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An unusual pattern of invertase activity development in the thermophilic fungus Thermomyces lanuginosus
FEMS Microbiology Letters 177 (1999) 39^45
An unusual pattern of invertase activity development in the thermophilic fungus Thermomyces lanuginosus Amitabha Chaudhuri 1 , Girish Bharadwaj, Ramesh Maheshwari * Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India Received 3 June 1999 ; received in revised form 7 June 1999 ; accepted 7 June 1999
Abstract In the thermophilic fungus Thermomyces lanuginosus, invertase displays an unusual pattern of development: the induced activity begins to diminish even before any substantial quantity of sucrose has been utilized or an appreciable amount of biomass has been produced. Despite this pattern of invertase activity, neither the growth rate nor the final mycelial yield is affected adversely. T. lanuginosus invertase is a thiol protein and the enzyme is active when specific sulfhydryl group(s) is in the reduced state. Measurements of reduced coenzyme and glutathione pools in sucrose-grown mycelia excluded oxidative stress as the primary reason for the observed decline in invertase activity. Rather, this unusual pattern of invertase is considered to be due to its localization in the hyphal tips. At the early stage of growth, the number of hyphal tips per unit mass of mycelium is maximum, whereas at later times their numbers do not increase in proportion to the biomass. As a result invertase activity shows an apparent inverse relationship with biomass. The enzyme activity disappears when the inducing carbon source is consumed and growth is completed. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Invertase ; Regulation; Redox status; Hyphal tip; Thermophilic fungus ; Thermomyces lanuginosus
1. Introduction Our earlier studies revealed that in contrast to yeasts and mesophilic molds, invertase in the thermophilic fungus Thermomyces lanuginosus was an induced enzyme, intracellular, highly unstable in the cell-free extracts [1] but stabilized by thiol compounds [2]. Moreover, the enzyme displayed a very
* Corresponding author. Fax: +91 (80) 334 1814; E-mail: [email protected] 1 Present address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
peculiar behavior: when T. lanuginosus was grown in a liquid medium containing sucrose as the carbon source, invertase activity was induced rapidly but the increase in enzyme activity was transient [1]. The enzyme activity began to diminish even before an appreciable utilization of sugar or any substantial increase in biomass had occurred. Surprisingly, despite the abrupt decline in invertase activity, neither the growth rate, nor the ¢nal mycelial yield was affected adversely [3]. By contrast, in the mesophilic fungi, for e.g. Neurospora crassa [4] and Aspergillus niger [1], mycelial invertase activity steadily increased with the age of cultures and was maintained at high levels even after the carbon source was completely
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exhausted. In the present investigation we have attempted to explain the unusual pattern of invertase activity in T. lanuginosus.
2. Materials and methods 2.1. Organism and culture conditions T. lanuginosus, strain RM-B, was isolated from horse-dung in our laboratory and has been deposited in the American Type Culture Collection (ATCC 44008). It was grown in a sucrose-asparagine medium in 150 ml medium in 500 ml Erlenmeyer £asks at 50³C with shaking at 240 rpm [3]. 2.2. Measurement of biomass and estimation of sucrose Samples of culture were removed aseptically at regular intervals, ¢ltered through a glass ¢ber ¢lter and washed. Growth was monitored as increase in mycelial dry weight. Sucrose in culture medium was estimated using yeast invertase as described previously [1]. 2.3. Assay of enzyme activities Mycelia were frozen in liquid nitrogen and powdered by grinding with acid-washed quartz in a mortar with pestle. The powder was stirred in 50 mM Na/K phosphate bu¡er (pH 6.0) containing 1 mM dithiothreitol (DTT) and 1 mM EDTA for 5^15 min at 4³C. The crude homogenate, or a clari¢ed extract prepared after centrifugation at 12 000Ug for 10 min, was used for the measurement of invertase activity by quantitating the reducing sugar liberated from the hydrolysis of 40 mM sucrose in 50 mM Na/K phosphate bu¡er (pH 6.5) for 5 min at 50³C. Reducing sugars were estimated by the method of Somogyi [5]. One unit of invertase activity was de¢ned as the amount of protein that produced 1 Wmol of glucose per min under the assay conditions. For trehalase assay, aliquots of the crude homogenate were used as the enzyme activity remained associated with the insoluble matter. Trehalase activity was measured using 2 mM trehalose in 50 mM Na acetate bu¡er, pH 5.5. Reaction was done at
50³C for 15 min with intermittent shaking, and the reducing sugar (glucose) released was estimated by the Somogyi method. One unit of trehalase activity was de¢ned as the amount of protein that produced 1 Wmol of glucose per min. For measurement of G6PDH activity, extracts were prepared as above in 50 mM Na/K phosphate bu¡er (pH 7.0) and clari¢ed by centrifugation before use. The reaction mixture in a total volume of 1 ml contained 12 mM glucose 6-phosphate, 0.1 mM NADP and 10 mM MgSO4 W7H2 O in 50 mM Na/ K phosphate bu¡er at pH 7.0. The reaction was initiated by adding di¡erent volumes of cell extract and monitoring the increase in A340 due to the formation of NADPH for 5 min at 50³C. One unit of enzyme activity was de¢ned as the amount of protein that produced 1 Wmol of NADPH per min under the assay conditions. 2.4. Estimation of total thiols, GSH and GSSG Mycelia (120^130 mg dry wt) were extracted in 5 ml of 5% sulfosalicylic acid for 10 min in a boiling water bath. The supernatant obtained after centrifugation was taken for the estimation of total thiols by reaction with 5,5P-dithio-bis(2-nitrobenzoic acid) (DTNB). Glutathione (GSH) and glutathione disul¢de (GSSG) were estimated by two methods. The ¢rst method was based on DTNB-GSSG reductase recycling assay [6]. The principle of this method is the oxidation of GSH by DTNB to give GSSG with stoichiometric formation of thionitrobenzoic acid (TNB) which is quantitated by measuring absorbance at 412 nm. GSSG was reduced to GSH by the action of glutathione reductase and NADPH resulting in the formation of more TNB, which was followed continuously at 412 nm. The amount of GSH in extracts was determined from a standard curve where GSH equivalents were plotted against the rate of change of absorbance at 412 nm. For the determination of GSSG, GSH in extract was blocked by reaction with N-ethylmaleimide (NEM). The unreacted NEM was removed by extractions with diethyl ether and sample was taken for assay as before. The second method was based on the reaction of thiols with monobromobimane (mBBr) to yield highly £uorescent thioether derivatives which are
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separated by HPLC and detected by a £uorimeter [7]. For the estimation of GSSG, GSH in extract was modi¢ed by reaction with NEM, followed by reduction of GSSG with DTT. The resulting GSH was derivatized by monobromobimane (mBBr) and samples were analyzed by HPLC. 2.5. Estimation of NADP+ and NADPH in mycelia The oxidized and the reduced pyridine nucleotides were estimated according to the method of Klingenberg [8]. The NADP in the neutralized extract was estimated enzymatically by the glucose 6-P dehydrogenase reaction. The reaction mixture in 50 mM Na/ K phosphate bu¡er (pH 7.0) contained 5 mM glucose 6-phosphate, 5 mM MgSO4 W7H2 O and di¡erent volumes of the extract in a total volume of 2.1 ml. The reaction was started by adding 10 units of glucose 6-phosphate dehydrogenase (G6PDH) (1 unit = 1 Wmol NADPH formed min31 ) and the increase in A340 due to NADPH was monitored for 20^25 min at 30³C. For the estimation of NADPH, mycelium (280^ 300 mg dry wt equivalent) was extracted in 4 ml of 0.5 M KOH in 50% ethanol for 10 min in a boiling water bath. After centrifugation, the clari¢ed extract was brought to pH 7.8 and NADPH was speci¢cally estimated by the glutathione reductase reaction. The reaction mixture in 50 mM Na/K phosphate bu¡er (pH 7.5) contained 0.025 mM GSSG and di¡erent volumes of the extract in a total volume of 2 ml. The reaction was started by adding 0.5 unit glutathione reductase (1 unit = 1 Wmol NADPH utilized min31 ) and the decrease in A340 due to conversion of NADPH was monitored for 5^10 min at 30³C. In the second method of NADPH estimation, the supernatant from alcoholic KOH extraction was adjusted to pH 7.8 and the reduced pyridine nucleotides were oxidized using glutamate dehydrogenase. The extract was deproteinized with 0.2 ml of 3 M HClO4 and the precipitated protein was removed by centrifugation. The supernatant was adjusted to pH 7.2 and taken for the estimation of NADP by the G6PDH reaction described earlier. 2.6. Activity of pentose phosphate pathway The activity of pentose phosphate pathway was
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measured based on the rationale that only the carbon-1 of glucose is decarboxylated through this pathway. Washed mycelia (1 g wet wt) were suspended in 50 ml medium containing 10 mM unlabelled glucose, 0.1% K2 HPO4 and 0.05% MgSO4 W7H2 O. One ml of the mycelial suspension was added to 2 ml of the above medium in Warburg £asks which contained a folded ¢lter paper soaked in 6 M KOH in the center well. After 2 min equilibration at 50³C in a shaker water bath, 14 C-1 glucose (0.25 WCi) was added to the suspension and the £asks were stoppered. After incubation for 30 min, the radioactivity due to 14 CO2 absorbed on the ¢lter paper was measured by scintillation counting. 2.7. Incorporation of 3 H-thymidine in mycelia T. lanuginosus was grown in a sucrose-asparagine medium and the mycelia were harvested at 6 h intervals. At each time point similar amount of mycelium (50 mg wet weight) was suspended in 10 ml of the spent medium and incubated in a shaker water bath at 50³C. 3 H-thymidine (10 WCi) was added and after 30 min incubation, the mycelium was washed successively with 5% TCA, ethanol and a mixture of chloroform:ethanol:diethyl ether (1:2:1 v/v) on glass ¢ber ¢lter. The radioactivity incorporated in mycelium was determined as described earlier [1].
3. Results 3.1. Distinct patterns of invertase and trehalase development Invertase activity in T. lanuginosus grown in a medium containing sucrose as the carbon source, expressed either as speci¢c activity or total activity, increased abruptly from undetectable levels to maximum level in 6 to 12 h (Fig. 1). Thereafter, the activity immediately began to fall although nearly 80^85% of sucrose was still available. Signi¢cant increase in biomass occurred during the time when invertase activity was declining. Despite this fall in enzyme activity, the rates of growth and utilization of sucrose were related. As seen from Fig. 1, the time of half-maximal utilization of sucrose and of halfmaximal growth was the same. The time of complete
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the label incorporated per unit mass of mycelia remained virtually constant although the biomass increased up to 36 h. The incorporation of radiolabelled thymidine did not parallel the increase in biomass. The time of maximal invertase activity (Fig. 1) closely approximated the time of DNA synthesis (Table 1) rather than the bulk increase in biomass. 3.3. Reciprocal modulation of invertase activity in extracts by reduced and oxidized glutathione Fig. 1. Patterns of development of invertase and trehalase activity in Thermomyces lanuginosus in relation to biomass and sucrose utilization. The fungus was grown in a medium containing 2% (w/v) sucrose in shake £asks at 50³C. Both enzyme activities were measured in crude mycelial homogenate. Enzyme activity in total mycelial biomass sampled (20 ml) is referred to as the total activity.
utilization of sucrose (36^42 h) coincided with the disappearance of invertase activity. In marked contrast, trehalase activity was low as long as carbon source remained in the medium and it was derepressed after carbon source was exhausted. 3.2. Invertase activity is correlated with the time of maximal DNA synthesis As invertase activity was not correlated with mycelial dry weight, we determined if it would relate to growth estimated by DNA synthesis. Maximum incorporation of 3 H-thymidine (cpm mg31 dry wt) occurred at approximately 6 h and decreased by 33% in 12 h, by 40% in 18 h and by 70% in 24 h. After 24 h,
In T. lanuginosus invertase is a thiol protein and its activity in cell-free extracts is modulated reciprocally by reduced and oxidized glutathione [2]. The redox status (SH/SS ratio) of the mycelium could, therefore, regulate the activity of invertase. To examine this, an in vitro approach was taken in which the response of the inactivated enzyme to mixtures of GSH and GSSG was studied. The activation of invertase depended on the ratio of [GSH]/[GSSG]; the enzyme activity increasing with the increasing ratio of SH/SS. Moreover, enzyme activation depended also on the absolute concentration of glutathione. At a higher absolute concentration of glutathione, 7.5 mM instead of 2.5 mM, a lower [GSH]/[GSSG] ratio resulted in higher recovery of activity. 3.4. Intracellular redox status The fall in invertase activity in T. lanuginosus after its early rise could be because the intracellular environment becomes oxidizing with time, i.e. the ratio GSH/GSSG becomes low, resulting in the oxidation
Table 1 Total thiols, GSH and GSSG in Thermomyces lanuginosus during growth in a medium containing sucrose Time of growth (h)
Total thiols (Wmole g dry wt31 )
GSH
GSSG
A
B
A
B
A
B
Average
6 12 18 24 30 36
6.8 þ 2.0 4.8 þ 1.0 4.4 þ 1.7 4.5 þ 0.3 n.d. 3.7 þ 0.1
6.1 þ 1.0 3.8 þ 0.5 3.6 þ 0.7 3.6 þ 0.5 n.d. 3.5 þ 0.5
4.8 4.3 2.5 3.6 3.5 3.4
0.05 þ 0.01 0.05 þ 0.01 0.07 þ 0.02 n.d. n.d. 0.06 þ 0.02
0.06 0.08 0.06 0.08 0.06 0.06
127 72 49 n.d. n.d. 59
80 54 41 45 58 56
104 63 45 45 58 58
A, estimations by DTNB-GSSG reductase recycling assay, in Wmol g31 dry weight. B, estimations by monobromobimane labelling method, in Wmol g31 dry weight. n.d., not determined.
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of invertase thiols and inactivation of the enzyme. Therefore, the concentrations of GSH and GSSG in mycelia were determined. Nearly similar values were found by both enzymatic and HPLC methods (Table 1). Throughout, glutathione constituted most of the total thiol compounds in the cell extracts. GSSG, which was detected in low amounts, did not vary signi¢cantly. A high GSH/GSSG ratio was observed at 6 h when invertase activity was increasing. This ratio was V50 or more during the growth period. 3.5. GSH levels, NADPH and activity of pentose phosphate pathway The cellular level of GSH is maintained by the glutathione reductase reaction that converts GSSG into GSH using NADPH as the reductant [9]. Whether the GSH/GSSG ratio is re£ected in the ratio of NADPH and NADP was determined. The concentration of NADPH was highest at 6 h and it declined by 2.5-fold between 6 and 36 h of growth although the level of NADP did not change signi¢cantly (Table 2). The NADPH/NADP ratio decreased 4-fold between these time points. Since NADPH is generated by the pentose phosphate pathway, we determined whether the activity of this pathway relates to the concentrations of reduced NADP coenzyme and of glutathione. The activity of pentose phosphate pathway during growth was estimated by two experimental approaches: by measuring the decarboxylation of glucose using 14 C-1 glucose and by determining the activity of G6PDH, a key enzyme of this pathway. As shown
43
in Table 2, both the decarboxylation of glucose as well as the activity of G6PDH were most active at 6 h and declined thereafter.
4. Discussion In fungi, two contrasting developmental patterns of invertase activity have been observed: (1) The (constitutive) enzyme activity increases steadily with time and is highest in mycelium when growth is completed and the carbon source has been utilized, as in N. crassa [4]. This pattern was explained on the basis that a major portion of invertase is distributed in the intramural space [10] and the increase in enzyme activity re£ects the amount of wall material in old mycelium. Similar pattern was also seen for the intracellular trehalases in N. crassa [11], and in T. lanuginosus (present study). (2) The (induced) invertase activity shows an inverse relationship with the amount of biomass and disappears upon the exhaustion of the carbon source (sucrose), as in T. lanuginosus (Fig. 1). The present investigation has attempted to understand the distinctive pattern of invertase in T. lanuginosus. Because the activity of T. lanuginosus invertase in cell extracts was modulated by the ratio of GSH and GSSG, it was suspected that a change from a reducing to an oxidizing environment in the hyphae could oxidize invertase thiol(s) and cause its inactivation. Several plant and animal enzymes are regulated by GSH/GSSG ratio [12,13]; however, no other invertase is regulated similarly. The ratio GSH/GSSG (Table 1) was maximum in mycelia sampled at 6 h
Table 2 Activity of pentose phosphate pathway and the levels of reductive power in Thermomyces lanuginosus during growth in sucrose mediuma Time of growth (h)
6 12 18 24 30 36 a b
Activity of pentose phosphate pathway as estimated bya C1 decarboxylation (cpm mg31 dry wt)
G6PDH (units g dry wt31 )
1029 310 129 76 41 326
26 20 17 15 14 17
NADP (nmol g dry wt31 )
NADPH (nmol g dry wt31 )
Ratio NADPH to NADP
20 þ 10 29 þ 10 18 þ 5 45 n.d.b 30 þ 10
80 þ 17 60 þ 17 30 þ 7 80 þ 16 n.d. 30 þ 6
4.0 2.1 1.7 1.7 n.d. 1.0
Average values of three determinations from a single experiment. n.d., not determined
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when invertase activity was increasing rapidly but growth was very small, though perceptible. This ratio is consistent with the NADPH/NADP ratio and the activity of the pentose phosphate pathway (Table 2) which generates reduced coenzyme required for glutathione reductase catalyzed reaction for GSH production (GSSG+NADPH+H C2GSH+NADP ). Although the GSH/GSSG ratio declined between 6 and 12 h, the invertase activity reduced after 12 h. Despite the perturbations, the GSH/GSSG ratio remained V50, and in fair agreement with that in other organisms [14]. A cautious view is that the intracellular environment in T. lanuginosus remained favorable for invertase. Why then did invertase activity show an inverse correlation with biomass? A revealing observation was that the time of maximum DNA synthesis (0^12 h) was related to the time of maximal growth rate of the fungus [3], and to the burst in invertase activity, but not to the time of maximal increase in biomass (8^16 h) [3]. In fungi, growth and nuclear division are con¢ned to hyphal tips [15,16]. The pattern of 3 H-thymidine incorporation suggested a higher frequency of branch initiation, the number of hyphal tips and of nuclear divisions at the early times. The increase in biomass mainly results from the deposition of cell wall polysaccharides in the elongating hyphal cells [16]. Therefore, for an enzyme that is localized in the apical region, rather than uniformly distributed in the hypha (as are invertase in N. crassa and trehalase in T. lanuginosus), its activity in mycelial samples will appear to diminish as the apical region is diluted by cell elongation and wall thickening in the proximal region of the hypha (Fig. 1). Our attempt to demonstrate invertase in the hypha by immuno£uorescence staining was thwarted by the failure to purify invertase [2]. A su¤cient quantity of mycelium for enzyme puri¢cation, from the early hours of culture, was di¤cult to obtain. The di¤culty in purifying invertase was exacerbated by the inactivation of enzyme during the puri¢cation steps. However, two other observations strongly support the view that in T. lanuginosus invertase is localized in the hyphal tips. One, the activities of both invertase and a protondriven sucrose transporter in T. lanuginosus followed a parallel course of induction and decline [17]. This suggests that invertase is restricted to the apical re-
gion where nutrient transporters have been postulated to be preferentially localized [18]. An association of sucrose transporter and invertase would provide T. lanuginosus a special advantage in scavenging and utilizing sucrose from the environment. Indeed, in mixtures of sucrose and glucose, the fungus utilizes sucrose faster than glucose [3]. Second, invertase synthesis in T. lanuginosus was dependent on growth and DNA synthesis [1], events that occur in the apical region [15,16]. Sucrose is not only necessary for the induced synthesis of sucrose transporter and invertase [1,17], but also for maintaining a reducing intracellular environment for invertase activity through the generation of reducing power (NADPH), which in turn promotes the reduction of GSSG to GSH. Among the other purposes for which the reducing power would be required in the growing hyphal tips are biosynthesis of membrane fatty acids and the conversion of ribonucleotides to deoxyribonucleotides for DNA synthesis. It needs to be emphasized that the measurements of glutathione and of reduced coenzyme describe the average property of mycelial sample, not their concentrations in the hyphal compartments. It is quite likely that throughout growth, the hyphal tip has a reducing environment. In conclusion, the distinctive pattern of invertase development in T. lanuginosus is a consequence of a combination of reasons: invertase being an induced enzyme; a thiol protein; it being dependent on a reducing intracellular environment for activity; and being localized in the hyphal tip. The study suggests that the activity of some redox-sensitive intracellular enzymes in fungi may be optimized by their induced synthesis as required and/or their placement in the most strategic location in the hypha.
Acknowledgements This work was supported by Department of Science and Technology, Government of India.
References [1] Maheshwari, R., Balasubramanyam, P.V. and Palanivelu, P. (1983) Distinctive behaviour of invertase in a thermophilic
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[2]
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fungus Thermomyces lanuginosus. Arch. Microbiol. 134, 255^ 260. Chaudhuri, A. and Maheshwari, R. (1996) A novel invertase from a thermophilic fungus Thermomyces lanuginosus: Its requirement of thiol and protein for activation. Arch. Biochem. Biophys. 327, 98^106. Maheshwari, R. and Balasubramanyam, P.V. (1988) Simultaneous utilization of glucose and sucrose by thermophilic fungi. J. Bacteriol. 170, 3274^3280. Hill, E.P. and Sussman, A.S. (1964) Development of trehalase and invertase activity in Neurospora. J. Bacteriol. 8, 1556^ 1566. Somogyi, M. (1952) Notes on sugar determination. J. Biol. Chem. 195, 19^23. Anderson, M.E. (1985) Determination of glutathione and glutathione disul¢de in biological samples. Methods Enzymol. 113, 548^555. Fahey, R.C. and Newton, G.L. (1987) Determination of lowmolecular weight thiols using monobromobimane £uorescent labelling and high performance liquid chromatography. Methods Enzymol. 143, 85^96. Klingenberg, M. (1987) Nicotinamide adenine dinucleotides and dinucleotide phosphates. In: Methods of Enzymatic Analysis (Bergmeyer, J. and GraMl, M., Eds.), Vol. 7, pp. 251^267. Verlag-Chemie, Weinheim. Meister, A. and Anderson, M.E. (1983) Glutathione. Annu. Rev. Biochem. 52, 711^760. Marzluf, G.A. and Metzenberg, R.L. (1967) Studies on the
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[14]
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functional signi¢cance of the transmembrane location of invertase in Neurospora crassa. Arch. Biochem. Biophys. 120, 487^496. Hanks, D.L. and Sussman, A.S. (1969) The relation between growth, conidiation and trehalase activity in Neurospora crassa. Am. J. Bot. 56, 1152^1159. Buchanan, B.B. (1991) Regulation of CO2 assimilation in oxygenic photosynthesis : The ferredoxin/thioredoxin system. Arch. Biochem. Biophys. 288, 1^9. Terada, T., Maeda, H., Okamoto, K., Nishinaka, T. and Mizoguchi, T. (1993) Modulation of glutathione S-transferase activity by a thiol/disul¢de exchange reaction and involvement of thiol transferase. Arch. Biochem. Biophys. 300, 495^500. Penninckx, M.J. and Elskens, M.T. (1993) Metabolism and functions of glutathione in microorganisms. Adv. Microbiol. Physiol. 34, 239^301. King, S.B. and Alexander, L.J. (1969) Nuclear behaviour, septation, and hyphal growth of Alternaria solani. Am. J. Bot. 56, 249^253. Wessels, J.G.H. (1986) Cell wall synthesis in apical hyphal growth. Int. Rev. Cytol. 104, 37^79. Palanivelu, P., Balasubramanyam, P.V. and Maheshwari, R. (1984) Co-induction of sucrose transport and invertase activities in a thermophilic fungus Thermomyces lanuginosus. Arch. Microbiol. 139, 44^47. Harold, F.M., Kropf, D.L. and Caldwell, J.J. (1985) Why do fungi drive electric currents through themselves? Exp. Mycol. 9, 183^186.
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An unusual pattern of invertase activity development in the thermophilic fungus Thermomyces lanuginosus Amitabha Chaudhuri 1 , Girish Bharadwaj, Ramesh Maheshwari * Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India Received 3 June 1999 ; received in revised form 7 June 1999 ; accepted 7 June 1999
Abstract In the thermophilic fungus Thermomyces lanuginosus, invertase displays an unusual pattern of development: the induced activity begins to diminish even before any substantial quantity of sucrose has been utilized or an appreciable amount of biomass has been produced. Despite this pattern of invertase activity, neither the growth rate nor the final mycelial yield is affected adversely. T. lanuginosus invertase is a thiol protein and the enzyme is active when specific sulfhydryl group(s) is in the reduced state. Measurements of reduced coenzyme and glutathione pools in sucrose-grown mycelia excluded oxidative stress as the primary reason for the observed decline in invertase activity. Rather, this unusual pattern of invertase is considered to be due to its localization in the hyphal tips. At the early stage of growth, the number of hyphal tips per unit mass of mycelium is maximum, whereas at later times their numbers do not increase in proportion to the biomass. As a result invertase activity shows an apparent inverse relationship with biomass. The enzyme activity disappears when the inducing carbon source is consumed and growth is completed. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Invertase ; Regulation; Redox status; Hyphal tip; Thermophilic fungus ; Thermomyces lanuginosus
1. Introduction Our earlier studies revealed that in contrast to yeasts and mesophilic molds, invertase in the thermophilic fungus Thermomyces lanuginosus was an induced enzyme, intracellular, highly unstable in the cell-free extracts [1] but stabilized by thiol compounds [2]. Moreover, the enzyme displayed a very
* Corresponding author. Fax: +91 (80) 334 1814; E-mail: [email protected] 1 Present address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
peculiar behavior: when T. lanuginosus was grown in a liquid medium containing sucrose as the carbon source, invertase activity was induced rapidly but the increase in enzyme activity was transient [1]. The enzyme activity began to diminish even before an appreciable utilization of sugar or any substantial increase in biomass had occurred. Surprisingly, despite the abrupt decline in invertase activity, neither the growth rate, nor the ¢nal mycelial yield was affected adversely [3]. By contrast, in the mesophilic fungi, for e.g. Neurospora crassa [4] and Aspergillus niger [1], mycelial invertase activity steadily increased with the age of cultures and was maintained at high levels even after the carbon source was completely
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 8 6 - 4
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exhausted. In the present investigation we have attempted to explain the unusual pattern of invertase activity in T. lanuginosus.
2. Materials and methods 2.1. Organism and culture conditions T. lanuginosus, strain RM-B, was isolated from horse-dung in our laboratory and has been deposited in the American Type Culture Collection (ATCC 44008). It was grown in a sucrose-asparagine medium in 150 ml medium in 500 ml Erlenmeyer £asks at 50³C with shaking at 240 rpm [3]. 2.2. Measurement of biomass and estimation of sucrose Samples of culture were removed aseptically at regular intervals, ¢ltered through a glass ¢ber ¢lter and washed. Growth was monitored as increase in mycelial dry weight. Sucrose in culture medium was estimated using yeast invertase as described previously [1]. 2.3. Assay of enzyme activities Mycelia were frozen in liquid nitrogen and powdered by grinding with acid-washed quartz in a mortar with pestle. The powder was stirred in 50 mM Na/K phosphate bu¡er (pH 6.0) containing 1 mM dithiothreitol (DTT) and 1 mM EDTA for 5^15 min at 4³C. The crude homogenate, or a clari¢ed extract prepared after centrifugation at 12 000Ug for 10 min, was used for the measurement of invertase activity by quantitating the reducing sugar liberated from the hydrolysis of 40 mM sucrose in 50 mM Na/K phosphate bu¡er (pH 6.5) for 5 min at 50³C. Reducing sugars were estimated by the method of Somogyi [5]. One unit of invertase activity was de¢ned as the amount of protein that produced 1 Wmol of glucose per min under the assay conditions. For trehalase assay, aliquots of the crude homogenate were used as the enzyme activity remained associated with the insoluble matter. Trehalase activity was measured using 2 mM trehalose in 50 mM Na acetate bu¡er, pH 5.5. Reaction was done at
50³C for 15 min with intermittent shaking, and the reducing sugar (glucose) released was estimated by the Somogyi method. One unit of trehalase activity was de¢ned as the amount of protein that produced 1 Wmol of glucose per min. For measurement of G6PDH activity, extracts were prepared as above in 50 mM Na/K phosphate bu¡er (pH 7.0) and clari¢ed by centrifugation before use. The reaction mixture in a total volume of 1 ml contained 12 mM glucose 6-phosphate, 0.1 mM NADP and 10 mM MgSO4 W7H2 O in 50 mM Na/ K phosphate bu¡er at pH 7.0. The reaction was initiated by adding di¡erent volumes of cell extract and monitoring the increase in A340 due to the formation of NADPH for 5 min at 50³C. One unit of enzyme activity was de¢ned as the amount of protein that produced 1 Wmol of NADPH per min under the assay conditions. 2.4. Estimation of total thiols, GSH and GSSG Mycelia (120^130 mg dry wt) were extracted in 5 ml of 5% sulfosalicylic acid for 10 min in a boiling water bath. The supernatant obtained after centrifugation was taken for the estimation of total thiols by reaction with 5,5P-dithio-bis(2-nitrobenzoic acid) (DTNB). Glutathione (GSH) and glutathione disul¢de (GSSG) were estimated by two methods. The ¢rst method was based on DTNB-GSSG reductase recycling assay [6]. The principle of this method is the oxidation of GSH by DTNB to give GSSG with stoichiometric formation of thionitrobenzoic acid (TNB) which is quantitated by measuring absorbance at 412 nm. GSSG was reduced to GSH by the action of glutathione reductase and NADPH resulting in the formation of more TNB, which was followed continuously at 412 nm. The amount of GSH in extracts was determined from a standard curve where GSH equivalents were plotted against the rate of change of absorbance at 412 nm. For the determination of GSSG, GSH in extract was blocked by reaction with N-ethylmaleimide (NEM). The unreacted NEM was removed by extractions with diethyl ether and sample was taken for assay as before. The second method was based on the reaction of thiols with monobromobimane (mBBr) to yield highly £uorescent thioether derivatives which are
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separated by HPLC and detected by a £uorimeter [7]. For the estimation of GSSG, GSH in extract was modi¢ed by reaction with NEM, followed by reduction of GSSG with DTT. The resulting GSH was derivatized by monobromobimane (mBBr) and samples were analyzed by HPLC. 2.5. Estimation of NADP+ and NADPH in mycelia The oxidized and the reduced pyridine nucleotides were estimated according to the method of Klingenberg [8]. The NADP in the neutralized extract was estimated enzymatically by the glucose 6-P dehydrogenase reaction. The reaction mixture in 50 mM Na/ K phosphate bu¡er (pH 7.0) contained 5 mM glucose 6-phosphate, 5 mM MgSO4 W7H2 O and di¡erent volumes of the extract in a total volume of 2.1 ml. The reaction was started by adding 10 units of glucose 6-phosphate dehydrogenase (G6PDH) (1 unit = 1 Wmol NADPH formed min31 ) and the increase in A340 due to NADPH was monitored for 20^25 min at 30³C. For the estimation of NADPH, mycelium (280^ 300 mg dry wt equivalent) was extracted in 4 ml of 0.5 M KOH in 50% ethanol for 10 min in a boiling water bath. After centrifugation, the clari¢ed extract was brought to pH 7.8 and NADPH was speci¢cally estimated by the glutathione reductase reaction. The reaction mixture in 50 mM Na/K phosphate bu¡er (pH 7.5) contained 0.025 mM GSSG and di¡erent volumes of the extract in a total volume of 2 ml. The reaction was started by adding 0.5 unit glutathione reductase (1 unit = 1 Wmol NADPH utilized min31 ) and the decrease in A340 due to conversion of NADPH was monitored for 5^10 min at 30³C. In the second method of NADPH estimation, the supernatant from alcoholic KOH extraction was adjusted to pH 7.8 and the reduced pyridine nucleotides were oxidized using glutamate dehydrogenase. The extract was deproteinized with 0.2 ml of 3 M HClO4 and the precipitated protein was removed by centrifugation. The supernatant was adjusted to pH 7.2 and taken for the estimation of NADP by the G6PDH reaction described earlier. 2.6. Activity of pentose phosphate pathway The activity of pentose phosphate pathway was
41
measured based on the rationale that only the carbon-1 of glucose is decarboxylated through this pathway. Washed mycelia (1 g wet wt) were suspended in 50 ml medium containing 10 mM unlabelled glucose, 0.1% K2 HPO4 and 0.05% MgSO4 W7H2 O. One ml of the mycelial suspension was added to 2 ml of the above medium in Warburg £asks which contained a folded ¢lter paper soaked in 6 M KOH in the center well. After 2 min equilibration at 50³C in a shaker water bath, 14 C-1 glucose (0.25 WCi) was added to the suspension and the £asks were stoppered. After incubation for 30 min, the radioactivity due to 14 CO2 absorbed on the ¢lter paper was measured by scintillation counting. 2.7. Incorporation of 3 H-thymidine in mycelia T. lanuginosus was grown in a sucrose-asparagine medium and the mycelia were harvested at 6 h intervals. At each time point similar amount of mycelium (50 mg wet weight) was suspended in 10 ml of the spent medium and incubated in a shaker water bath at 50³C. 3 H-thymidine (10 WCi) was added and after 30 min incubation, the mycelium was washed successively with 5% TCA, ethanol and a mixture of chloroform:ethanol:diethyl ether (1:2:1 v/v) on glass ¢ber ¢lter. The radioactivity incorporated in mycelium was determined as described earlier [1].
3. Results 3.1. Distinct patterns of invertase and trehalase development Invertase activity in T. lanuginosus grown in a medium containing sucrose as the carbon source, expressed either as speci¢c activity or total activity, increased abruptly from undetectable levels to maximum level in 6 to 12 h (Fig. 1). Thereafter, the activity immediately began to fall although nearly 80^85% of sucrose was still available. Signi¢cant increase in biomass occurred during the time when invertase activity was declining. Despite this fall in enzyme activity, the rates of growth and utilization of sucrose were related. As seen from Fig. 1, the time of half-maximal utilization of sucrose and of halfmaximal growth was the same. The time of complete
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the label incorporated per unit mass of mycelia remained virtually constant although the biomass increased up to 36 h. The incorporation of radiolabelled thymidine did not parallel the increase in biomass. The time of maximal invertase activity (Fig. 1) closely approximated the time of DNA synthesis (Table 1) rather than the bulk increase in biomass. 3.3. Reciprocal modulation of invertase activity in extracts by reduced and oxidized glutathione Fig. 1. Patterns of development of invertase and trehalase activity in Thermomyces lanuginosus in relation to biomass and sucrose utilization. The fungus was grown in a medium containing 2% (w/v) sucrose in shake £asks at 50³C. Both enzyme activities were measured in crude mycelial homogenate. Enzyme activity in total mycelial biomass sampled (20 ml) is referred to as the total activity.
utilization of sucrose (36^42 h) coincided with the disappearance of invertase activity. In marked contrast, trehalase activity was low as long as carbon source remained in the medium and it was derepressed after carbon source was exhausted. 3.2. Invertase activity is correlated with the time of maximal DNA synthesis As invertase activity was not correlated with mycelial dry weight, we determined if it would relate to growth estimated by DNA synthesis. Maximum incorporation of 3 H-thymidine (cpm mg31 dry wt) occurred at approximately 6 h and decreased by 33% in 12 h, by 40% in 18 h and by 70% in 24 h. After 24 h,
In T. lanuginosus invertase is a thiol protein and its activity in cell-free extracts is modulated reciprocally by reduced and oxidized glutathione [2]. The redox status (SH/SS ratio) of the mycelium could, therefore, regulate the activity of invertase. To examine this, an in vitro approach was taken in which the response of the inactivated enzyme to mixtures of GSH and GSSG was studied. The activation of invertase depended on the ratio of [GSH]/[GSSG]; the enzyme activity increasing with the increasing ratio of SH/SS. Moreover, enzyme activation depended also on the absolute concentration of glutathione. At a higher absolute concentration of glutathione, 7.5 mM instead of 2.5 mM, a lower [GSH]/[GSSG] ratio resulted in higher recovery of activity. 3.4. Intracellular redox status The fall in invertase activity in T. lanuginosus after its early rise could be because the intracellular environment becomes oxidizing with time, i.e. the ratio GSH/GSSG becomes low, resulting in the oxidation
Table 1 Total thiols, GSH and GSSG in Thermomyces lanuginosus during growth in a medium containing sucrose Time of growth (h)
Total thiols (Wmole g dry wt31 )
GSH
GSSG
A
B
A
B
A
B
Average
6 12 18 24 30 36
6.8 þ 2.0 4.8 þ 1.0 4.4 þ 1.7 4.5 þ 0.3 n.d. 3.7 þ 0.1
6.1 þ 1.0 3.8 þ 0.5 3.6 þ 0.7 3.6 þ 0.5 n.d. 3.5 þ 0.5
4.8 4.3 2.5 3.6 3.5 3.4
0.05 þ 0.01 0.05 þ 0.01 0.07 þ 0.02 n.d. n.d. 0.06 þ 0.02
0.06 0.08 0.06 0.08 0.06 0.06
127 72 49 n.d. n.d. 59
80 54 41 45 58 56
104 63 45 45 58 58
A, estimations by DTNB-GSSG reductase recycling assay, in Wmol g31 dry weight. B, estimations by monobromobimane labelling method, in Wmol g31 dry weight. n.d., not determined.
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of invertase thiols and inactivation of the enzyme. Therefore, the concentrations of GSH and GSSG in mycelia were determined. Nearly similar values were found by both enzymatic and HPLC methods (Table 1). Throughout, glutathione constituted most of the total thiol compounds in the cell extracts. GSSG, which was detected in low amounts, did not vary signi¢cantly. A high GSH/GSSG ratio was observed at 6 h when invertase activity was increasing. This ratio was V50 or more during the growth period. 3.5. GSH levels, NADPH and activity of pentose phosphate pathway The cellular level of GSH is maintained by the glutathione reductase reaction that converts GSSG into GSH using NADPH as the reductant [9]. Whether the GSH/GSSG ratio is re£ected in the ratio of NADPH and NADP was determined. The concentration of NADPH was highest at 6 h and it declined by 2.5-fold between 6 and 36 h of growth although the level of NADP did not change signi¢cantly (Table 2). The NADPH/NADP ratio decreased 4-fold between these time points. Since NADPH is generated by the pentose phosphate pathway, we determined whether the activity of this pathway relates to the concentrations of reduced NADP coenzyme and of glutathione. The activity of pentose phosphate pathway during growth was estimated by two experimental approaches: by measuring the decarboxylation of glucose using 14 C-1 glucose and by determining the activity of G6PDH, a key enzyme of this pathway. As shown
43
in Table 2, both the decarboxylation of glucose as well as the activity of G6PDH were most active at 6 h and declined thereafter.
4. Discussion In fungi, two contrasting developmental patterns of invertase activity have been observed: (1) The (constitutive) enzyme activity increases steadily with time and is highest in mycelium when growth is completed and the carbon source has been utilized, as in N. crassa [4]. This pattern was explained on the basis that a major portion of invertase is distributed in the intramural space [10] and the increase in enzyme activity re£ects the amount of wall material in old mycelium. Similar pattern was also seen for the intracellular trehalases in N. crassa [11], and in T. lanuginosus (present study). (2) The (induced) invertase activity shows an inverse relationship with the amount of biomass and disappears upon the exhaustion of the carbon source (sucrose), as in T. lanuginosus (Fig. 1). The present investigation has attempted to understand the distinctive pattern of invertase in T. lanuginosus. Because the activity of T. lanuginosus invertase in cell extracts was modulated by the ratio of GSH and GSSG, it was suspected that a change from a reducing to an oxidizing environment in the hyphae could oxidize invertase thiol(s) and cause its inactivation. Several plant and animal enzymes are regulated by GSH/GSSG ratio [12,13]; however, no other invertase is regulated similarly. The ratio GSH/GSSG (Table 1) was maximum in mycelia sampled at 6 h
Table 2 Activity of pentose phosphate pathway and the levels of reductive power in Thermomyces lanuginosus during growth in sucrose mediuma Time of growth (h)
6 12 18 24 30 36 a b
Activity of pentose phosphate pathway as estimated bya C1 decarboxylation (cpm mg31 dry wt)
G6PDH (units g dry wt31 )
1029 310 129 76 41 326
26 20 17 15 14 17
NADP (nmol g dry wt31 )
NADPH (nmol g dry wt31 )
Ratio NADPH to NADP
20 þ 10 29 þ 10 18 þ 5 45 n.d.b 30 þ 10
80 þ 17 60 þ 17 30 þ 7 80 þ 16 n.d. 30 þ 6
4.0 2.1 1.7 1.7 n.d. 1.0
Average values of three determinations from a single experiment. n.d., not determined
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when invertase activity was increasing rapidly but growth was very small, though perceptible. This ratio is consistent with the NADPH/NADP ratio and the activity of the pentose phosphate pathway (Table 2) which generates reduced coenzyme required for glutathione reductase catalyzed reaction for GSH production (GSSG+NADPH+H C2GSH+NADP ). Although the GSH/GSSG ratio declined between 6 and 12 h, the invertase activity reduced after 12 h. Despite the perturbations, the GSH/GSSG ratio remained V50, and in fair agreement with that in other organisms [14]. A cautious view is that the intracellular environment in T. lanuginosus remained favorable for invertase. Why then did invertase activity show an inverse correlation with biomass? A revealing observation was that the time of maximum DNA synthesis (0^12 h) was related to the time of maximal growth rate of the fungus [3], and to the burst in invertase activity, but not to the time of maximal increase in biomass (8^16 h) [3]. In fungi, growth and nuclear division are con¢ned to hyphal tips [15,16]. The pattern of 3 H-thymidine incorporation suggested a higher frequency of branch initiation, the number of hyphal tips and of nuclear divisions at the early times. The increase in biomass mainly results from the deposition of cell wall polysaccharides in the elongating hyphal cells [16]. Therefore, for an enzyme that is localized in the apical region, rather than uniformly distributed in the hypha (as are invertase in N. crassa and trehalase in T. lanuginosus), its activity in mycelial samples will appear to diminish as the apical region is diluted by cell elongation and wall thickening in the proximal region of the hypha (Fig. 1). Our attempt to demonstrate invertase in the hypha by immuno£uorescence staining was thwarted by the failure to purify invertase [2]. A su¤cient quantity of mycelium for enzyme puri¢cation, from the early hours of culture, was di¤cult to obtain. The di¤culty in purifying invertase was exacerbated by the inactivation of enzyme during the puri¢cation steps. However, two other observations strongly support the view that in T. lanuginosus invertase is localized in the hyphal tips. One, the activities of both invertase and a protondriven sucrose transporter in T. lanuginosus followed a parallel course of induction and decline [17]. This suggests that invertase is restricted to the apical re-
gion where nutrient transporters have been postulated to be preferentially localized [18]. An association of sucrose transporter and invertase would provide T. lanuginosus a special advantage in scavenging and utilizing sucrose from the environment. Indeed, in mixtures of sucrose and glucose, the fungus utilizes sucrose faster than glucose [3]. Second, invertase synthesis in T. lanuginosus was dependent on growth and DNA synthesis [1], events that occur in the apical region [15,16]. Sucrose is not only necessary for the induced synthesis of sucrose transporter and invertase [1,17], but also for maintaining a reducing intracellular environment for invertase activity through the generation of reducing power (NADPH), which in turn promotes the reduction of GSSG to GSH. Among the other purposes for which the reducing power would be required in the growing hyphal tips are biosynthesis of membrane fatty acids and the conversion of ribonucleotides to deoxyribonucleotides for DNA synthesis. It needs to be emphasized that the measurements of glutathione and of reduced coenzyme describe the average property of mycelial sample, not their concentrations in the hyphal compartments. It is quite likely that throughout growth, the hyphal tip has a reducing environment. In conclusion, the distinctive pattern of invertase development in T. lanuginosus is a consequence of a combination of reasons: invertase being an induced enzyme; a thiol protein; it being dependent on a reducing intracellular environment for activity; and being localized in the hyphal tip. The study suggests that the activity of some redox-sensitive intracellular enzymes in fungi may be optimized by their induced synthesis as required and/or their placement in the most strategic location in the hypha.
Acknowledgements This work was supported by Department of Science and Technology, Government of India.
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