Intracellular pH homeostasis in the filamentous fungus Aspergillus niger

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).

Different control mechanisms regulate glucoamylase and protease gene transcription in Aspergillus oryzae in solid-state and submerged fermentation

“…Consequently, proton fluxes occur across cellular membranes to maintain intracellular pH homeostasis (Hesse et al 2002). Upon transfer of growing A. oryzae biomass to 2% WLM, the intracellular pH decreases due to metabolic activity and, as a consequence of proton fluxes across the extra cellular membrane, the growth medium started to acidify.…”

High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

“…The pKa of the Venus chromophore is reported to be 6.0 [19]. At pH of 7.6, which is a reasonable estimate for the pH in fungal cells [32], there would be 2.5% of protonated acceptor.…”

Organic Acid Production by Filamentous Fungi

“…The acidophilic nature of A. niger is demonstrated by the finding that even under the extremely low ambient pH conditions of citric acid fermentation, the cytosolic and vacuolar (mitochondrial) compartments retain pH values of 7.5 and 6.1, respectively (Hesse et al, 2000(Hesse et al, , 2002. Additional investigations demonstrated that intracellular pH declines as dissolved oxygen concentration falls (Legiša and Grdadolnik, 2002).…”