No abstract

The effects of long-term exposure (5 h) of Phycomyces blakesleeanus mycelium to 5 mM KCN on respiration and phosphate metabolites were tested. Exposure to cyanide, antimycin A and azide lead to a decrease in the activity of cyanide-sensitive respiration (CSR), and the ratio of core polyphosphates (PPc) and inorganic phosphates (Pi), which is a good indicator of the metabolic state of a cell. After 5 h of incubation, the activity of CSR returned to control values. For this, the recovery of cytochrome c oxidase (COX) was required. In addition, the PPc/Pi ratio started to recover shortly after initiation of COX recovery, but never reached control values. This led us to conclude that the regulation of polyphosphate (PPn) levels in the cell is tightly coupled to respiratory chain functioning. In addition, acutely applied cyanide caused two different responses, observed by 31P NMR spectroscopy, that were probably mediated through the mechanism of glycolytic oscillations, triggered by the effect of cyanide on mitochondria. [Projekat Ministarstva nauke Republike Srbije, br. 173040]

Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 and 500 µM NFA for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleeanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33±5 and 21±3 % of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.

Vanadium speciation in the fungus Phycomyces blakesleeanus was examined by X-ray absorption near-edge structure (XANES) spectroscopy, enabling assessment of oxidation states and related molecular symmetries of this transition element in the fungus. The exposure of P. blakesleeanus to two physiologically important vanadium species (V(5+) and V(4+)) resulted in the accumulation of this metal in central compartments of 24 h old mycelia, most probably in vacuoles. Tetrahedral V(5+), octahedral V(4+), and proposed intracellular complexes of V(5+) were detected simultaneously after addition of a physiologically relevant concentration of V(5+) to the mycelium. A substantial fraction of the externally added V(4+) remained mostly in its original form. However, observable variations in the pre-edge-peak intensities in the XANES spectra indicated intracellular complexation and corresponding changes in the molecular coordination symmetry. Vanadate complexation was confirmed by (51)V NMR and Raman spectroscopy, and potential binding compounds including cell-wall constituents (chitosan and/or chitin), (poly)phosphates, DNA, and proteins are proposed. The evidenced vanadate complexation and reduction could also explain the resistance of P. blakesleeanus to high extracellular concentrations of vanadium.

The biological and chemical basis of vanadium action in fungi is relatively poorly understood. In the present study, we investigate the influence of vanadate (V5+) on phosphate metabolism of Phycomyces blakesleeanus. Addition of V5+ caused increase of sugar phosphates signal intensities in 31P NMR spectra in vivo. HPLC analysis of mycelial phosphate extracts demonstrated increased concentrations of glucose 6 phosphate, fructose 6 phosphate, fructose 1, 6 phosphate and glucose 1 phosphate after V5+ treatment. Influence of V5+ on the levels of fructose 2, 6 phosphate, glucosamine 6 phosphate and glucose 1, 6 phosphate (HPLC), and polyphosphates, UDPG and ATP (31P NMR) was also established. Increase of sugar phosphates content was not observed after addition of vanadyl (V4+), indicating that only vanadate influences its metabolism. Obtained results from in vivo experiments indicate catalytic/inhibitory vanadate action on enzymes involved in reactions of glycolysis and glycogenesis i.e., phosphoglucomutase, phosphofructokinase and glycogen phosphorylase in filamentous fungi.