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Transport of amino acids and ammonium in mycelium of Agaricus bisporus
Biochimica et Biophysica Acta 1428 (1999) 260^272 www.elsevier.com/locate/bba
Transport of amino acids and ammonium in mycelium of Agaricus bisporus Monique A.S.H. Kersten a; *, Michel J.C. Arninkhof a , Huub J.M. Op den Camp a , Leo J.L.D. Van Griensven b , Chris van der Drift a a
Department of Microbiology and Evolutionary Biology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands b Mushroom Experimental Station, P.O. Box 6042, NL-5960 AA Horst, The Netherlands Received 31 March 1999; received in revised form 25 May 1999; accepted 3 June 1999
Abstract Mycelium of Agaricus bisporus took up methylamine (MA), glutamate, glutamine and arginine by high-affinity transport systems following Michaelis^Menten kinetics. The activities of these systems were influenced by the nitrogen source used for mycelial growth. Moreover, MA, glutamate and glutamine uptakes were derepressed by nitrogen starvation, whereas arginine uptake was repressed. The two ammonium-specific transport systems with different affinities and capacities were inhibited by NH 4 , with a Ki of 3.7 WM for the high-velocity system. The Km values for glutamate, glutamine and arginine transport were 124, 151 and 32 WM, respectively. Inhibition of arginine uptake by lysine and histidine showed that they are competitive inhibitors. MA, glutamate and glutamine uptake was inversely proportional to the intracellular NH 4 concentration. Moreover, increase of the intracellular NH 4 level caused by PPT (DL-phosphinotricin) resulted in an immediate cessation of MA, glutamine and glutamate uptake. It seems that the intracellular NH 4 concentration regulates its own influx by feedback-inhibition of the uptake system and probably also its efflux which becomes apparent when mycelium is grown on protein. Addition of extracellular NH 4 did not inhibit glutamine uptake, suggesting that NH4 and glutamine are equally preferred nitrogen sources. The physiological importance of these uptake systems for the utilization of nitrogen compounds by A. bisporus is discussed. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Arginine; Glutamate; Glutamine; Methylamine ; Transport; (Agaricus bisporus)
1. Introduction
Abbreviations: MA, methylamine; DNP, 2,4-dinitrophenol; GS, glutamine synthetase; NADP-GDH, NADP-dependent glutamate dehydrogenase; NAD-GDH, NAD-dependent glutamate dehydrogenase; GOGAT, glutamate synthase; CE, compost extract ; AAM, amino acid mixture; MES, 2-(N-morpholino)ethanesulfonic acid; CEA, compost extract agar; PPT, DL-phosphinotricin ; AZS, azaserine; MSX, L-methionine-S-sulfoximine * Corresponding author. Fax: +31-24-355-3450; E-mail: [email protected]
Agaricus bisporus, the white button mushroom, is commercially grown in large amounts on a substrate which consists of a fermented mixture of horse manure, wheat straw, chicken manure and gypsum [1]. Through a two-phase composting process a selective substrate is obtained which is rich in organic sources of nitrogen and low in free NH3 , the latter being inhibitory to growth of A. bisporus. Although the basic nutritional requirements of A. bisporus are es-
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 0 9 3 - 8
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sentially known [2] and the composted substrate can easily ful¢l the demands, yields of mushrooms can be increased substantially by adding products with high protein contents to the colonized compost [3]. Since the yield increase is due to the protein added, amino acids have to play an important role in mushroom nutrition. Nutrient acquisition is a highly regulated process that includes digestion, transport, and subsequent metabolism. With regard to nitrogen metabolism in A. bisporus (reviewed by Baars et al. [4]), the main function of the catabolic enzymes is to produce small solute molecules that can be transported across the cellular membrane. Under laboratory conditions growth of A. bisporus is supported by a number of single amino acids or NH 4 as a single N source and glucose as C source [5]. A. bisporus also grew readily on protein as sole source of C and N [6]. In contrast to the repressing e¡ect of NH 4 on the use of alternative N sources in many yeasts and fungi [7,8], utilization of protein in A. bisporus appeared not to be a¡ected by the addition of NH 4 to the medium [9]. Amino acid transport in fungi has been studied most extensively in Aspergillus nidulans, Neurospora crassa, Penicillium chrysogenum and Saccharomyces cerevisiae, and appears to represent a compromise between bacterial transport systems, with speci¢cities for individual amino acids, and animal cell systems, with broad speci¢cities for classes of structurally related amino acids [10^13]. Furthermore, di¡erent transport systems are expressed at di¡erent stages of development of the fungi and under di¡erent environmental conditions. In contrast to the analogous systems in bacterial and animal cells, the transport systems in fungi generally mediate only unidirectional £uxes from the external media into the cell and their activities are regulated by transinhibition [14]. Amino acid transport of fungi is an active process which occurs via H symport, a universal mechanism for coupling transport to ATP hydrolysis, which is required for proton expulsion [15]. As is the case for amino acids, NH 4 ions are transported actively across the cellular membrane, most likely via NH 4 uniport [16,17]. The most convenient method for measuring NH transport is with 4 14 [ C]methylamine, an NH4 analogue which is transported by the same system. Kleiner [16] describes transport systems with very high a¤nity for NH 4
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in P. chrysogenum (Km 0.25 WM), A. nidulans (Km 2.8 WM), N. crassa (Km 7 WM) and S. cerevisiae (Km 1 WM). Nonspeci¢c di¡usion of NH3 was only observed when NH 4 transport systems were repressed. Furthermore, the neutral molecule NH3 is a weak base and protonates rapidly to yield NH 4 . At the near neutral pH more than 99% of the total ammonia will be in the protonated form. Next to exogenous NH 4 , ammonium ions are also produced intracellularly as a result of the turn-over of cellular components. Once inside the cell, NH 4 can be incorporated into the amino acids glutamate and glutamine. The enzymes glutamine synthetase (GS; EC 6.3.1.2) [18], glutamate synthase (GOGAT; EC 1.4.7.1) [5], NADP -glutamate dehydrogenase (NADP-GDH; EC 1.4.1.4) [19] and NAD -glutamate dehydrogenase (NAD-GDH; EC 1.4.1.2) [20] are all primarily involved in nitrogen metabolism in A. bisporus and at least GS is directly involved in NH 4 assimilation [21]. Puri¢cation and isolation of their corresponding genes (glnA [22]; gdhA [23]; gdhB [20]) enabled a study of the regulation of an important part of the nitrogen metabolism. In order to investigate an additional level of control that regulates the quantities of NH 4 and the products of its assimilation, we studied the uptake of 14 C-nitrogen compounds by A. bisporus mycelium. Also, these uptake mechanisms can provide important clues for factors that in£uence the utilization of nitrogen compounds as nutrients. In this paper we report the transport of NH 4 and the amino acids glutamate, glutamine and arginine. 2. Materials and methods 2.1. Organism and growth conditions Agaricus bisporus strain Horst U1 was obtained from the collection of the Mushroom Experimental Station, Horst, The Netherlands. Stock cultures were maintained at 4³C on slants of wheat agar. Mycelium was grown at 24³C in static cultures (shaken once a day) using Fernbach £asks containing 225 ml of liquid medium. Di¡erent media were used. Compost extract (CE) medium was prepared according to Rainey [24]. The other media contained 100 mM glucose, a variable amount of an appropriate
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nitrogen source, 7.8 mM K2 HPO4 , 4.6 mM NaH2 PO4 cH2 O, 1.6 mM MgSO4 c7H2 O, 0.5 mM CaCl2 , 0.134 mM Na2 EDTA, 25 WM FeSO4 , 5 WM MnSO4 , 4.8 WM H3 BO3 , 2.4 WM KI, 52 nM Na2 MoO4 , 4 nM CuSO4 , 4 nM CoCl2 , 0.5 WM thiamincHCl and 0.1 WM D(+)-biotin, 0.5 ml/l Tween-80. The pH was adjusted to 6.8. The concentrations of the nitrogen sources were adjusted to the amount of nitrogen they contained so as to reach 20 mM concentrations of nitrogen atoms. (NH4 )2 HPO4 , glutamate, glutamine, allantoin, albumin and an amino acids mixture (AAM) were used as nitrogen sources. AAM medium contained 1 mM of each essential amino acids except tyrosine. In case of NH 4 , albumin and AAM, the media were bu¡ered with 10 g/l 2-(N-morpholino)ethanesulfonic acid (MES). The media were sterilized at 121³C for 20 min. Glutamine, allantoin, albumin and AAM were ¢lter-sterilized. For inoculation preparation mycelium was grown for 7 days on agar plates containing CE medium solidi¢ed with 1.5% (w/v) Bacto-agar (CEA), overlaid with a cellophane disc. Plates were inoculated with seven inoculation points per dish. The mycelium discs were fragmented in a Waring blender for 30 s and 25-ml aliquots of the homogenate were used to inoculate the liquid media. Cultures were harvested in the logarithmic growth phase by ¢ltration over nylon gauze (100 Wm pore size) after which the medium pH was measured. 2.2. Preparation of mycelium for transport studies Mycelium from 250 ml of culture was washed twice by resuspension in 500 ml 10 mM potassium phosphate bu¡er (pH 6.8) and ¢ltration and ¢nally resuspended in about 100 ml uptake medium (glucose, phosphates, MgSO4 , trace elements and vitamins as described above). In case of nitrogen or carbon starvation, cultures were resuspended in 250 ml uptake medium without nitrogen or carbon source. In addition, penicillin G (50 Wg/ml) and streptomycin (50 Wg/ml) were added to prevent bacterial growth. Every 24 h and just before uptake experiments, the cultures were washed again. Aliquots (15 ml) were taken from the 100 ml uptake suspension for dry weight determination and the rest was immediately used for transport assays.
2.3. Transport assays Transport was studied at 24³C in a total volume of 15 ml containing mycelium in uptake medium. At t = 0 the 14 C-labelled N source (55.5 kBq) and if appropriate the non-labelled N source and inhibitors were added. At given time intervals, two 1-ml samples were taken and ¢ltered immediately on paper ¢lters (Whatman grade 1, q25 mm) under suction (Millipore). Filters were washed once with 5 ml of ice-cold water and twice with 5 ml of ice-cold 50 mM unlabelled N source (glutamate, glutamine, arginine or NH4 Cl; pH 6.8). The ¢lter discs with mycelium were placed in vials and mixed with 5 ml of scintillation £uid (OptiPhase `Hisafe' 3, Wallac). After overnight incubation, the amount of radioactivity in the mycelium was determined with a liquid scintillation counter (Wallac-1409). Results are expressed as Wmol substrate transported/g dry wt. mycelium per minute. The 14 C-labelled N sources (Amersham) had the following speci¢c activities: L[U-14 C]glutamate 9.21 GBq/mmol; L-[U-14 C]glutamine 10.25 GBq/mmol; L-[U-14 C]arginine 10.93 GBq/mmol; L-[U-14 C]methylaminecHCl 2.11 GBq/ mmol. Inhibitors were dissolved in uptake medium with a ¢nal pH of 6.8 before addition to the uptake assay. Ammonium was added as (NH4 )2 HPO4 . When inhibitors were added during the transport assay, a same volume of uptake medium was added to the control (transport assay without inhibitors). 2.4. Respiration rate 14 L-[U- C]glutamate
(55.5 kBq) was added to 15 ml nitrogen-starved mycelium in uptake medium and the mixture was divided in 2-ml portions in stoppered vials. The vials were equipped with a disposable centre well containing 0.3 ml of ethanolamine/ ethylene glycol (1:2, v/v) to trap 14 CO2 produced. Routinely, incubations were terminated at time intervals by the addition of 0.5 ml of 3 M perchloric acid to the medium, followed by a second incubation for 18 h at 4³C to ensure complete volatilization of CO2 . Radioactivity of 14 CO2 was measured in 10 ml of toluene/methanol (2:1, v/v) containing 0.4% Omni£uor (New England Nuclear Research Products).
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2.5. Analytical procedures The harvested mycelium was washed exhaustively with 0.15 M NaCl and double-distilled water and was immediately freeze-dried (Lyovac GT2). NH 4 was extracted twice from 50 mg dried mycelium with 2 ml double-distilled water at 90³C for 30 min, and determined by the procedure of Bergmeyer and Beutler [25]. For extraction of amino acids, about 100 mg dried mycelium was ground and extracted three times in 6 ml of ice-cold ethanol (96%)/water/ thiodiglycol (81:30:1, v/v/v), containing 0.64 g/l citric acid. The homogenates were centrifuged (10 000Ug; 5 min; 4³C) and the supernatants were combined. The solution was washed with 2 volumes of icecold chloroform and the waterphase was dried at 40³C using a rotavapor (Bu«chi). The samples were dissolved in 0.1 M HCl (100 mg dry wt./10 ml) and ¢ltered (0.45 Wm) to remove high molecular mass material. An amount of 0.5 Wmol (High Sensitivity Method; HSM) or 5 Wmol (Standard Sensitivity Method; SSM) of the internal standards norvaline and sarcosine were added to the mycelia before the extraction procedure. Samples were analysed on a Hewlett^Packard (HP) AminoQuant amino acid analyser (HP 1090 Liquid Chromatograph equipped with an autosampler, coupled to a HP 1046 A Fluorescence detector and a Diode-Array detector). After derivatization with ortho-phthaldialdehyde (OPA) and 9-£uorenyl-methylchloroformate (FMOC) the amino acids were separated at a £ow rate of 0.45 ml/min on a reversed-phase C-18 AminoQuant column (200U2.1 mm, HP) according to the method described in the operators handbook (HP AminoQuant Series II).
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su¤cient medium for all uptake experiments. Although some other media were tested, the tendency to form hyphal aggregates led to problems in most cases. We tested also if A. bisporus could grow on AAM medium without an additional carbon source. Some growth occurred but was ¢nally inhibited probably by the degradation products formed. Mycelial yield on AAM medium was too low to quantify uptake rates accurately. At the end of growth the medium turned yellow-brown and we measured NH 4 and pH values in the medium of 14.7 þ 2.5 mM (n = 3) and 7.8 þ 0.3 pH (n = 3), respectively. NH 4 secretion, to about 2 to 4 mM, was observed before, when mycelium was grown on casein or albumin as sole N and C source [22]. 3.2. Method development for uptake studies As mentioned by Burgstaller [17], each speci¢ed fungus calls for its own method. As already noted, a homogeneous suspension of A. bisporus mycelium could best be achieved by growth on CE. To avoid rupturing the hyphal tips, mycelium was harvested by ¢ltration without the use of vacuum. The uptake medium was the same as the de¢ned growth medium except for the omission of the nitrogen source. All growth parameters like pH and temperature were the same as with normal growth. Fragmentation of mycelium in a Waring blender prior to uptake experiments, or shaking during uptake resulted in lower
3. Results 3.1. Growth of mycelium on di¡erent media Growth of A. bisporus on liquid compost extract (CE) medium resulted in the ¢nest `hairy' grown hyphae compared to growth on the other media. Moreover, mycelium grown on CE could already be harvested after 7 days. In view of these practical reasons and the fact that CE resembles the natural substrate of A. bisporus, we used CE as the `general' nitrogen-
Fig. 1. Arginine uptake (50 WM external substrate concentration) in albumin/glucose-grown mycelium. The regression line represents the average of the duplicate samples (b and F).
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Fig. 2. Concentration dependence of initial uptake rates of methylamine (a,b), L-glutamate (c), L-glutamine (d) and L-arginine (e) by A. bisporus mycelium grown on nutrient-su¤cient or nitrogen-de¢cient medium. Insets show the corresponding Lineweaver^Burk plots. Glutamate uptake (c) is shown both on nutrient-su¤cient (F) and nutrient-de¢cient (b) medium.
uptake rates compared to static uptake mixtures. This is probably the result of damaged mycelium. A mycelial density of 2^5 mg dry weight/ml is favoured because in this range the rate of transport is directly proportional to the dry weight (results not shown). Cessation of all £uxes and removal of extracellular label could best be achieved by rapid ¢ltration, washing and cooling at the same time with a large volume (15 ml) of washing solution. Smaller volumes resulted in large di¡erences between duplicate samples.
3.3. Respiration rate Uptake of the 14 C-labelled substrates into the mycelium was linear for at least 60 min but declined slightly after 75 min. A maximum incubation time of 30 min was chosen to avoid underestimation of the 14 C-labelled substrate uptake due to subsequent metabolism and to 14 CO2 production. A typical uptake ¢gure is shown in Fig. 1. The respiratory loss of carbon from glutamate accounted for 5.4% of the total absorbed 14 C after a 30-min incubation period.
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Initial uptake rates were calculated during the ¢rst minutes. 3.4. Methylamine and amino acid uptake Uptake of MA and amino acids by A. bisporus was measured at di¡erent concentrations in the range 2^ 1000 WM and showed Michaelis^Menten saturation kinetics (Fig. 2). MA uptake exhibited complex saturation curves suggesting the presence of more than one saturable component. Analyses of MA uptake in Lineweaver^Burk plots showed biphasic kinetics. Values for the Michaelis constants (Km ) and maximum velocities (Vmax ), estimated from the Lineweaver^Burk plots, are given in Table 1. Table 2 shows the e¡ect on the levels of the MA, glutamate, glutamine and arginine transport activities of the mycelium when the nitrogen source used for growth is varied. All of the nitrogen sources used, supported good growth. The uptake activities show some variation with the di¡erent nitrogen sources but are more or less in the same range. However, uptake of glutamate could only be measured after nitrogen starvation. MA and glutamine uptake also increased after nitrogen starvation. In contrast, arginine transport activity decreased after nitrogen starvation. The e¡ect of the nitrogen source on intracellular pools of ammonium, glutamate, glutamine and arginine is shown in Table 3. NH 4 secretion during growth was observed in some cases. The accumulation ratios can be determined by assuming that about 80% of the mycelial wet weight (conversion factor wet weight/dry weight is about 10) is intracellular
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water and that there is no intracellular compartmentation. For example, 10 WM of initial MA concentration was taken up by CE-grown mycelium with a intracellular NH 4 concentration of 2350 WM. This represents a concentration gradient of 240:1 between the mycelium and external environment. The uptake of the other tested nitrogen sources at micromolar concentrations were also performed against a gradient of several orders of magnitude. 3.5. Methylamine uptake MA transport by nutrient-su¤cient mycelium is extremely low at low external concentrations (0^ 150 WM) (Fig. 2). A second transport system is active above 150 WM MA. After nitrogen starvation, the uptake exhibits biphasic kinetics in the same ranges of MA concentrations. In the 0^150 WM range, the Vmax increased a 100-fold whereas the Vmax increased a 10-fold in the 150^1000 WM range. The a¤nity for MA increased only slightly. MA uptake was maximal after 44 h of nitrogen starvation (Fig. 3). The intracellular NH 4 concentration decreased during nitrogen starvation and was minimal after 44 h. Addition of cycloheximide (50 Wg/ml) to mycelial cultures before nitrogen starvation prevented the increase of transport activity. Addition of NH 4 to nitrogen-starved mycelium resulted in an inhibition of MA uptake. Dixon plots showed that the inhibitions were competitive (Fig. 4) with a Ki of 3.7 þ 0.8 WM. After a few minutes, the inhibition was released as a result of the removal of NH 4 from the incubation medium by the mycelium. If we assume that the length of the lag period is a
Table 1 Kinetic parameters for uptake of methylamine, L-glutamate, L-glutamine and L-arginine by Agaricus bisporus mycelium Nitrogen compound Glutamate Glutamine Arginine Methylaminea
Vmax (Wmol/g dry wt./min)
Class Acidic amino acid Neutral amino acid Basic amino acid Inorganic N compound (ammonium analogue)
b
0.049 0.10 0.06 0.003 0.08 0.40b 0.91b
Km (WM) 136b 151 38 24 316 63b 425b
The Km and Vmax values were estimated from Lineweaver^Burk plots. The values are the means of two independent experiments. Individual values did not di¡er more than 10%. a This uptake showed biphasic kinetics. b Measured under nitrogen-starvation conditions.
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Table 2 Uptake of methylamine, L-glutamate, L-glutamine and L-arginine by A. bisporus mycelium grown on di¡erent media Uptake rate (Wmol/g dry wt./min) of radiolabelled compoundsa
Culture condition
Methylamine
L-Glutamate
L-Glutamine
L-Arginine
Nitrogen-su¤cient media Compost Extract NH 4 /glucose Glutamate/glucose Glutamine/glucose Allantoin/glucose Albumin/glucose AAM/glucose
0.036 0.015 0.063 0.053 0.294 n.d. n.d.
b.d. b.d. b.d. b.d. n.d. b.d. b.d.
0.034 0.021 0.053 0.057 0.211 n.d. n.d.
0.045 0.026 n.d. n.d. n.d. 0.042 0.026
Nitrogen-starvedb
0.336
0.055
0.141
0.008
Values are the means of two independent experiments. Individual values did not di¡er more than 10%. b.d., below detection (values 6 0.004 Wmol/g dry wt./min); n.d., not determined; AAM, amino acid mixture (1 mM of each essential amino acid except tyrosine). a Transport rates were measured at an initial external substrate concentration of 250 WM (methylamine); 500 WM (glutamate); 100 WM (glutamine); 100 WM (arginine). b CE-grown mycelium was starved for nitrogen for 44 h.
measure of the time required to transport NH 4 into the mycelium, we can make a rough estimate of the NH 4 transport rate. The Vmax , determined at 100 WM NH 4 , was about 0.78 Wmol/g dry wt./min NH4 also inhibited MA uptake by nitrogen-su¤cient mycelium. Glutamate, glutamine and arginine (5 mM ¢nal
concentration in uptake solution) did not inhibit MA uptake (250 WM) by nitrogen-su¤cient or -starved mycelium. In contrast, addition of 5 mM PPT (DL-phosphinotricin), which inhibits the amidation of glutamate by GS [21,26], resulted in complete inhibition within 5 min. Addition of PPT 15 min after the start of the uptake experiment showed not
Table 3 Ammonium concentration in culture medium and extractable amino acid and ammonium pool sizes in A. bisporus mycelium grown on di¡erent media Culture condition
Compost extract Compost extract (nitrogen-starved) NH 4 /glucose Glutamate/glucose Glutamine/glucose Allantoin/glucose Albumin/glucose AAM/glucose
NH 4 concentration (mM) in medium
Mycelium concentrations (Wmol/g dry wt.)
before mycelium growth
after mycelium growth
NH 4
Glu
Gln
Arg
total amino acidsa
0.06 þ 0.006 (n = 5) 0
1.1 þ 0.16 (n = 5) 0
4.9 þ 0.6 (n = 3) 0.7
3.9 þ 0.3 (n = 3) 0.7
4.2 þ 0.9 (n = 3) 2.2
37.8 10.2
20.8 0 2.6
16.5 0 1.8
11.8 167.6 26.8
9.8 3.2 118.1
10.4 11.9 12.0
78.9 248.7 277.6
0 0.2 0.07
0 1.3 0.92
18.8 þ 1.7 (n = 5) 11.5 þ 1.4 (n = 4) 38.7 11.3 45.1 þ 12.8 (n = 3) 4.6 15.1 18.0
n.d. 15.1 n.d.
n.d. 5.3 n.d.
n.d. 10.8 n.d.
n.d. 120.4 n.d.
Values are the means of 2 to 5 independent experiments. n.d., not determined; AAM, amino acid mixture (1 mM of each essential amino acid except tyrosine). a Includes additional amino acids which are not given in the Table.
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only inhibition but also excretion of labelled MA (results not shown). 3.6. Glutamate uptake Addition of NH 4 or PPT to the uptake mixture resulted in inhibition of glutamate uptake and excre tion of NH 4 in the case of PPT (Fig. 5). NH4 inhibits the uptake within 10 min whereas PPT addition caused a decreased uptake after 20 min. Addition of cycloheximide to mycelial cultures before nitrogen starvation prevents development of transport activity. 3.7. Arginine uptake
Fig. 4. Dixon plots of the inhibition of methylamine (MA) uptake by ammonium. MA concentrations were 250 (F), 500 (b) and 1000 (R) WM. Ammonium was added at concentrations of 50 and 100 WM or omitted. The reciprocal uptake rate is plotted against the competitor concentration.
The speci¢city of the arginine uptake system was estimated from the extent of inhibition of arginine uptake by addition of 100-fold excess of NH 4 and some amino acids (Table 4). Addition of a large excess of L-lysine almost completely inhibited arginine uptake. Arginine uptake was also strongly inhibited by L-histidine, while the other amino acids inhibited only slightly. On the basis of the observed initial velocity of arginine uptake at two concentrations and various concentrations of unlabelled L-lysine
(Fig. 6a) and L-histidine (Fig. 6b), a Ki of 31 WM and 230 WM was calculated for the inhibition of arginine transport by lysine and histidine, respectively. Addition of NH 4 to the uptake mixture did not result in inhibition of arginine uptake.
Fig. 3. E¡ect of nitrogen starvation on methylamine (MA) transport activity (left axis) and intracellular ammonium pool (a, right axis). MA uptake was measured at 100 WM (b) and 250 WM (F) external substrate concentration. The e¡ect of cycloheximide on the development of MA transport activity (measured at 250 WM external concentration) was tested by addition of 50 Wg/ml cycloheximide to the cultures at t = 0 (R).
Fig. 5. E¡ect of ammonium and PPT on glutamate transport (40 WM initial concentration) by nitrogen-starved mycelium of A. bisporus. At t = 20, NH 4 (b) or PPT (R) was added at a concentration of 5 mM or omitted (F).
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Table 4 Uptake of L-arginine in the presence of unlabelled substrates into A. bisporus mycelium grown on compost extract Inhibitor
Uptake of L-arginine (% of level without inhibitor)
None
100 0 6.5 28.2 95.3 70.2 93.4 64.0 71.2 100
L-Arginine L-Lysine
L-Histidine
L-Glutamate L-Glutamine L-Leucine L-Valine L-Serine
Ammonium
Inhibitors were added at a concentration of 1 mM. L-[U-14 C]arginine was used at a concentration of 3.7 kBq/ml and unlabelled L-arginine was added to reach a ¢nal concentration of 10 WM. 100% values are equal to 0.017 Wmol/g dry wt./min.
3.8. Glutamine uptake Addition of PPT (5 mM) or azaserine (AZS; 1 mM) completely inhibited glutamine uptake (25 WM) by nitrogen-su¤cient mycelium (data not shown). As with glutamate, these inhibitors need 10 to 20 min to act. Inhibition of glutamate synthase by AZS, a glutamine analogue, blocks the transfer of amide nitrogen from glutamine to glutamate [5,27]. In contrast, 5 mM glutamate or NH 4 did not inhibit glutamine uptake. 3.9. Energy coupling As can be seen in Table 5, C-starvation resulted in a decrease of glutamate uptake activity. The activity was not directly in£uenced by the presence or ab-
Fig. 6. Dixon plots of the inhibition of arginine uptake by lysine (a) and histidine (b). Arginine concentrations were 10 (F) and 30 (b) WM. Lysine and histidine were added at concentrations of 100, 500 and 1000 WM or omitted. The reciprocal uptake rate is plotted against the competitor concentration.
Table 5 E¡ect of glucose on glutamate uptake Culture conditiona
Glucose in uptake medium
Uptake rateb (% of normal level)
N-starvation (normal assay) N-starvation N/C-starvation N/C-starvation
+ 3 + 3
100 100 þ 8.5 (n = 3) 51.7 þ 2.1 (n = 2) 53.4 þ 7.2 (n = 3)
100% value is equal to 0.012 Wmol/g dry wt./min. a Compost-extract grown mycelium was starved for nitrogen and/or glucose for 44 h. b Transport rates were measured at an initial external substrate concentration of 40 WM.
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sence of glucose in the uptake medium. Uptake of MA and amino acids was inhibited when inhibitors of energy metabolism such as 2,4-dinitrophenol (DNP) or NaN3 were added. A ¢nal concentration of 1 mM of these inhibitors resulted in 70^100% inhibition of the tested substrates (results not shown). 4. Discussion Studies on nitrogen metabolism in Agaricus bisporus have been focused on NH 4 assimilation, on the characterization of the enzymes involved and on their corresponding genes [4,20,22,23]. Little is known about the uptake and utilization of nitrogen compounds when mycelium is grown on its commercial substrate, horse manure compost. This study was performed to gain insight into the ammonium and amino acid transport capacities of A. bisporus. The available information on transport of amino acids and NH 4 stems from only a few fungi and this knowledge has been used as a guideline for A. bisporus [11,13,14,16]. Our results indicate the presence of several transport systems in A. bisporus. Uptake is markedly in£uenced by the nitrogen su¤ciency of the mycelium. Some of the transport systems have their highest activity under conditions of rapid growth, perhaps serving mainly to provide amino acids for net protein synthesis. Other systems have their highest activity under starvation conditions, perhaps serving mainly as sources of nitrogen. 4.1. Energy coupling As in other ¢lamentous fungi investigated so far, uptake of NH 4 and amino acids in A. bisporus displays characteristic features of active transport. Transport exhibited Michaelis-Menten saturation kinetics, was eliminated by the metabolic inhibitors NaN3 and DNP, and occurred against a concentration gradient. The e¡ect of glucose on glutamate transport suggests that glucose is not directly involved in transport but probably acts as an energy source. Similar inhibitory e¡ects of DNP and NaN3 have been demonstrated for amino acid uptake by Saccharomyces cerevisiae [28], Penicillium cyclopium [29] and Paxillus involutus [30] and for MA transport by Stemphylium botryosum [31].
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4.2. Methylamine uptake We obtained evidence for two MA transport systems, one with higher a¤nity (Km ) and lower capacity (Vmax ) than the other, both of which are derepressed under nitrogen starvation, resulting in an increase of Vmax . The inhibition by the protein synthesis inhibitor cycloheximide suggests that the increase in activity of the high-velocity system (Km 425 WM; Vmax 0.91 Wmol/g dry wt./min) during nitrogen starvation requires de novo synthesis of a protein component of the transport system. Because of the low Ki (3.7 WM) by NH 4 of the high-velocity system, which can be assumed to be identical to the Km for transport, NH 4 is most likely the transported species. Multiplicity of ammonium transporters is also found in S. cerevisiae, which possesses three systems which have also been characterized at the molecular level [32,33]. These authors suggest, on the basis of database analysis, that families of NH 4 transporters exist in bacteria, as well as in plants and animals. In contrast, only a single transport system has been found in P. chrysogenum [34], A. nidulans [35], S. botryosum [31]. Ammonium and amino acid transport systems of eukaryotic microorganisms are regulated at two distinct levels: (1) at the level of synthesis of transport proteins (repression/induction) and (2) at the level of their activities (inactivation, transinhibition, substrate inhibition and compartmentalization) [14]. In A. bisporus, analysis of the intracellular NH 4 pool (Fig. 3 and Tables 2 and 3) revealed an inverse correlation of NH 4 and MA transport activity, suggesting involvement of NH 4 in the repression of the carrier. Furthermore, the inhibition by PPT suggests that the activity was directly controlled by the intracellular NH 4 level. The glutamate analogue PPT inhibits the enzyme activity of GS and as a result the intracellular pool of NH 4 increases while the glutamine pool decreases [21,26]. However, because of the speed of the response, it cannot be ruled out that this glutamate analogue also interacts directly with the uptake system. Like in A. bisporus, NH 4 transport is derepressed during nitrogen starvation in P. chrysogenum [34] and A. nidulans [36], whereas the activity changed very little in S. cerevisiae [37] and decreased in S. botryosum [31]. Moreover, all of these uptake systems are repressed by NH 4 . However, in contrast
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to A. bisporus, the NH 4 uptake systems in the other fungi were also repressed by glutamine and/or asparagine (A. nidulans, S. cerevisiae, S. botryosum) or almost all amino acids (P. chrysogenum). We found no inverse correlation between intracellular glutamine or asparagine levels and NH 4 transport activity. Furthermore, the amino acids glutamate, glutamine and arginine did not inhibit uptake. So the regulation of the NH 4 transport system seems to be di¡erent compared to the other fungi. It seems that the intracellular NH 4 concentration regulates its own in£ux by feedback-inhibition of the uptake system and probably also its e¥ux which becomes apparent when mycelium is grown on protein. Similar results have been found in the alga Chara australis [38]. An additional level of (genetic) control seems to be present in A. bisporus under N-starvation conditions. The results might explain why A. bisporus mycelium can grow on extracellular NH 4 concentrations as high as 40 mM, which is toxic to many organisms. Furthermore, the fact that PPT completely blocks NH 4 uptake has consequences for the conclusions drawn by Baars et al. [21]. They based their conclusion that NH 4 assimilation proceeds via the GS/GOGAT pathway, under the conditions used, on the fact that PPT inhibits NH 4 incorporation. However, because 15 NH does not enter the cell, it can never 4 be measured. The increase of intracellular NH 4 results in NH excretion but also in an increase of the 4 glutamate pool. Therefore, it cannot be excluded that NH 4 assimilation also takes place via the activity of NADP-GDH. The experiment should be repeated in a manner that allows a build up of intracellular 15 NH 4 before adding the inhibitor PPT. 4.3. Amino acid uptake Our results show that glutamate uptake is inhibited by the extracellular or intracellular NH 4 concentration. The fact that PPT needs more time to act than NH 4 suggests that PPT is taken up and inhibits via the accumulation of intracellular NH 4 , which is the actual repressor. The kinetics of glutamate transport in A. bisporus show that only one glutamate transport system was operating over the range 20 WM to 800 WM which is probably an acidic amino acid system. In N. crassa ¢ve distinct amino acid transport systems have been genetically and bio-
chemically characterized [11,13,14]. Glutamate uptake in this fungus takes place by an acidic system, which is active under nitrogen starvation conditions and has a Km of 16 WM [39]. Glutamate is also transported by general and neutral systems when pH is 3.8. However, this system is arti¢cial and a low pH imposes an unnatural £ux of acidic amino acids [11]. Because transport in A. bisporus was measured at pH 6.8, only a very small portion of glutamate exists in its protonated form. Regulation of the acidic system by derepression under nitrogen de¢ciency and NH 4 repression is also found in some other fungi [40,41]. The inhibition of glutamate uptake by NH 4 also explains the results of Baars et al. [42]. They found that feeding of cultures with both NH 4 and glutamate resulted in GS, NADP-GDH and NAD-GDH enzyme activities typical for growth on NH 4 alone, even if NH was present in low concentrations com4 pared to glutamate. The inhibition kinetics of arginine uptake by lysine and histidine (Fig. 6) are very similar to typical competitive inhibition. Arginine and lysine appeared to be equivalent substrates for a basic amino acid permease, while a lower a¤nity for histidine was found. N. crassa and P. chrysogenum also transport arginine via a basic amino acid permease (Km = 2.4 WM and 6 WM, respectively). However, in contrast to A. bisporus, these fungi also express a general uptake system under nitrogen starvation conditions, which is repressed by NH 4 and regulated by transinhibition [40,43]. Like with MA uptake, glutamine uptake seems to be inversely regulated by the intracellular NH 4 pool (Tables 2 and 3). Furthermore, the inhibition by PPT suggests that the activity was directly controlled by the intracellular NH 4 level. Addition of extracellular NH did not inhibit glutamine uptake suggesting 4 that NH4 and glutamine are equally preferred nitrogen sources, their uptake being mediated by di¡erent transport systems. AZS inhibits GOGAT and as a result the intracellular glutamine pool increases [21]. The inhibition of glutamine uptake, however, occurs probably via the expected increase of intracellular NH 4 because a high glutamine pool did not result in inhibition of uptake when mycelium was grown on glutamine/glucose (Tables 2 and 3). It is also possible that AZS competes with glutamine for the same binding site at the uptake carrier. Gluta-
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mine uptake by P. involutus is via a general amino acid permease (Km = 11 WM) which is una¡ected by exogenously supplied NH 4 but is inhibited by glutamate, MSX and most other amino acids [30]. In N. crassa uptake is mediated by neutral and general amino acid systems (Km = 13 WM and 268 WM) which are regulated by transinhibition [14]. In S. cerevisiae uptake is mediated by at least three transporters [44]. The general system is inactivated by NH 4 and its synthesis is repressed by glutamine while a speci¢c high a¤nity (Km = 590 WM) system is also expressed on rich N sources. P. cyclopium and P. chrysogenum transport glutamine via a general amino acid system which is only active under N de¢ciency [13,29]. The results in this paper indicate that in A. bisporus L-glutamate, L-glutamine, L-arginine and MA are actively transported by di¡erent transport systems. Confusion over transport of individual amino acids has usually resulted from the presence of multiple transport systems with overlapping substrate specificities. However, it has usually been possible to resolve this confusion by genetic studies, isolating mutant strains de¢cient in one or more transport systems [11]. Recently, a transformation system for A. bisporus has been developed [45]. Development of mutants will be helpful with future studies.
References
[8]
[9] [10] [11] [12]
[13]
[14] [15] [16] [17] [18]
[19]
[20]
[21]
[1] T.R. Fermor, P.E. Randle, J.F. Smith, in: P.B. Flegg, D.M. Spencer, D.A. Wood (Eds.), The Biology and Technology of the Cultivated Mushroom, Wiley, Chichester, 1985, pp. 81^ 109. [2] D.A. Wood, T.R. Fermor, in: P.B. Flegg, D.M. Spencer, D.A. Wood (Eds.), The Biology and Technology of the Cultivated Mushroom, Wiley, Chichester, 1985, pp. 43^ 61. [3] J.P.G. Gerrits, in: L.J.L.D. Van Griensven (Ed.), The Cultivation of Mushrooms, Darlington Mushroom Laboratories Ltd., Rustington, 1988, pp. 29^72. [4] J.J.P. Baars, H.J.M. Op den Camp, C. van der Drift, A.S.M. Sonnenberg, L.J.L.D. Van Griensven, CMR Newslett. 3 (1996) 1^15. [5] J.J.P. Baars, H.J.M. Op den Camp, J.M.H. Hermans, V. Mikes, C. van der Drift, L.J.L.D. Van Griensven, G.D. Vogels, Microbiology 140 (1994) 1161^1168. [6] H.M. Kalisz, D. Moore, D.A. Wood, Trans. Br. Mycol. Soc. 86 (1986) 519^525. [7] D.H. Jennings, in: L. Boddy, R. Marchant, D.J. Read
[22]
[23]
[24] [25]
[26] [27] [28] [29] [30]
271
(Eds.), Nitrogen, Phosphorus and Sulphur Utilization by Fungi, Cambridge University Press, Cambridge, 1988, pp. 1^31. I. Ahmad, J.A. Hellebust, in: J.R. Norris, D.J. Read, A.K. Varma (Eds.), Methods in Microbiology vol. 23, Academic Press, London, 1991, pp. 181^202. H.M. Kalisz, D.A. Wood, D. Moore, Trans. Br. Mycol. Soc. 88 (1987) 221^227. A. Whitaker, Trans. Br. Mycol. Soc. 67 (1976) 365^376. L. Wol¢nbarger, in: J.W. Payne (Ed.), Microorganisms and Nitrogen Sources, Wiley, London, 1980, pp. 63^87. M.O. Garraway, R.C. Evans, in: M.O. Garraway, R.C. Evans (Eds.), Fungal Nutrition and Physiology, Wiley, New York, 1984, pp. 96^123. D.H. Jennings, in: D.H. Jennings (Ed.), The Physiology of Fungal Nutrition, University Press, Cambridge, 1995, pp. 229-250. J. Hora¨k, Biochim. Biophys. Acta 864 (1986) 223^256. D.H. Gri¤n, in: D.H. Gri¤n (Ed.), Fungal Physiology, Wiley-Liss, New York, 1994, pp. 159^188. D. Kleiner, Biochim. Biophys. Acta 639 (1981) 41^52. W. Burgstaller, Crit. Rev. Microbiol. 23 (1997) 1^46. J.J.P. Baars, H.J.M. Op den Camp, J.W.G. Paalman, V. Mikes, C. van der Drift, L.J.L.D. Van Griensven, G.D. Vogels, Curr. Microbiol. 31 (1995) 108^113. J.J.P. Baars, H.J.M. Op den Camp, A.H.A.M. Van Hoek, C. van der Drift, L.J.L.D. Van Griensven, J. Visser, G.D. Vogels, Curr. Microbiol. 30 (1995) 211^217. M.A.S.H. Kersten, Y. Mu«ller, J.J.P. Baars, A.S.M. Sonnenberg, J. Visser, P.J. Schaap, C. van der Drift, L.J.L.D. Van Griensven, H.J.M. Op den Camp, in: L.J.L.D. Van Griensven, J. Visser (Eds.), Proceedings of the Fourth Meeting on the Genetics and Cellular Biology of Basidiomycetes, 1998, pp. 48^57. J.J.P. Baars, H.J.M. Op den Camp, C. van der Drift, J.J.M. Joordens, S.S. Wijmenga, L.J.L.D. Van Griensven, G.D. Vogels, Biochim. Biophys. Acta 1310 (1995) 74^80. M.A.S.H. Kersten, Y. Mu«ller, H.J.M. Op den Camp, G.D. Vogels, L.J.L.D. Van Griensven, J. Visser, P.J. Schaap, Mol. Gen. Genet. 256 (1997) 179^186. P.J. Schaap, Y. Mu«ller, J.J.P. Baars, H.J.M. Op den Camp, A.S.M. Sonnenberg, L.J.L.D. Van Griensven, J. Visser, Mol. Gen. Genet. 250 (1996) 339^347. P.B. Rainey, New Zealand Nat. Sci. 16 (1989) 109^112. H.U. Bergmeyer, H.O. Beutler, in: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, vol. 8, Weinheim, Basel, Switzerland, 1985, pp. 454^461. P.J. Lea, K.W. Joy, J.L. Ramos, M.G. Guerrero, Phytochemistry 23 (1984) 1^6. B. Botton, M. Chalot, Methods Microbiol. 23 (1991) 204^ 244. E.H. Ramos, L.C. De Bongioanni, A.O.M. Stoppani, Biochim. Biophys. Acta 599 (1980) 214^231. W. Roos, Biochim. Biophys. Acta 978 (1989) 119^133. M. Chalot, A. Brun, B. Botton, B. So«derstro«m, Microbiology 142 (1996) 1749^1756.
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[31] A. Breiman, I. Barash, J. Gen. Microbiol. 116 (1980) 201^ 206. [32] E. Dubois, M. Grenson, Mol. Gen. Genet. 175 (1979) 67^76. [33] A. Marini, S. Soussi-Boudekou, S. Vissers, B. Andre, Mol. Cell. Biol. 17 (1997) 4282^4293. [34] S.L. Hackette, G.E. Skye, C. Burton, I.H. Segel, J. Biol. Chem. 245 (1970) 4241^4250. [35] R.J. Cook, C. Anthony, J. Gen. Microbiol. 109 (1978) 265^ 274. [36] J.A. Pateman, E. Dunn, J.R. Kinghorn, E.C. Forbes, Mol. Gen. Genet. 133 (1974) 225^236. [37] R.J. Roon, J.S. Levy, F. Larimore, J. Biol. Chem. 252 (1977) 3599^3604. [38] P.R. Ryan, N.A. Walker, J. Exp. Bot. 45 (1994) 1057^1067.
[39] M.L. Pall, Biochim. Biophys. Acta 211 (1970) 513^520. [40] D.R. Hunter, I.H. Segel, Arch. Biochem. Biophys. 144 (1971) 168^183. [41] J.A. Pateman, J.R. Kinghorn, E. Dunn, J. Bacteriol. 119 (1974) 534^542. [42] J.J.P. Baars, H.J.M. Op den Camp, C. van der Drift, L.J.L.D. Van Griensven, G.D. Vogels, Curr. Microbiol. 31 (1995) 345^350. [43] R.H. Davis, Microbiol. Rev. 50 (1986) 280^313. [44] X. Zhu, J. Garrett, J. Schreve, T. Michaeli, Curr. Genet. 30 (1996) 107^114. [45] M.J.A. De Groot, P. Bundock, P.J.J. Hooykaas, A.G.M. Beijersbergen, Nat. Biotechnol. 16 (1998) 839^842.
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Transport of amino acids and ammonium in mycelium of Agaricus bisporus Monique A.S.H. Kersten a; *, Michel J.C. Arninkhof a , Huub J.M. Op den Camp a , Leo J.L.D. Van Griensven b , Chris van der Drift a a
Department of Microbiology and Evolutionary Biology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands b Mushroom Experimental Station, P.O. Box 6042, NL-5960 AA Horst, The Netherlands Received 31 March 1999; received in revised form 25 May 1999; accepted 3 June 1999
Abstract Mycelium of Agaricus bisporus took up methylamine (MA), glutamate, glutamine and arginine by high-affinity transport systems following Michaelis^Menten kinetics. The activities of these systems were influenced by the nitrogen source used for mycelial growth. Moreover, MA, glutamate and glutamine uptakes were derepressed by nitrogen starvation, whereas arginine uptake was repressed. The two ammonium-specific transport systems with different affinities and capacities were inhibited by NH 4 , with a Ki of 3.7 WM for the high-velocity system. The Km values for glutamate, glutamine and arginine transport were 124, 151 and 32 WM, respectively. Inhibition of arginine uptake by lysine and histidine showed that they are competitive inhibitors. MA, glutamate and glutamine uptake was inversely proportional to the intracellular NH 4 concentration. Moreover, increase of the intracellular NH 4 level caused by PPT (DL-phosphinotricin) resulted in an immediate cessation of MA, glutamine and glutamate uptake. It seems that the intracellular NH 4 concentration regulates its own influx by feedback-inhibition of the uptake system and probably also its efflux which becomes apparent when mycelium is grown on protein. Addition of extracellular NH 4 did not inhibit glutamine uptake, suggesting that NH4 and glutamine are equally preferred nitrogen sources. The physiological importance of these uptake systems for the utilization of nitrogen compounds by A. bisporus is discussed. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Arginine; Glutamate; Glutamine; Methylamine ; Transport; (Agaricus bisporus)
1. Introduction
Abbreviations: MA, methylamine; DNP, 2,4-dinitrophenol; GS, glutamine synthetase; NADP-GDH, NADP-dependent glutamate dehydrogenase; NAD-GDH, NAD-dependent glutamate dehydrogenase; GOGAT, glutamate synthase; CE, compost extract ; AAM, amino acid mixture; MES, 2-(N-morpholino)ethanesulfonic acid; CEA, compost extract agar; PPT, DL-phosphinotricin ; AZS, azaserine; MSX, L-methionine-S-sulfoximine * Corresponding author. Fax: +31-24-355-3450; E-mail: [email protected]
Agaricus bisporus, the white button mushroom, is commercially grown in large amounts on a substrate which consists of a fermented mixture of horse manure, wheat straw, chicken manure and gypsum [1]. Through a two-phase composting process a selective substrate is obtained which is rich in organic sources of nitrogen and low in free NH3 , the latter being inhibitory to growth of A. bisporus. Although the basic nutritional requirements of A. bisporus are es-
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 0 9 3 - 8
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sentially known [2] and the composted substrate can easily ful¢l the demands, yields of mushrooms can be increased substantially by adding products with high protein contents to the colonized compost [3]. Since the yield increase is due to the protein added, amino acids have to play an important role in mushroom nutrition. Nutrient acquisition is a highly regulated process that includes digestion, transport, and subsequent metabolism. With regard to nitrogen metabolism in A. bisporus (reviewed by Baars et al. [4]), the main function of the catabolic enzymes is to produce small solute molecules that can be transported across the cellular membrane. Under laboratory conditions growth of A. bisporus is supported by a number of single amino acids or NH 4 as a single N source and glucose as C source [5]. A. bisporus also grew readily on protein as sole source of C and N [6]. In contrast to the repressing e¡ect of NH 4 on the use of alternative N sources in many yeasts and fungi [7,8], utilization of protein in A. bisporus appeared not to be a¡ected by the addition of NH 4 to the medium [9]. Amino acid transport in fungi has been studied most extensively in Aspergillus nidulans, Neurospora crassa, Penicillium chrysogenum and Saccharomyces cerevisiae, and appears to represent a compromise between bacterial transport systems, with speci¢cities for individual amino acids, and animal cell systems, with broad speci¢cities for classes of structurally related amino acids [10^13]. Furthermore, di¡erent transport systems are expressed at di¡erent stages of development of the fungi and under di¡erent environmental conditions. In contrast to the analogous systems in bacterial and animal cells, the transport systems in fungi generally mediate only unidirectional £uxes from the external media into the cell and their activities are regulated by transinhibition [14]. Amino acid transport of fungi is an active process which occurs via H symport, a universal mechanism for coupling transport to ATP hydrolysis, which is required for proton expulsion [15]. As is the case for amino acids, NH 4 ions are transported actively across the cellular membrane, most likely via NH 4 uniport [16,17]. The most convenient method for measuring NH transport is with 4 14 [ C]methylamine, an NH4 analogue which is transported by the same system. Kleiner [16] describes transport systems with very high a¤nity for NH 4
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in P. chrysogenum (Km 0.25 WM), A. nidulans (Km 2.8 WM), N. crassa (Km 7 WM) and S. cerevisiae (Km 1 WM). Nonspeci¢c di¡usion of NH3 was only observed when NH 4 transport systems were repressed. Furthermore, the neutral molecule NH3 is a weak base and protonates rapidly to yield NH 4 . At the near neutral pH more than 99% of the total ammonia will be in the protonated form. Next to exogenous NH 4 , ammonium ions are also produced intracellularly as a result of the turn-over of cellular components. Once inside the cell, NH 4 can be incorporated into the amino acids glutamate and glutamine. The enzymes glutamine synthetase (GS; EC 6.3.1.2) [18], glutamate synthase (GOGAT; EC 1.4.7.1) [5], NADP -glutamate dehydrogenase (NADP-GDH; EC 1.4.1.4) [19] and NAD -glutamate dehydrogenase (NAD-GDH; EC 1.4.1.2) [20] are all primarily involved in nitrogen metabolism in A. bisporus and at least GS is directly involved in NH 4 assimilation [21]. Puri¢cation and isolation of their corresponding genes (glnA [22]; gdhA [23]; gdhB [20]) enabled a study of the regulation of an important part of the nitrogen metabolism. In order to investigate an additional level of control that regulates the quantities of NH 4 and the products of its assimilation, we studied the uptake of 14 C-nitrogen compounds by A. bisporus mycelium. Also, these uptake mechanisms can provide important clues for factors that in£uence the utilization of nitrogen compounds as nutrients. In this paper we report the transport of NH 4 and the amino acids glutamate, glutamine and arginine. 2. Materials and methods 2.1. Organism and growth conditions Agaricus bisporus strain Horst U1 was obtained from the collection of the Mushroom Experimental Station, Horst, The Netherlands. Stock cultures were maintained at 4³C on slants of wheat agar. Mycelium was grown at 24³C in static cultures (shaken once a day) using Fernbach £asks containing 225 ml of liquid medium. Di¡erent media were used. Compost extract (CE) medium was prepared according to Rainey [24]. The other media contained 100 mM glucose, a variable amount of an appropriate
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nitrogen source, 7.8 mM K2 HPO4 , 4.6 mM NaH2 PO4 cH2 O, 1.6 mM MgSO4 c7H2 O, 0.5 mM CaCl2 , 0.134 mM Na2 EDTA, 25 WM FeSO4 , 5 WM MnSO4 , 4.8 WM H3 BO3 , 2.4 WM KI, 52 nM Na2 MoO4 , 4 nM CuSO4 , 4 nM CoCl2 , 0.5 WM thiamincHCl and 0.1 WM D(+)-biotin, 0.5 ml/l Tween-80. The pH was adjusted to 6.8. The concentrations of the nitrogen sources were adjusted to the amount of nitrogen they contained so as to reach 20 mM concentrations of nitrogen atoms. (NH4 )2 HPO4 , glutamate, glutamine, allantoin, albumin and an amino acids mixture (AAM) were used as nitrogen sources. AAM medium contained 1 mM of each essential amino acids except tyrosine. In case of NH 4 , albumin and AAM, the media were bu¡ered with 10 g/l 2-(N-morpholino)ethanesulfonic acid (MES). The media were sterilized at 121³C for 20 min. Glutamine, allantoin, albumin and AAM were ¢lter-sterilized. For inoculation preparation mycelium was grown for 7 days on agar plates containing CE medium solidi¢ed with 1.5% (w/v) Bacto-agar (CEA), overlaid with a cellophane disc. Plates were inoculated with seven inoculation points per dish. The mycelium discs were fragmented in a Waring blender for 30 s and 25-ml aliquots of the homogenate were used to inoculate the liquid media. Cultures were harvested in the logarithmic growth phase by ¢ltration over nylon gauze (100 Wm pore size) after which the medium pH was measured. 2.2. Preparation of mycelium for transport studies Mycelium from 250 ml of culture was washed twice by resuspension in 500 ml 10 mM potassium phosphate bu¡er (pH 6.8) and ¢ltration and ¢nally resuspended in about 100 ml uptake medium (glucose, phosphates, MgSO4 , trace elements and vitamins as described above). In case of nitrogen or carbon starvation, cultures were resuspended in 250 ml uptake medium without nitrogen or carbon source. In addition, penicillin G (50 Wg/ml) and streptomycin (50 Wg/ml) were added to prevent bacterial growth. Every 24 h and just before uptake experiments, the cultures were washed again. Aliquots (15 ml) were taken from the 100 ml uptake suspension for dry weight determination and the rest was immediately used for transport assays.
2.3. Transport assays Transport was studied at 24³C in a total volume of 15 ml containing mycelium in uptake medium. At t = 0 the 14 C-labelled N source (55.5 kBq) and if appropriate the non-labelled N source and inhibitors were added. At given time intervals, two 1-ml samples were taken and ¢ltered immediately on paper ¢lters (Whatman grade 1, q25 mm) under suction (Millipore). Filters were washed once with 5 ml of ice-cold water and twice with 5 ml of ice-cold 50 mM unlabelled N source (glutamate, glutamine, arginine or NH4 Cl; pH 6.8). The ¢lter discs with mycelium were placed in vials and mixed with 5 ml of scintillation £uid (OptiPhase `Hisafe' 3, Wallac). After overnight incubation, the amount of radioactivity in the mycelium was determined with a liquid scintillation counter (Wallac-1409). Results are expressed as Wmol substrate transported/g dry wt. mycelium per minute. The 14 C-labelled N sources (Amersham) had the following speci¢c activities: L[U-14 C]glutamate 9.21 GBq/mmol; L-[U-14 C]glutamine 10.25 GBq/mmol; L-[U-14 C]arginine 10.93 GBq/mmol; L-[U-14 C]methylaminecHCl 2.11 GBq/ mmol. Inhibitors were dissolved in uptake medium with a ¢nal pH of 6.8 before addition to the uptake assay. Ammonium was added as (NH4 )2 HPO4 . When inhibitors were added during the transport assay, a same volume of uptake medium was added to the control (transport assay without inhibitors). 2.4. Respiration rate 14 L-[U- C]glutamate
(55.5 kBq) was added to 15 ml nitrogen-starved mycelium in uptake medium and the mixture was divided in 2-ml portions in stoppered vials. The vials were equipped with a disposable centre well containing 0.3 ml of ethanolamine/ ethylene glycol (1:2, v/v) to trap 14 CO2 produced. Routinely, incubations were terminated at time intervals by the addition of 0.5 ml of 3 M perchloric acid to the medium, followed by a second incubation for 18 h at 4³C to ensure complete volatilization of CO2 . Radioactivity of 14 CO2 was measured in 10 ml of toluene/methanol (2:1, v/v) containing 0.4% Omni£uor (New England Nuclear Research Products).
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2.5. Analytical procedures The harvested mycelium was washed exhaustively with 0.15 M NaCl and double-distilled water and was immediately freeze-dried (Lyovac GT2). NH 4 was extracted twice from 50 mg dried mycelium with 2 ml double-distilled water at 90³C for 30 min, and determined by the procedure of Bergmeyer and Beutler [25]. For extraction of amino acids, about 100 mg dried mycelium was ground and extracted three times in 6 ml of ice-cold ethanol (96%)/water/ thiodiglycol (81:30:1, v/v/v), containing 0.64 g/l citric acid. The homogenates were centrifuged (10 000Ug; 5 min; 4³C) and the supernatants were combined. The solution was washed with 2 volumes of icecold chloroform and the waterphase was dried at 40³C using a rotavapor (Bu«chi). The samples were dissolved in 0.1 M HCl (100 mg dry wt./10 ml) and ¢ltered (0.45 Wm) to remove high molecular mass material. An amount of 0.5 Wmol (High Sensitivity Method; HSM) or 5 Wmol (Standard Sensitivity Method; SSM) of the internal standards norvaline and sarcosine were added to the mycelia before the extraction procedure. Samples were analysed on a Hewlett^Packard (HP) AminoQuant amino acid analyser (HP 1090 Liquid Chromatograph equipped with an autosampler, coupled to a HP 1046 A Fluorescence detector and a Diode-Array detector). After derivatization with ortho-phthaldialdehyde (OPA) and 9-£uorenyl-methylchloroformate (FMOC) the amino acids were separated at a £ow rate of 0.45 ml/min on a reversed-phase C-18 AminoQuant column (200U2.1 mm, HP) according to the method described in the operators handbook (HP AminoQuant Series II).
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su¤cient medium for all uptake experiments. Although some other media were tested, the tendency to form hyphal aggregates led to problems in most cases. We tested also if A. bisporus could grow on AAM medium without an additional carbon source. Some growth occurred but was ¢nally inhibited probably by the degradation products formed. Mycelial yield on AAM medium was too low to quantify uptake rates accurately. At the end of growth the medium turned yellow-brown and we measured NH 4 and pH values in the medium of 14.7 þ 2.5 mM (n = 3) and 7.8 þ 0.3 pH (n = 3), respectively. NH 4 secretion, to about 2 to 4 mM, was observed before, when mycelium was grown on casein or albumin as sole N and C source [22]. 3.2. Method development for uptake studies As mentioned by Burgstaller [17], each speci¢ed fungus calls for its own method. As already noted, a homogeneous suspension of A. bisporus mycelium could best be achieved by growth on CE. To avoid rupturing the hyphal tips, mycelium was harvested by ¢ltration without the use of vacuum. The uptake medium was the same as the de¢ned growth medium except for the omission of the nitrogen source. All growth parameters like pH and temperature were the same as with normal growth. Fragmentation of mycelium in a Waring blender prior to uptake experiments, or shaking during uptake resulted in lower
3. Results 3.1. Growth of mycelium on di¡erent media Growth of A. bisporus on liquid compost extract (CE) medium resulted in the ¢nest `hairy' grown hyphae compared to growth on the other media. Moreover, mycelium grown on CE could already be harvested after 7 days. In view of these practical reasons and the fact that CE resembles the natural substrate of A. bisporus, we used CE as the `general' nitrogen-
Fig. 1. Arginine uptake (50 WM external substrate concentration) in albumin/glucose-grown mycelium. The regression line represents the average of the duplicate samples (b and F).
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Fig. 2. Concentration dependence of initial uptake rates of methylamine (a,b), L-glutamate (c), L-glutamine (d) and L-arginine (e) by A. bisporus mycelium grown on nutrient-su¤cient or nitrogen-de¢cient medium. Insets show the corresponding Lineweaver^Burk plots. Glutamate uptake (c) is shown both on nutrient-su¤cient (F) and nutrient-de¢cient (b) medium.
uptake rates compared to static uptake mixtures. This is probably the result of damaged mycelium. A mycelial density of 2^5 mg dry weight/ml is favoured because in this range the rate of transport is directly proportional to the dry weight (results not shown). Cessation of all £uxes and removal of extracellular label could best be achieved by rapid ¢ltration, washing and cooling at the same time with a large volume (15 ml) of washing solution. Smaller volumes resulted in large di¡erences between duplicate samples.
3.3. Respiration rate Uptake of the 14 C-labelled substrates into the mycelium was linear for at least 60 min but declined slightly after 75 min. A maximum incubation time of 30 min was chosen to avoid underestimation of the 14 C-labelled substrate uptake due to subsequent metabolism and to 14 CO2 production. A typical uptake ¢gure is shown in Fig. 1. The respiratory loss of carbon from glutamate accounted for 5.4% of the total absorbed 14 C after a 30-min incubation period.
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Initial uptake rates were calculated during the ¢rst minutes. 3.4. Methylamine and amino acid uptake Uptake of MA and amino acids by A. bisporus was measured at di¡erent concentrations in the range 2^ 1000 WM and showed Michaelis^Menten saturation kinetics (Fig. 2). MA uptake exhibited complex saturation curves suggesting the presence of more than one saturable component. Analyses of MA uptake in Lineweaver^Burk plots showed biphasic kinetics. Values for the Michaelis constants (Km ) and maximum velocities (Vmax ), estimated from the Lineweaver^Burk plots, are given in Table 1. Table 2 shows the e¡ect on the levels of the MA, glutamate, glutamine and arginine transport activities of the mycelium when the nitrogen source used for growth is varied. All of the nitrogen sources used, supported good growth. The uptake activities show some variation with the di¡erent nitrogen sources but are more or less in the same range. However, uptake of glutamate could only be measured after nitrogen starvation. MA and glutamine uptake also increased after nitrogen starvation. In contrast, arginine transport activity decreased after nitrogen starvation. The e¡ect of the nitrogen source on intracellular pools of ammonium, glutamate, glutamine and arginine is shown in Table 3. NH 4 secretion during growth was observed in some cases. The accumulation ratios can be determined by assuming that about 80% of the mycelial wet weight (conversion factor wet weight/dry weight is about 10) is intracellular
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water and that there is no intracellular compartmentation. For example, 10 WM of initial MA concentration was taken up by CE-grown mycelium with a intracellular NH 4 concentration of 2350 WM. This represents a concentration gradient of 240:1 between the mycelium and external environment. The uptake of the other tested nitrogen sources at micromolar concentrations were also performed against a gradient of several orders of magnitude. 3.5. Methylamine uptake MA transport by nutrient-su¤cient mycelium is extremely low at low external concentrations (0^ 150 WM) (Fig. 2). A second transport system is active above 150 WM MA. After nitrogen starvation, the uptake exhibits biphasic kinetics in the same ranges of MA concentrations. In the 0^150 WM range, the Vmax increased a 100-fold whereas the Vmax increased a 10-fold in the 150^1000 WM range. The a¤nity for MA increased only slightly. MA uptake was maximal after 44 h of nitrogen starvation (Fig. 3). The intracellular NH 4 concentration decreased during nitrogen starvation and was minimal after 44 h. Addition of cycloheximide (50 Wg/ml) to mycelial cultures before nitrogen starvation prevented the increase of transport activity. Addition of NH 4 to nitrogen-starved mycelium resulted in an inhibition of MA uptake. Dixon plots showed that the inhibitions were competitive (Fig. 4) with a Ki of 3.7 þ 0.8 WM. After a few minutes, the inhibition was released as a result of the removal of NH 4 from the incubation medium by the mycelium. If we assume that the length of the lag period is a
Table 1 Kinetic parameters for uptake of methylamine, L-glutamate, L-glutamine and L-arginine by Agaricus bisporus mycelium Nitrogen compound Glutamate Glutamine Arginine Methylaminea
Vmax (Wmol/g dry wt./min)
Class Acidic amino acid Neutral amino acid Basic amino acid Inorganic N compound (ammonium analogue)
b
0.049 0.10 0.06 0.003 0.08 0.40b 0.91b
Km (WM) 136b 151 38 24 316 63b 425b
The Km and Vmax values were estimated from Lineweaver^Burk plots. The values are the means of two independent experiments. Individual values did not di¡er more than 10%. a This uptake showed biphasic kinetics. b Measured under nitrogen-starvation conditions.
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Table 2 Uptake of methylamine, L-glutamate, L-glutamine and L-arginine by A. bisporus mycelium grown on di¡erent media Uptake rate (Wmol/g dry wt./min) of radiolabelled compoundsa
Culture condition
Methylamine
L-Glutamate
L-Glutamine
L-Arginine
Nitrogen-su¤cient media Compost Extract NH 4 /glucose Glutamate/glucose Glutamine/glucose Allantoin/glucose Albumin/glucose AAM/glucose
0.036 0.015 0.063 0.053 0.294 n.d. n.d.
b.d. b.d. b.d. b.d. n.d. b.d. b.d.
0.034 0.021 0.053 0.057 0.211 n.d. n.d.
0.045 0.026 n.d. n.d. n.d. 0.042 0.026
Nitrogen-starvedb
0.336
0.055
0.141
0.008
Values are the means of two independent experiments. Individual values did not di¡er more than 10%. b.d., below detection (values 6 0.004 Wmol/g dry wt./min); n.d., not determined; AAM, amino acid mixture (1 mM of each essential amino acid except tyrosine). a Transport rates were measured at an initial external substrate concentration of 250 WM (methylamine); 500 WM (glutamate); 100 WM (glutamine); 100 WM (arginine). b CE-grown mycelium was starved for nitrogen for 44 h.
measure of the time required to transport NH 4 into the mycelium, we can make a rough estimate of the NH 4 transport rate. The Vmax , determined at 100 WM NH 4 , was about 0.78 Wmol/g dry wt./min NH4 also inhibited MA uptake by nitrogen-su¤cient mycelium. Glutamate, glutamine and arginine (5 mM ¢nal
concentration in uptake solution) did not inhibit MA uptake (250 WM) by nitrogen-su¤cient or -starved mycelium. In contrast, addition of 5 mM PPT (DL-phosphinotricin), which inhibits the amidation of glutamate by GS [21,26], resulted in complete inhibition within 5 min. Addition of PPT 15 min after the start of the uptake experiment showed not
Table 3 Ammonium concentration in culture medium and extractable amino acid and ammonium pool sizes in A. bisporus mycelium grown on di¡erent media Culture condition
Compost extract Compost extract (nitrogen-starved) NH 4 /glucose Glutamate/glucose Glutamine/glucose Allantoin/glucose Albumin/glucose AAM/glucose
NH 4 concentration (mM) in medium
Mycelium concentrations (Wmol/g dry wt.)
before mycelium growth
after mycelium growth
NH 4
Glu
Gln
Arg
total amino acidsa
0.06 þ 0.006 (n = 5) 0
1.1 þ 0.16 (n = 5) 0
4.9 þ 0.6 (n = 3) 0.7
3.9 þ 0.3 (n = 3) 0.7
4.2 þ 0.9 (n = 3) 2.2
37.8 10.2
20.8 0 2.6
16.5 0 1.8
11.8 167.6 26.8
9.8 3.2 118.1
10.4 11.9 12.0
78.9 248.7 277.6
0 0.2 0.07
0 1.3 0.92
18.8 þ 1.7 (n = 5) 11.5 þ 1.4 (n = 4) 38.7 11.3 45.1 þ 12.8 (n = 3) 4.6 15.1 18.0
n.d. 15.1 n.d.
n.d. 5.3 n.d.
n.d. 10.8 n.d.
n.d. 120.4 n.d.
Values are the means of 2 to 5 independent experiments. n.d., not determined; AAM, amino acid mixture (1 mM of each essential amino acid except tyrosine). a Includes additional amino acids which are not given in the Table.
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only inhibition but also excretion of labelled MA (results not shown). 3.6. Glutamate uptake Addition of NH 4 or PPT to the uptake mixture resulted in inhibition of glutamate uptake and excre tion of NH 4 in the case of PPT (Fig. 5). NH4 inhibits the uptake within 10 min whereas PPT addition caused a decreased uptake after 20 min. Addition of cycloheximide to mycelial cultures before nitrogen starvation prevents development of transport activity. 3.7. Arginine uptake
Fig. 4. Dixon plots of the inhibition of methylamine (MA) uptake by ammonium. MA concentrations were 250 (F), 500 (b) and 1000 (R) WM. Ammonium was added at concentrations of 50 and 100 WM or omitted. The reciprocal uptake rate is plotted against the competitor concentration.
The speci¢city of the arginine uptake system was estimated from the extent of inhibition of arginine uptake by addition of 100-fold excess of NH 4 and some amino acids (Table 4). Addition of a large excess of L-lysine almost completely inhibited arginine uptake. Arginine uptake was also strongly inhibited by L-histidine, while the other amino acids inhibited only slightly. On the basis of the observed initial velocity of arginine uptake at two concentrations and various concentrations of unlabelled L-lysine
(Fig. 6a) and L-histidine (Fig. 6b), a Ki of 31 WM and 230 WM was calculated for the inhibition of arginine transport by lysine and histidine, respectively. Addition of NH 4 to the uptake mixture did not result in inhibition of arginine uptake.
Fig. 3. E¡ect of nitrogen starvation on methylamine (MA) transport activity (left axis) and intracellular ammonium pool (a, right axis). MA uptake was measured at 100 WM (b) and 250 WM (F) external substrate concentration. The e¡ect of cycloheximide on the development of MA transport activity (measured at 250 WM external concentration) was tested by addition of 50 Wg/ml cycloheximide to the cultures at t = 0 (R).
Fig. 5. E¡ect of ammonium and PPT on glutamate transport (40 WM initial concentration) by nitrogen-starved mycelium of A. bisporus. At t = 20, NH 4 (b) or PPT (R) was added at a concentration of 5 mM or omitted (F).
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Table 4 Uptake of L-arginine in the presence of unlabelled substrates into A. bisporus mycelium grown on compost extract Inhibitor
Uptake of L-arginine (% of level without inhibitor)
None
100 0 6.5 28.2 95.3 70.2 93.4 64.0 71.2 100
L-Arginine L-Lysine
L-Histidine
L-Glutamate L-Glutamine L-Leucine L-Valine L-Serine
Ammonium
Inhibitors were added at a concentration of 1 mM. L-[U-14 C]arginine was used at a concentration of 3.7 kBq/ml and unlabelled L-arginine was added to reach a ¢nal concentration of 10 WM. 100% values are equal to 0.017 Wmol/g dry wt./min.
3.8. Glutamine uptake Addition of PPT (5 mM) or azaserine (AZS; 1 mM) completely inhibited glutamine uptake (25 WM) by nitrogen-su¤cient mycelium (data not shown). As with glutamate, these inhibitors need 10 to 20 min to act. Inhibition of glutamate synthase by AZS, a glutamine analogue, blocks the transfer of amide nitrogen from glutamine to glutamate [5,27]. In contrast, 5 mM glutamate or NH 4 did not inhibit glutamine uptake. 3.9. Energy coupling As can be seen in Table 5, C-starvation resulted in a decrease of glutamate uptake activity. The activity was not directly in£uenced by the presence or ab-
Fig. 6. Dixon plots of the inhibition of arginine uptake by lysine (a) and histidine (b). Arginine concentrations were 10 (F) and 30 (b) WM. Lysine and histidine were added at concentrations of 100, 500 and 1000 WM or omitted. The reciprocal uptake rate is plotted against the competitor concentration.
Table 5 E¡ect of glucose on glutamate uptake Culture conditiona
Glucose in uptake medium
Uptake rateb (% of normal level)
N-starvation (normal assay) N-starvation N/C-starvation N/C-starvation
+ 3 + 3
100 100 þ 8.5 (n = 3) 51.7 þ 2.1 (n = 2) 53.4 þ 7.2 (n = 3)
100% value is equal to 0.012 Wmol/g dry wt./min. a Compost-extract grown mycelium was starved for nitrogen and/or glucose for 44 h. b Transport rates were measured at an initial external substrate concentration of 40 WM.
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sence of glucose in the uptake medium. Uptake of MA and amino acids was inhibited when inhibitors of energy metabolism such as 2,4-dinitrophenol (DNP) or NaN3 were added. A ¢nal concentration of 1 mM of these inhibitors resulted in 70^100% inhibition of the tested substrates (results not shown). 4. Discussion Studies on nitrogen metabolism in Agaricus bisporus have been focused on NH 4 assimilation, on the characterization of the enzymes involved and on their corresponding genes [4,20,22,23]. Little is known about the uptake and utilization of nitrogen compounds when mycelium is grown on its commercial substrate, horse manure compost. This study was performed to gain insight into the ammonium and amino acid transport capacities of A. bisporus. The available information on transport of amino acids and NH 4 stems from only a few fungi and this knowledge has been used as a guideline for A. bisporus [11,13,14,16]. Our results indicate the presence of several transport systems in A. bisporus. Uptake is markedly in£uenced by the nitrogen su¤ciency of the mycelium. Some of the transport systems have their highest activity under conditions of rapid growth, perhaps serving mainly to provide amino acids for net protein synthesis. Other systems have their highest activity under starvation conditions, perhaps serving mainly as sources of nitrogen. 4.1. Energy coupling As in other ¢lamentous fungi investigated so far, uptake of NH 4 and amino acids in A. bisporus displays characteristic features of active transport. Transport exhibited Michaelis-Menten saturation kinetics, was eliminated by the metabolic inhibitors NaN3 and DNP, and occurred against a concentration gradient. The e¡ect of glucose on glutamate transport suggests that glucose is not directly involved in transport but probably acts as an energy source. Similar inhibitory e¡ects of DNP and NaN3 have been demonstrated for amino acid uptake by Saccharomyces cerevisiae [28], Penicillium cyclopium [29] and Paxillus involutus [30] and for MA transport by Stemphylium botryosum [31].
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4.2. Methylamine uptake We obtained evidence for two MA transport systems, one with higher a¤nity (Km ) and lower capacity (Vmax ) than the other, both of which are derepressed under nitrogen starvation, resulting in an increase of Vmax . The inhibition by the protein synthesis inhibitor cycloheximide suggests that the increase in activity of the high-velocity system (Km 425 WM; Vmax 0.91 Wmol/g dry wt./min) during nitrogen starvation requires de novo synthesis of a protein component of the transport system. Because of the low Ki (3.7 WM) by NH 4 of the high-velocity system, which can be assumed to be identical to the Km for transport, NH 4 is most likely the transported species. Multiplicity of ammonium transporters is also found in S. cerevisiae, which possesses three systems which have also been characterized at the molecular level [32,33]. These authors suggest, on the basis of database analysis, that families of NH 4 transporters exist in bacteria, as well as in plants and animals. In contrast, only a single transport system has been found in P. chrysogenum [34], A. nidulans [35], S. botryosum [31]. Ammonium and amino acid transport systems of eukaryotic microorganisms are regulated at two distinct levels: (1) at the level of synthesis of transport proteins (repression/induction) and (2) at the level of their activities (inactivation, transinhibition, substrate inhibition and compartmentalization) [14]. In A. bisporus, analysis of the intracellular NH 4 pool (Fig. 3 and Tables 2 and 3) revealed an inverse correlation of NH 4 and MA transport activity, suggesting involvement of NH 4 in the repression of the carrier. Furthermore, the inhibition by PPT suggests that the activity was directly controlled by the intracellular NH 4 level. The glutamate analogue PPT inhibits the enzyme activity of GS and as a result the intracellular pool of NH 4 increases while the glutamine pool decreases [21,26]. However, because of the speed of the response, it cannot be ruled out that this glutamate analogue also interacts directly with the uptake system. Like in A. bisporus, NH 4 transport is derepressed during nitrogen starvation in P. chrysogenum [34] and A. nidulans [36], whereas the activity changed very little in S. cerevisiae [37] and decreased in S. botryosum [31]. Moreover, all of these uptake systems are repressed by NH 4 . However, in contrast
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to A. bisporus, the NH 4 uptake systems in the other fungi were also repressed by glutamine and/or asparagine (A. nidulans, S. cerevisiae, S. botryosum) or almost all amino acids (P. chrysogenum). We found no inverse correlation between intracellular glutamine or asparagine levels and NH 4 transport activity. Furthermore, the amino acids glutamate, glutamine and arginine did not inhibit uptake. So the regulation of the NH 4 transport system seems to be di¡erent compared to the other fungi. It seems that the intracellular NH 4 concentration regulates its own in£ux by feedback-inhibition of the uptake system and probably also its e¥ux which becomes apparent when mycelium is grown on protein. Similar results have been found in the alga Chara australis [38]. An additional level of (genetic) control seems to be present in A. bisporus under N-starvation conditions. The results might explain why A. bisporus mycelium can grow on extracellular NH 4 concentrations as high as 40 mM, which is toxic to many organisms. Furthermore, the fact that PPT completely blocks NH 4 uptake has consequences for the conclusions drawn by Baars et al. [21]. They based their conclusion that NH 4 assimilation proceeds via the GS/GOGAT pathway, under the conditions used, on the fact that PPT inhibits NH 4 incorporation. However, because 15 NH does not enter the cell, it can never 4 be measured. The increase of intracellular NH 4 results in NH excretion but also in an increase of the 4 glutamate pool. Therefore, it cannot be excluded that NH 4 assimilation also takes place via the activity of NADP-GDH. The experiment should be repeated in a manner that allows a build up of intracellular 15 NH 4 before adding the inhibitor PPT. 4.3. Amino acid uptake Our results show that glutamate uptake is inhibited by the extracellular or intracellular NH 4 concentration. The fact that PPT needs more time to act than NH 4 suggests that PPT is taken up and inhibits via the accumulation of intracellular NH 4 , which is the actual repressor. The kinetics of glutamate transport in A. bisporus show that only one glutamate transport system was operating over the range 20 WM to 800 WM which is probably an acidic amino acid system. In N. crassa ¢ve distinct amino acid transport systems have been genetically and bio-
chemically characterized [11,13,14]. Glutamate uptake in this fungus takes place by an acidic system, which is active under nitrogen starvation conditions and has a Km of 16 WM [39]. Glutamate is also transported by general and neutral systems when pH is 3.8. However, this system is arti¢cial and a low pH imposes an unnatural £ux of acidic amino acids [11]. Because transport in A. bisporus was measured at pH 6.8, only a very small portion of glutamate exists in its protonated form. Regulation of the acidic system by derepression under nitrogen de¢ciency and NH 4 repression is also found in some other fungi [40,41]. The inhibition of glutamate uptake by NH 4 also explains the results of Baars et al. [42]. They found that feeding of cultures with both NH 4 and glutamate resulted in GS, NADP-GDH and NAD-GDH enzyme activities typical for growth on NH 4 alone, even if NH was present in low concentrations com4 pared to glutamate. The inhibition kinetics of arginine uptake by lysine and histidine (Fig. 6) are very similar to typical competitive inhibition. Arginine and lysine appeared to be equivalent substrates for a basic amino acid permease, while a lower a¤nity for histidine was found. N. crassa and P. chrysogenum also transport arginine via a basic amino acid permease (Km = 2.4 WM and 6 WM, respectively). However, in contrast to A. bisporus, these fungi also express a general uptake system under nitrogen starvation conditions, which is repressed by NH 4 and regulated by transinhibition [40,43]. Like with MA uptake, glutamine uptake seems to be inversely regulated by the intracellular NH 4 pool (Tables 2 and 3). Furthermore, the inhibition by PPT suggests that the activity was directly controlled by the intracellular NH 4 level. Addition of extracellular NH did not inhibit glutamine uptake suggesting 4 that NH4 and glutamine are equally preferred nitrogen sources, their uptake being mediated by di¡erent transport systems. AZS inhibits GOGAT and as a result the intracellular glutamine pool increases [21]. The inhibition of glutamine uptake, however, occurs probably via the expected increase of intracellular NH 4 because a high glutamine pool did not result in inhibition of uptake when mycelium was grown on glutamine/glucose (Tables 2 and 3). It is also possible that AZS competes with glutamine for the same binding site at the uptake carrier. Gluta-
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mine uptake by P. involutus is via a general amino acid permease (Km = 11 WM) which is una¡ected by exogenously supplied NH 4 but is inhibited by glutamate, MSX and most other amino acids [30]. In N. crassa uptake is mediated by neutral and general amino acid systems (Km = 13 WM and 268 WM) which are regulated by transinhibition [14]. In S. cerevisiae uptake is mediated by at least three transporters [44]. The general system is inactivated by NH 4 and its synthesis is repressed by glutamine while a speci¢c high a¤nity (Km = 590 WM) system is also expressed on rich N sources. P. cyclopium and P. chrysogenum transport glutamine via a general amino acid system which is only active under N de¢ciency [13,29]. The results in this paper indicate that in A. bisporus L-glutamate, L-glutamine, L-arginine and MA are actively transported by di¡erent transport systems. Confusion over transport of individual amino acids has usually resulted from the presence of multiple transport systems with overlapping substrate specificities. However, it has usually been possible to resolve this confusion by genetic studies, isolating mutant strains de¢cient in one or more transport systems [11]. Recently, a transformation system for A. bisporus has been developed [45]. Development of mutants will be helpful with future studies.
References
[8]
[9] [10] [11] [12]
[13]
[14] [15] [16] [17] [18]
[19]
[20]
[21]
[1] T.R. Fermor, P.E. Randle, J.F. Smith, in: P.B. Flegg, D.M. Spencer, D.A. Wood (Eds.), The Biology and Technology of the Cultivated Mushroom, Wiley, Chichester, 1985, pp. 81^ 109. [2] D.A. Wood, T.R. Fermor, in: P.B. Flegg, D.M. Spencer, D.A. Wood (Eds.), The Biology and Technology of the Cultivated Mushroom, Wiley, Chichester, 1985, pp. 43^ 61. [3] J.P.G. Gerrits, in: L.J.L.D. Van Griensven (Ed.), The Cultivation of Mushrooms, Darlington Mushroom Laboratories Ltd., Rustington, 1988, pp. 29^72. [4] J.J.P. Baars, H.J.M. Op den Camp, C. van der Drift, A.S.M. Sonnenberg, L.J.L.D. Van Griensven, CMR Newslett. 3 (1996) 1^15. [5] J.J.P. Baars, H.J.M. Op den Camp, J.M.H. Hermans, V. Mikes, C. van der Drift, L.J.L.D. Van Griensven, G.D. Vogels, Microbiology 140 (1994) 1161^1168. [6] H.M. Kalisz, D. Moore, D.A. Wood, Trans. Br. Mycol. Soc. 86 (1986) 519^525. [7] D.H. Jennings, in: L. Boddy, R. Marchant, D.J. Read
[22]
[23]
[24] [25]
[26] [27] [28] [29] [30]
271
(Eds.), Nitrogen, Phosphorus and Sulphur Utilization by Fungi, Cambridge University Press, Cambridge, 1988, pp. 1^31. I. Ahmad, J.A. Hellebust, in: J.R. Norris, D.J. Read, A.K. Varma (Eds.), Methods in Microbiology vol. 23, Academic Press, London, 1991, pp. 181^202. H.M. Kalisz, D.A. Wood, D. Moore, Trans. Br. Mycol. Soc. 88 (1987) 221^227. A. Whitaker, Trans. Br. Mycol. Soc. 67 (1976) 365^376. L. Wol¢nbarger, in: J.W. Payne (Ed.), Microorganisms and Nitrogen Sources, Wiley, London, 1980, pp. 63^87. M.O. Garraway, R.C. Evans, in: M.O. Garraway, R.C. Evans (Eds.), Fungal Nutrition and Physiology, Wiley, New York, 1984, pp. 96^123. D.H. Jennings, in: D.H. Jennings (Ed.), The Physiology of Fungal Nutrition, University Press, Cambridge, 1995, pp. 229-250. J. Hora¨k, Biochim. Biophys. Acta 864 (1986) 223^256. D.H. Gri¤n, in: D.H. Gri¤n (Ed.), Fungal Physiology, Wiley-Liss, New York, 1994, pp. 159^188. D. Kleiner, Biochim. Biophys. Acta 639 (1981) 41^52. W. Burgstaller, Crit. Rev. Microbiol. 23 (1997) 1^46. J.J.P. Baars, H.J.M. Op den Camp, J.W.G. Paalman, V. Mikes, C. van der Drift, L.J.L.D. Van Griensven, G.D. Vogels, Curr. Microbiol. 31 (1995) 108^113. J.J.P. Baars, H.J.M. Op den Camp, A.H.A.M. Van Hoek, C. van der Drift, L.J.L.D. Van Griensven, J. Visser, G.D. Vogels, Curr. Microbiol. 30 (1995) 211^217. M.A.S.H. Kersten, Y. Mu«ller, J.J.P. Baars, A.S.M. Sonnenberg, J. Visser, P.J. Schaap, C. van der Drift, L.J.L.D. Van Griensven, H.J.M. Op den Camp, in: L.J.L.D. Van Griensven, J. Visser (Eds.), Proceedings of the Fourth Meeting on the Genetics and Cellular Biology of Basidiomycetes, 1998, pp. 48^57. J.J.P. Baars, H.J.M. Op den Camp, C. van der Drift, J.J.M. Joordens, S.S. Wijmenga, L.J.L.D. Van Griensven, G.D. Vogels, Biochim. Biophys. Acta 1310 (1995) 74^80. M.A.S.H. Kersten, Y. Mu«ller, H.J.M. Op den Camp, G.D. Vogels, L.J.L.D. Van Griensven, J. Visser, P.J. Schaap, Mol. Gen. Genet. 256 (1997) 179^186. P.J. Schaap, Y. Mu«ller, J.J.P. Baars, H.J.M. Op den Camp, A.S.M. Sonnenberg, L.J.L.D. Van Griensven, J. Visser, Mol. Gen. Genet. 250 (1996) 339^347. P.B. Rainey, New Zealand Nat. Sci. 16 (1989) 109^112. H.U. Bergmeyer, H.O. Beutler, in: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, vol. 8, Weinheim, Basel, Switzerland, 1985, pp. 454^461. P.J. Lea, K.W. Joy, J.L. Ramos, M.G. Guerrero, Phytochemistry 23 (1984) 1^6. B. Botton, M. Chalot, Methods Microbiol. 23 (1991) 204^ 244. E.H. Ramos, L.C. De Bongioanni, A.O.M. Stoppani, Biochim. Biophys. Acta 599 (1980) 214^231. W. Roos, Biochim. Biophys. Acta 978 (1989) 119^133. M. Chalot, A. Brun, B. Botton, B. So«derstro«m, Microbiology 142 (1996) 1749^1756.
BBAGEN 24847 27-7-99
272
M.A.S.H. Kersten et al. / Biochimica et Biophysica Acta 1428 (1999) 260^272
[31] A. Breiman, I. Barash, J. Gen. Microbiol. 116 (1980) 201^ 206. [32] E. Dubois, M. Grenson, Mol. Gen. Genet. 175 (1979) 67^76. [33] A. Marini, S. Soussi-Boudekou, S. Vissers, B. Andre, Mol. Cell. Biol. 17 (1997) 4282^4293. [34] S.L. Hackette, G.E. Skye, C. Burton, I.H. Segel, J. Biol. Chem. 245 (1970) 4241^4250. [35] R.J. Cook, C. Anthony, J. Gen. Microbiol. 109 (1978) 265^ 274. [36] J.A. Pateman, E. Dunn, J.R. Kinghorn, E.C. Forbes, Mol. Gen. Genet. 133 (1974) 225^236. [37] R.J. Roon, J.S. Levy, F. Larimore, J. Biol. Chem. 252 (1977) 3599^3604. [38] P.R. Ryan, N.A. Walker, J. Exp. Bot. 45 (1994) 1057^1067.
[39] M.L. Pall, Biochim. Biophys. Acta 211 (1970) 513^520. [40] D.R. Hunter, I.H. Segel, Arch. Biochem. Biophys. 144 (1971) 168^183. [41] J.A. Pateman, J.R. Kinghorn, E. Dunn, J. Bacteriol. 119 (1974) 534^542. [42] J.J.P. Baars, H.J.M. Op den Camp, C. van der Drift, L.J.L.D. Van Griensven, G.D. Vogels, Curr. Microbiol. 31 (1995) 345^350. [43] R.H. Davis, Microbiol. Rev. 50 (1986) 280^313. [44] X. Zhu, J. Garrett, J. Schreve, T. Michaeli, Curr. Genet. 30 (1996) 107^114. [45] M.J.A. De Groot, P. Bundock, P.J.J. Hooykaas, A.G.M. Beijersbergen, Nat. Biotechnol. 16 (1998) 839^842.
BBAGEN 24847 27-7-99