No abstract

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 Ca 2+ -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 DpH 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 DpH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger DpH 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 lM CCCP), transient (2 lM CCCP) or permanent (10 lM CCCP) collapse of the vacuolar membrane DpH. 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 acidsecreting capacity, pH cyt and pH vac values were still well maintained (pH 7.5 and 6.4, respectively).

The growth of the filamentous fungus Aspergillus niger, a common food spoilage organism, is inhibited by the weak acid preservative sorbic acid (trans-trans-2,4-hexadienoic acid). Conidia inoculated at 10 5 /ml of medium showed a sorbic acid MIC of 4.5 mM at pH 4.0, whereas the MIC for the amount of mycelia at 24 h developed from the same spore inoculum was threefold lower. The MIC for conidia and, to a lesser extent, mycelia was shown to be dependent on the inoculum size. A. niger is capable of degrading sorbic acid, and this ability has consequences for food preservation strategies. The mechanism of action of sorbic acid was investigated using 31 P nuclear magnetic resonance (NMR) spectroscopy. We show that a rapid decline in cytosolic pH (pH cyt ) by more than 1 pH unit and a depression of vacuolar pH (pH vac ) in A. niger occurs in the presence of sorbic acid. The pH gradient over the vacuole completely collapsed as a result of the decline in pH cyt . NMR spectra also revealed that sorbic acid (3.0 mM at pH 4.0) caused intracellular ATP pools and levels of sugar-phosphomonoesters and -phosphodiesters of A. niger mycelia to decrease dramatically, and they did not recover. The disruption of pH homeostasis by sorbic acid at concentrations below the MIC could account for the delay in spore germination and retardation of the onset of subsequent mycelial growth.