Cadmium-Responsive Thiols in the Ectomycorrhizal Fungus Paxillus involutus

Molecular and cellular mechanisms underlying the sustained metal tolerance of ectomycorrhizal fungi are largely unknown. Some of the main mechanisms involved in metal detoxification appear to involve the chelation of metal ions in the cytosol with thiol-containing compounds, such as glutathione, phytochelatins, or metallothioneins. We used an improved high-performance liquid chromatography method for the simultaneous measurement of thiol-containing compounds from cysteine and its derivatives (γ-glutamylcysteine, glutathione) to higher-molecular-mass compounds (phytochelatins). We found that glutathione and γ-glutamylcysteine contents increased when the ectomycorrhizal fungus Paxillus involutus was exposed to cadmium. An additional compound with a 3-kDa molecular mass, most probably related to a metallothionein, increased drastically in mycelia exposed to cadmium. The relative lack of phytochelatins and the presence of a putative metallothionein suggest that ectomycorrhizal fungi may use a different means to tolerate heavy metals, such as Cd, than do their plant hosts.     

Interaction between the Ectomycorrhizal Fungus Paxillus involutus, Damping-off Fungi and Pinus resinosa Seedlings

Paxillus involutus, an ectomycorrhizal fungus, had an inhibitory effect on the root pathogenic fungus Fusarium moniliforme and two isolates of F. oxysporum when grown in paired cultures on modified Melin Norkrans’ medium. In contrast, one isolate of F. oxysporum was not inhibited and another damping-off fungus, Cylindrocarpon destructans inhibited growth of Pax. involutus in similar paried cultures. Survival of Pinus resinosa (red pine) seedlings was increased significantly when they were grown in vitro concomitantly with either Pax. involutus and F. moniliforme or Pax. involutus and the three isolates of F. oxysporum, compared with seedlings inoculated with either F. moniliforme or F. oxysporum isolates alone. pax. involutus showed no protective effect against C. destructans. The number of colony forming units of Fusarium spp. was reduced significantly in the root extract and rhizosphere substrate of P. resinosa seedlings inoculated with Pax. involutus. Spore germination of Fusarium spp. was reduced significantly when treated with culture filtrate of Pax. involutus and root extract of P. resinosa seedlings inoculated with Pax. involutus. Neither colony forming units nor spore germination of C. destructans was affected either by culture filtrate of Pax. involutus or root extract of P. resinosa seedlings inoculated with Pax. involutus.     

Zinc Phosphate Transformations by the Paxillus involutus/Pine Ectomycorrhizal Association

In this research, we investigate zinc phosphate transformations by Paxillus involutus/pine ectomycorrhizas using zinc-resistant and zinc-sensitive strains of the ectomycorrhizal fungus under high- and low-phosphorus conditions to further understand fungal roles in the transformation of toxic metal minerals in the mycorrhizosphere. Mesocosm experiments with ectomycorrhizas were performed under sterile conditions with zinc phosphate localized in cellophane bags: zinc and phosphorus mobilization and uptake by the ectomycorrhizal biomass were analyzed. In the presence of a phosphorus source, an ectomycorrhizal association with a zinc-resistant strain accumulated the least zinc compared to a zinc-sensitive ectomycorrhizal association and non-mycorrhizal plants. Under low-phosphorus conditions, mycorrhizal seedlings infected with the zinc-resistant strain increased the dissolution of zinc phosphate and zinc accumulation by the plant. Extended X-ray absorption fine structure analysis of both mycorrhizal and nonmycorrhizal roots showed octahedral coordination of zinc by oxygen-containing ligands such as carboxylates or phosphate. We conclude that zinc phosphate solubilization and zinc and phosphorus uptake by the association depend on ectomycorrhizal infection, strain of the mycobiont, and the phosphorus status of the matrix.     

Involutin Is an Fe3+ Reductant Secreted by the Ectomycorrhizal Fungus Paxillus involutus during Fenton-Based Decomposition of Organic Matter

Ectomycorrhizal fungi play a key role in mobilizing nutrients embedded in recalcitrant organic matter complexes, thereby increasing nutrient accessibility to the host plant. Recent studies have shown that during the assimilation of nutrients, the ectomycorrhizal fungus Paxillus involutus decomposes organic matter using an oxidative mechanism involving Fenton chemistry (Fe²⁺ + H₂O₂ + H⁺ → Fe³⁺ + ˙OH + H₂O), similar to that of brown rot wood-decaying fungi. In such fungi, secreted metabolites are one of the components that drive one-electron reductions of Fe³⁺ and O₂, generating Fenton chemistry reagents. Here we investigated whether such a mechanism is also implemented by P. involutus during organic matter decomposition. Activity-guided purification was performed to isolate the Fe³⁺-reducing principle secreted by P. involutus during growth on a maize compost extract. The Fe³⁺-reducing activity correlated with the presence of one compound. Mass spectrometry and nuclear magnetic resonance (NMR) identified this compound as the diarylcyclopentenone involutin. A major part of the involutin produced by P. involutus during organic matter decomposition was secreted into the medium, and the metabolite was not detected when the fungus was grown on a mineral nutrient medium. We also demonstrated that in the presence of H₂O₂, involutin has the capacity to drive an in vitro Fenton reaction via Fe³⁺ reduction. Our results show that the mechanism for the reduction of Fe³⁺ and the generation of hydroxyl radicals via Fenton chemistry by ectomycorrhizal fungi during organic matter decomposition is similar to that employed by the evolutionarily related brown rot saprotrophs during wood decay.     

Cadmium-responsive thiols in the ectomycorrhizal fungus Paxillus involutus

Molecular and cellular mechanisms underlying the sustained metal tolerance of ectomycorrhizal fungi are largely unknown. Some of the main mechanisms involved in metal detoxification appear to involve the chelation of metal ions in the cytosol with thiol-containing compounds, such as glutathione, phytochelatins, or metallothioneins. We used an improved high-performance liquid chromatography method for the simultaneous measurement of thiol-containing compounds from cysteine and its derivatives (gamma-glutamylcysteine, glutathione) to higher-molecular-mass compounds (phytochelatins). We found that glutathione and gamma-glutamylcysteine contents increased when the ectomycorrhizal fungus Paxillus involutus was exposed to cadmium. An additional compound with a 3-kDa molecular mass, most probably related to a metallothionein, increased drastically in mycelia exposed to cadmium. The relative lack of phytochelatins and the presence of a putative metallothionein suggest that ectomycorrhizal fungi may use a different means to tolerate heavy metals, such as Cd, than do their plant hosts.     

Polyamine biosynthesis in the ectomycorrhizal fungus Paxillus involutus exposed to zinc

Following exposure of the ectomycorrhizal fungus Paxillus involutus to 3·75 μg ml^-1 or 15 μg ml^-1 zinc for 2 weeks, growth was unaffected, putrescine and spermidine concentrations remained unaltered, while spermine concentration increased substantially. This was accompanied by small changes in the activities of ornithine decarboxylase and S -adenosylmethionine decarboxylase. There was, however, reduced synthesis of aminopropylcadaverine and N,N' bis (3-aminopropyl)cadaverine at the higher zinc concentration. It is possible that the increased spermine concentration may have resulted from displacement of the amine from the cell wall by the metal ion.     

Endogenous polyamine content and metabolism in the ectomycorrhizal fungus Paxillus involutus

Endogenous polyamine content of the ectomycorrhizal fungus Paxillus involutus , as well as the activity of its biosynthetic enzymes in relation to mycelia ageing were investigated in this work. Polyamines in free, PCA-soluble and insoluble conjugated forms, are present in Paxillus involutus mycelia in relatively high amounts and the ratio of putrescine to spermidine is age-dependent. Both arginine- and ornithine-decarboxylases are present, but putrescine biosynthesis proceeds mostly via ornithine decarboxylase and decreases with the age of mycelia. There was a large release of free polyamines from mycelia which showed age-dependent features. Clear polyamine uptake was observed in 2-wk-old mycelia and no competition between putrescine and cadaverine was detected. Putrescine uptake seems to reduce ornithine decarboxylase activity, but does not affect arginine decarboxylase.     

Biosynthesis of L-[15N]aspartic acid and L-[15N]alanine by immobilized bacteria

Glycoproteins in nitrocellulose transfers of electrophoretically separated mixtures of cellular and viral proteins are rapidly and sensitively located by sequential incubation with the lectin concanavalin A and the enzymatically active glycoprotein horseradish peroxidase. The bound enzyme is located by incubation with a substrate which is converted to a highly insoluble colored product. The specificity of the method is demonstrated by the abolition of concanavalin A binding in the presence of α-methyl mannoside. The method is capable of detecting as little as 60 ng of a purified model glycoprotein after electrophoresis. It has been applied to the analysis of the glycoproteins of purified Lassa virus and of the virus-specific glycoproteins in Japanese encephalitis virus-infected cells.     

Efficient syntheses of [3-15N]uracil and [3-15N]thymine.

Regiospecific syntheses of [3-15N]uracil and [3-15N]thymine are described using [15N]ammonium sulfate as a source of labeled nitrogen. The overall yields are excellent, and the reactions are amenable to production of multigram quantities of labeled material.     

Size and complexity of the nuclear genome of the ectomycorrhizal fungus Paxillus involutus

The basidiomycete Paxillus involutus is forming ectomycorrhizal symbiosis with a broad range of forest trees. Reassociation kinetics on P. involutus nuclear DNA indicated a haploid genome size of 23 Mb including 11% of repetitive DNA. A similar genome size (20 Mb) was estimated by genomic reconstruction analysis using three single copy genes. To assess the gene density in the P. involutus genome, a cosmid containing a 33-kb fragment of genomic DNA was sequenced and used to identify putative open reading frames (ORFs). Twelve potential ORFs were predicted, eight displayed significant sequence similarities to known proteins found in other organisms and notably, several homologues to the Podospora anserina vegetative incompatibility protein (HetE1) were found. By extrapolation, we estimate the total number of genes in the P. involutus haploid genome to approximately 7700.     

Simultaneous acquisition of [13C,15N]- and [15N,15N]-separated 4D gradient-enhanced NOESY spectra in proteins

Summary The simultaneous acquisition of a 4D gradient-enhanced and sensitivity-enhanced [¹³C,¹⁵N]/[¹⁵N,¹⁵N]-separated NOESY is presented for the 74-residue [¹³C,¹⁵N]-labeled N-terminal SH3 domain of mGrb2 complexed with a peptide gragment from mSOS-2 in 90% H₂O. The method readily accommodates different ¹³C and ¹⁵N spectral widths, but requires that the same number of increments be collected for both ¹³C and ¹⁵N in the simultaneous dimension (F₂). For purposes of display and analysis, the two 4D spectra can be deconvolved during the processing stage by the appropriate linear combination of separately stored FIDs. Compared to collecting each of these two 4D data sets separately, the presented method is a factor (2)^1/2 more efficient in sensitivity per unit acquisition time. The interleaved nature of this method may also lead to improved peak registration between the two 4D spectra.     

Ammonium and methylamine transport by the ectomycorrhizal fungus Paxillus involutus and ectomycorrhizas

Using [(14)C]methylamine as an analogue of ammonium, the kinetics and the energetics of NH(4)(+) transport were studied in the ectomycorrhizal fungus, Paxillus involutus (Batsch) Fr. The apparent half-saturation constant (K(m)) and the maximum uptake rate (V(max)) for the carrier-mediated transport derived from the Eadie-Hofstee transformation were 180 μM and 380 nmol (mg dry wt)(-1) min(-1,) respectively. Both pH dependence and inhibition by protonophores indicate that methylamine transport in P. involutus was dependent on the electrochemical H(+) gradient. Both long-term and short-term uptake experiments were consistent with regulation of ammonium/methylamine transport processes by the presence of an organic nitrogen source. Analysis of methylamine uptake by different P. involutus isolates revealed no obvious trend in the uptake capacities in relation to N deposition at the collection site. Kinetic parameters were determined in P. involutus/Betula pendula (Roth.) axenic association and in detached mycorrhizal roots isolated from forest sites. Enhanced methylamine uptake in the presence of the fungal symbiont was demonstrated. Homogeneous V(max) values were found for axenic and detached mycorrhizas, whereas K(m) values showed greater variations.     

Metabolism of [13N]ammonia in rat lung

Bolus injection of [13N]ammonia into the femoral vein of pentobarbital-anesthetized rats was followed by rapid clearance from the blood and first-pass extraction of nearly 30% by the lungs. Of the label present in the lungs at 6 s after injection (about 27% of the dose), more than 20% was in metabolized form. Of the label present in the lungs at 2 min after injection (about 10% of the dose), 18-25% was in ammonia, about 75% was in glutamine (amide) and less than 1% was in glutamate and aspartate. Thus, despite the presence of significant amounts of glutamate dehydrogenase, the overwhelming route for metabolism of ammonia entering the rat lung in vivo was the glutamine synthetase reaction. Lung tissue that was removed 6 s after intravenous injection of [13N]ammonia and incubated in Krebs-Ringer glucose medium at 37 degrees C for 20 min, showed a significant increase (more than one-third), compared to unincubated lung tissue in the quantity of label in glutamine. Between 6s and 2 min after injection, during which time the total 13N content of the lungs decreased by more than 60%, the maintenance of a quasi-steady state in the concentration of labeled glutamine suggested a short-term balance between formation from extracted ammonia and loss of glutamine into the circulation. Our data support the concept that the lungs are a source of circulating glutamine in the rat. Despite the large fractional extraction of blood-borne [13N]ammonia by the lungs, only minute amounts of tracer (0.2-0.6 ppm of the injected dose) were detected in the expired air within the first 5 min after administration of [13N]ammonia to anesthetized rats, so that pulmonary excretion was not a significant pathway of ammonia elimination. The present findings emphasize the importance of the lungs in the maintenance of whole-body nitrogen homeostasis and suggest the use of [13N]ammonia and 13N-labeled amino acids as non-invasive probes in the study of normal and diseased lung metabolism.     

Nitrous oxide production by the ectomycorrhizal fungi Paxillus involutus and Tylospora fibrillosa

Nitrous oxide (N(2)O) production by filamentous fungi has been demonstrated in pure culture and has been estimated indirectly in soils. However, it is unknown whether ectomycorrhizal fungi can also produce N(2)O. We demonstrate for the first time the ability of nitrogen (N)-tolerant ectomycorrhizal fungi (Paxillus involutus and Tylospora fibrillosa), found in forest soils under moderate to high rates of N deposition, to produce N(2)O from nitrate reduction. The N(2)O concentrations from the ectomycorrhizal fungal treatments after a 10-day pure culture experiment were 0.0117±0.00015 (P. involutus) and 0.0114±0.0003 (T. fibrillosa), and 0.0114±0.00043 μmol N(2)O L(-1) from a known fungal denitrifier (Fusarium lichenicola). No N(2)O was detected in the control treatment. Our results indicate the potential for these two N-tolerant ectomycorrhizal fungi to contribute to N(2)O production. Given that these species are abundant in many forest soils, the strength and regulation of fungal N(2)O production should now be verified in situ.     

Microbial preparation of L-[15N]tyrosine and [15N]tyramine and their gas chromatographic-mass spectrometric analyses

The preparation of L-[15N]tyrosine and [15N]tyramine by microbial synthesis is described. Immobilized Erwinia herbicola cells were added to a reaction mixture containing phenol, pyruvic acid, and 15NH4Cl. The reaction was driven by excess nonlabeled pyruvate and phenol. Under these denaturing concentrations of phenol, immobilized cells were more effective than free ones. Gram quantities of L-[15N]tyrosine were obtained without label dilution. The conversion of this L-[15N]tyrosine into [15N]tyramine by Streptococcus faecalis was performed at maximal efficiency. Gas chromatographic-mass spectrometric studies and 1H and 15N NMR analyses of the labeled compounds are reported.