<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.     

OXYGEN CONSUMPTION ASSOCIATED WITH FERRIC REDUCTASE ACTIVITY AND IRON UPTAKE BY IRON-LIMITED CELLS OF CHLORELLA KESSLERI (CHLOROPHYCEAE)

Plasma membrane ferric reductase activity was enhanced 5-fold under iron limitation in the unicellular green alga Chlorella kessleri Fott et Nováková. Furthermore, ferric reductase activity in iron-limited cells was approximately 50% higher in the light than in the dark. In contrast, iron uptake rates of iron-limited cells were unaffected by light versus dark treatments. Rates of iron uptake were much lower than rates of ferric reduction, averaging approximately 2% of the dark ferric reduction rate. Ferric reduction was associated with an increased rate of O₂ consumption in both light and dark, the increase in the light being approximately 1.5 times as large as in the dark. The increased rate of O₂ consumption could be decreased by half by the addition of catalase, indicating that H₂O₂ is the product of the O₂ consumption and that the increased O₂ consumption is nonrespiratory. The stimulation of O₂ consumption was almost completely abolished by the addition of bathophenanthroline disulfonate, a strong chelator of Fe^{2 +} . Anaerobic conditions or the presence of exogenous superoxide dismutase affected neither ferric reduction nor iron uptake. We suggest that the O₂ consumption associated with ferric reductase activity resulted from superoxide formation from the aerobic oxidation of Fe^{2 +} , which is the product of ferric reductase activity. At saturating concentrations of Fe^{3 +} chelates, ferric reductase activity is much greater than the iron uptake rate, leading to rapid oxidation of Fe^{2 +} and superoxide generation. Therefore, O₂ consumption is not an integral part of the iron assimilation process.     

Intra- and intercellular iron trafficking and subcellular compartmentation within roots

Iron is abundant in most soils, but ferric compounds are almost insoluble. Therefore, plant roots use as tools acidification and enzymatic reduction of iron at the outer cell surface (strategy I) or solubilization by phytosiderophores, which are specific ferric chelators (strategy II). In the first case, iron is taken up as Fe²⁺ into the root symplast, and in the latter one, iron is taken up as Fe(III) complex. The path of iron from the root surface, up to the point of the xylem vessels within the central cylinder, may be completely symplasmic. However, a part of this route also may be an apoplasmic one, through the free space of the cell walls of rhizodermis and cortex (apoplast). In the endodermis, the Casparian band forms a strict barrier for apoplasmic transport; to move past this site, all ions must enter the symplast. During symplasmic transport, the intracellular environment is protected against the reactive species of iron by handling of iron in chelated forms. A promising candidate for this purpose is the plant-endogenous chelator nicotianamine. At the apoplasmic site, iron can be oxidized followed by precipitation as hydroxide or phosphate compounds. Thus, a pool of apoplastic iron can be formed, as shown by reductive mobilization or by proton-induced X-ray emission. This pool may be remobilized when iron deficiency takes place. During radial transport to the vessels, vacuoles may compete with the transport stream forming an iron store. When there is an iron excess, as in plants growing in waterlogged soils or by experimental techniques, plants can escape the deleterious effects of free iron by depositing it in phytoferritin, a storage protein inducible under iron excess. Also, nicotianamine forms a pool of metabolically available iron. Thus, in roots cells of the nicotianamine-free tomato mutant chloronerva iron precipitations occur as evidenced by energy dispersive X-ray analysis and the electron microscopic energy loss technique of energy spectroscopic imaging. Future research concerning the plant root's iron metabolism are needed to clarify the function of nicotianamine in intra- and intercellular iron trafficking and to identify the so-called iron-sensor which mediates the regulation of iron acquisition reactions of rhizodermal cells in response to the iron nutritional status of the plant.     

Microbial depassivation of Fe(0) for contaminant removal under semi-aerobic conditions

Increasing evidence has shown that the reaction of zero-valent iron [Fe(0)] by oxygen can produce strong oxidants and rapidly oxidize the tractable contaminants. However, Fe(0) is vulnerable to passivation in the presence of oxygen, which significantly decreases its surface reactivity towards the removal of refractory contaminants. Microorganisms capable of reducing ferric iron in the presence of oxygen are expected to overcome the limitation of Fe(0) passivation. However, no studies to date have shown that microorganisms are able to depassivate Fe(0) for the removal of recalcitrant compounds in the presence of oxygen. In this study, we demonstrated that the carotenoid-producing Sphingobium hydrophobicum C1 was able to significantly enhance the removal of deca-brominated diphenyl ether by depassivating Fe(0) and subsequently removing the newly formed metabolites under semi-aerobic conditions (> 4 mg/L oxygen). S. hydrophobicum C1 effectively depassivated Fe(0) and regenerated its reactivity by reducing ferric iron under semi-aerobic conditions. Some unique characteristics of S. hydrophobicum C1, including the presence of membrane-integrated carotenoids and certain cell proteins, were essential for the ferric iron reduction of S. hydrophobicum C1 in the presence of oxygen. Our results may provide new insights into the bioremediation of persistent pollutants and will contribute to future studies to enhance our understanding of microbial iron reduction.

     

Siderophore-mediated iron (III) transport in the mycelia of the cultivated fungus, Agaricus bisporus.

Three structurally diverse iron (III) sequestering compounds (siderophores) were isolated from the supernatants of early stationary phase iron-deficient cultures of vegetative mycelia of the cultivated mushroom, Agaricus bisporus (ATCC 36416). The compounds were purified as their ferric chelates to homogeneity by gel permeation, cation exchange, and low-pressure reversed phase C18 chromatographies, and characterized as trihydroxamic acids. The chelates were identified as ferrichrome, ferric fusarinine C, and an unusual compound, des (diserylglycyl) ferrirhodin (DDF) by HPTLC cochromatography and electrophoresis against authentic samples, hydrolysis and amino acid analysis, and FAB-MS and 1H NMR spectroscopy. The iron transport activities of the three compounds (and of some structurally similar exogenous compounds) in young mycelial cells were determined by time- and concentration-dependent kinetic assays and inhibition experiments (CN-, N3-) using 55Fe(3+)-labeled chelates. 55Iron (III) uptake mediated by all three compounds was found to be via high affinity, energy-dependent processes; transport effectiveness was in the order: ferrichrome > DDF > ferric fusarinine C. The relative uptake of iron by lambda-cis ferrichromes was: ferrichrome > ferrirhodin > ferrichrome A; transport activity by the delta-cis fusarinines was: ferric fusarinine C > tris cis-(and trans-) fusarinine iron (III) > ferric N1-triacetylfusarinine C.     

Influence of electron donor/acceptor concentrations on hydrous ferric oxide (HFO) bioreduction

Dissimilatory metal-reducing bacteria (DMRB) facilitate the reduction of Feand Mn oxides in anoxic soils and sediments and play an important role inthe cycling of these metals and other elements such as carbon in aqueousenvironments. Previous studies investigating the reduction of Fe(III) oxidesby DMRB focused on reactions under constant initial electron donor (lactate)and electron acceptor (Fe oxide) concentrations. Because the concentrationsof these reactants can vary greatly in the environment and would be expectedto influence the rate and extent of oxide reduction, the influence of variableelectron acceptor and donor concentrations on hydrous ferric oxide (HFO)bioreduction was investigated. Batch experiments were conducted in pH 7 HCO₃– buffered media using Shewanella putrefaciens strain CN32. In general, the rate of Fe(III) reduction decreased with increasing HFO:lactateratios, resulting in a relatively greater proportion of crystalline Fe(III) oxidesof relatively low availability for DMRB. HFO was transformed to a variety ofcrystalline minerals including goethite, lepidocrocite, and siderite but was almostcompletely dissolved at high lactate to HFO ratios. These results indicate thatelectron donor and acceptor concentrations can greatly impact the bioreductionof HFO and the suite of Fe minerals formed as a result of reduction. The respirationdriven rate of Fe(II) formation from HFO is believed to be a primary factor governingthe array of ferrous and ferric iron phases formed during reduction.     

Physiology of Organotrophic and Lithotrophic Growth of the Thermophilic Iron-Reducing Bacteria Thermoterrabacterium ferrireducens and Thermoanaerobacter siderophilus

Growth physiology of the iron-reducing bacteria Thermoterrabacterium ferrireducens and Thermoanaerobacter siderophilus was investigated. The stimulation of the organotrophic growth of T. ferrireducens and T. siderophilusin the presence of Fe(III) was shown to be due to the utilization of ferric iron as an electron acceptor in catabolic processes and not to the effect exerted on the metabolism by Fe(II) or by changes in the redox potential. It was established that Fe(III) reduction in T. ferrireducens is not a detoxication strategy. In T. siderophilus, this process is carried out to alleviate the inhibitory effect of hydrogen. T. ferrireducens was shown to be capable of lithoautotrophic growth with molecular hydrogen as an electron donor and amorphous ferric oxide as an electron acceptor, in the absence of any organic substances. The minimum threshold of H₂ consumption was 3 × 10^–5 vol % of H₂. The presence of CO dehydrogenase activity in T. ferrireducens suggests that CO₂ fixation in this organism involves the anaerobic acetyl-CoA pathway. T. siderophilus failed to grow under lithoautotrophic conditions. The fact that T. ferrireducens contains c-type cytochromes and T. siderophilus lacks them confirms the operation of different mechanisms of ferric iron reduction in these species.     

Characterization of Fe(III) Reduction by Chlororespiring Anaeromxyobacter dehalogenans

Anaeromyxobacter dehalogenans strain 2CP-C has been shown to grow by coupling the oxidation of acetate to the reduction of ortho-substituted halophenols, oxygen, nitrate, nitrite, or fumarate. In this study, strain 2CP-C was also found to grow by coupling Fe(III) reduction to the oxidation of acetate, making it one of the few isolates capable of growth by both metal reduction and chlororespiration. Doubling times for growth of 9.2 and 10.2 h were determined for Fe(III) and 2-chlorophenol reduction, respectively. These were determined by using the rate of [¹⁴C]acetate uptake into biomass. Fe(III) compounds used by strain 2CP-C include ferric citrate, ferric pyrophosphate, and amorphous ferric oxyhydroxide. The addition of the humic acid analog anthraquinone 2,6-disulfonate (AQDS) increased the reduction rate of amorphous ferric iron oxide, suggesting AQDS was used as an electron shuttle by strain 2CP-C. The addition of chloramphenicol to fumarate-grown cells did not inhibit Fe(III) reduction, indicating that the latter activity is constitutive. In contrast, the addition of chloramphenicol inhibited dechlorination activity, indicating that chlororespiration is inducible. The presence of insoluble Fe(III) oxyhydroxide did not significantly affect dechlorination, whereas the presence of soluble ferric pyrophosphate inhibited dechlorination. With its ability to respire chlorinated organic compounds and metals such as Fe(III), strain 2CP-C is a promising model organism for the study of the interaction of these potentially competing processes in contaminated environments.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in continuous cultures

The oxidation and growth kinetics of ferrous iron with Thiobacillus ferrooxidans in continuous cultures was examined at several total iron concentrations. On-line off-gas analyses of O₂ and CO₂ were used to measure the oxygen and carbon dioxide consumption rates in the culture. Off-line respiration measurements in a biological oxygen monitor (BOM) were used to measure directly the maximum specific oxygen consumption rate, qO₂,max, of cells grown in continuous culture. It was shown that these reproducibly measured values of qO₂,max vary with the dilution rate. The biomass-specific oxygen consumption rate, qO₂, is dependent on the ratio of the ferric and ferrous iron concentrations in the culture. The oxidation kinetics was accurately described with a rate equation for competitive ferric iron inhibition, using the value of qO₂,max measured in the BOM. Accordingly, only the kinetic constant Ks/K _i needed to be fitted from the measurements. A new method was introduced to determine the steady-state kinetics of a cell suspension in a batch culture that only takes a few hours. The batch culture was set up by terminating the feeding of a continuous culture at its steady state. The kinetic constant K _s/K _i determined in this batch culture agreed with the value determined in continuous cultures at various steady states. Received: 8 February 1999 / Accepted: 17 February 1999     

FERRIC CHELATE REDUCTASE ACTIVITY AS AFFECTED BY THE IRON‐LIMITED GROWTH RATE IN FOUR SPECIES OF UNICELLULAR GREEN ALGAE (CHLOROPHYTA)1

Four species of green algae (Chlorella kessleri Fott et Nováková, Chlorococcum macrostigmatum Starr, Haematococcus lacustris[Girod‐Chantrans] Rostaf., Stichococcus bacillaris Näg.) were grown in iron‐limited chemostats and under phosphate limitation and iron (nutrient) sufficiency. For all four species, steady‐state culture density declined with decreasing degree of iron limitation (increasing iron‐limited growth rate), whereas chl per cell or biovolume increased. Plasma membrane ferric chelate reductase activity was enhanced by iron limitation in all species and suppressed by phosphate limitation and iron sufficiency. These results confirm previous work that C. kessleri uses a reductive mechanism of iron acquisition and also suggest that the other three species use the same mechanism. Although imposition of iron limitation led to enhanced activities of ferric chelate reductase in all species, the relationship between ferric chelate reductase activity and degree of iron limitation varied. Ferric chelate reductase activity in C. macrostigmatum and S. bacillaris was an inverse function of the degree of iron limitation, with the most rapidly growing iron‐limited cells exhibiting the highest ferric chelate reductase activity. In contrast, ferric chelate reductase activity was only weakly affected by the degree of iron limitation in C. kessleri and H. lacustris. Calculation of ferric reductase activity per unit chl allowed a clear differentiation between iron‐limited and iron‐sufficient cells. The possible extension of the ferric chelate reductase assay to investigate the absence or presence of iron limitation in natural waters may be feasible, but it is unlikely that the assay could be used to estimate the degree of iron limitation.     

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).     

Overexpression and purification of ferric enterobactin esterase from Escherichia coli. Demonstration of enzymatic hydrolysis of enterobactin and its iron complex.

The Escherichia coli ferric enterobactin esterase gene (fes) was cloned into the vector pGEM3Z under the control of the T7 gene 10 promoter and overexpressed to approximately 15% of the total cellular protein. The ferric enterobactin esterase (Fes) enzyme was purified as a 43-kDa monomer by gel filtration chromatography. Purified Fes preparations were examined for esterase activity on enterobactin and its metal complexes and for iron reduction from ferric complexes of enterobactin and 1,3,5-tris(N,N',N"-2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM), a structural analog lacking ester linkages. Fes effectively catalyzed the hydrolysis of both enterobactin and its ferric complex, exhibiting a 4-fold greater activity on the free ligand. It also cleaved the aluminum (III) complex at a rate similar to the ferric complex, suggesting that ester hydrolysis of the ligand backbone is independent of any reductive process associated with the bound metal. Ferrous iron was released from the enterobactin complex at a rate similar to ligand cleavage indicating that hydrolysis and iron reduction are tightly associated. However, no detectable release of ferrous iron from the MECAM complex implies that, with these in vitro preparations, metal reduction depends upon, and is subsequent to, the esterase activity of Fes. These observations are discussed in relation to studies which show that such enterobactin analogs can supply growth-promoting iron concentrations to E. coli.     

Organic matter mineralization with the reduction of ferric iron: A review

A review of the literature indicates that numerous microorganisms can reduce ferric iron during the metabolism of organic matter. In most cases, the reduction of ferric iron appears to be enzymatically catalyzed and, in some instances, may be coupled to an electron transport chain that could generate ATP. However, the physiology and biochemistry of ferric iron reduction are poorly understood. In pure culture, ferric iron‐reducing organisms metabolize fermentable substrates, such as glucose, primarily to typical fermentation products, and transfer only a minor portion of the electron equivalents in the fermentable substrates to ferric iron. However, fermentation products, especially hydrogen and acetate, may be important electron donors for ferric iron reduction in natural environments. The ability of some organisms to couple the oxidation of fermentation products to the reduction of ferric iron means that it is possible for a food chain of iron‐reducing organisms to completely mineralize nonrecalcitrant organic matter with ferric iron as the sole electron acceptor. The rate and extent of ferric iron reduction depend on the forms of ferric iron that are available. Most of the ferric iron in sediments is resistant to microbial reduction. Ferric iron‐reducing organisms can exclude sulfate reduction and methane production from the zone of ferric iron reduction in sediments by outcompeting sulfate‐reducing and methanogenic food chains for organic matter when ferric iron is available as amorphic ferric oxyhydroxide. There are few quantitative estimates of the rates of ferric iron reduction in natural environments, but there is evidence that ferric iron reduction can be an important pathway for organic matter decomposition in some environments. There is a strong need for further study on all aspects of microbial reduction of ferric iron.     

Effects of ferric soils on arthropod abundance and herbivory on <Emphasis Type="Italic">Tibouchina heteromalla</Emphasis> (Melastomataceae): is fluctuating asymmetry a good indicator of environmental stress?

High concentration of heavy metals in the soils represents an important factor of physiological stress that influences the normal functioning of plants through oxidation processes, and negatively affects insect performance and leaf consumption by herbivorous insects. One useful indicator to evaluate environmental stress in plants by heavy metals and herbivory is the fluctuating asymmetry, which describes the random differences in size or shape between the two sides of a bilateral character in organisms and it is a widely used measure of developmental instability in plants. We evaluated under natural conditions, the effects of variation of heavy metals in the soils on herbivore patterns, fluctuating asymmetry and arthropod abundance in Tibouchina heteromalla in rupestrian grasslands along the Espinhaço chain in Brazil. We selected two study areas, the first characterized by the presence of soils with low concentration of heavy metals (quartzite soils). In the second area, the soils are characterized by the presence of high concentration of heavy metals such as iron (ferric soils). We found that leaf thickness was higher in ferric soils than in quartzite soils. Conversely, total leaf area was greater in quartzite soils in comparison to ferric soils. Plants in soils with heavy metals had both lower herbivory levels and arthropod abundance than plants in soils with low concentrations of heavy metals. Fluctuating asymmetry levels were significantly greater in individuals from quartzite soils compared to individuals from ferric soils. Herbivory was positively related with individual fluctuating asymmetry in quartzite soils. Our results suggest that T. heteromalla presents tolerance to soils with heavy metals suggesting an acclimatization to these environmental conditions, and therefore, ferric soils may not represent a factor of environmental stress.     

Is nitrate reduced to ammonium in waterlogged acid sulfate soil?

Summary A study of transformations of added nitrate nitrogen in an acid sulfate soil under waterlogged conditions indicated that chemical reduction of NO₃–N to NH₄–N is effected by high concentrations of ferrous iron, released in the soil following flooding and reduction of ferric to ferrous iron.     

Iron reductase systems on the plant plasma membrane—A review

Higher plant roots, leaf mesophyll tissue, protoplasts as well as green algae are able to reduce extra-cellular ferricyanide and ferric chelates. In roots of dicotyledonous and nongraminaceous, monocotyledonous plants, the rate of ferric reduction is increased by iron deficiency. This reduction is an obligatory prerequisite for iron uptake and is mediated by redox systems localized on the plasma membrane. Plasma membrane-bound iron reductase systems catalyze the transmembrane electron transport from cytosolic reduced pyridine nucleotides to extracellular iron compounds. Natural and synthetic ferric complexes can act as electron acceptors.This paper gives an overview about the present knowledge on iron reductase systems at the plant plasma membrane with special emphasis on biochemical characteristics and localisation.     

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.     

Porewater oxidation,dissolved phosphate and the iron curtain

The process of dissolved phosphate removal from aqueous solution, which occurs during oxidation of soluble ferrous compounds to insoluble ferric forms, was examined in soils of two tidal freshwater marshes. Sites of amorphous iron deposition and sorption or co-precipitation of phosphate were found to be in surface soils and along creekbanks, where both ion diffusion and porewater advection move dissolved iron and phosphate from reduced to oxidized regions. Profiles of extractable iron and total phosphorus from creekbank and interior soils were consistent with hypothesized differences between a high and a low marsh. Porewater concentrations of dissolved phosphate were higher in creekbank soils of the high marsh, compared with water actually discharging from the creekbank during tidal exposure. We propose that an iron curtain of ferric hydroxides functions as a barrier to diffusive and advective movement of dissolved phosphate along surfaces of tidal freshwater marshes, and has important implications for the distribution and availability of phosphorus in other types of wetlands and aqueous systems.     

A widespread mechanism in ectomycorrhizal fungi to access nitrogen from mineral-associated proteins

A large fraction of nitrogen (N) in forest soils is present in mineral-associated proteinaceous compounds. The strong association between proteins and minerals limits microbial accessibility to this source, which is a relatively stable reservoir of soil N. We have shown that the ectomycorrhizal (ECM) fungus Paxillus involutus can acquire N from iron oxide-associated proteins. Using tightly controlled isotopic, spectroscopic and chromatographic experiments, we demonstrated that the capacity to access N from iron oxide-associated bovine serum albumin (BSA) is shared with the ECM fungi Hebeloma cylindrosporum and Piloderma olivaceum. Despite differences in evolutionary history, growth rates, exploration types and the decomposition mechanisms of organic matter, their N acquisition mechanisms were similar to those described for P. involutus. The fungi released N from mineral-associated BSA by direct action of extracellular aspartic proteases on the mineral-associated BSA, without initial desorption of the protein. Hydrolysis was suppressed by the adsorption of proteases to minerals, but this adverse effect was counteracted by the secretion of compounds that conditioned the mineral surface. These data suggest that the enzymatic exudate-driven mechanism to access N from mineral-associated proteins is found in ECM fungi of multiple lineages and exploration types.