. 2,5-DMHQ readily reduces Fe3+ at a rate constant of 4.5 x 10(3) M(-1)s(-1) at pH 4.0. Fe2+ is also very stable at a low pH. H2O2 generation results from the autoxidation of the semiquinone radical and was observed only when 2,5-DMHQ was incubated with Fe3+. Consistent with this conclusion, lipid peroxidation occurred only in incubation mixtures containing both 2,5-DMHQ and Fe3+. Catalase and hydroxyl radical scavengers were effective inhibitors of lipid peroxidation, whereas superoxide dismutase caused no inhibition. At a low concentration of oxalate (50 micro M), ferric ion reduction and lipid peroxidation are enhanced. Thus, the enhancement of both ferric ion reduction and lipid peroxidation may be due to oxalate increasing the solubility of the ferric ion. Increasing the oxalate concentration such that the oxalate/ferric ion ratio favored formation of the 2:1 and 3:1 complexes resulted in inhibition of iron reduction and lipid peroxidation. Our results confirm that hydroxyl radical formation occurs via the 2,5-DMBQ redox cycle.     

Effect of pH and oxalic acid on the reduction of Fe3+ by a biomimetic chelator and on Fe3+ desorption/adsorption onto wood: Implications for brown-rot decay

Fenton reaction is thought to play an important role in wood degradation by brown-rot fungi. In this context, the effect of oxalic acid and pH on iron reduction by a biomimetic fungal chelator and on the adsorption/desorption of iron to/from wood was investigated. The results presented in this work indicate that at pH 2.0 and 4.5 and in the presence of oxalic acid, the phenolate chelator 2,3-dihydroxybenzoic acid (2,3-DHBA) is capable of reducing ferric iron only when the iron is complexed with oxalate to form Fe³⁺-mono-oxalate (Fe(C₂O₄)⁺). Within the pH range tested in this work, this complex formation occurs when the oxalate:Fe³⁺ molar ratio is less than 20 (pH 2.0) or less than 10 (pH 4.5). When aqueous ferric iron was passed through a column packed with milled red spruce (Picea rubens) wood equilibrated at pH 2.0 and 4.5, it was observed that ferric iron binds to wood at pH 4.5 but not at pH 2.0, and the bound iron could then be released by application of oxalic acid at pH 4.5. The release of bound iron was dependent on the amount of oxalic acid applied in the column. When the amount of oxalate was at least 20-fold greater than the amount of iron bound to the wood, all bound iron was released. When Fe–oxalate complexes were applied to the milled wood column equilibrated in the pH range of 2–4.5, iron from Fe–oxalate complexes was bound to the wood only when the pH was 3.6 or higher and the oxalate:Fe³⁺ molar ratio was less than 10. When 2,3-DHBA was evaluated for its ability to release iron bound to the milled wood, it was found that 2,3-DHBA possessed a greater affinity for ferric iron than the wood as 2,3-DHBA was capable of releasing the ferric iron bound to the wood in the pH range 3.6–5.5. These results further the understanding of the mechanisms employed by brown-rot fungi in wood biodegradation processes.     

<Emphasis Type="Italic">Synechocystis</Emphasis> ferredoxin-NADP<Superscript>+</Superscript> oxidoreductase is capable of functioning as ferric reductase and of driving the Fenton reaction in the absence or presence of free flavin

We purified free flavin-independent NADPH oxidoreductase from Synechocystis sp. PCC6803 based on NADPH oxidation activity elicited during reduction of t-butyl hydroperoxide in the presence of Fe(III)-EDTA. The N-terminal sequencing of the purified enzyme revealed it to be ferredoxin-NADP⁺ oxidoreductase (FNR _S ). The purified enzyme reacted with cytochrome c, ferricyanide and 2,6-dichloroindophenol (DCIP). The substrate specificity of the enzyme was similar to the known FNR. DNA degradation occurring in the presence of NADPH, Fe(III)-EDTA and hydrogen peroxide was potently enhanced by the purified enzyme, indicating that Synechocystis FNR _S may drive the Fenton reaction. The Fenton reaction by Synechocystis FNR _S in the presence of natural chelate iron compounds tended to be considerably lower than that in the presence of synthetic chelate iron compounds. The Synechocystis FNR _S is considered to reduce ferric iron to ferrous iron when it evokes the Fenton reaction. Although Synechocystis FNR _S was able to reduce iron compounds in the absence of free flavin, the ferric reduction by the enzyme was enhanced by the addition of free flavin. The enhancement was detected not only in the presence of natural chelate iron compounds but also synthetic chelate iron compounds.     

Lactobionic acid as an iron chelator: a rationale for its effectiveness as an organ preservant

Lactobionic acid, a major constituent of a solution used to preserve organs prior to transplantation, can chelate ferric iron. This is evident by its ability to solubilize iron as well as changes that occur in the UV-VIS spectra of iron in its presence. Relative to iron (III) chelated to EDTA, the lactobionic acid-iron (III) complex is less able to participate in the Fenton reaction as measured by formaldehyde generation from DMSO and bleaching of p-N,N-dimethylnitrosoaniline. Similar effects are seen with citrate and ATP, two substances which also appear to be able to ameliorate ischemia/reperfusion injury. These findings present a rationale for the effectiveness of lactobionic acid as an organ preservant.     

Synechocystis DrgA protein functioning as nitroreductase and ferric reductase is capable of catalyzing the Fenton reaction

In order to identify an enzyme capable of Fenton reaction in Synechocystis, we purified an enzyme catalyzing one-electron reduction of t-butyl hydroperoxide in the presence of FAD and Fe(III)-EDTA. The enzyme was a 26 kDa protein, and its N-terminal amino acid sequencing revealed it to be DrgA protein previously reported as quinone reductase [Matsuo M, Endo T and Asada K (1998) Plant Cell Physiol39, 751-755]. The DrgA protein exhibited potent quinone reductase activity and, furthermore, we newly found that it contained FMN and highly catalyzed nitroreductase, flavin reductase and ferric reductase activities. This is the first demonstration of nitroreductase activity of DrgA protein previously identified by a drgA mutant phenotype. DrgA protein strongly catalyzed the Fenton reaction in the presence of synthetic chelate compounds, but did so poorly in the presence of natural chelate compounds. Its ferric reductase activity was observed with both natural and synthetic chelate compounds with a better efficiency with the latter. In addition to small molecular-weight chemical chelators, an iron transporter protein, transferrin, and an iron storage protein, ferritin, turned out to be substrates of the DrgA protein, suggesting it might play a role in iron metabolism under physiological conditions and possibly catalyze the Fenton reaction under hyper-reductive conditions in this microorganism.     

Autoxidation and MAO-mediated metabolism of dopamine as a potential cause of oxidative stress: role of ferrous and ferric ions

The autoxidation and monoamine oxidase (MAO)-mediated metabolism of dopamine (3-hydroxytyramine; DA) cause a continuous production of hydroxyl radical (*OH), which is further enhanced by the presence of iron (ferrous iron, Fe(2+) and ferric ion, Fe(3+)). The accumulation of hydrogen peroxide (H2O2) in the presence of Fe(2+) appears to discard the involvement of the Fenton reaction in this process. It has been found that the presence of DA significantly reduces the formation of thiobarbituric acid reagent substances (TBARS), which under physiological conditions takes place in mitochondrial preparations. The presence of DA is also able to reduce TBARS formation in mitochondrial preparations even in the presence of iron (Fe(2+) and Fe(3+)). However, DA boosted the carbonyl content of mitochondrial proteins, which was further increased in the presence of iron (Fe(2+) and Fe(3+)). This latter effect is also accompanied by a significant reduction in thiol content of mitochondrial proteins. It has also been observed how the pre-incubation of mitochondria with pargyline, an acetylenic MAO inhibitor, reduces the production of *OH and increases the formation of TBARS. Although, the MAO-mediated metabolism of DA increases MAO-B activity, the presence of iron inhibits both MAO-A and MAO-B activities. Consequently, DA has been shown to be a double-edged sword, because it displays antioxidant properties in relation to both the Fenton reaction and lipid peroxidation and exhibits pro-oxidant properties by causing both generation *OH and oxidation of mitochondrial proteins. Evidently, these pro-oxidant properties of DA help explain the long-term side effects derived from l-DOPA treatment of Parkinson's disease and its exacerbation by the concomitant use of DA metabolism inhibitors.     

Protection by tropolones against H2O2-induced DNA damage and apoptosis in cultured Jurkat cells

Tropolones, the naturally occurring compounds responsible for the durability of heartwood of several cupressaceous trees, have been shown to possess both metal chelating and antioxidant properties. However, little is known about the ability of tropolone and its derivatives to protect cultured cells from oxidative stress-mediated damage. In this study, the effect of tropolones on hydrogen peroxide-induced DNA damage and apoptosis was investigated in cultured Jurkat cells. Tropolone, added to the cells 15 min before the addition of glucose oxidase, provided a dose dependent protection against hydrogen peroxide induced DNA damage. The IC₅₀ value observed was about 15 μM for tropolone. Similar dose dependent protection was also observed with three other tropolone derivatives such as trimethylcolchicinic acid, purpurogallin and β-thujaplicin (the IC₅₀ values were 34, 70 and 74 μM, respectively), but not with colchicine and tetramethyl purpurogallin ester. Hydrogen peroxide-induced apoptosis was also inhibited by tropolone. However, in the absence of exogenous H₂O₂ but in the presence of non-toxic concentrations of exogenous iron (100 μM Fe³⁺), tropolone dramatically increased the formation of single strand breaks at molar ratios of tropolone to iron lower than 3 to 1, while, when the ratio increased over 3, no toxicity was observed. In conclusion, the results presented in this study indicate that the protection offered by tropolone against hydrogen peroxide-induced DNA damage and apoptosis was due to formation of a redox-inactive iron complex, while its enhancement of iron-mediated DNA damage at ratios of [tropolone]/[Fe³⁺] lower than 3, was due to formation of a lipophilic iron complex which facilitates iron transport through cell membrane in a redox-active form.     

Inhibition of mushroom tyrosinase by tropolone

Tropolone inhibits both mono- and o-dihydroxyphenolase activity of mushroom tyrosinase. Most of the inhibition exerted by tropolone was reversed by dialysis or by excess CU²⁺. The data indicate that tropolone and o-dihydroxyphenols compete for binding to the copper at the active site of the enzyme. Comparison between the effectiveness of various copper chelators showed that tropolone is one of the most potent inhibitors of mushroom tyrosinase; 50% inhibition was observed with 0.4 × 10^?6 M tropolone.     

Inhibition of the erythrocyte (Ca2+ + Mg2+)-ATPase by nonheme iron.

The erythrocyte calmodulin-stimulated (Ca2+ + Mg2+)-ATPase (CaM-ATPase), an integral membrane protein, is inhibited in different types of congenital hemolytic anemias for which oxidative processes appear as a common feature. The oxidation of hemoglobin and its degradation lead to the accumulation of ferric heme (hemin) and nonheme iron in the red cell. We have shown previously that hemin inhibits the activity of the enzyme of normal erythrocyte (Leclerc et al. (1988) Biochim. Biophys. Acta, 946, 49-56) involving an oxidation of thiol groups. The present study demonstrates that nonheme iron also inhibits the CaM-ATPase activity. In contrast with hemin, the inhibition of the enzyme induced by the nonheme treatment is prevented by butylated hydroxytoluene, a protecting agent of unsaturated phospholipid peroxidations, while dithiothreitol, a reducing agent of protein disulfide bridges, does not restore the activity of the enzyme. We conclude that nonheme iron inhibits the enzyme at least in part, through the peroxidation of phospholipids of the membrane bilayer.     

Metschnikowia strains isolated from botrytized grapes antagonize fungal and bacterial growth by iron depletion

Noble-rotted grapes are colonized by complex microbial populations. I isolated pigment-producing Metschnikowia strains from noble-rotted grapes that had antagonistic activity against filamentous fungi, yeasts, and bacteria. A red-maroon pigment was formed from a diffusible colorless precursor released by the cells into the medium. The conversion of the precursor required iron and could occur both in the cells (red colonies) and in the medium (red halos around colonies). The intensity of pigmentation was correlated with the intensity of the antimicrobial activity. Mutants that did not form pigment also lacked antifungal activity. Within the pigmented halos, conidia of the sensitive fungi did not germinate, and their hyphae did not grow and frequently lysed at the tips. Supplementation of the medium with iron reduced the size of the halos and the inhibition zones, while it increased the pigment accumulation by the colonies. The iron-binding agent tropolone had a similar effect, so I hypothesize that pigmented Metschnikowia isolates inhibit the growth of the sensitive microorganisms by pigment formation, which depletes the free iron in the medium. As the pigment is a large nondiffusible complex produced in the presence of both low and high concentrations of ferric ions, the proposed mechanism is different from the mechanisms operating in microbes that release siderophores into the environment for iron acquisition.     

Lytic polysaccharide monooxygenases promote oxidative cleavage of lignin and lignin–carbohydrate complexes during fungal degradation of lignocellulose

Overcoming lignocellulosic biomass recalcitrance, especially the cleavage of cross-linkages in lignin–carbohydrate complexes (LCCs) and lignin, is essential for both the carbon cycle and industrial biorefinery. Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in fungal polysaccharide oxidative degradation. Nevertheless, comprehensive analysis showed that LPMOs from a white-rot fungus, Pleurotus ostreatus, correlated well with the Fenton reaction and were involved in the degradation of recalcitrant nonpolysaccharide fractions in this research. Thus, LPMOs participated in the extracellular Fenton reaction by enhancing iron reduction in quinone redox cycling. A Fenton reaction system consisting of LPMOs, hydroquinone, and ferric iron can efficiently produce hydroxy radicals and then cleave LCCs or lignin linkages. This finding indicates that LPMOs are underestimated auxiliary enzymes in eliminating biomass recalcitrance.     

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction

Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP⁺ reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k _cat/K _m value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k _cat/K _m value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.     

Low-level iron-dependent mutants of Listeria monocytogenes and their virulence in macrophages

Listeria monocytogenes is an opportunistic intracellular pathogen capable of growth that requires iron for growth within phagocytic cells and virulence expression. In the presence of an appropriate concentration tropolone, an iron-chelating agent, growth of L. monocytogenes is completely inhibited. However, this inhibition can be relieved by addition of dopamine, norepinephrine, or ferric citrate. By selection on streptonigrin medium supplemented with tropolone and norepinephrine, we have obtained two spontaneous mutants, Lm-8 and Lm-15, with the same iron dependence but lower iron dependence than the wild-type Lm-B38. The association between iron requirement and virulence of the two mutants and the wild type was studied in the J774 macrophage cell line. One hour after phagocytosis by the J774 macrophage cell line, the two mutants and the parental strain displayed no difference in the number of phagocytosed bacteria. Twenty-four hours after phagocytosis, the number of bacteria within the surviving macrophages was identical for the wild strain and the two clones. However, only 40% of macrophage cells infected with Lm-8 and 90% of those infected with Lm-15 were alive after 24 h in comparison with macrophage cells infected with the parental strain Lm-B38. These data demonstrate that there is no direct correlation between iron requirement and virulence of L. monocytogenes in the J774 macrophage cell line.     

Ceriporic acid B, an extracellular metabolite of Ceriporiopsis subvermispora, suppresses the depolymerization of cellulose by the Fenton reaction

The white rot fungus, Ceriporiopsis subvermispora, is able to degrade lignin in wood without intensive damage to cellulose. Since lignin biodegradation by white rot fungi proceeds by radical reactions, accompanied by the production of a large amount of Fe3+-reductant phenols and reductive radical species in the presence of iron ions, molecular oxygen, and H2O2, C. subvermispora has been proposed to possess a biological system which suppresses the production of a cellulolytic active oxygen species, *OH, by the Fenton reaction. In the present paper, we demonstrate that 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B), an extracellular metabolite of C. subvermispora, strongly inhibited *OH production and the depolymerization of cellulose by the Fenton reaction in the presence of iron ions, cellulose, H2O2, and a reductant for Fe3+, hydroquinone (HQ), at the physiological pH of the fungus.     

A selective lignin-degrading fungus,Ceriporiopsis subvermispora,produces alkylitaconates that inhibit the production of a cellulolytic active oxygen species,hydroxyl radical in the presence of iron and H(2)O(2)

A cellulolytic active oxygen species, hydroxyl radicals (.OH), play a leading role in the erosion of wood cell walls by brown-rot and non-selective white-rot fungi. In contrast, selective white-rot fungi have been considered to possess unknown systems for the suppression of .OH production due to their wood decay pattern with a minimum loss of cellulose. In the present paper, we first report that 1-nonadecene-2,3-dicarboxylic acid, an alkylitaconic acid (ceriporic acid B) produced by the selective white-rot fungus Ceriporiopsis subvermispora intensively inhibited .OH production by the Fenton reaction by direct interaction with Fe ions, while non-substituted itaconic acid promoted the Fenton reaction. Suppression of the Fenton reaction by the alkylitaconic acid was observed even in the presence of the Fe(3+) reductants, cysteine and hydroquinone. The inhibition of .OH production by the diffusible fungal metabolite accounts for the extracellular system of the fungus that attenuates the formation of .OH in the presence of iron, molecular oxygen, and free radicals produced during lignin biodegradation.     

Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by peroxides

Hydrogen peroxide and organic hydroperoxides react with haemoglobin to release iron which can be complexed to apotransferrin, bleomycin and desferrioxamine. This released iron promotes deoxyribose degradation by a Fenton reaction, DNA degradation in the presence of bleomycin and stimulates lipid peroxidation. It is likely that iron released from haemoglobin is the true generator of hydroxyl radicals in the Fenton reaction.     

Copper catechol-driven Fenton reactions and their potential role in wood degradation

Metals can potentially play a role in the non-enzymatic processes involved in wood biodegradation. Dihydroxybenzenes reduce Cu(II)–Cu(I), which then react with H₂O₂ driving a Fenton reaction. In this work the degradation of veratryl alcohol (VA), the simplest non-phenolic lignin model compound, via a cuprous Fenton reaction mediated by 1,2-dihydroxybenzene (catechol, CAT) was studied. A factorial experimental design was performed to assess the impact of several experimental variables including, pH, and CAT, CuCl₂ and H₂O₂ concentrations on VA degradation. Optimized conditions were determined using a response surface modeling methodology (RSM). The greatest amount of VA degradation occurred at a CAT:CuCl₂:H₂O₂ ratio of 0.287:0.313:4.062, a pH of 3.6. A time-course measurement for VA degradation was performed under these experimental conditions and after an 8 h reaction period, 31% of the VA was degraded. Under the same experimental conditions, VA degradation by an iron CAT-driven Fenton reaction was more effective than the copper CAT-driven Fenton reaction. In a similar experiment, carboxymethyl cellulose (CMC) depolymerization was also determined. Only the iron CAT-driven Fenton reaction was found to depolymerize CMC. We suggest that the greater redox potential of the Fe(III)CAT complex compared to the Cu(II)CAT complex would dictate that under most environmental conditions, degradation of VA would occur by the iron complex only. This research has important implications for the mechanisms of brown rot fungal degradation in wood because it eliminates a pathway that had previously been proposed as a mechanism explaining free radical generation in the oxidative depolymerization of cellulose in the cell wall.     

Effect of pH and Oxalate on Hydroquinone-Derived Hydroxyl Radical Formation during Brown Rot Wood Degradation

The redox cycle of 2,5-dimethoxybenzoquinone (2,5-DMBQ) is proposed as a source of reducing equivalent for the regeneration of Fe²⁺ and H₂O₂ in brown rot fungal decay of wood. Oxalate has also been proposed to be the physiological iron reductant. We characterized the effect of pH and oxalate on the 2,5-DMBQ-driven Fenton chemistry and on Fe³⁺ reduction and oxidation. Hydroxyl radical formation was assessed by lipid peroxidation. We found that hydroquinone (2,5-DMHQ) is very stable in the absence of iron at pH 2 to 4, the pH of degraded wood. 2,5-DMHQ readily reduces Fe³⁺ at a rate constant of 4.5 × 10³ M⁻¹s⁻¹ at pH 4.0. Fe²⁺ is also very stable at a low pH. H₂O₂ generation results from the autoxidation of the semiquinone radical and was observed only when 2,5-DMHQ was incubated with Fe³⁺. Consistent with this conclusion, lipid peroxidation occurred only in incubation mixtures containing both 2,5-DMHQ and Fe³⁺. Catalase and hydroxyl radical scavengers were effective inhibitors of lipid peroxidation, whereas superoxide dismutase caused no inhibition. At a low concentration of oxalate (50 μM), ferric ion reduction and lipid peroxidation are enhanced. Thus, the enhancement of both ferric ion reduction and lipid peroxidation may be due to oxalate increasing the solubility of the ferric ion. Increasing the oxalate concentration such that the oxalate/ferric ion ratio favored formation of the 2:1 and 3:1 complexes resulted in inhibition of iron reduction and lipid peroxidation. Our results confirm that hydroxyl radical formation occurs via the 2,5-DMBQ redox cycle.     

Iron uptake from ferric citrate by Vibrio anguillarum

We report here that Vibrio anguillarum possesses a non-inducible active transport system which can efficiently supply iron to the cell from ferric citrate, independently of the siderophore-based mechanisms. The strains tested were able to grow in CM9 medium in iron-restricted conditions when ferric citrate was present in the medium. Moreover, the presence of ferric citrate inhibited the production of siderophores in the strains tested. V. anguillarum cells and isolated membranes could incorporate ⁵⁵Fe³⁺ complexed by citrate, without a difference between cells grown in the presence or absence of ferric citrate. The presence of 2,4-dinitrophenol, ferrozine, ferricyanide, trypsin, as well as low temperature produced a marked decrease or total inhibition of ⁵⁵Fe³⁺ uptake by the cells. All these results suggest that iron uptake from ferric citrate in V. anguillarum must be an energy-dependent process not induced by the presence of iron or citrate in the medium, mediated by a membrane protein(s), which may require an iron reduction step to function.