- Email: [email protected]
Allosamidin inhibits the fragmentation of Acremonium chrysogenum but does not influence the cephalosporin-C production of the fungus
FEMS Microbiology Letters 164 (1998) 231^236
Allosamidin inhibits the fragmentation of Acremonium chrysogenum but does not in£uence the cephalosporin-C production of the fungus Erzseèbet Saèndor a , Tuënde Pusztahelyi a , Levente Kara¡a a , Zsolt Karaènyi b , Imre Poècsi a , Saèndor Biroè a , Attila Szentirmai a , Istvaèn Poècsi a; * a
Department of Microbiology and Biotechnology, Kossuth Lajos University, P.O. Box 63, H-4010 Debrecen, Hungary b 1st Department of Medicine, University Medical School of Debrecen, P.O. Box 19, H-4012 Debrecen, Hungary Received 1 April 1998; revised 7 May 1998; accepted 10 May 1998
Abstract The pseudotrisaccharide allosamidin, a potent inhibitor of chitinases, retarded the fragmentation of hyphae but did not affect the fungal growth and cephalosporin-C production in Acremonium chrysogenum. In vitro inhibition of A. chrysogenum cell-bound chitinase(s) by allosamidin revealed that about 47% of the soluble intracellular chitinase activity was resistant to the inhibitory effect of allosamidin. On the other hand, about 76% of the total chitinase activity localised in both the soluble and insoluble enzyme fractions was effectively inhibited by allosamidin. All the chitinase activities were measured using a new procedure based on purified A. chrysogenum chitin as substrate. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Acremonium chrysogenum; Fragmentation ; Chitinase ; Allosamidin; Cephalosporin-C production
1. Introduction The growth of the ¢lamentous fungus Acremonium chrysogenum, which is the main fungal producer of the widely used antibiotic cephalosporin-C, is characterised by dimorphism [1^5]. In the idiophase, A. chrysogenum hyphae swell and fragment into spherical cells called `yeast-like' forms or `arthrospores', and this conversion coincides with the maximum rate of cephalosporin-C production [1^5]. In present paper we report on the role that chiti* Corresponding author. Tel.: +36 (52) 316666, ext. 2061; Fax: +36 (52) 310936; E-mail: [email protected]
nolytic enzymes play in the breakage of mycelial hyphae in A. chrysogenum cultures during the switch from the rapid growth to the production phase. Moreover, the signi¢cance of the fragmentation in the antibiotic production is also discussed.
2. Materials and methods 2.1. Organism and culture conditions A. chrysogenum ATCC 46117 (synonym: Cephalosporium acremonium W 532553) was grown using standard cultivation methods as described elsewhere
0378-1097 / 98 / $19.00 ß 1998 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 8 ) 0 0 2 0 9 - 2
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Fig. 1. Typical morphological forms of A. chrysogenum at 65 h of incubation in the absence (A) and in the presence (B) of 6.0 Wg ml31 allosamidin.
[5]. All the fermentations were performed in 500 ml unbu¡ered Erlenmeyer £asks containing 50 ml aliquots of a minimal culture medium which was
supplied, in g l31 , with glucose, 20.0; (NH4 )2 SO4 , 15.0; methionine, 6.0; NaNO3 , 3.0, K2 HPO4 , 1.0; KCl, 0.5; MgSO4 .7H2 O, 0.5; CaCl2 , 0.4; and was
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233
Fig. 2. The average cell numbers in hyphae of A. chrysogenum during fermentation in the absence (8) and in the presence of 4.0 Wg ml31 (F) or 6.0 Wg ml31 (O) allosamidin. Arrow indicates the addition of allosamidin.
also supplied with 2.0 ml trace element solution (3.8 g MnSO4 H2 O, 2.5 g ZnSO4 , 2.5 g CuSO4 and 0.05 g FeSO4 dissolved in 200 ml distilled water) per litre of fermentation medium. To each £ask was also added 0.1 ml Silicone SAG 471 (Union Carbide Europe, Versoix, Switzerland) as an antifoam agent. The cultures were incubated at 200 rpm at 28³C.
2.2. Preparation of allosamidin solutions Allosamidin (Eli Lilly Company, Indianapolis, IN, USA) stock solutions were prepared in 0.005 M acetic acid in a concentration of 2.0 mg ml31 . For in vivo studies, this solution was diluted with distilled water, sterilised by ¢ltration and was added to the culture media at 12 h of incubation as required. In in
Fig. 3. In vitro inhibition of the soluble (F) and the total (8) cell-bound chitinase activities by allosamidin. A. chrysogenum cells were grown in the absence of allosamidin and were harvested at 46 h incubation time.
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vitro enzyme inhibition studies the stock solution was diluted with 0.1 M citric acid-sodium citrate (pH 5.5) bu¡er.
by boiling for 2 min. Finally, the concentration of the released N-acetyl-D-glucosamine was determined spectrophotometrically [7].
2.3. Preparation of enzyme samples
2.5. Puri¢cation of A. chrysogenum chitin
A. chrysogenum cells from 80 ml aliquots of culture £uids were harvested by ¢ltration in the stationary phase of growth at 46 h of cultivation. The cells were washed, resuspended in 40 ml of 0.1 M citric acid-sodium citrate (pH 5.5) bu¡er and were frozen immediately at 370³C. Following was that the cells were disrupted with a type X25 French-press equipment (AB Biox, Goëteborg, Sweden), and were allowed to thaw at 37³C. After addition of phenylmethylsulfonyl £uoride [6], the samples were either used directly for the determination of the total cellbound chitinase activity or centrifuged at 10 000Ug for 15 min. In the latter case only the supernatants were used in the chitinase assay to estimate the soluble cell-bound enzyme activity. Extracellular chitinase, N-acetyl-L-D-hexosaminidase and protease activities were determined in the ¢ltrates of the cultures. The allosamidin content of the samples was removed by bu¡er exchange on PD10 columns (Pharmacia Biotech, Uppsala, Sweden) before activity measurements. Enzyme samples used in protease determinations did not contain phenylmethylsulfonyl £uoride.
Fungal chitin was puri¢ed from exponentially growing A. chrysogenum at 20 h fermentation time. A quantity of 200 g wet weight mycelium was exposed to 4% NaOH overnight and concentrated HCl for 1 h at room temperature. Crude chitin was precipitated by ¢ltration into ice-cold water and was further re¢ned by subsequent agitation in 4% NaOH and 7% HCl solutions, and by exhaustive dialysis against distilled water. Yield: 0.4 g chitin which consisted of 91.0% N-acetyl-D-glucosamine and 9.0% D-glucosamine as calculated from elemental analysis data.
2.4. The chitinase assay A new two-step chitinase assay was elaborated. In Step 1, 0.9 ml enzyme sample was added to 1.0 ml chitin suspension containing 3.0 mg colloidal A. chrysogenum chitin in 0.1 M citric acid-sodium citrate (pH 5.5) bu¡er. The reaction volumes were completed to 2.0 ml with 0.5 Wg ml31 allosamidin solution and/or the citrate bu¡er as required. The reaction mixtures were incubated with shaking for 8 h at 25³C, boiled for 5 min, and were centrifuged at 10 000Ug for 10 min. In Step 2, 0.6 nkat activity of puri¢ed Penicillium chrysogenum N-acetyl-L-Dhexosaminidase (chitobiase) [6] was added to 0.9 ml aliquots of the supernatants from Step 1, and the reaction volumes were adjusted to 1 ml with 0.1 M citric acid-sodium citrate (pH 5.5) bu¡er. After incubation at 37³C for 1 h, the reactions were stopped
2.6. Determination of growth, fragmentation, proportion of yeast-like forms, CPC production, N-acetyl-L-D-hexosaminidase and protease activities The microbial growth was characterised by the changes in the dry cell weight [8], the fragmentation process was quanti¢ed by the average cell numbers in hyphae [5], yeast-like cells were counted analysing the cell lengths and diameters on photomicrographs [4] and the production of cephalosporin-C was monitored by HPLC [8]. N-Acetyl-L-D-hexosaminidase and protease activities were measured using p-nitrophenyl-N-acetyl-LD-glucosaminide and azocasein substrates, respectively [7]. Protease activities were characterised by K, the velocity constant of the enzymic reaction as before [7]. 2.7. Reproducibility and statistics In terms of reproducibility, all the experiments described in this report are the mean values of 3^5 independent measurements. The S.D. values of the means were always less than 15%. For the statistical analysis of fragmentation the SAS for Windows system, Version 6.12 was used. The signi¢cance of the di¡erences between cell counts changing in time and as a function of the applied allosamidin concentra-
FEMSLE 8217 9-7-98
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tions was examined by the two-way analysis of variance. Only the probability levels of P 9 0.005 were considered as indicative of the statistical signi¢cance.
3. Results and discussion All chitin-containing fungi that have been investigated so far produce chitinases, which are thought to have autolytic, nutritional and morphogenetic functions [9]. In ¢lamentous fungi they probably have a role in hyphal growth, branching and in spore germination [9,10]. Earlier enzyme inhibition studies have demonstrated that chitinases are also involved in the cell separation of the yeasts Saccharomyces cerevisiae [11,12] and Candida albicans [9]. Allosamidin, which is an e¡ective inhibitor of fungal chitinases [13^15], did not have any e¡ect on the growth of A. chrysogenum (data not shown) similar to S. cerevisiae [11,12] and C. albicans [13]. In terms of morphology, the pseudotrisaccharide signi¢cantly hindered the fragmentation of A. chrysogenum hyphae even at concentrations of as low as 4.0 Wg ml31 (6.4 WM) or 6.0 Wg ml31 (9.6 WM) (Figs. 1 and 2). That is, chitinolytic enzymes played an essential role in the breakage of hyphae in addition to mechanical forces [16] and the physiological state of the cells [17]. On the other hand, the swelling of slender hyphal ¢laments was not in£uenced by allosamidin and, hence, the quantity of spherical cells in hyphae was similar to that of yeast-like cells in control cultures (Fig. 1) [5]. Consequently, the formation of swollen hyphal fragments might be triggered by either low molecular mass arthrospore-inducing factors [18] or increases in the thiol concentrations of the cells [19] rather than by the action of chitinases. Most interestingly, the cephalosporin-C production of A. chrysogenum, which was about 0.5 g l31 in control cultures [5], was not in£uenced by the addition of allosamidin, which challenges any possible causal connection between the antibiotic production and the fragmentation of the fungus. In A. chrysogenum culture ¢ltrates, a very high, K = 0.08^0.10, protease activity was detected between 40 and 110 h incubation times. In comparison, the maximum proteolytic activity in ageing P. chrysogenum cultures was only K = 0.015 as reported by Pusztahelyi et al. [7]. As a possible consequence of the
235
action of proteases, no extracellular chitinase and Nacetyl-L-D-hexosaminidase activities were observed at any fermentation time in A. chrysogenum cultures. Nevertheless, signi¢cant soluble and insoluble cellbound chitinase activities (Fig. 3) were detected in the stationary phase of growth of A. chrysogenum when the fragmentation had started and was progressing [4,5]. As demonstrated in Fig. 3, about 47% (36 pkat mg31 dryweight ) of the soluble cell-bound chitinase activity (77 pkat mg31 dryweight ) was resistant to the inhibitory e¡ect of allosamidin. On the other hand, about 76% (113 pkat mg31 dryweight ) of the total chitinase activity (149 pkat mg31 dryweight ) localised in both the soluble (41 pkat mg31 dryweight ) and insoluble (72 pkat mg31 dryweight ) enzyme fractions was inhibited e¡ectively by the pseudotrisaccharide. Obviously, the allosamidin sensitive enzyme activity, which was detected in both the soluble and insoluble cell-bound enzyme fractions, played a crucial role in the fragmentation process. The enzymological features and the physiological function of the allosamidin resistant chitinolytic enzyme(s), which were localised exclusively in the soluble cell-bound fraction as shown in Fig. 3, has remained yet to be elucidated. Interestingly, allosamidin resistant chitinases have only been detected in bacteria and plants thus far [20].
Acknowledgments This project was supported by the OTKA (grant reference number: T 019484), the Foundation for the Hungarian Higher Education and Research (AMFK 639/96) and by the Hungarian Creative Art Foundation (13-914-97/P1). The allosamidin was generously supplied by the Lilly Research Laboratories, Indianapolis, IN, USA; we especially thank the valuable help of Dr. H.A. Kirst and Mrs. M.H. Niedenthal.
References [1] Nash, C.H. and Huber, F.M. (1971) Antibiotic synthesis and morphological di¡erentiation of Cephalosporium acremonium. Appl. Microbiol. 22, 6^10.
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[2] Matsumura, M., Imanaka, T., Yoshida, T. and Taguchi, H. (1980) Morphological di¡erentiation in relation to Cephalosporin C synthesis by Cephalosporium acremonium. J. Ferment. Technol. 58, 197^204. [3] Bartoshevich, Yu.E., Zaslavskaya, P.L., Novak, M.J. and Yudina, O.D. (1990) Acremonium chrysogenum di¡erentiation and biosynthesis of cephalosporin. J. Basic Microbiol. 30, 313^320. [4] Kara¡a, L., Saèndor, E., Kozma, J. and Szentirmai, A. (1996) Cephalosporin-C production, morphology and alternative respiration of Acremonium chrysogenum in glucose-limited chemostat. Biotechnol. Lett. 18, 701^706. [5] Kara¡a, L., Saèndor, E., Kozma, J. and Szentirmai, A. (1997) Methionine enhances sugar consumption, fragmentation, vacuolation and cephalosporin-C production in Acremonium chrysogenum. Process Biochem. 32, 495^499. [6] Poècsi, I., Pusztahelyi, T., Bogaèti, M.Sz. and Szentirmai, A. (1993) The formation of N-acetyl-L-D-hexosaminidase is repressed by glucose in Penicillium chrysogenum. J. Basic Microbiol. 33, 259^267. [7] Pusztahelyi, T., Poècsi, I. and Szentirmai, A. (1997) Ageing of Penicillium chrysogenum cultures under carbon starvation. II. Protease and N-acetyl-L-D-hexosaminidase production. Biotechnol. Appl. Biochem. 25, 87^93. [8] Kozma, J. and Kara¡a, L. (1996) E¡ect of oxygen on the respiratory system and cephalosporin-C production in Acremonium chrysogenum. J. Biotechnol. 48, 59^66. [9] Gooday, G.W., Zhu, W.-Y. and O'Donnell, R.W. (1992) What are the roles of chitinases in the growing fungus ? FEMS Microbiol. Lett. 100, 387^392. [10] Rast, D.M., Horsch, M., Furter, R. and Gooday, G.W. (1991) A complex chitinolytic system in exponentially growing mycelium of Mucor rouxii: properties and function. J. Gen. Microbiol. 137, 2797^2810. [11] Cabib, E., Silverman, S.J. and Shaw, J.A. (1992) Chitinase
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
and chitin synthase 1: counterbalancing activities in cell separation of Saccharomyces cerevisiae. J. Gen. Microbiol. 138, 97^102. Izumida, H., Nishijima, M., Takadera, T., Nomoto, A.M. and Sano, H. (1995) The e¡ect of chitinase inhibitors, cyclo(ArgPro) against cell separation of Saccharomyces cerevisiae and the morphological change of Candida albicans. J. Antibiot. 49, 829^831. Dickinson, K., Keer, V., Hitchcock, C.A. and Adams, D.J. (1989) Chitinase activity from Candida albicans and its inhibition by allosamidin. J. Gen. Microbiol. 135, 1417^1421. McNab, R. and Glover, L.A. (1991) Inhibition of Neurospora crassa cytosolic chitinase by allosamidin. FEMS Microbiol. Lett. 82, 79^82. Isakurada, M., Morgavi, D.P., Komatani, K., Tomita, Y. and Onodera, R. (1996) Puri¢cation and characteristics of cytosolic chitinase from Piromyces communis OTS1. FEMS Microbiol. Lett. 137, 75^78. Makagiansar, H.Y., Ayazi Shamlou, P., Thomas, C.R. and Lilly, M.D. (1993) The in£uence of mechanical forces on the morphology and penicillin production of Penicillium chrysogenum. Bioprocess Eng. 9, 83^90. Paul, G.C., Kent, C.A. and Thomas, C.R. (1994) Hyphal vacuolation and fragmentation in Penicillium chrysogenum. Biotechnol. Bioeng. 44, 655^660. Natsume, M. and Marumo, S. (1984) Arthrospore-inducing substances from Acremonium chrysogenum which stimulate cephalosporin C production. Agric. Biol. Chem. 48, 567^569. Crabbe, M.J.C. (1988) The e¡ect of thiols and the Ca2 ionophore A23817 on growth and antibiotic production in Cephalosporium acremonium. FEMS Microbiol. Lett. 56, 71^78. Koga, D., Isogai, A., Sakuda, S., Matsumoto, S., Suzuki, A., Kimura, S. and Ide, A. (1987) Speci¢c inhibition of Bombyx mori chitinase by allosamidin. Agric. Biol. Chem. 51, 471^476.
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Allosamidin inhibits the fragmentation of Acremonium chrysogenum but does not in£uence the cephalosporin-C production of the fungus Erzseèbet Saèndor a , Tuënde Pusztahelyi a , Levente Kara¡a a , Zsolt Karaènyi b , Imre Poècsi a , Saèndor Biroè a , Attila Szentirmai a , Istvaèn Poècsi a; * a
Department of Microbiology and Biotechnology, Kossuth Lajos University, P.O. Box 63, H-4010 Debrecen, Hungary b 1st Department of Medicine, University Medical School of Debrecen, P.O. Box 19, H-4012 Debrecen, Hungary Received 1 April 1998; revised 7 May 1998; accepted 10 May 1998
Abstract The pseudotrisaccharide allosamidin, a potent inhibitor of chitinases, retarded the fragmentation of hyphae but did not affect the fungal growth and cephalosporin-C production in Acremonium chrysogenum. In vitro inhibition of A. chrysogenum cell-bound chitinase(s) by allosamidin revealed that about 47% of the soluble intracellular chitinase activity was resistant to the inhibitory effect of allosamidin. On the other hand, about 76% of the total chitinase activity localised in both the soluble and insoluble enzyme fractions was effectively inhibited by allosamidin. All the chitinase activities were measured using a new procedure based on purified A. chrysogenum chitin as substrate. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Acremonium chrysogenum; Fragmentation ; Chitinase ; Allosamidin; Cephalosporin-C production
1. Introduction The growth of the ¢lamentous fungus Acremonium chrysogenum, which is the main fungal producer of the widely used antibiotic cephalosporin-C, is characterised by dimorphism [1^5]. In the idiophase, A. chrysogenum hyphae swell and fragment into spherical cells called `yeast-like' forms or `arthrospores', and this conversion coincides with the maximum rate of cephalosporin-C production [1^5]. In present paper we report on the role that chiti* Corresponding author. Tel.: +36 (52) 316666, ext. 2061; Fax: +36 (52) 310936; E-mail: [email protected]
nolytic enzymes play in the breakage of mycelial hyphae in A. chrysogenum cultures during the switch from the rapid growth to the production phase. Moreover, the signi¢cance of the fragmentation in the antibiotic production is also discussed.
2. Materials and methods 2.1. Organism and culture conditions A. chrysogenum ATCC 46117 (synonym: Cephalosporium acremonium W 532553) was grown using standard cultivation methods as described elsewhere
0378-1097 / 98 / $19.00 ß 1998 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 8 ) 0 0 2 0 9 - 2
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Fig. 1. Typical morphological forms of A. chrysogenum at 65 h of incubation in the absence (A) and in the presence (B) of 6.0 Wg ml31 allosamidin.
[5]. All the fermentations were performed in 500 ml unbu¡ered Erlenmeyer £asks containing 50 ml aliquots of a minimal culture medium which was
supplied, in g l31 , with glucose, 20.0; (NH4 )2 SO4 , 15.0; methionine, 6.0; NaNO3 , 3.0, K2 HPO4 , 1.0; KCl, 0.5; MgSO4 .7H2 O, 0.5; CaCl2 , 0.4; and was
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233
Fig. 2. The average cell numbers in hyphae of A. chrysogenum during fermentation in the absence (8) and in the presence of 4.0 Wg ml31 (F) or 6.0 Wg ml31 (O) allosamidin. Arrow indicates the addition of allosamidin.
also supplied with 2.0 ml trace element solution (3.8 g MnSO4 H2 O, 2.5 g ZnSO4 , 2.5 g CuSO4 and 0.05 g FeSO4 dissolved in 200 ml distilled water) per litre of fermentation medium. To each £ask was also added 0.1 ml Silicone SAG 471 (Union Carbide Europe, Versoix, Switzerland) as an antifoam agent. The cultures were incubated at 200 rpm at 28³C.
2.2. Preparation of allosamidin solutions Allosamidin (Eli Lilly Company, Indianapolis, IN, USA) stock solutions were prepared in 0.005 M acetic acid in a concentration of 2.0 mg ml31 . For in vivo studies, this solution was diluted with distilled water, sterilised by ¢ltration and was added to the culture media at 12 h of incubation as required. In in
Fig. 3. In vitro inhibition of the soluble (F) and the total (8) cell-bound chitinase activities by allosamidin. A. chrysogenum cells were grown in the absence of allosamidin and were harvested at 46 h incubation time.
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vitro enzyme inhibition studies the stock solution was diluted with 0.1 M citric acid-sodium citrate (pH 5.5) bu¡er.
by boiling for 2 min. Finally, the concentration of the released N-acetyl-D-glucosamine was determined spectrophotometrically [7].
2.3. Preparation of enzyme samples
2.5. Puri¢cation of A. chrysogenum chitin
A. chrysogenum cells from 80 ml aliquots of culture £uids were harvested by ¢ltration in the stationary phase of growth at 46 h of cultivation. The cells were washed, resuspended in 40 ml of 0.1 M citric acid-sodium citrate (pH 5.5) bu¡er and were frozen immediately at 370³C. Following was that the cells were disrupted with a type X25 French-press equipment (AB Biox, Goëteborg, Sweden), and were allowed to thaw at 37³C. After addition of phenylmethylsulfonyl £uoride [6], the samples were either used directly for the determination of the total cellbound chitinase activity or centrifuged at 10 000Ug for 15 min. In the latter case only the supernatants were used in the chitinase assay to estimate the soluble cell-bound enzyme activity. Extracellular chitinase, N-acetyl-L-D-hexosaminidase and protease activities were determined in the ¢ltrates of the cultures. The allosamidin content of the samples was removed by bu¡er exchange on PD10 columns (Pharmacia Biotech, Uppsala, Sweden) before activity measurements. Enzyme samples used in protease determinations did not contain phenylmethylsulfonyl £uoride.
Fungal chitin was puri¢ed from exponentially growing A. chrysogenum at 20 h fermentation time. A quantity of 200 g wet weight mycelium was exposed to 4% NaOH overnight and concentrated HCl for 1 h at room temperature. Crude chitin was precipitated by ¢ltration into ice-cold water and was further re¢ned by subsequent agitation in 4% NaOH and 7% HCl solutions, and by exhaustive dialysis against distilled water. Yield: 0.4 g chitin which consisted of 91.0% N-acetyl-D-glucosamine and 9.0% D-glucosamine as calculated from elemental analysis data.
2.4. The chitinase assay A new two-step chitinase assay was elaborated. In Step 1, 0.9 ml enzyme sample was added to 1.0 ml chitin suspension containing 3.0 mg colloidal A. chrysogenum chitin in 0.1 M citric acid-sodium citrate (pH 5.5) bu¡er. The reaction volumes were completed to 2.0 ml with 0.5 Wg ml31 allosamidin solution and/or the citrate bu¡er as required. The reaction mixtures were incubated with shaking for 8 h at 25³C, boiled for 5 min, and were centrifuged at 10 000Ug for 10 min. In Step 2, 0.6 nkat activity of puri¢ed Penicillium chrysogenum N-acetyl-L-Dhexosaminidase (chitobiase) [6] was added to 0.9 ml aliquots of the supernatants from Step 1, and the reaction volumes were adjusted to 1 ml with 0.1 M citric acid-sodium citrate (pH 5.5) bu¡er. After incubation at 37³C for 1 h, the reactions were stopped
2.6. Determination of growth, fragmentation, proportion of yeast-like forms, CPC production, N-acetyl-L-D-hexosaminidase and protease activities The microbial growth was characterised by the changes in the dry cell weight [8], the fragmentation process was quanti¢ed by the average cell numbers in hyphae [5], yeast-like cells were counted analysing the cell lengths and diameters on photomicrographs [4] and the production of cephalosporin-C was monitored by HPLC [8]. N-Acetyl-L-D-hexosaminidase and protease activities were measured using p-nitrophenyl-N-acetyl-LD-glucosaminide and azocasein substrates, respectively [7]. Protease activities were characterised by K, the velocity constant of the enzymic reaction as before [7]. 2.7. Reproducibility and statistics In terms of reproducibility, all the experiments described in this report are the mean values of 3^5 independent measurements. The S.D. values of the means were always less than 15%. For the statistical analysis of fragmentation the SAS for Windows system, Version 6.12 was used. The signi¢cance of the di¡erences between cell counts changing in time and as a function of the applied allosamidin concentra-
FEMSLE 8217 9-7-98
E. Saèndor et al. / FEMS Microbiology Letters 164 (1998) 231^236
tions was examined by the two-way analysis of variance. Only the probability levels of P 9 0.005 were considered as indicative of the statistical signi¢cance.
3. Results and discussion All chitin-containing fungi that have been investigated so far produce chitinases, which are thought to have autolytic, nutritional and morphogenetic functions [9]. In ¢lamentous fungi they probably have a role in hyphal growth, branching and in spore germination [9,10]. Earlier enzyme inhibition studies have demonstrated that chitinases are also involved in the cell separation of the yeasts Saccharomyces cerevisiae [11,12] and Candida albicans [9]. Allosamidin, which is an e¡ective inhibitor of fungal chitinases [13^15], did not have any e¡ect on the growth of A. chrysogenum (data not shown) similar to S. cerevisiae [11,12] and C. albicans [13]. In terms of morphology, the pseudotrisaccharide signi¢cantly hindered the fragmentation of A. chrysogenum hyphae even at concentrations of as low as 4.0 Wg ml31 (6.4 WM) or 6.0 Wg ml31 (9.6 WM) (Figs. 1 and 2). That is, chitinolytic enzymes played an essential role in the breakage of hyphae in addition to mechanical forces [16] and the physiological state of the cells [17]. On the other hand, the swelling of slender hyphal ¢laments was not in£uenced by allosamidin and, hence, the quantity of spherical cells in hyphae was similar to that of yeast-like cells in control cultures (Fig. 1) [5]. Consequently, the formation of swollen hyphal fragments might be triggered by either low molecular mass arthrospore-inducing factors [18] or increases in the thiol concentrations of the cells [19] rather than by the action of chitinases. Most interestingly, the cephalosporin-C production of A. chrysogenum, which was about 0.5 g l31 in control cultures [5], was not in£uenced by the addition of allosamidin, which challenges any possible causal connection between the antibiotic production and the fragmentation of the fungus. In A. chrysogenum culture ¢ltrates, a very high, K = 0.08^0.10, protease activity was detected between 40 and 110 h incubation times. In comparison, the maximum proteolytic activity in ageing P. chrysogenum cultures was only K = 0.015 as reported by Pusztahelyi et al. [7]. As a possible consequence of the
235
action of proteases, no extracellular chitinase and Nacetyl-L-D-hexosaminidase activities were observed at any fermentation time in A. chrysogenum cultures. Nevertheless, signi¢cant soluble and insoluble cellbound chitinase activities (Fig. 3) were detected in the stationary phase of growth of A. chrysogenum when the fragmentation had started and was progressing [4,5]. As demonstrated in Fig. 3, about 47% (36 pkat mg31 dryweight ) of the soluble cell-bound chitinase activity (77 pkat mg31 dryweight ) was resistant to the inhibitory e¡ect of allosamidin. On the other hand, about 76% (113 pkat mg31 dryweight ) of the total chitinase activity (149 pkat mg31 dryweight ) localised in both the soluble (41 pkat mg31 dryweight ) and insoluble (72 pkat mg31 dryweight ) enzyme fractions was inhibited e¡ectively by the pseudotrisaccharide. Obviously, the allosamidin sensitive enzyme activity, which was detected in both the soluble and insoluble cell-bound enzyme fractions, played a crucial role in the fragmentation process. The enzymological features and the physiological function of the allosamidin resistant chitinolytic enzyme(s), which were localised exclusively in the soluble cell-bound fraction as shown in Fig. 3, has remained yet to be elucidated. Interestingly, allosamidin resistant chitinases have only been detected in bacteria and plants thus far [20].
Acknowledgments This project was supported by the OTKA (grant reference number: T 019484), the Foundation for the Hungarian Higher Education and Research (AMFK 639/96) and by the Hungarian Creative Art Foundation (13-914-97/P1). The allosamidin was generously supplied by the Lilly Research Laboratories, Indianapolis, IN, USA; we especially thank the valuable help of Dr. H.A. Kirst and Mrs. M.H. Niedenthal.
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