Báo cáo Y học: Intracellular pH homeostasis in the filamentous fungus Aspergillus niger pdf

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger Stephan J. A. Hesse 1 , George J. G. Ruijter 2 , Cor Dijkema 1 and Jaap Visser 2, † 1 Department of Biophysics, Wageningen University, the Netherlands; 2 Department of Microbiology, section Fungal Genomics, Wageningen University, the Netherlands Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31 PNMR 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 l M CCCP), transient (2 l M CCCP) or perma- nent (10 l M CCCP) collapse of the vacuolar membrane DpH. Nonlethal levels of the metabolic inhibitor azide (N 3 – , 0.1 m M ) 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 PNMR 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). Keywords: Aspergillus niger; intracellular pH; pH homeo- stasis; 31 P NMR; perfusion. During operation of cellular metabolism under aerobic conditions net intracellular production of protons takes place mainly by formation of tricarboxylic acid cycle acids, CO 2 /H 2 CO 3 and protein synthesis [1]. Consequently, tight control of proton fluxes, in combination with the ability to maintain pH gradients across cellular membranes, is a crucial aspect of cellular energetics. Large deviations of cytoplasmic pH (pH cyt ) need to be avoided to keep control of fundamental intracellular processes which are sensitive to pH, such as DNA transcription, protein synthesis and enzyme activities [2]. In order to ensure optimal activity of major metabolic pathways, constant removal of free protons from the cytoplasm is required [3]. In lower eukaryotes and plants this process is mediated through the action of the plasma membrane P-ATPase at the expense of ATP hydrolysis, which results in pH and electrical potential differences across the plasma membrane. The P-ATPase is involved in intracellular pH (pH in ) regulation, maintenance of a proper ion balance and generation of the electrochemi- cal proton gradient (proton motive force, Dp) across the cytoplasmic membrane which drives an array of secondary transport systems [4]. In Saccharomyces cerevisiae, intracel- lular pH is thought to be additionally regulated through the action of alkali-cation/H + antiporters, such as the Nha1 antiporter with a H + /K + (Na + ) exchange mechanism [5]. Intracellular pH homeostasis in the filamentous fungus Neurospora crassa has been suggested to be achieved by parallel operation of the H + -extruding P-ATPase and a high-affinity proton symport uptake system for K + , yielding a net 1 : 1 exchange of K + for cytoplasmic H + [6]. Other major ATPases in fungal cells are the vacuolar membrane V-ATPase and the mitochondrial membrane F 1 F 0 -ATPase. The action of the former, ATP-dependent transport of protons into the vacuole, is thought to contribute to cytoplasmic pH homeostasis as well [7]. The resulting electrochemical proton potential is able to drive amino acid and ion transport across the vacuolar mem- brane, probably through proton antiport systems. The F 1 F 0 -ATPase uses the proton motive force generated by the electron transport chain across the inner mitochondrial membrane to drive phosphorylation of ADP to ATP. Consequently, intracellular pH and pH gradients are directly linked to cellular energy levels and metabolism [8]. The response of intracellular pH values to different conditions, especially variation in extracellular pH (pH ex ), reveals clues to mechanisms of pH regulation [9]. In a previous study we reported on a system based on long-term acquisition of 31 P NMR spectra of constantly perfused and well-oxygenated immobilized mycelium for the determin- ation of compartmental pH values in the filamentous fungus Aspergillus niger [10]. A. niger is industrially important for Correspondence to S. J. A. Hesse, Department of Biophysics, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, the Netherlands. Fax: +31 317 484 011, Tel.: +31 317 484 692, E-mail: stephan.hesse@algemeen.mgim.wau.nl Abbreviations: CCCP, chlorophenylhydrazone. Present address: Postbus 396, 6700 AJ Wageningen, the Netherlands. (Received 12 April 2002, accepted 30 May 2002) Eur. J. Biochem. 269, 3485–3494 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03042.x large-scale production of organic acids (e.g. citric acid) due to a high intrinsic excretion capacity for this compound [11]. Despite this capacity, quantitative analysis of metabolism using kinetic models and metabolic engineering, comple- mentary to traditional strain improvement, is still a promising approach to increase citric acid production or to shorten fermentation times [12]. However, a more predictive accuracy for kinetic (mechanistic) models requires a more detailed description of the conditions under which the enzymes involved operate in vivo. With the perfusion system developed, problems related to fungal morphology during long-term in vivo NMR measurements have been overcome. With this method it is now possible to obtain more information on intracellular pH, one of the key parameters affecting enzyme activity, and its homeostasis. Also, the ability of A. niger to acidify its medium to pH values below 2.0 during production of large quantities of organic acids implies that a very efficient pH-homeostatic system exists in these cells. As protein synthesis and intracellular enzyme activities are sensitive to pH, mainten- ance of intracellular pH under extreme conditions (especi- ally low pH ex ) is crucial to ensure optimal cellular activity during (industrial) fermentations. So far, however, data on intracellular pH in filamentous fungi have been scarce. A few reports have dealt with cytoplasmic pH of citric acid- producing A. niger mycelium [13,14]. More detailed know- ledge about intracellular pH homeostasis in filamentous fungi is to date only available for N. crassa [1,15,16]. To investigate the ability of A. niger to maintain cellular energy levels, cells were subjected to several stresses like low pH ex , citric acid producing conditions, increased proton permeab- ility by an uncoupler and inhibition of ATP synthesis by sodium azide. Also tested was the effect of different carbon sources on steady-state DpH values. Together our results provide reliable data on intracellular pH under various extracellular conditions, and demonstrate the ability of the fungus to maintain cellular energetics under extreme conditions with a formidable tolerance towards extracellular acidity. EXPERIMENTAL PROCEDURES Strain, immobilization of conidia and culture conditions Condiospores of A. niger NW131 (cspA1 goxC17) lacking glucose oxidase activity [17] were propagated at 30 °C on complete medium [18] solidified by 1.5% agar and contain- ing 1% D -glucose. Conidiospores were harvested with a solution containing 0.9% NaCl and 0.05% (v/v) Tween-80. Immobilization of conidia in Ca 2+ -alginate beads (diameter 1 mm) was performed as described previously [10]. A 2.5% solution of Manugel DJX (ISP Alginates, Tadworth, Surrey, UK) was used in all immobilization experiments. After harvesting and washing with demineralized water, immobilized conidia were cultured in 500-mL Erlenmeyer flasks. Immobilized mycelium was obtained by incubating the beads (10 g) for 40–44 h in a rotary shaker at 250 r.p.m and 30 °C in 100 mL minimal medium (2 gÆL )1 NH 4 NO 3 , 1.5 gÆL )1 KH 2 PO 4 ,0.5gÆL )1 KCl, 0.5 gÆL )1 MgSO 4 Æ7H 2 O pH 6.0), supplemented with 1.5% D -glucose, 0.02% (v/v) of a trace element solution [19] and 0.05% yeast extract. To obtain immobilized mycelium under citric acid- producing conditions a 500-mL bubble column reactor was filled with 125 mL immobilized conidia and 375 mL medium optimized for citric acid production (140 gÆL )1 decationized glucose, 0.2 gÆL )1 KH 2 PO 4 ,1.25gÆL )1 (NH 4 ) 2 SO 4 ,0.25gÆL )1 MgSO 4 Æ7H 2 O, 1.3 mgÆL )1 ZnSO 4 Æ 7H 2 O, 6.5 mgÆL )1 FeSO 4 Æ7H 2 O). Under these conditions no growth of biomass outside of the beads occurred. The culture pH was not regulated and was initially set at 3.5. Cultures were sparged with 1.5 LÆmin )1 air, and after 24 h 15 lL of a solution containing 30% (v/v) polypropylene- glycol in alcohol was added to the reactor to prevent excessive foaming. The reactors were run for 2 or 7 days at 30 °C. The fermentation volume was periodically adjusted to 0.5 L with double-distilled water. Perfusion conditions Immobilized biomass from shake-flask cultures was har- vested, washed with a buffer (30 °C) containing 25 m M sodium citrate pH 5.8, 0.25 gÆL )1 NH 4 NO 3 ,0.2gÆL )1 KCl, 0.2 gÆL )1 MgSO 4 Æ7H 2 O, 0.2 m M KH 2 PO 4 ,0.3m M CaCl 2 , and perfused within the NMR tube with 1 L of the same buffer saturated with oxygen. The extracellular pH was varied by using perfusion buffer with pH values ranging from 1.0 to 7.0 or by direct titration of the buffer reservoir with 2 M HCl. The same buffer supplemented with 50 m M Tris was used under alkaline extracellular conditions (pH ex , 7.0–9.0). The effect of the presence of various sugars on intracellular pH values was tested after a 2-h transfer of immobilized biomass to 150 mL of perfusion buffer pH 5.8 supplemented with 10 m M of sugar ( D -glucose, D -fructose, D -xylose or L -arabinose). Subsequently, the beads were perfused with the same buffer saturated with oxygen for 3 h. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and sodium azide (NaN 3 ) were directly added to the buffer reservoir (pH 5.8) from a 10-m M stock solution in ethanol and a 10% stock solution in water, respectively. Oxygen consumption during perfusion (DO 2 ) was expressed as the percentage of oxygen removed from an oxygen saturated buffer after passage through the immobilized cell plug. Immobilized citric acid producing mycelium from 2- and 7-day-old fermentations was directly transferred from the bubble column reactor to the NMR tube and perfused with filtered culture medium (500 mL) from 2-day- and 7-day- old fermentations, respectively. In all cases, a 4-cm high plug of immobilized biomass (± 12.5 mL beads) was perfused at arateof15mLÆmin )1 . 31 P NMR spectroscopy 31 P NMR spectra were recorded at 121.5 MHz at 30 °C on a AMX300 wide-bore spectrometer (Bruker, Germany), equipped with a 20-mm switchable 31 P/ 13 C probe tuned at the 31 P nucleus, and collected in 15, 20 or 60-min blocks (4500, 5700 or 18000 FIDs, respectively) using acquisition parameters described previously [10]. Methylene diphos- phonic acid (0.2 M , pH 8.9), contained in an in situ capillary, was used as an internal reference, resonating at 16.92 p.p.m. relative to 85% H 3 PO 4 (0 p.p.m). Analyses Cytoplasmic and vacuolar pH values were determined by comparing the pH-sensitive chemical shifts of cytoplasmic 3486 S. J. A. Hesse et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (P cyt ) and vacuolar inorganic phosphate (P vac )witha calibration curve for inorganic phosphate (P i ). This curve has been referred to in many other NMR studies on both yeasts and fungi [16,20,21], and shows the pH dependence of the chemical shift of P i measured in a medium made up to approximate concentrations of the major cationic compo- nents in yeast [22]. The perfusion buffer pH (pH ex )was monitored by pH electrode measurements. Intracellular pH values in the presence of various carbon sources and in mycelium producing high levels of citric acid were deter- mined from 12–16 and 5–7 different spectra, respectively; final values represent mean values that were statistically analysed by a two-tailed Student’s t-test (a ¼ 5%). Relative increases in polyphosphate levels were determined from 31 P NMR spectra by relating the integral of the polyphosphate peak to the integral of the internal reference peak. Citrate, ammonium and phosphate levels in culture filtrate samples of citric acid fermentations were determined as described before [17]. Perfusion buffer and transfer medium samples were analysed for sugars and organic acids by HPLC using an HPX-87H column (Bio-Rad) eluted with 25 m M HCl at 50 °C with UV (210 nm) and refractive index detection, and for polyols using a Carbopac MA-1 column (Dionex) eluted with 480 m M NaOH at 20 °C with pulsed amperometric detection. Dry weight determinations on immobilized biomass were carried out by dissolving the Ca 2+ -alginate beads in a 100-m M solution of the Ca 2+ -scavenger sodium hexametaphosphate (Fluka AG, Buchs, Switzerland). Mycelium was then collected by filtration, washed with demineralized water, frozen in liquid nitrogen, lyophilized and weighed. RESULTS The dependence of intracellular pH values on ambient pH A. niger has the ability to acidify its environment to values as low as pH 1.5 [17]. To investigate to what extent extracellular pH (pH ex ) affects intracellular pH (pH in ) values, we determined pH cyt and pH vac as a function of ambient pH using 31 P NMR as described previously [10]. Surprisingly, the cells were able to maintain pH cyt and pH vac at 7.6 and 6.2, respectively, when pH ex was varied between 1.5 and 7.0, implying that a very steep DpH over the cytoplasmic membrane of 6.1 can be sustained by the cells (Fig. 1A). The DpH over the vacuolar membrane was maintained at a constant value of 1.4. At pH ex 1.5, this gradient was about 0.1 pH unit larger due to a slightly more acidic vacuole. Further acidification to pH ex 1.0 caused pH cyt to drop to pH 7.4, in parallel to an even larger vacuolar acidification than at pH ex 1.5. A comparable observation was made at pH ex 8.0: pH cyt was still reason- ably well regulated, whereas the vacuoles became more alkaline (pH vac 6.5). Interestingly, in the pH ex range of 1.0– 7.0 in the presence of 25 m M citrate, cells consumed the same amount of oxygen once a steady-state was reached (DO 2 ¼ 8–9%). Cellular metabolism collapsed and O 2 consumption rapidly dropped to 0% when pH ex reached a value of about 0.7–0.8. Cells only moderately increased their oxygen consumption (from 9 to 11%) just before the collapse. A representative 31 P NMR spectrum of immobi- lized A. niger mycelium perfused in the presence of 25 m M citrate pH 1.5 and 0.2 m M P i isshowninFig.1B. Fig. 1. The dependence of A. niger NW131 cytoplasmic pH (pH cyt ) and vacuolar pH (pH vac ) on extracellular pH (pH ex ) in the presence of oxygen- saturated buffer (A) and typical 31 P NMR spectrum of immobilized A. niger NW131 mycelium (B). (A) Buffer contained 25 m M citrate (pH ex 1.0–7.0) or 25 m M citrate and 50 m M Tris (pH ex 7.0–8.5). Values are the mean of two experiments. (B) Mycelium cultured for 42 h and perfused with oxygen-saturated buffer containing 25 m M citrate at pH ex 1.5. Abbreviations and chemical shifts of the assignments: SME: sugar phospho- monoesters, 4.9 p.p.m.; SDE: sugar phosphodiesters, 4.5 p.p.m.; P cyt : cytoplasmic inorganic phosphate, 2.9 p.p.m.; P vac : vacuolar inorganic phosphate, 1.2 p.p.m.; P ex : extracellular inorganic phosphate, 0.6 p.p.m.; c-ATP: ) 4.9 p.p.m.; P 2 : pyrophosphate and terminal phosphate of polyphosphate, )5.8 p.p.m.; a-ATP: )9.9 p.p.m.; NAD(H): )10.6 p.p.m.; UDPG: uridine diphosphoglucose, )10.6 and )12.3 p.p.m.; P 3 and P 4 : penultimate phosphates of polyphosphate, )17.7 and )19.7 p.p.m., respectively; a-ATP: )18.6 p.p.m.; P n : polyphosphate, )22.5 p.p.m. Data were collected in a 60-min block. The internal reference is not shown. Ó FEBS 2002 Intracellular pH homeostasis in A. niger (Eur. J. Biochem. 269) 3487 Different bioenergetic states with different sugars For the yeast Candida tropicalis it was demonstrated by 31 P NMR that cells aerobically metabolizing glucose were more energized than xylose-fed cells [23]. Besides higher UDPG and polyphosphate levels and higher rates of P i assimilation, cells metabolizing glucose had a slightly higher pH cyt and a slightly lower pH vac . To investigate to what extent different nutritional conditions result in different steady-state intracellular pH values in A. niger, mycelium was perfused for 3 h with perfusion buffer pH 5.8 containing one of the following sugars: L -arabi- nose, D -xylose, D -fructose or D -glucose. In this sequence these sugars represent poor to good carbon sources for A. niger. The initial oxygen consumption (DO 2 )ofperfused biomass was 10–15% (using buffers saturated with O 2 ). No steady oxygen consumption was reached for any of the sugars tested during 3 h of data acquisition. Instead, a gradual increase in oxygen consumption was observed. In the absence of sugar, citrate was the only available carbon source, and under these circumstances the initial oxygen consumption was lower and remained constant throughout the experiment (8–9%). Catabolism of glucose, fructose and xylose resulted in a more pronounced cytoplasmic alkalinization compared to arabinose or citrate only, whereas a clearly stronger vacuolar acidification was observed only in the presence of glucose and fructose (Table 1). Although the differences between the deter- mined pH in values were only small, an increased oxygen consumption coincided with higher pH cyt and lower pH vac values. As inferred from the 31 P NMR spectra, however, no significant differences in ATP levels could be observed for the conditions tested (spectra not shown). Replacement ofcitratebya25m M Mes buffer (pH 5.8) resulted in similar pH values in the case of glucose (results not shown), indicating that the effect of citrate on pH in is only minor. HPLC analysis of perfusion buffer samples showed that no polyols or organic acids were excreted during 3 h of perfusion, ensuring constant extracellular conditions during data acquisition. Besides generating ATP and establishing pH gradients, an alternative way for the cells to store energy generated by catabolism may be polyphos- phate synthesis. The relative increase in polyphosphate levels during 3 h of perfusion appeared to coincide with increased pH gradients over the cytoplasmic and vacuolar membrane (Table 1). Younger mycelium (18–24 h old) contained hardly any polyphosphate (spectra not shown). The effect of CCCP on intracellular pH A strong argument in favour of Mitchell’s chemiosmotic theory [24] was the fact that it could explain the mode of action of lipid-soluble weak acids that are able to carry out electrogenic proton transport across biological and artificial membranes, thereby dissipating both the electrical mem- brane potential (DY) and the proton gradient (DpH). These uncouplers abolish the tight coupling of electron transport to oxidative phosphorylation and allow respiration to proceed without control by phosphorylation. The decreased ability of the cytoplasmic and vacuolar membrane to maintain transmembrane proton gradients in vivo in the presence of increasing extracellular concentrations of the uncoupler CCCP is shown in Fig. 2. It should be noted that after transfer of the cells to the perfusion system, pH in values (especially pH vac ) reached their steady-state values only after 1.5–2 h of perfusion. To shorten experimental times, CCCP (and azide, see below) were added before this time point, resulting in slightly different initial pH cyt and pH vac values in Figs 2 and 3. Addition of 1 l M CCCP to the perfusion buffer caused pH cyt to drop from 7.6 to 7.1 and pH vac to increase from 6.3 to 6.6 during the first 45 min after addition (Fig. 2A). At the same time, ATP and vacuolar phosphate levels decreased and cytoplasmic phosphate levels increased (spectra not shown). During this period oxygen consumption increased from approximately 9 to 21%. A complete collapse of the vacuolar membrane pH gradient did not occur. The cells started to recover 60– 75 min after the addition. At this point initial ATP levels were nearly restored. The absolute pH in values determined after recovery were slightly higher than initial values, in particular pH cyt . Consequently, the re-established pH gradient over the vacuolar membrane (1.5) was somewhat higher than before addition (1.3). In the presence of 2 l M CCCP essentially the same changes were observed as described for 1 l M CCCP. In this case, however, the vacuolar membrane pH gradient was completely dissipated (Fig. 2B). After 30 min no distinct P cyt or P vac could be recorded. Instead, both peaks had merged into one large intracellular phosphate resonance, corresponding to an intracellular pH value of 6.9. The cells reacted to the Table 1. Steady-state pH in values, increase in oxygen consumption and relative increase in polyphosphate levels during 3 h of perfusion in the presence of oxygen-saturated buffer containing 25 m M citrate pH 5.8, supplemented with 10 m M sugar. Sugar pH cyt pH vac Increase in O 2 consumption a [polyphosphate] b Glucose 7.76 ± 0.02 6.05 ± 0.04 24% 1.82 Fructose 7.73 ± 0.02 6.08 ± 0.02 21% 1.48 Xylose 7.72 ± 0.03 6.15 ± 0.04 17% 1.39 Arabinose 7.64 ± 0.02 6.19 ± 0.03 10% 1.31 — 7.58 ± 0.01 6.21 ± 0.02 0% 1.02 a Expressed as the percentage of oxygen removed from an oxygen-saturated buffer after passage through the immobilized cell plug. Initial oxygen consumption in the presence of sugar: 10–15%; in the presence of citrate only: 8–9%. b Relative increases in polyphosphate levels were determined from 31 P NMR spectra by relating the integral of the polyphosphate peak to the integral of the internal reference peak at t ¼ 0 and t ¼ 3h. 3488 S. J. A. Hesse et al. (Eur. J. Biochem. 269) Ó FEBS 2002 imposed stress condition once more by increasing their oxygen consumption from 9 to 30% within 60 min of addition. Surprisingly, 90 min after CCCP was added the large intracellular phosphate resonance split up again into two separate resonances: P cyt , shifting to the left (indicating alkalinization within that compartment) and P vac ,shifting to the right (indicating acidification within that compart- ment). Again, the onset of pH in recovery coincided with the (partial) restoration of original ATP levels (spectra not shown). With 10 l M CCCP, an irreversible collapse of the vacuolar membrane pH gradient was observed within 15 min (Fig. 2C). The intracellular pH, deduced from the chemical shift of a large intracellular phosphate resonance, became 6.7 and remained around that value during another 105 min of perfusion. The oxygen consumption increased during the first 5 min following the CCCP addition, but then rapidly dropped to 0%, indicating cell death. Con- comitantly, a rapid loss of ATP was observed, whereas UDPG, cofactor and polyphosphate levels gradually decreased during the 120 min that spectra were acquired. A complete collapse of the residual DpH across the cytoplasmic membrane (approximately 0.9 pH unit) did not occur, even when CCCP levels were doubled to 20 l M . The effect of azide on intracellular pH The inhibitory mode of action of azide on cellular ATP synthesis is twofold by inhibiting both the mitochondrial F 1 F 0 -ATPase and cytochrome-c-oxidase in the terminal part of the electron transport chain. The effect of transient and permanent depletion of ATP due to azide addition on pH in is shown in Fig. 3. In the former case (0.1 m M N 3 – ,Fig.3A)a transient decrease in pH cyt was observed with a minimal value of 7.0 after 30 min. In contrast with CCCP, the drop in pH cyt was paralleled by a decrease in pH vac from 6.3 to 5.9. Spectra showed that ATP levels dropped sharply, whereas both cytoplasmic and vacuolar phosphate levels were slightly higher compared to the original situation (Fig. 4A and B). At the same time, cells increased their oxygen consumption from 9 to 23%. Interestingly, the observed rise in both pH cyt and pH vac after 45 min was accompanied by a small increase in ATP level (Fig. 4C). In the new steady-state, pH in values were identical to those observed before the addition of the inhibitor, although ATP and UDPG levels were somewhat lower (Fig. 4D). Polyphosphate levels remained unchanged during 2.5 h of perfusion. Cells lost all of their ATP permanently when 0.5 m M azide was used. Moreover, an effective and permanent dissipation of the vacuolar mem- brane pH gradient was observed (Fig. 3B), visualized in the spectra as one large intracellular phosphate resonance (data not shown). Lethal azide concentrations were much more effective in dissipating the pH gradient across the cytoplasmic membrane than lethal doses of uncoupler. The residual pH gradient observed 2 h after azide addition (0.5 m M ) was 0.3– 0.4 pH unit, which is considerably lower than when CCCP was used (0.9 pH unit). Eventually, pH in became equal to Fig. 2. The effect of CCCP addition on cytoplasmic pH (pH cyt ) and vacuolar pH (pH vac ) in immobilized A. niger NW131 mycelium, grown for 42 h and perfused with oxygen-saturated buffer containing 25 m M citrate pH 5.8. CCCP was added at t ¼ 0, and the applied concen- trations were 1 l M (A), 2 l M (B) and 10 l M (C). Addition occurred before pH cyt and pH vac reached steady-state values, resulting in slightly different initial pH values (see text). Data points represent the mean of two experiments. Ó FEBS 2002 Intracellular pH homeostasis in A. niger (Eur. J. Biochem. 269) 3489 pH ex after 8 h of perfusion. During this whole period only a very small breakdown of polyphosphate could be observed. The bioenergetic state of citric acid producing mycelium A. niger has the capacity to produce high levels of citric acid from hexoses and disaccharides in traditional citric acid producing processes when two important criteria are met [25]: a low pH (< 2) and absence of manganese ions (Mn 2+ ). Low pH is necessary to avoid production of gluconic acid and oxalic acid. In our experiments interfer- ence by gluconic acid production was prevented by using an A. niger N400 derivative, strain NW131, lacking glucose oxidase activity. The amount of citric acid accumulated from glucose in 7 days by immobilized biomass in a bubble column reactor was approximately 50 gÆL )1 .Inatypical fermentation the pH dropped from 3.5 to 1.8 in  3days, and remained around that value. The cells consumed all NH 4 + and PO 4 3– during the first 24 h (data not shown), and no citrate was produced in this period. Dry weight determinations of 2- and 7-day-old cultures indicated a small decrease in biomass content during the fermentation (17.4 and 16.1 gÆL )1 , respectively). It was decided to Fig. 3. The effect of sodium azide addition on cytoplasmic pH (pH cyt ) and vacuolar pH (pH vac ) in immobilized A. niger NW131 mycelium, grown for 42 h and perfused with oxygen-saturated buffer containing 25 m M citrate pH 5.8. Azide was added at t ¼ 0, and the applied concentrations were 0.1 m M (A) and 0.5 m M (B). Addition occurred before pH cyt and pH vac reached steady-state values, resulting in slightly different initial values. Data points represent the mean of two experiments. Fig. 4. 31 P NMR spectra of immobilized A. niger NW131 mycelium in the presence of 25 m M citrate pH 5.8, showing the effect of addition of 0.1 m M sodium azide. (A) The situation before addition. P cyt ,cyto- plasmic inorganic phosphate resonance; P vac , vacuolar inorganic phosphate resonance. The internal reference is not shown. (B) After 30 min both P cyt and P vac had moved to a lower chemical shift (i.e. compartmental acidification), accompanied by a sharp decrease in intensities of the c-ATP and the a-ATP peaks as indicated by the arrows. (C) After 45 min ATP levels started to restore again, and P cyt and P vac moved to a higher chemical shift again (i.e. compartmental alkalinization). (D) In the new steady-state (t ¼ 120 min) compart- mental pH values were identical to those observed before addition. See Fig. 3A for corresponding pH values. 3490 S. J. A. Hesse et al. (Eur. J. Biochem. 269) Ó FEBS 2002 investigate a younger (2-day old) and an older (7-day old) culture. To mimic citric acid production conditions as closely as possible, immobilized cell mass was perfused with filtered medium of 2-day- and 7-day-old cultures, respect- ively, saturated with oxygen. The specific citric acid production rates of mycelium after transfer to the perfusion set up were 0.20 gÆLh )1 (2-day-old mycelium) and 0.32 gÆLh )1 (7-day-old mycelium), whereas in the bioreactor values of 0.37 gÆLh )1 (2-day-old mycelium) and 0.19 gÆLh )1 (7-day-old mycelium) were obtained. The values obtained for perfused mycelium confirmed that spectra were acquired under true citric acid producing conditions, although transfer to the perfusion system appeared to have an effect upon specific production rates. 31 P NMR spectra of 2- and 7-day-old-citric acid-producing mycelium showed a remark- able constancy with respect to metabolite levels (including polyphosphate) and pH in values during the first 4–6 h of perfusion (spectra not shown). For correct assignment of P cyt and P vac ,CCCP(2l M ) was used to partially collapse the DpH between these two compartments. No difference could be detected between steady-state pH cyt values of 2- and 7-day-old mycelium (7.53 ± 0.05 and 7.54 ± 0.04, respectively) although cytoplasmic phosphate, sugar phos- phate and ATP levels were clearly higher in 2-day-old mycelium. Compared to pH cyt values obtained in the presence of various carbon sources (Table 1) or under extreme extracellular acidity (Fig. 1A, pH ex 1.5–2.0), citric acid-producing mycelium appeared to have only a slightly more acidic cytoplasm. The vacuoles of 2-day-old mycelium were relatively alkaline (pH 6.41 ± 0.03), whereas an even higher pH vac was found in vacuoles of 7-day-old mycelium (pH 6.50 ± 0.04). DISCUSSION The proper functioning of cells relies on maintenance of their intracellular pH within relatively narrow limits, as large deviations of pH from normal values would be severely inhibitory to metabolism based on pH optima of cytoplasmic enzymes [1]. Our results show that A. niger is indeed capable of tightly maintaining its intracellular pH values within a narrow range. In the presence of various carbon sources at pH ex 5.8, pH cyt varied only from 7.58 (citrate) to 7.76 (glucose), whereas pH vac ranged from 6.05 (glucose) to 6.19 (citrate) (Table 1). As no significant differences in ATP levels could be observed in the 31 P spectra, an energetically more favourable carbon source may lead to a larger availability of ATP to the P-ATPase and V-ATPase due to increased ATP turnover, reflected by a higher oxygen consumption and larger pH gradients. Of the carbon sources tested, glucose has the largest carbon catabolite repressing effect in A. niger,andcitratethe lowest (glucose > fructose > xylose > arabinose > cit- rate; G. J. G. Ruijter, Wageningen, the Netherlands, personal communication). Thus, the carbon catabolite repressing capacity of the carbon source could be linked to steady-state DpH values. Interestingly, larger pH gradients and a higher oxygen consumption coincided with increased polyphosphate syn- thesis in A. niger (Table 1). Polyphosphate has been suggested to function as a cellular phosphate or high-energy reserve. Although polyphosphate may have additional functions (e.g. chelation of cations, or a pH-homeostatic function as sequestrator or donor of protons), its exact physiological role is not yet fully understood. Our results showed that with increased cellular energy levels polyphos- phate accumulated to higher levels, suggesting a role in cellular energy storage for the polymer. Recent studies on E. coli cells revealed a more regulatory role for polyphos- phate [26]. Cells deficient in polyphosphate were unable to express many genes that are needed for adaptation to deficiencies and environmental stresses during the stationary phase, and lost their viability relatively quickly. Increased polyphosphate synthesis may therefore greatly enhance the chances of survival in stationary phase cells. If so, it is crucial for cells to accumulate as much polyphosphate as possible in times of energy excess. Our results are in agreement with this hypothesis as polyphosphate accumu- lation was maximal in the presence of glucose at pH ex 5.8. In the presence of a poor carbon source at the same pH ex (citrate), no accumulation of polyphosphate was observed. In the presence of glucose at pH ex 1.0, no increase in polyphosphate levels occurred either (results not shown). A key role for polyphosphate in stationary phase cells is further corroborated by the fact that polyphosphate was practically absent in younger (18–24-h-old) mycelium. In N. crassa, the ratio of polyphosphate to orthophosphate in vacuoles increased from 2.4 in early log phase cells to 13.5 in stationary phase cells [15]. When early log phase cells were exposed to a hypo-osmotic shock, both pH cyt and pH vac increased and cells lost 95% of their total polyphos- phate content. In contrast, hypo-osmotic shock of station- ary phase cells did not cause any changes in intracellular pH or polyphosphate levels, showing that these cells were much more effective in handling osmotic stress. A striking observation was the ability of A. niger to maintain constant intracellular pH values during extracel- lular acidification to pH values as low as 1.0 without having to change its steady-state oxygen consumption. In yeast, filamentous fungi and higher plant cells, the proton pumping activity of the plasma membrane P-ATPase has been recognized as the major factor responsible for pH cyt homeostasis [27–29], possibly in combination with high affinity potassium uptake in symport with protons. Oper- ating in parallel in N. crassa, these two systems yield a net 1 : 1 exchange of K + for cytoplasmic H + [6]. In N. crassa, changes of pH ex between 3.9 and 9.3 affect pH cyt linearly with a slope of approximately 0.1 unit pH cyt per unit pH ex [1]. In S. cerevisiae, both pH cyt and pH vac became more acidic at pH ex 3.5 compared with pH ex 6.5 whether glucose was present or not [30]. Intracellular pH homeostasis in respiring Escherichia coli cells was good (pH cyt 7.6 ± 0.2) over a pH ex range of about 5.5–9.0 [31]. Finally, in sycamore (Acer pseudoplatanus L) cells pH cyt and pH vac values were maintained when pH ex was varied from 4.5 to 7.5 [29]. Oxygen consumption measurements of these cells in a perfusion setup revealed a progressive acceleration of the rate of O 2 consumption towards the uncoupled O 2 uptake rate (+1l M FCCP) as pH ex decreased from 6.5 to 4.5. Our results clearly show that after addition of CCCP (2 l M , pH ex 5.8) a much higher oxygen consumption could be achieved (DO 2 ¼ 25–30%) than the steady-state oxygen consumption (DO 2 ¼ 8–9%) observed in the presence of citrate only (25 m M ,pH ex 1.0–7.0). Apparently A. niger can maintain its intracellular pH while keeping its oxygen uptake far from the uncoupled value. An obvious Ó FEBS 2002 Intracellular pH homeostasis in A. niger (Eur. J. Biochem. 269) 3491 explanation for this high tolerance towards extreme extracellular acidity would be to contribute this behaviour to plasma membranes with an unusual lipid composition, rendering them highly impermeable to protons. For acido- philic prokaryotes (both bacteria and archaea) it has been shown that a link exists between the lipid composition of their plasma membranes and an acidophilic mode of existence [32]. The proton permeability (P, cmÆs )1 )in biological membranes has been found to be extremely pH dependent, with values ranging from 10 )3 to 10 )6 cmÆs )1 [33]. Based on results obtained by Sanders and Slayman [1], Burgstaller argued that the proton permeability of N. crassa plasma membranes is probably much lower than 10 )3 to explain their results [33]. Using lipid bilayer membranes composed of bacterial phosphatidylethanolamine, Gutkn- echt found a 10 6 times lower P at pH 2 compared to pH 7, indicating much lower values for P at low pH [34]. Using a value for P of 10 )6 cmÆs )1 for A. niger at pH ex 1.5, and assuming hydrolysis of 1 ATP/H + expelled by the P- ATPase, the ATP requirement to maintain a pH cyt value of 7.6 can be calculated from the passive proton flux J H + (molÆgdw )1 Æs )1 ) ¼ PÆAÆD[H + ]. For A (m 2 Ægdw )1 ), a value of 2.9 has been found for A. niger NW131 (B. R. Poulsen, Department of Microbiology, Fungal Genomics Section, Wageningen University, the Netherlands, personal commu- nication). Using these values the ATP turnover for pH homeostasis is 10 )6 molÆgdw )1 Æs )1 . This value is within the same range of ATP turnover necessary for cellular main- tenance (2 · 10 )6 molÆgdw )1 Æs )1 ), assuming a maintenance coefficient m of 0.034 g glucoseÆgdw )1 Æh )1 and 38 mol ATP formed per mole glucose (B. R. Poulsen, personal commu- nication). Although the exact value for P in fungal membranes at low pH is not known, these values suggest that with a low intrinsic proton permeability the energy costs to maintain such large DpHs are relatively low. This means that physical protection by the cytoplasmic mem- brane alone may be sufficient to keep pH cyt close to neutrality in an extremely acidic environment. If we assume DY to be 0 mV, then the proton motive force (Dp) generated at pH ex 1.0 would be around )400 mV (Dp ¼ DY ) ZDpH). This is near the theoretical limit if we assume the maximal amount of energy available to the P-ATPase to result from ATP hydrolysis and if a stoichiometry of 1 H + expelled per ATP hydrolysed is assumed [35]. Compared to the pH-homeostatic properties of the organisms mentioned above, A. niger behaves like a typical acidophile. So far, bacteria and archaea have been the main focus of studies on intracellular pH homeostasis in acido- philes. Comparable to A. niger, the acidophile Thiobacillus ferrooxidans is capable of maintaining its intracellular pH constant at a value of 6.5 over a range of pH ex from 1.0 to 8.0 [36]. Acidophiles are able to sustain such large cytoplasmic DpH values by counteracting the large inwardly directed H + gradient with a positive-inside DY that results from Donnan or H + diffusion potentials [37]. Thus, the proton motive force across the cytoplasmic membrane is reduced in order to decrease the proton leak into the cells and to decrease the back-pressure for H + extrusion by the P-ATPase. In this way H + influx eventually becomes self- limiting with an increasing positive-inside DY.AK + /H + symport uptake system operating in parallel with the P-ATPase, combined with a low intrinsic cation permeab- ility of the plasma membrane, offers the acidophile a possibility to generate and sustain a positive-inside DY. Indications for such a mechanism have been reported for two acidophilic eukaryotes: the alga Dunaliella acidophila and the yeast Metschnikowia reukaufii [38]. Whether A. niger relies on generation of a positive-inside DY, a low plasma membrane proton permeability at low pH, or a combination of both, still remains to be investigated. The capacity of the various energy-transducing mem- branes to maintain proton gradients appeared to be quite different. CCCP was much more efficient in dissipating DpH across the vacuolar membrane than across the cytoplasmic membrane, a finding that had already been reported for S. cerevisiae [39]. This could mean that the ability of the V-ATPase to be stimulated by increased proton leak is rather poor. ATP levels (and pH gradients) could be maintained or restored as long as the respiratory rate could still be stimulated after uncoupler addition, even when a temporary collapse of the vacuolar membrane DpH occurred. A complete collapse of DpH across the cytoplas- mic membrane in the presence of lethal CCCP levels (10–20 l M ) could not be observed. A similar result has been reported for S. cerevisiae [39], and the authors suggested that besides ATP-dependent ion pumps an additional role in pH homeostasis may be reserved for the cytoplasmic buffering capacity. However, Sanders and Slayman [1] have clearly shown that a large involvement of the cytoplasmic buffering capacity in intracellular pH homeostasis is not very likely. A more obvious explanation would be that, although true uncouplers are able to reduce the H + electrochemical potential difference across a membrane to zero when the concentration applied is high enough, the pH difference can only be dissipated if the charge imbalance as a result of the H + transport is compensated by the movement of other ions [40]. Hence, if the permeability of the A. niger plasma membrane for other ions is low, a lack of compensating charge fluxes could account for the observed residual DpH. Lethal levels of azide resulted in a smaller residual cytoplasmic membrane DpH than lethal levels of CCCP (0.3 and 0.9 pH units, respectively). Besides cytoplasmic acidification due to specific inhibition of ATP synthesis, azide has been suggested to have additional uncoupling abilities [41]. This combined effect may account for the observed difference. However, as lethal doses of CCCP also lead to a complete loss of ATP, it is more tempting to speculate that azide, upon uptake into the cell, is able to alter ion conductivity in the cytoplasmic membrane. As a consequence, larger compensating ion fluxes may occur that allow a larger dissipation of cytoplasmic membrane DpH. Indeed, azide was found to have a specific effect on ion transport (probably K + /H + exchange) in plasma membranes of S. cerevisiae [42]. In the presence of nonlethal azide concentrations, chan- ges in pH vac followed a course that was similar to pH cyt . Vacuolar H + influx in response to increased cytoplasmic H + levels indicates a role in pH cyt homeostasis for this organelle. These results are in accordance with observations made in S. cerevisiae [30] and in higher plant cells (Acer pseudoplatanus L.) [29]. The observed recovery of A. niger from nonlethal CCCP levels, even under conditions of complete vacuolar mem- brane DpH dissipation, may be attributed to induced expression of membrane-bound ATP-dependent transpor- 3492 S. J. A. Hesse et al. (Eur. J. Biochem. 269) Ó FEBS 2002 ters of the ATP-binding cassette superfamily involved in multidrug resistance. Transcription of two ATP-binding cassette transporter encoding genes (atrAandatrB) in A. nidulans was shown to be enhanced within a few minutes of treatment with several drugs, including antibio- tics, azole fungicides and plant defence toxins [43]. A subsequent energy-dependent drug efflux activity may also confer such resistance to A. niger after CCCP addition, provided that a certain minimal ATP level can be maintained within the cell. Industrial-scale production of citric acid is performed mainly by fermentation processes using A. niger [12]. Previously reported values for pH cyt in citric acid producing A. niger mycelium vary from 6.5 [13] to 6.8 [14]. We report here a considerable higher pH cyt of 7.5 (in both 2- and 7-day-old-mycelium) under fully oxygenated conditions. The low pH cyt values reported earlier probably resulted from difficulties to keep the mycelium well-energized during spectra acquisition. With metabolic engineering and mod- elling studies as a promising approach to improve produc- tivity [44], a detailed description of pH in during citric acid production provides valuable additional information about the conditions under which the enzymes involved operate in vivo. To reach a higher degree of accuracy for Aspergillus kinetic models, more information on the internal metabolic changes that take place during the transition to citric acid producing mycelium is needed in the future. 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