The interactions of vanadate monomer with the mycelium of fungus Phycomyces blakesleeanus: reduction or uptake?

The possibility of reduction of vanadate monomer in the mycelium of fungus Phycomyces blakesleeanus was investigated in this study by means of polarography. Control experiments were performed with vanadyl [V(IV)] and vanadate [V(V)] in 10 mM Hepes, pH 7.2. Addition of P. blakesleeanus mycelium resulted in disappearance of all V(IV) polarographic waves recorded in the control. This points to the uptake of all available V(IV) by the mycelium, up to 185 µmol/gFW, and suggests P. blakesleeanus as a potential agent in V(IV) bioremediation. Polarographic measurements of mycelium with low concentrations (0.1-1 mM) of V(V), that only allows the presence of monomer, showed that fungal mycelia removes around 27% of V(V) from the extracellular solution. Uptake was saturated at 104 ± 2 µmol/gFW which indicates excellent bioaccumulation capability of P. blakesleeanus. EPR, 51V NMR and polarographic experiments showed no indications of any measurable extracellular complexation of V(V) monomer with fungal exudates, reduction by the mycelium or adsorption to the cell wall. Therefore, in contrast to vanadium oligomers, vanadate monomer interactions with the mycelium are restricted to its transport into the fungal cell, probably by a phosphate transporter.

(51)V NMR investigation of cell-associated vanadate species in Phycomyces blakesleeanus mycelium.

(51)V NMR spectroscopy was used for detection and identification of cell-associated vanadate (V(5+)) species after exposure of Phycomyces blakesleeanus mycelium, in exponential phase of growth, to sodium orthovanadate. Complete disappearance of monomer and dimer signals and decreased intensity of the tetramer signal were observed about 40 min after treatment. Simultaneously, a signal at -532 ppm, with increasing intensity, was detected in spectra. The time-dependent rise in this signal was connected to a decrease in the extracellular monomer signal, indicating its transport into the cell. The signal at -532 ppm did not belong to any known simple oxido-vanadate species, nor to a complex with any of the components of experimental medium. This signal was the only one present in spectrum of the mycelium washed 35 min after treatment, and the only one observed in mycelium cultivated on vanadate-contained medium. Therefore, its appearance can be attributed to intracellular complexation, and may represent an important detoxification mechanism of the cell exposed to a physiologically relevant concentration of vanadate. Experiments ((51)V NMR and polarography) performed with Cd-pretreated mycelium (inhibitor of an enzyme responsible for V(5+) reduction) and ferricyanide-preincubated mycelium excluded the possibility of V(5+) tetramer's entry into the cell.

Oxygen regulation of alternative respiration in fungus Phycomyces blakesleeanus: connection with phosphate metabolism.

Environmental changes can often result in oxygen deficiency which influences cellular energy metabolism, but such effects have been insufficiently studied in fungi. The effects of oxygen deprivation on respiration and phosphate metabolites in Phycomyces blakesleeanus were investigated by oxygen electrode and (31)P NMR spectroscopy. Mycelium was incubated in hypoxic and anoxic conditions for 1.5, 3 and 5 h and then reoxygenated. Participation of alternative oxidase (AOX) in total respiration increased gradually in both treatments and after 5 h of anoxia exceeded a value 50% higher than in control. Shortly after reintroduction of oxygen into the system AOX level decreased close to the control level. Oxygen deprivation also caused a reversible decrease of polyphosphate/inorganic phosphate ratio (PPc/Pi), which was strongly correlated with the increase of AOX participation in total respiration. Unexpectedly, ATP content remained almost constant, probably due to the ability of PolyP to sustain energy and phosphate homeostasis of the cell under stress conditions. This was further substantiated by the effects of azide, a cytochrome c oxidase inhibitor, which also decreased PPc/Pi ratio, but to a smaller extent in oxygen deprived than control and reoxygenated specimens.