Characterization of an Inducible Citrate Uptake System in Penicillium Simplicissimum

FEMS Microbiology Letters 213 (2002) 21^26 www.fems-microbiology.org

Characterization of an inducible citrate uptake system in Penicillium simplicissimum

Martin Síimkovic› a, Michal Kalin›a¤k a, Wolfgang Burgstaller b, L’udov|¤t Varec›ka a;

a Department of Biochemistry and Microbiology, Slovak University of Technology, Radlinske¤ho 9, 81102 Bratislava, Slovak Republic Downloaded from https://academic.oup.com/femsle/article/213/1/21/516581 by guest on 23 September 2021 b Institute of Microbiology, Leopold-Franzens-University Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria

Received 20 March 2002; received in revised form 4 May 2002; accepted 10 May 2002

First published online 3 June 2002

Abstract

When citrate was used as a sole source of carbon, citrate uptake by Penicillium simplicissimum increased 267-fold (if glucose-grown mycelium was adapted to citrate) or 1400-fold (if the fungus was grown on citrate) compared to glucose-grown mycelium. Inhibition of macromolecular synthesis prevented this stimulation of citrate uptake. Citrate uptake by glucose-grown mycelium was low (0.0015 nmol 31 31 31 min (mg DW) ) and most probably due to diffusion of undissociated citric acid. Citrate-adapted mycelium had a KM of 65 Wmol l 31 31 31 31 31 and a Vmax of 0.34 nmol min (mg DW) . In citrate-grown mycelium KM was 318 Wmol l and Vmax was 8.5 nmol min (mg DW) . Citrate uptake was inhibited by sodium azide and uncouplers (TCS, 3,3P,4P,5-tetrachlorosalicylanilide; FCCP, carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone). Because of this we postulate that the induced citrate uptake must be an active transport process. The pH optimum of citrate uptake was between pH 6 and 7. EDTA and Mg2þ,Mn2þ,Cu2þ,Zn2þ,Fe2þ,Ca2þ only weakly influenced the induced citrate uptake. The properties of citrate uptake by Aspergillus niger and P. simplicissimum are compared. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Penicillium simplicissimum; Citrate; Uptake; E¥ux; Induction

1. Introduction Citrate excretion by Penicillium simplicissimum can be used for the solubilization of metals in biohydrometallur- Citrate is a compound which is frequently present in gical processes [8]. P. simplicissimum excretes citrate if fungal growth media (either added or excreted by the fun- growth is limited and glucose is in excess [9]. Excretion gus), and which is also available for fungi in soil [1,2]. of citrate by P. simplicissimum is mediated by a transport Although there have been few investigations, it is reason- protein [10]. Uptake of citrate was also observed with able to assume that many fungi can use citrate as a sole P. simplicissimum, because the fungus could use citrate source of carbon. Besides being a carbon source, citrate as a sole carbon source [11]. This raises the question forms complexes with a variety of metals [3,4]. These com- whether the excretion of citrate is mediated by an inverse plexes in£uence the toxicity of metals and can help to take citrate uptake system or if there are two di¡erent transport up a metal ion, for instance iron [5] and manganese [6,7]. proteins, as is the case, for instance, with glutamate uptake In spite of these multiple functions of citrate, there is a and excretion by Corynebacterium glutamicum [12]. To lack of studies of fungal citrate uptake systems. With re- answer this question one necessary step is to characterize spect to mitosporic fungi only the citrate uptake system of the uptake system for citrate in P. simplicissimum. The Aspergillus niger ATCC 11414 has been investigated in results presented extend those published by Gallmetzer et detail [7]. al. [10] and show that adaptation to and/or growth of the fungus with citrate leads to appearance of the transport process with di¡erent properties than those of the citrate- naive fungus. Further aims of this study were to characterize the citrate uptake system in P. simplicissimum, to com-

* Corresponding author. Tel.: +421 (2) 5932 5514; pare it with citrate uptake by A. niger, and to discuss Fax: +421 (2) 5292 3198. whether P. simplicissimum excretes citrate by an inverse E-mail address: [email protected] (L. Varec›ka). citrate uptake system.

FEMSLE 10536 12-7-02 22 M. Síimkovic› et al. / FEMS Microbiology Letters 213 (2002) 21^26

2. Materials and methods otherwise; speci¢c radioactivity about 1000 cpm nmol31). The suspension was immediately vortexed and 1-ml ali- 2.1. Culture conditions quots were withdrawn, ¢ltered through a membrane ¢lter (Whatman GF/A) and washed with 2U3 ml of 1 mol l31 The medium for submerged cultivation of P. simplicis- citric acid (pH 7.4). When the kinetics was measured, ali- 31 simum consisted of (g l ): 80 glucose, 0.84 (NH4)2SO4, quots were withdrawn at the times indicated in the ¢gures, 0.68 NH4Cl, 0.8 KH2PO4, 0.4 MgSO4W7H2O. These com- otherwise after 20 min. The membrane ¢lters were trans- ponents were dissolved into 500 ml of distilled water and ferred to scintillation vials and the radioactivity was mea- the pH was titrated with 1 mol l31 NaOH to 7.4. Then, 20 sured by liquid scintillation counting. All experiments were ml of a solution of trace elements was added (g l31): 0.5 carried out in triplicate. The results are expressed as aver- FeSO4W7H2O, 0.33 MnSO4W5H2O, 0.42 ZnSO4W7H2O, 0.05 age of triplicates þ S.D. of a representative of three experi- CuSO4W5H2O, 0.3 CaCl2W2H2O. Two hundred milliliters of ments.

the liquid medium were inoculated with a conidial suspen- Downloaded from https://academic.oup.com/femsle/article/213/1/21/516581 by guest on 23 September 2021 sion to a ¢nal density of 8U106 conidia ml31. Submerged 2.4. Uptake of unlabeled citric acid cultivation was carried out for 24 h at 30‡C in the dark on a rotary shaker (250 rpm). When citrate excretion had to Ten milliliters of a culture grown with citrate as carbon be induced, the cultivation was carried out in the same source were ¢ltered through a nylon net (20 Wm), washed medium, except that 1 mol l31 Tris bu¡er (pH 8) was with 2U10 ml of 50 mmol l31 MES bu¡er (pH 6.0) and included and the cultivation was prolonged for 3 days. suspended into 100 ml of the same bu¡er. This suspension For growth on citrate, citric acid (100 mmol l31) was was incubated for 15 min and the procedure was repeated. used instead of glucose and the medium was adjusted The ¢nal suspension was supplemented with citric acid with solid NaOH to pH 5.0. Other conditions were iden- (¢nal concentration 0.1, 0.5 or 1 mmol l31, plus the sub- tical to those described above. stance to be tested for its in£uence on the citrate transport). Immediately (time 0), and at time intervals indicated 2.2. Adaptation of glucose-grown mycelium to citrate as a in the ¢gures, two aliquots (each 10 ml) were taken and sole carbon source ¢ltered. The ¢ltrate was immediately frozen and later used for the analysis of citrate. Citrate was determined by A 24-h culture was centrifuged for 10 min at 2500Ug HPLC as described by Burgstaller et al. [13]. Intracellular under sterile conditions and the supernatant was removed citrate was extracted as described by Gallmetzer et al. [10]. by means of a micropipette. The mycelium was suspended into 150^200 ml of fresh medium which contained no glu- 2.5. Chemicals cose, but 50 or 250 mmol l31 citric acid titrated to pH 5.0 with Tris base. Centrifugation and re-suspension were re- The chemicals used were from the following sources: peated three times. To the ¢nal suspension a sterile ampi- [14C]citric acid from Radiochemical Centre, Amersham, cillin solution was added (¢nal concentration 100 Wgml31) UK; dimethylsulfoxide (DMSO) was from Merck, Darm- and the suspension was incubated for another 48 h. Adap- stadt, Germany; Good bu¡ers from Serva, Heidelberg, tation was terminated by ¢ltration of the mycelium Germany (MES, HEPES) and from Fluka, Buchs, Swit- through a nylon net. The mycelium was washed three zerland (PIPES, MOPS); carbonyl cyanide p-tri£uorome- times with 150 ml of the medium containing citric acid thoxy-phenylhydrazone (FCCP) from Sigma, St. Louis, and re-suspended in 100 ml of the medium without glucose MO, USA; 3,3P,4P,5-tetrachlorosalicylanilide (TCS) from and citric acid, at a density of 4^10 mg dry weight per ml. Eastman-Kodak, Rochester, MN, USA; tris-(hydroxyme- Uptake experiments were started after 15 min of incuba- thylamino) methane was purchased from Medika, Brati- tion at ambient temperature. The following mixture of slava, Slovakia. All other reagents were purchased from inhibitors was used to test whether the synthesis of macro- Lachema, Brno, Czech Republic. molecules was necessary for the induction of citrate uptake: chloramphenicol and erythromycin (inhibitors of mitochondrial protein synthesis; each 30 Wgml31), cyclo- 3. Results heximide (inhibitor of cytoplasmic protein synthesis; 60 Wg ml31), rifamycin and rifampicin (inhibitors of RNA syn- Hyphae grown on glucose as a sole carbon source were thesis; 30 and 15 Wgml31, respectively) and ampicillin essentially devoid of the ability to transport citrate: at an (100 Wgml31). extracellular citrate concentration of 0.1 mmol l31 the uptake rate was 0.0015 nmol min31 (mg DW)31 (Fig. 1A). 2.3. Uptake of [14C]citric acid This uptake had a pH optimum between pH 3 and 4 (Fig. 1B). Uptake increased with citrate concentration, Uptake was started by the addition of radiolabeled cit- with no sign of saturation (Fig. 1C). Uncouplers only ric acid (¢nal concentration 0.1 mmol l31, if not indicated weakly inhibited citrate uptake (the maximal inhibition

FEMSLE 10536 12-7-02 M. Síimkovic› et al. / FEMS Microbiology Letters 213 (2002) 21^26 23 Downloaded from https://academic.oup.com/femsle/article/213/1/21/516581 by guest on 23 September 2021

Fig. 1. Properties of citrate uptake by glucose-grown mycelium. Kinetics of transport (A), pH dependence (B), dependence of uptake on citrate concentration (C) and the e¡ect of uncoupler TCS (D). A: Transport was measured at 30‡C for the time indicated in the presence of 0.1 mmol l31 citrate in the culture medium without glucose. B: Transport was measured for 20 min at 30‡C in the presence of 0.1 mmol l 31 citrate in media without glucose and with 50 mmol l31 of di¡erent Good bu¡er (HEPES, MOPS, MES, PIPES) adjusted to the pH indicated in the ¢gure. Values are corrected for radioactivity measured at time zero. C: Transport was measured in the medium without glucose at 30‡C in the presence of 0.05, 1 and 10 mmol l31 citrate for 20 min. Values are corrected for radioactivity measured at time zero. D: Transport was measured at 30‡C for 20 min in the presence of 0.1 mmol l31 citrate in the medium without glucose in the presence of indicated concentrations of TCS (note the exponential scale of abscissa) and the same volume of methanol in control (0.3% v/v). Values are corrected for radioactivity measured at time zero. Data shown in A^D were obtained from independent experiments.

Fig. 2. Properties of citrate uptake by citrate-adapted mycelium. Kinetics of transport (A), pH dependence (B), the dependence of the uptake on the citrate concentration (C) and the e¡ect of uncoupler (D). The conditions were identical to those described in Fig. 1 except that citrate-adapted mycelium was used. Data shown in A^D were obtained from independent experiments.

FEMSLE 10536 12-7-02 24 M. Síimkovic› et al. / FEMS Microbiology Letters 213 (2002) 21^26

A further ¢ve-fold stimulation of citrate uptake ^ to 2.1 nmol min31 (mg DW)31 at 0.1 mmol l31 citrate (Fig. 5B) ^ was observed if the fungus was grown with citrate as the sole carbon source. With this mycelium Vmax 31 31 was 8.46 nmol min (mg DW) and KM was 318 Wmol l31 (Fig. 5A). It should be noted that the biomass yield during growth on citrate was up to 10 times lower as compared to glucose as the sole carbon source. The uptake of citrate was abolished by the presence of equimolar concentrations of succinate or glucose (Fig. 5B). The level of intracellular citrate remained constant during the transport measurement (Fig. 5C). This indicated that the trans-

Fig. 3. Further characteristics of induced citrate uptake. Inhibition of port measurements were carried out under conditions of Downloaded from https://academic.oup.com/femsle/article/213/1/21/516581 by guest on 23 September 2021 the induction of citrate uptake by inhibitors of macromolecular synthe- constant metabolic £ux through the pathways contributing sis (A) and citrate uptake by mycelium grown under conditions favoring citrate e¥ux (B). A: Citrate uptake by citrate-adapted mycelium to the citrate level in the hyphae. (circles), with inhibitors present during the adaptation period (dia- monds), and by glucose-grown mycelium (triangles). B: Citrate uptake by glucose-grown mycelium and by mycelium which was grown under 4. Discussion conditions favoring citrate e¥ux. The time course was measured at 30‡C in glucose-grown mycelium suspended in the cultivation medium (squares) and in mycelium grown and suspended in the Tris-bu¡ered The presented results show that in P. simplicissimum, cultivation medium at pH 8 (circles). Data shown in A and Bwere ob- the citrate uptake system is induced if citrate is the sole tained from independent experiments. source of carbon and energy. Compared to glucose-grown mycelium, citrate uptake increased 267-fold with citrate- adapted mycelium and 1400-fold with citrate-grown myce- at a high uncoupler concentration was about 30%; lium. The induced citrate uptake must be an active trans- Fig. 1D). port process because it was inhibited by uncouplers and Signi¢cant changes in citric acid uptake were observed if sodium azide. The absence of the inhibition of citrate up- glucose-grown mycelium was incubated in a medium con- take by 10 mmol l31 cyanide could be explained by the taining citrate as the sole carbon source. The citrate up- release of HCN from the acidic cultivation medium. The take rate rose by two orders of magnitude, to 0.4 nmol min31 (mg DW)31 at 0.1 mmol l31 citrate (Fig. 2A). The properties of uptake also changed. The pH dependence was biphasic with a pH optimum between pH 6 and 7, with a small depression on the acidic side and an inhibition at alkaline pH values (Fig. 2B). The dependence of citrate uptake on citrate concentration showed saturation 35 31 in the concentration range of 10 mol l (KM 65 þ 5.0 31 31 31 Wmol l ; Vmax 0.34 þ 0.13 nmol min (mg DW) ; Fig. 2C). The uncouplers TCS (Fig. 2D) and FCCP (not shown) completely inhibited citrate uptake at a concentration of less than 10 Wmol l31. Azide (1 mmol l31 ; inhibitor of F0F1-ATPase) decreased citrate uptake into citrate- adapted mycelium to 7.5%, mucidine (50 Wmol l31 ; inhibitor of respiration) to 8.8%, but cyanide (10 mmol l31 ; inhibitor of respiration) only to 91.7% (not shown). The temperature optimum was about 37‡C and the tempera- Fig. 4. E¡ect of metal cations and EDTA on citrate uptake by glucose- ture dependence could be characterized by a Q10 of 3.0 grown (A) and citrate-adapted mycelium (B). A: Transport was mea- 31 and an activation energy of 42.7 þ 3.5 kJ mol31 kg31 be- sured in the cultivation medium (control), medium with 20 mmol l EDTA (+EDTA), or with modi¢ed medium where Mg2þ (3Mg) or tween 12 and 38‡C. Thus, adaptation to citrate increased Mn2þ (3Mn) was omitted. Transport was measured for 20 min at 30‡C citrate uptake strongly and elicited a dependence on met- and the results were corrected for the radioactivity measured at zero abolic energy. The presence of inhibitors of macromolec- time. B: Transport was measured in the cultivation medium without ular synthesis prevented almost completely the stimulation glucose (Con), or in this medium containing 20 mmol l31 EDTA 2þ 3 2þ 3 of citrate uptake (Fig. 3). Citrate uptake was only margin- (+EA), or in modi¢ed media where Mg ( Mg), or Mn ( Mn), or Cu2þ (3Cu), or Zn2þ (3Zn), or Fe2þ (3Fe) were omitted. In another ally a¡ected by EDTA or by omitting cations (Fig. 4). The control experiment, glucose (440 mmol l31) was present (Glc). Other 2þ omission of Zn stimulated citrate uptake up to 20% and conditions as in A. Data shown in A and Bwere obtained from inde- the omission of Fe2þ caused an inhibition of about 20%. pendent experiments.

FEMSLE 10536 12-7-02 M. Síimkovic› et al. / FEMS Microbiology Letters 213 (2002) 21^26 25 Downloaded from https://academic.oup.com/femsle/article/213/1/21/516581 by guest on 23 September 2021

Fig. 5. Properties of citrate uptake by citrate-grown mycelium. The decrease of citrate uptake in the medium was measured by HPLC and citrate uptake was calculated from these data. A: Dependence of uptake on citrate concentration. The citrate uptake has been measured for 20 min at 30‡C with the concentrations of citrate indicated in the ¢gure. B: Kinetics of citrate uptake by citrate-grown mycelium (squares) and the e¡ect of succinate (inverted triangles) and of glucose (circles). Transport was measured in the presence of 0.1 mmol l31 citrate at 30‡C for the time indicated. Glucose and succinate were present at the same concentration as citrate. C: Citrate levels in citrate-grown mycelium during the transport measurement. Transport was measured at the conditions indicated in B. Data shown in A^C were obtained from independent experiments. synthesis of macromolecules was necessary for the induc- system: pH 2^4 in A. niger, pH 6^7 in P. simplicissimum. tion of this transport system as con¢rmed by the e¡ect of In addition, strong e¡ects of EDTA and manganese, inhibitors of RNA and protein synthesis. The low rate of which were characteristic of A. niger ATCC 11414, were citrate uptake by glucose-grown mycelium of P. simplicis- not found in P. simplicissimum: 20 mmol l31 EDTA in- simum was most probably due to simple di¡usion of un- hibited citrate uptake only by 10% and the withdrawal of dissociated citric acid. We draw this conclusion because Mn2þ increased citrate uptake only by about 10%. of two observations: (i) citrate uptake showed a linear Can P. simplicissimum excrete citrate via an inverse up- increase over a wide range of citrate concentrations; take system? The obtained results do not support this (ii) the uncouplers TCS and FCCP had only little e¡ect assumption. Firstly, no citrate uptake was observed by on citrate uptake. There was also a clear di¡erence be- mycelium which excreted citrate (the same was observed tween citrate-adapted and citrate-grown mycelium. Citrate- with A. niger) [7]. This means that the transport system for grown mycelium showed a ¢ve-fold higher KM value and citrate e¥ux cannot take up citrate. Secondly, the citrate a 25-fold higher Vmax value. This could mean that citrate- uptake system is an active transport system and thus needs grown mycelium contained a higher number of citrate up- a proton motive force. If citrate e¥ux proceeded via an take systems per unit area. inverse citrate uptake system, abolishing the proton mo- The only other ¢lamentous fungus in which citrate tive force should lead to an increased citrate e¥ux. This transport has been characterized is A. niger ATCC 11414 was observed, for instance, with Candida utilis [14]. How- [7]. In citrate-grown mycelium of A. niger, the Vmax was ever, uncouplers and sodium azide only decreased citrate 31 31 31 8.9 nmol min (mg DW) and the KM was 280 Wmol l . uptake but did not increase citrate e¥ux. Thus, the kinetic properties of the induced citrate uptake systems are similar in both fungi. Another similarity is that citrate uptake was inhibited by uncouplers and sodium azide in both fungi. However, there are also Acknowledgements di¡erences. Glucose-grown mycelium of P. simplicissimum showed a much lower citrate uptake rate compared to This work has been in part supported by Grant 1/7342/ glucose-grown mycelium of A. niger (Vmax 3.6 nmol 20 of the Slovak Grant Agency VEGA and by a grant to 31 31 31 min (mg DW) and KM 220 Wmol l ). A further di¡er- M.K. from the bilateral grant program Austria^Slovakia ence was the pH optimum of the induced citrate uptake (Fellowship No. B-1710).

FEMSLE 10536 12-7-02 26 M. Síimkovic› et al. / FEMS Microbiology Letters 213 (2002) 21^26

References Solubilization of zinc oxide from ¢lter dust with Penicillium simplicissimum: bioreactor leaching and stoichiometry. Environ. Sci. Tech- [1] Jones, D.L. (1998) Organic acids in the rhizosphere ^ a critical re- nol. 26, 340^346. view. Plant Soil 205, 25^44. [9] Gallmetzer, M. and Burgstaller, W. (2001) Citrate e¥ux in glucose- [2] Gadd, G.M. (1999) Fungal production of citric and oxalic acid: limited and glucose-su⁄cient chemostat culture of Penicillium simpli- importance in metal speciation, physiology and biogeochemical pro- cissimum. Antonie van Leeuwenhoek 79, 81^87. cesses. Adv. Microb. Physiol. 41, 47^92. [10] Gallmetzer, M., Mu«ller, B. and Burgstaller, W. (1998) Net e¥ux of [3] Kwack, H. and Veech, R.L. (1992) Citrate: its relation to free mag- citrate in Penicillium simplicissimum is mediated by a transport pro- nesium ion concentration and cellular energy. Curr. Top. Cell. Regul. tein. Arch. Microbiol. 169, 353^359. 33, 195^207. [11] Schinner, F., Brunner, H., Burgstaller, W., PernfuM, B. and Strasser [4] Errecalde, O. and Campbell, P.G.C. (2000) Cadmium and zinc bio- H. (1990) Mikrobielle Laugung von Zwischenprodukten und Erzen availability to Selenastrum capricornutum (Chlorophyceae): acciden- der Montanwerke Brixlegg. Bericht fu«r das Projektjahr 1989/1990 (in tal metal uptake and toxicity in the presence of citrate. J. Phycol. 36, German). 473^483. [12] Hoischen, C. and Kra«mer, R. (1989) Evidence for an e¥ux carrier [5] Winkelmann, G. (Ed.) (2001) Microbial Transport Systems. Wiley- system involved in the secretion of glutamate by Corynebacterium VCH, Weinheim. glutamicum. Arch. Microbiol. 151, 342^347. Downloaded from https://academic.oup.com/femsle/article/213/1/21/516581 by guest on 23 September 2021 [6] Auling, G. (1994) Manganese: function and transport in fungi. In: [13] Burgstaller, W., Zanella, A. and Schinner, F. (1994) Bu¡er-stimulated Metal Ions in Fungi (Winkelmann, G. and Winge, D.R., Eds.), pp. citrate-e¥ux in Penicillium simplicissimum: an alternative charge bal- 215^236. Dekker, New York. ancing ion £ow in case of reduced proton back£ow? Arch. Microbiol. [7] Netik, A., Torres, N.V., Riol, J.-M. and Kubicek, C.P. (1997) Uptake 161, 75^81. and export of citric acid by Aspergillus niger is reciprocally regulated [14] Cassio, F. and Leao, C. (1991) Low- and high-a⁄nity transport sys- by manganese ions. Biochim. Biophys. Acta 1326, 287^294. tems for citric acid in the yeast Candida utilis. Appl. Environ. Micro- [8] Burgstaller, W., Strasser, H., Wo«bking, H. and Schinner, F. (1992) biol. 57, 3623^3628.

FEMSLE 10536 12-7-02