Intracellular pH homeostasis in the filamentous fungus Aspergillus niger

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by (31) P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+ -alginate beads. The fungus maintained constant cytoplasmic pH (pH(cyt) ) and vacuolar pH (pH(vac) ) values of 7.6 and 6.2, respectively, when the extracellular pH (pH(ex) ) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a DeltapH over the cytoplasmic membrane of 6.6-6.7 was reached (pH(ex) 0.7-0.8). Maintenance of these large pH differences was possible without increased respiration compared to pH(ex) 5.8. Perfusion in the presence of various hexoses and pentoses (pH(ex) 5.8) revealed that the magnitude of DeltapH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger DeltapH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m -chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 mum CCCP), transient (2 mum CCCP) or permanent (10 mum CCCP) collapse of the vacuolar membrane DeltapH. Nonlethal levels of the metabolic inhibitor azide (N-3 (-) , 0.1 mm) caused a transient decrease in pH(cyt) that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, (31) P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pH(ex) (1.8) and a high acid-secreting capacity, pH(cyt) and pH(vac) values were still well maintained (pH 7.5 and 6.4, respectively).