Perfusion in the presence of various hexoses and pentoses pHex5.8 revealed that the magnitude of DpH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catab
Trang 1Intracellular pH homeostasis in the filamentous fungus
Aspergillus niger
Stephan J A Hesse1, George J G Ruijter2, Cor Dijkema1and Jaap Visser2,†
1
Department of Biophysics, Wageningen University, the Netherlands;2Department of Microbiology, section Fungal Genomics, Wageningen University, the Netherlands
Intracellular pH homeostasis in the filamentous fungus
Aspergillus nigerwas measured in real time by31P NMR
during perfusion in the NMR tube of fungal biomass
immobilized in Ca2+-alginate beads The fungus maintained
constant cytoplasmic pH (pHcyt) and vacuolar pH (pHvac)
values of 7.6 and 6.2, respectively, when the extracellular pH
(pHex) 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
(pHex0.7–0.8) Maintenance of these large pH differences
was possible without increased respiration compared to
pHex5.8 Perfusion in the presence of various hexoses and
pentoses (pHex5.8) 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 lMCCCP), transient (2 lMCCCP) or perma-nent (10 lM CCCP) collapse of the vacuolar membrane DpH Nonlethal levels of the metabolic inhibitor azide (N3, 0.1 mM) caused a transient decrease in pHcytthat 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,31P NMR spectra of citric acid producing A.niger mycelium showed that despite a combination of low pHex(1.8) and a high acid-secreting capacity, pHcyt and pHvac values were still well maintained (pH 7.5 and 6.4, respectively)
Keywords: Aspergillus niger; intracellular pH; pH homeo-stasis;31P 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,
CO2/H2CO3and 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 (pHcyt) 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 (pHin) 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
F1F0-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
F1F0-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 (pHex), reveals clues to mechanisms of pH regulation [9] In a previous study we reported on a system based on long-term acquisition of31P 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)
Trang 2large-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 pHex) 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 pHex,
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
E X P E R I M E N T A L P R O C E D U R E S
Strain, immobilization of conidia and culture conditions
Condiospores of A.niger NW131 (cspA1 goxC17) lacking
glucose oxidase activity [17] were propagated at 30Con
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 Ca2+-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 30Cin 100 mL minimal medium (2 gÆL)1NH4NO3,
1.5 gÆL)1KH2PO4, 0.5 gÆL)1KCl, 0.5 gÆL)1MgSO4Æ7H2O
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 KH2PO4, 1.25 gÆL)1 (NH4)2SO4, 0.25 gÆL)1 MgSO4Æ7H2O, 1.3 mgÆL)1 ZnSO4Æ 7H2O, 6.5 mgÆL)1FeSO4Æ7H2O) 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)1air, 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
30C 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 (30C) containing 25 mM
sodium citrate pH 5.8, 0.25 gÆL)1NH4NO3, 0.2 gÆL)1KCl, 0.2 gÆL)1MgSO4Æ7H2O, 0.2 mMKH2PO4, 0.3 mM CaCl2, 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 2MHCl The same buffer supplemented with 50 mM
Tris was used under alkaline extracellular conditions (pHex, 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 mMof 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 (NaN3) were directly added to the buffer reservoir (pH 5.8) from a 10-mMstock solution in ethanol and a 10% stock solution in water, respectively Oxygen consumption during perfusion (DO2) 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
a rate of 15 mLÆmin)1
31P NMR spectroscopy
31P NMR spectra were recorded at 121.5 MHz at 30Con
a AMX300 wide-bore spectrometer (Bruker, Germany), equipped with a 20-mm switchable31P/13Cprobe tuned at the31P 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 (0.2M, 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% H3PO4(0 p.p.m)
Analyses Cytoplasmic and vacuolar pH values were determined by comparing the pH-sensitive chemical shifts of cytoplasmic
Trang 3(Pcyt) and vacuolar inorganic phosphate (Pvac) with a
calibration curve for inorganic phosphate (Pi) 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 Pimeasured in a medium made up to
approximate concentrations of the major cationic
compo-nents in yeast [22] The perfusion buffer pH (pHex) 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 (a¼ 5%) Relative
increases in polyphosphate levels were determined from31P
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 HPLCusing
an HPX-87H column (Bio-Rad) eluted with 25 mMHCl at
50Cwith UV (210 nm) and refractive index detection, and
for polyols using a Carbopac MA-1 column (Dionex) eluted
with 480 mM NaOH at 20Cwith pulsed amperometric
detection Dry weight determinations on immobilized
biomass were carried out by dissolving the Ca2+-alginate
beads in a 100-mMsolution of the Ca2+-scavenger sodium
hexametaphosphate (Fluka AG, Buchs, Switzerland)
Mycelium was then collected by filtration, washed with
demineralized water, frozen in liquid nitrogen, lyophilized
and weighed
R E S U L T S
The dependence of intracellular pH values on ambient pH A.nigerhas the ability to acidify its environment to values
as low as pH 1.5 [17] To investigate to what extent extracellular pH (pHex) affects intracellular pH (pHin) values, we determined pHcyt and pHvacas a function of ambient pH using31P NMR as described previously [10] Surprisingly, the cells were able to maintain pHcytand pHvac
at 7.6 and 6.2, respectively, when pHexwas 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 pHex1.5, this gradient was about 0.1 pH unit larger due to a slightly more acidic vacuole Further acidification to pHex 1.0 caused
pHcyt to drop to pH 7.4, in parallel to an even larger vacuolar acidification than at pHex 1.5 A comparable observation was made at pHex8.0: pHcytwas still reason-ably well regulated, whereas the vacuoles became more alkaline (pHvac6.5) Interestingly, in the pHexrange of 1.0– 7.0 in the presence of 25 mM citrate, cells consumed the same amount of oxygen once a steady-state was reached (DO2¼ 8–9%) Cellular metabolism collapsed and O2
consumption rapidly dropped to 0% when pHexreached a value of about 0.7–0.8 Cells only moderately increased their oxygen consumption (from 9 to 11%) just before the collapse A representative31P NMR spectrum of immobi-lized A.niger mycelium perfused in the presence of 25 mM
citrate pH 1.5 and 0.2 mMPiis shown in Fig 1B
Fig 1 The dependence of A niger NW131 cytoplasmic pH (pH cyt ) and vacuolarpH (pH vac ) on extracellular pH (pH ex ) in the presence of oxygen-saturated buffer (A) and typical31P 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.
Trang 4Different bioenergetic states with different sugars
For the yeast Candida tropicalis it was demonstrated by31P
NMR that cells aerobically metabolizing glucose were
more energized than xylose-fed cells [23] Besides higher
UDPG and polyphosphate levels and higher rates of Pi
assimilation, cells metabolizing glucose had a slightly
higher pHcytand a slightly lower pHvac 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 (DO2) of perfused
biomass was 10–15% (using buffers saturated with O2)
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 pHin values were only small, an increased oxygen
consumption coincided with higher pHcytand lower pHvac
values As inferred from the31P NMR spectra, however,
no significant differences in ATP levels could be observed
for the conditions tested (spectra not shown) Replacement
of citrate by a 25 mM 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 pHinis only
minor HPLCanalysis 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 polyphospolyphos-phate
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, pHinvalues (especially pHvac) 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 pHcytand pHvac values in Figs 2 and 3 Addition of 1 lM CCCP to the perfusion buffer caused pHcytto drop from 7.6 to 7.1 and
pHvacto 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 pHinvalues determined after recovery were slightly higher than initial values, in particular pHcyt 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 lM
CCCP essentially the same changes were observed as described for 1 lM CCCP In this case, however, the vacuolar membrane pH gradient was completely dissipated (Fig 2B) After 30 min no distinct Pcyt or Pvac 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.
Increase in
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%.bRelative 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 3 h.
Trang 5imposed 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: Pcyt, shifting to the left (indicating
alkalinization within that compartment) and Pvac, shifting
to the right (indicating acidification within that
compart-ment) Again, the onset of pHinrecovery coincided with the
(partial) restoration of original ATP levels (spectra not
shown) With 10 lMCCCP, 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 lM
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
F1F0-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 pHinis
shown in Fig 3 In the former case (0.1 mMN3, Fig 3A) a
transient decrease in pHcyt was observed with a minimal
value of 7.0 after 30 min In contrast with CCCP, the drop in
pHcytwas paralleled by a decrease in pHvacfrom 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 pHcytand
pHvacafter 45 min was accompanied by a small increase in
ATP level (Fig 4C) In the new steady-state, pHin 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 mM 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 mM) was 0.3– 0.4 pH unit, which is considerably lower than when CCCP was used (0.9 pH unit) Eventually, pHinbecame equal to
Fig 2 The effect of CCCP addition on cytoplasmic pH (pH cyt ) and
vacuolarpH (pH vac ) in immobilized A niger NW131 mycelium, grown
for42 h and perfused with oxygen-saturated buffercontaining 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.
Trang 6pHexafter 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.nigerhas 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
(Mn2+) 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 In a typical fermentation the pH dropped from 3.5 to 1.8 in 3 days, and remained around that value The cells consumed all
NH4+and PO43–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 vacuolarpH (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.
Trang 7investigate 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.31P 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 pHinvalues during the first 4–6 h of
perfusion (spectra not shown) For correct assignment of
Pcytand Pvac, C C C P (2 lM) was used to partially collapse
the DpH between these two compartments No difference
could be detected between steady-state pHcyt 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 pHcyt values obtained in the
presence of various carbon sources (Table 1) or under
extreme extracellular acidity (Fig 1A, pHex1.5–2.0), 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 pHvacwas found in vacuoles of 7-day-old mycelium
(pH 6.50 ± 0.04)
D I S C U S S I O N
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 pHex5.8, pHcytvaried only from 7.58
(citrate) to 7.76 (glucose), whereas pHvacranged from 6.05
(glucose) to 6.19 (citrate) (Table 1) As no significant
differences in ATP levels could be observed in the 31P
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, and citrate the
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.colicells revealed a more regulatory role for polyphos-phate [26] Cells deficient in polyphospolyphos-phate 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 pHex5.8 In the presence of a poor carbon source at the same pHex (citrate), no accumulation of polyphosphate was observed
In the presence of glucose at pHex 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 pHcyt and
pHvacincreased 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 pHcyt 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 pHexbetween 3.9 and 9.3 affect pHcytlinearly with a slope of approximately 0.1 unit pHcytper unit pHex [1] In S.cerevisiae, both pHcyt and pHvacbecame more acidic at pHex3.5 compared with pHex6.5 whether glucose was present or not [30] Intracellular pH homeostasis in respiring Escherichia coli cells was good (pHcyt7.6 ± 0.2) over a pHexrange of about 5.5–9.0 [31] Finally, in sycamore (Acer pseudoplatanus L) cells pHcytand pHvacvalues were maintained when pHex 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 O2consumption towards the uncoupled O2uptake rate (+ 1 lMFCCP) as pHexdecreased from 6.5 to 4.5 Our results clearly show that after addition of CCCP (2 lM,
pHex 5.8) a much higher oxygen consumption could be achieved (DO2¼ 25–30%) than the steady-state oxygen consumption (DO2¼ 8–9%) observed in the presence of citrate only (25 mM, pHex1.0–7.0) Apparently A.niger can maintain its intracellular pH while keeping its oxygen uptake far from the uncoupled value An obvious
Trang 8explanation 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)3to 10)6cmÆs)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)3to
explain their results [33] Using lipid bilayer membranes
composed of bacterial phosphatidylethanolamine,
Gutkn-echt found a 106times 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)6cmÆs)1 for A.niger at pHex 1.5, and
assuming hydrolysis of 1 ATP/H+ expelled by the
P-ATPase, the ATP requirement to maintain a pHcytvalue of
7.6 can be calculated from the passive proton flux JH+
(molÆg dw)1Æs)1)¼ PÆAÆD[H+] For A (m2Æg dw)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)6molÆg dw)1Æs)1 This value is within the
same range of ATP turnover necessary for cellular
main-tenance (2· 10)6molÆg dw)1Æs)1), assuming a maintenance
coefficient m of 0.034 g glucoseÆg dw)1Æh)1and 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 pHcyt close to
neutrality in an extremely acidic environment If we assume
DY to be 0 mV, then the proton motive force (Dp) generated
at pHex 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
ferrooxidansis capable of maintaining its intracellular pH
constant at a value of 6.5 over a range of pHexfrom 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 A K+/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 lM) 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 pHvacfollowed a course that was similar to pHcyt Vacuolar H+influx in response to increased cytoplasmic
H+ levels indicates a role in pHcyt homeostasis for this organelle These results are in accordance with observations made in S.cerevisiae [30] and in higher plant cells (Acer pseudoplatanusL.) [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
Trang 9transpor-ters of the ATP-binding cassette superfamily involved in
multidrug resistance Transcription of two ATP-binding
cassette transporter encoding genes (atrA and atrB) 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 pHcytin citric acid producing
A.nigermycelium vary from 6.5 [13] to 6.8 [14] We report
here a considerable higher pHcyt of 7.5 (in both 2- and
7-day-old-mycelium) under fully oxygenated conditions
The low pHcyt 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 pHinduring 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 Our results
have shown that, at least with respect to intracellular pH,
these changes are minor: the combination of low pHexand a
high acid-secreting capacity only led to a slightly lower
cytoplasmic pH
A C K N O W L E D G E M E N T S
The authors acknowledge financial support from the ECin the
framework of the Eurofung Cell Factory project (QTRK3-1999–
00729).
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