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Elaboration of pullulan by spheroplasts of Aureobasidium pullulans
[ 45 1 Trans. Br, mycol, Soc. 76 (3) 451-456 (1981)
]
Printed in Great Britain
ELABORATION OF PULLULAN BY SPHEROPLASTS OF AUREOBASIDIUM PULLULANS By BRIAN
J. CAT LEY AND ANNE HUTCHISON
Department of Brewing and Biological Sciences, Heriot-Watt University, Edinburgh
Using Zymolyase, a carbohydrase preparation derived from Arthrobacter luteus, spheroplasts of Aureobasidium pullulans have been prepared and their ability to produce the extracellular glucan pull ulan has been examined. Partial lysis of the cell wall afforded preparations that were not impaired in their ability to absorb glucose, but which had partially lost their facility to elaborate polysaccharide. It is concluded that the assembly or excretion of pullulan is associated with the cell wall, the plasma membrane and possibly the neighbouring periplasmic space. The walls of micro-organisms are involved in many aspects of cell physiology. Although the plasma membrane and the cell wall are distinct structures there is a close functional co-operation between them. Thus, the apical growth of fungal hyphae probably occurs by controlled and local lysis of cell-wall components brought about by lytic enzymes discharged through the plasma membrane (Bartnicki-Garcia, 1973). The cascade of chitin synthetase zymogen, active chitin synthetase and related inhibitor, that is responsible for the assembly and deposition of chitin in bud-scar tissue, is intimately associated with the plasmalemma of Saccharomyces cerevisiae Meyen ex Hansen, where the zymogen has been located on the inner surface (Duran, Bowers & Cabib, 1975). The assembly of some yeast cell-wall polysaccharides at sites external to the plasmalemma is thought to be catalysed by membrane-located synthetases. Thus, whilst most of the D-mannosyl transferase activity found in membranous preparations derived from S. cerevisiae is associated with internal membranes, at least 20 % has been located in the plasma membrane and is thought to catalyse the assembly of the cell-wall mannoprotein (Santos, Villanueva & Sentandreu, 1978) in addition to the further glycosylation of extracellular enzymes. Other peripheral enzymes partially located in the periplasmic space (Santos, Sanchez, Villanueva & Nombela, 1979) are the derepressible p-glucosidases responsible for the mobilization of P-l,3 glucan in the cell wall of Penicillium italicum Wehmer and the cell-wall P-l,3-g1ucanases that have been reported in Schizosaccharomyces Lindner (Fleet & Phaff, 1974). Pullulan is an a-glucan secreted by AurelJbasidium pullulans (de Bary) Arnaud (Wallenfels, Keilich, Bechtler & Freudenberger, 1965). Studies
of the physiology and biochemistry of synthesis (Carley, 1979) showed that glucose is an adequate donor for the production of polysaccharide by the whole cell (Catley, 1971a); that the pH of the environment affects this elaboration (Carley, 1971b); and that lipid intermediates may be involved in the assembly process (Taguchi et al., 1973). These observations suggest that later stages of pull ulan biosynthesis may occur on the outer surface of the plasmalemma or in the periplasmic space. The purpose of this communication is to describe the removal of the cell wall surrounding the blastospore of A. pullulans and to demonstrate the subsequent modulation of polysaccharide excretion. MATERIALS AND METHODS
Growth of organism and fractionation of cells Aureobasidium pullulans (ATCC 9348) was grown as described previously (Kelly & Catley, 1977) but
without the addition of trace elements. Singlecelled forms, blastospores, were separated from the mycelium by filtration through nylon mesh (45 [tm pore size, supplied by Henry Simon Ltd, Stockport) and washed three times by centrifugation in distilled water. Production of spheroplasts
Samples of washed cells, 50-100 mg dry weight, were suspended in 0'1 M phosphate buffer (10'0 ml); pH 7'4 made up to 0'7 M with KCl. To this was added z-mercaptoethanol (zo ul) and Zymolyase 60000 (100,al at concentrations of 1 mg, 0-2 mg and 0-06 mg ml- 1) . Cells were incubated for 1 h at 37°C and the lytic reagents removed by washing the spheroplasts with uptake medium. The treatment of washed cells with pullulanase 16-2
0007-1536/81/2828-7450 $01.00 © 1981 The British Mycological Society
452
Pullulan elaboration by spheroplasts of Aureobasidium pullulans
(pullulan-6-glucanohydrolase, E.C. 3.2.1.41) was accomplished by suspending 50 mg dry weight of cells in 0'1 M citrate-phosphate buffer (4'0 ml), pH 5'4 made up to 0'7 M with KCl. Pullulanase (o-oj zekatals) was added and the cells were incubated and shaken at 37° for 1 h before being washed and prepared for electron microscopy. Similarly, a comparable concentration of washed cells was incubated with a mixture of z-mercaptoethanol (0'05 M) and EDTA (4Na)-ethylenediamine tetraacetate (0'05 M) at 22° for 1 h.
Polymer production and cellular incorporation of glucose The uptake medium used was identical to the growth medium except that NH 4Cl and yeast extract were omitted, the concentration of glucose was 10 mM, the pH adjusted to 5'5 and KCI added to a final concentration of 0'7 M. [14C-U]-nglucose, 0'2 ,umoles containing 9'2 x 104Bq of 14C isotope were introduced to the uptake medium just prior to the addition of cells, and the suspension was agitated in a shaking water bath at 27°. Excreted polymer was measured by transferring aliquots (200,u1) of cell-free medium on to 2 x 2 em squares of filter paper (Whatman, 3 mm). These were extracted three times in 67 % aqueous ethanol, then squares were removed and dried with infra-red illumination. Counts were made in a scintillant comprising 0'5 % PPO (2,5-diphenyloxazole) and 0'01 % POPOP (1,4-bis[2-phenyloxazolyl] benzene) in toluene. The polysaccharide product was identified as pull ulan by co-precipitation with unlabelled pullulan, treatment with pullulanase and identification of the released [14C]-maltotriose using paper chromatography (Catley, 1971a). The concentration of glucose present in the uptake medium was measured using the glucose oxidase reagent (Lloyd & Whelan, 1969). Electron microscopy Cells were suspended in 2 % aqueous KMnO 4 for at least 2 h at room temperature, then washed in distilled water until all traces of colour had been removed from the washings. Dehydration in a series of ethanol washes (15 min in each of 50 %, 70 %, 90 %-twice and 100 %-twice, vIv) was completed by rinses in epoxy propane (10 min twice), then acetone (10 min) before suspension in 50 % resin (Epon Brz-acetone). After 1 h this was replaced by undiluted resin and the preparation heated at 100°. Samples were sectioned with an LKB microtome and examined using an AEI 802 transmission electron microscope. Photographs were taken using Ilford SP 353 film.
Chemicals All chemicals used were Analar or of the highest purity available commercially. Zymolyase 60000 was purchased from the Kirin Brewery Co. Ltd, Takasaka, Gumma Pref., Japan and pullulanase was supplied by Boehringer Corporation (London) Ltd. RESUL TS
The extent to which components of the cell wall have been removed by a range of treatments is revealed in Fig. 1. Comparison of Fig. 1 a with Fig. 1 b shows that little, if any, encroachment has been made by exposure of the cells to pullulanase. The effect of z-mercaptoethanol/Bfr'I'A on the integrity of the cell wall is shown in Fig. 1c. The sulphydryl reagent complements the enzymic treatment and probably acts by reducing the disulphide bonds of constituent cell-wall proteins (Larnpen, 1968). Some dissolution of the wall is seen but it is not extensive. Nevertheless the presence of mercaptoethanol in the enzymic digest must aid the removal of polysaccharide components by increasing their accessibility. Fig. 1 d illustrates the ability of Zymolyase to remove most of the cell wall. Preparations of spheroplasts using Zymolyase at these concentrations (10,ug ml- 1) afforded cells that were incapable of polymer production or glucose utilization (Fig. 2). Reduction of the carbohydrase concentrations to 2'0 and 0'6,ug ml" produced spheroplasts that fully maintained their ability to assimilate glucose but were impaired in their rate of pull ulan production to some 45 % of the untreated cells. Cross-sections of these cells revealed walls (Fig. 1 e, f) that had been damaged, but to a lesser extent than those (Fig. 1 d) subjected to the higher concentration of enzyme. It therefore appears that a controlled removal of the cell wall is possible. DISCUSSION
The polymorphic forms of A. pullulans, and the influence of the environment on them, have been described as a number of cycles (Ramos & GardaAcha, 1975a). Studies of the chemical composition of cell walls (Brown & Nickerson, 1965; Brown & Lindberg, 1967a, b; Brown, Hanic & Hsiao, 1973; Dominguez, GoiH & Uruburu, 1978) have been reported, and a comparison of extracellular polysaccharides with those in the cell wall has been published (Kikuchi et al., 1973). Recent examinations of cell ultrastructure have revealed the budding process in both single-cell and mycelial forms (Ramos, Garcia-Acha & Peberdy,
B. J. Gatley and Anne Hutchison
453
(a ) ...._ _
,f
(c)
.....- - - - - - - 1 1 •
.
,
. . . ... .
. ',
.'
Fig. 1, Ultra-thin sections of blastospores of A. pullulans, (a) Untreated control. (b) Pullulanase treatment. (c) 2-mercaptoethanoljEDTA treatment. Zymolyase treatment at final concentrations of (d) 10 /lg ml" l ) (II) 2 /lg ml-\ (f) 0·6 /kgml", Bar marker represents 1 /lm.
454
Pullulan elaboration by spheroplasts of Aureobasidium pullulans
1975); the hyphae have been studied using highvoltage electron microscopy and reconstruction techniques (Crang & Pechak, 1978) ; and scanning electron microscopy has been used to visualize the sequence of developmental stages (Passmore, 1973; Pechak & Crang, 1977). Freeze-etching and negative staining have revealed details of the yeast cell-wall architecture and the relationship between the external face of the membrane and the innermost layer of the cell wall (Ramos & Garcia-Acha, 1975 b). The preparation of spheroplasts and protoplasts from A . pullulans has been achieved using a number of lytic preparations. Hydrolases used in these digestions have been isolated from Streptomyces venezuelae Ehrlich, Gottlieb, Burkholder, Anderson & Pridham RA and Micromonospora chalcea (Foulerton) 0rskov (Ramos & Garcia-Acha, 1972), Oerskovia xanthineolytica and a Basidiomycete QM 806 (Jeffries et al., 1977). The lytic agent used in the present investigation is Zymolyase, a carbohydrase preparation obtained from the culture filtrate of Arthrobacter luteus (Kitamura, Kaneko & Yamamoto, 1971; Kaneko, Kitamura & Yamamoto, 1973; Kuo & Yamamoto, 1975) and containing endo P-(1 ..... 3) glucanase activity which is active against the majority of P-(1 ..... 3) glucans (Kitamura & Yamamoto, 1972). The action of pullulanase was also investigated, for it had been suggested that pull ulan is not only an extracellular polysaccharide that is elaborated into the medium but is also a minor component of the cell wall (Kikuchi et al., 1973; Dominguez et al., 1978). However, whilst it has been observed that whole cell walls are susceptible to a-amylase (Dominguez et al., 1978) and extracted cell-wall polymers are hydrolysed by pullulanase (Kikuchi et al., 1973) there is no unequivocal evidence that these enzymes catalyse the hydrolysis of glucosidic bonds in pullulan originally located in the cell wall. Under the conditions of the present investigation no destruction of the cell wall by pullulanase could be detected (Fig. 1 b). Digestion with Zymolyase yielded cells that were contained by a variable residuum of cell wall (Fig. 1 d-f). In examining spheroplast formation in Saccharomyces cerevisiae it has been observed that a small proportion of the remaining cell wall is sufficient to maintain the original cell shape (Darling, Theilade & Birch-Andersen, 1969; Pringle, Forsdyke & Rose, 1979). The intermediate stage, en route to the spheroplast and protoplast, has been termed the prospheroplast. Both spheroplasts and prospheroplasts derived from S. cerevisiae are osmotically sensitive. Contrasting with this fragility, the spheroplasts of A. pullulans did not lyse when suspended in
10
12
-
•
.i ~
10
~
'J
6
~
4
x 8
o
60
120
ISO
T i me (min)
Fig. 2. Consumption of glucose C-, e, A, 'f' ) and elaboration of pullulan CD, 0, 6, 17) by cells treated with Zymolyase at final concentrations of 10 pg ml'! C_, D ); 2/~g rnl" ce, 0) and Q'6pg ml " ! CA, 6 ), and by untreated cells C'f', 17 ).
hypotonic medium. However, despite their resistance to lysis those prepared using the highest concentration of Zymolyase (Fig. 1 d) did not incorporate labelled glucose, even in a stabilizing medium of 0'7 M-KCl, and it is possible that protein complexes in the plasmalemma, responsible for the binding and assimilation of glucose into the cell (Burger, Hejmova & Kleinzeller, 1959 ; de la Fuente & Sols, 1962), may have been perturbed or lost by the remo val of neighbouring cell-wall structures. The concern of the present investigation was to examine the ability of the yeast cell to elaborate pullulan subsequent to the removal of part or all of the cell wall. Clearly any modification to the peripheral architecture must still allow assimilation and metabolism of the glucose carbon source. Accordingly a series of cell-wall digestions was designed wherein partial removal of the cell wall was achieved. The extent of digestion is revealed in Fig. 1 e, f and the corresponding changes in glucose utilization and glucan excretion are presented in Fig. 2. It is apparently possible to remove ceJl wall to the extent where glucose absorption remains undiminished but polymer elaboration is appreciably attenuated. It must be concluded that components of the cell wall playa role in the elaboration of pullulan and that the Zymolyase preparation can modify or destroy them. There is no evidence to indicate what this loss may be attributed to. It could be explained as a loss or inhibition of peri plasmically located
B.J. Catley and Anne Hutchison polymerase. Some evidence suggests that lipidphosphoglucose intermediates may be involved in the biosynthesis of pullulan (Taguchi et al., 1973). The location of polysaccharide synthetases, or enzymes modifying existing polysaccharides, on the external face of some microbial plasma membranes is established (Larsen & Haug, 1970, 1971; Brown, Wilson & Richardson, 1976; Zaar, 1979), and if lipid intermediates are involved in pullulan biosynthesis it is possible that polysaccharide assembly may be located in the periplasmic region of the cell.
REFERENCES
BARTNICKI-GARCIA, S. (1973). Fundamental aspects of hyphal morphogenesis. In Microbial Differentiation, pp. 245-267. Cambridge University Press. BROWN, R. G. & NICKERSON, W. J. (1965). Cornposittion and structure of cell walls of filamentous and yeast-like forms of Aureobasidium (Pullularia) pullulans, Proceedings of the American Society for Microbiology, p. 26. BROWN, R. G. & LINDBERG, B. (1967 a). Polysaccharides from cell walls of Aureobasidium (Pullularia) pullulans. Part I. Glucans. Acta Chemica Scandinavica zt, 2379-2382. BROWN, R. G. & LINDBERG, B. (1967 b). Polysaccharides from cell walls of Aureobasidium (Pullularia) pullulans. Part II. Heteropolysaccharide. Acta Chemica Scandinavica 21, 2383-2389. BROWN, R. G., HANIC, L. A. & HSIAO, M. (1973). Structure and chemical composition of yeast chlamydospores of Aureobasidium pullulans, Canadian Journal of Microbiology 19, 163-168. BROWN, R. M., WILSON, J. H. M. & RICHARDSON, C. L. (1976). Cellulose biosynthesis in Acetobacter xylinum, Visualization of the site of synthesis and direct measurement of the in vivo process. Proceedings of the National Academy of Sciences U.S.A. 73, 4565-45 69. BURGER, M., HEJMovA, L. & KLEINZELLER, A. (1959). Transport of some mono- and di-saccharides into yeast cells. Biochemical Journal 71, 233-242. CATLEY, B. J. (1971 a). Utilization of carbon sources by Pullularia pullulans for the elaboration of extracellular polysaccharides. Applied Microbiology zz, 64 1-649. CATLEY, B. J. (1971 b). Role of pH and nitrogen limitation in the elaboration of the extracellular polysaccharide pullulan by Pullularia pullulans. Applied Microbiology zz, 650-654. CATLEY, B. J. (1979). Pullulan synthesis by Aureobasidium pullulans. In Microbial Polysaccharides and Polysaccharidases, pp. 69-84. Academic Press. CRANG, R. E. & PECHAK, D. G. (1978). Serial section reconstruction of the black yeast, Aureobasidium pullulans by means of high voltage electron microscopy. Protoplasma 96, 225-234. DARLING, S., THEILADE, J. & BIRCH-ANDERSEN, A. (1969). Kinetic and morphological observations on
455
Saccharomyces cereoisiae during spheroplast formation. Journal of Bacteriology 98, 797-810. DE LA FUENTE, G. & SOLS, A. (1962). Transport of sugars in yeasts. II. Mechanisms of utilization of disaccharides and related glycosides. Biochimica et Biophysica Acta 56, 49-62. DOMINGUEZ, J. B., GONI, F. M. & URUBURU, F. (1978). The transition from yeast-like to chlamydospore cells in Pullularia pullulans. Journal of General Microbiology 108, 111-117. DuRAN, A., BOWERS, B. & CABIB, E. (1975). Chitin synthetase zymogen is attached to the yeast plasma membrane. Proceedings of the National Academy of Sciences, U.S.A. 71,,3952-3955. FLEET, G. H. & PHAFF, H. J. (1974). Glucanases in Schizosaccharomyces. Isolation and properties of the cell-wall associated jf-(l -+ 3)-glucanases. Journal of Biological Chemistry 249, 1717-1728. JEFFRIES, T. W., EVELEIGH, D. E., MAcMILLAN, J. D., PARRISH, F. W. & REESE, E. T. (1977). Enzymatic hydrolysis of the walls of yeast cells and germinated fungal spores. Biochimica et Biophysica Acta 499, 10-23. KANEKO, T., KITAMURA, K. & YAMAMOTO, Y. (1973). Susceptibilities of yeasts to yeast cell wall lytic enzymes of Arthrobacter luteus. Agricultural and Biological Chemistry 37, 2295-2302. KELLY, P. J. & CATLEY, B. J. (1977). The effect of ethidium bromide mutagenesis on dimorphism, extracellular metabolism and cytochrome levels in Aureobasidium pullulans. Journal of General Microbiology 101" 249-254. KIKUCHI, Y., TAGUCHl, R., SAKANO, Y. & KOBAYASHI, T. (1973). Comparison of extracellular polysaccharide produced by Pullularia pullulans with polysaccharides in the cells and cell wall. Agricultural and Biological Chemistry 37, 1751-1753. KITAMURA, K., KANEKO, T. & YAMAMOTO, Y. (1971). Lysis of viable yeast cells by enzymes of Arthrobacter luteus. Archives of Biochemistry and Biophysics 145, 402-404. KITAMURA, K. & YAMAMOTO, Y. (1972). Purification and properties of an enzyme, Zymolyase, which lyses viable yeast cells. Archives of Biochemistry and Biophysics 153, 403-406. Kuo, S.-C. & YAMAMOTO, S. (1975). Preparation and growth of protoplasts, Methods in Cell Biology 11, 169- 183. LAMPEN, J. O. (1968). External enzymes of yeast: their nature and formation. Antonie van Leeuwenhoek 34, 1-18. LARSEN, B. & HAUG, A. (1970). Biosynthesis of alginate. Part 1. Composition and structure of alginate produced by Azotobacter vinelandii (Lipman). Carbohydrate Research 17, 287-296. LARSEN, B. & HAUG, A. (1971). Biosynthesis of alginate. Part III. Tritium incorporation with polymannuronic acid 5-epimerase from Azotobacter oinelandii. Carbohydrate Research 1,0, 225-232. LLOYD, J. B. & WHELAN, W. J. (1969). An improved method for enzymic determination of glucose in the presence of maltose. Analytical Biochemistry 30, 467-470.
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Pullulan elaboration by spheroplasts of Aureobasidium pullulans S. M. (1973). Scanning electron microSANTOS, E., VILLANUEVA, J. R. &
PASSMORE, scopy of yeast colonies. Journal of The Institute of Brewing 79, 237-243. PECHAK, D. G. & CRANG, R E. (1977). An analysis of Aureobasidium pullulans developmental stages by means of scanning electron microscopy. Mycologia 69, 7 83-792. PRINGLE, A. T., FORSDYKE, J. & ROSE, A. H. (1979). Scanning electron microscopy study of Saccharomyces cerevisiae spheroplast formation. Journal of Bacteriology 140, 289-293. RAMos, S. & GARciA-AcHA, I. (1972). Production and regeneration of protoplasts of Pullularia pullulans, In 3rd International Symposium on Yeast Protoplasts-« Salamanca, p. 57. RAMOS, S. & GARciA-AcHA, I. (1975a). A vegetative cycle of Pullularia pullulans. Transactions of the British Mycological Society 64, 129-135. RAMos, S. & GARcfA-AcHA, I. (1975b). Cell wall enzymatic lysis of the yeast form of Pullularia pullulans and wall regeneration by protoplasts. Archives of Microbiology 1°4,271-277. RAMos, S., GARciA-ACHA, I. & PEBERDY, J. F. (1975). Wall structure and the budding process in Pullularia pullulans. Transactions of the British Mycological Society 64, 283-288.
SENTANDREU, R (1978). The plasma membrane of Saccharomyces cerevisiae, isolation and properties. Biochimica et Biophysica Acta 508, 39-54. SANTOS, T., SANCHEZ, M., VILLANUEVA, J. R & NOMBELA, C. (1979). Derepression of P-1,3glucanases in Penicillium italicum: localization of the various enzymes and correlation with cell wall glucan mobilization and autolysis. Journal of Bacteriology 137,6-12. TAGUCHI, R, SAKANO, Y., KIKUCHI, Y., SAKUMA, M. & KOBAYASHI, T. (1973). Synthesis of pullulan by acetone-dried cells and cell-free enzyme from Pullularia pullulans, and the participation of lipid intermediate. Agricultural and Biological Chemistry 37, 1635- 1641. WALLENFELS, K., KEILlCH, G., BECHTLER, G. & FREUDENBERGER, D. (1965). Untersuchungen an Pullulan. IV. Die Klarung des Strukturproblems mit physikalischen, chemischen und enzymatischen Methoden. Biochemische Zeitschrift 341, 433-450. ZAAR, K. (1979). Visualization of pores (export sites) correlated with cellulose production in the envelope of the gram negative bacterium Acetobacter xylinum, Journal of Cell Biology 80, 773-777.
(Received for publication 29 May 1980)
]
Printed in Great Britain
ELABORATION OF PULLULAN BY SPHEROPLASTS OF AUREOBASIDIUM PULLULANS By BRIAN
J. CAT LEY AND ANNE HUTCHISON
Department of Brewing and Biological Sciences, Heriot-Watt University, Edinburgh
Using Zymolyase, a carbohydrase preparation derived from Arthrobacter luteus, spheroplasts of Aureobasidium pullulans have been prepared and their ability to produce the extracellular glucan pull ulan has been examined. Partial lysis of the cell wall afforded preparations that were not impaired in their ability to absorb glucose, but which had partially lost their facility to elaborate polysaccharide. It is concluded that the assembly or excretion of pullulan is associated with the cell wall, the plasma membrane and possibly the neighbouring periplasmic space. The walls of micro-organisms are involved in many aspects of cell physiology. Although the plasma membrane and the cell wall are distinct structures there is a close functional co-operation between them. Thus, the apical growth of fungal hyphae probably occurs by controlled and local lysis of cell-wall components brought about by lytic enzymes discharged through the plasma membrane (Bartnicki-Garcia, 1973). The cascade of chitin synthetase zymogen, active chitin synthetase and related inhibitor, that is responsible for the assembly and deposition of chitin in bud-scar tissue, is intimately associated with the plasmalemma of Saccharomyces cerevisiae Meyen ex Hansen, where the zymogen has been located on the inner surface (Duran, Bowers & Cabib, 1975). The assembly of some yeast cell-wall polysaccharides at sites external to the plasmalemma is thought to be catalysed by membrane-located synthetases. Thus, whilst most of the D-mannosyl transferase activity found in membranous preparations derived from S. cerevisiae is associated with internal membranes, at least 20 % has been located in the plasma membrane and is thought to catalyse the assembly of the cell-wall mannoprotein (Santos, Villanueva & Sentandreu, 1978) in addition to the further glycosylation of extracellular enzymes. Other peripheral enzymes partially located in the periplasmic space (Santos, Sanchez, Villanueva & Nombela, 1979) are the derepressible p-glucosidases responsible for the mobilization of P-l,3 glucan in the cell wall of Penicillium italicum Wehmer and the cell-wall P-l,3-g1ucanases that have been reported in Schizosaccharomyces Lindner (Fleet & Phaff, 1974). Pullulan is an a-glucan secreted by AurelJbasidium pullulans (de Bary) Arnaud (Wallenfels, Keilich, Bechtler & Freudenberger, 1965). Studies
of the physiology and biochemistry of synthesis (Carley, 1979) showed that glucose is an adequate donor for the production of polysaccharide by the whole cell (Catley, 1971a); that the pH of the environment affects this elaboration (Carley, 1971b); and that lipid intermediates may be involved in the assembly process (Taguchi et al., 1973). These observations suggest that later stages of pull ulan biosynthesis may occur on the outer surface of the plasmalemma or in the periplasmic space. The purpose of this communication is to describe the removal of the cell wall surrounding the blastospore of A. pullulans and to demonstrate the subsequent modulation of polysaccharide excretion. MATERIALS AND METHODS
Growth of organism and fractionation of cells Aureobasidium pullulans (ATCC 9348) was grown as described previously (Kelly & Catley, 1977) but
without the addition of trace elements. Singlecelled forms, blastospores, were separated from the mycelium by filtration through nylon mesh (45 [tm pore size, supplied by Henry Simon Ltd, Stockport) and washed three times by centrifugation in distilled water. Production of spheroplasts
Samples of washed cells, 50-100 mg dry weight, were suspended in 0'1 M phosphate buffer (10'0 ml); pH 7'4 made up to 0'7 M with KCl. To this was added z-mercaptoethanol (zo ul) and Zymolyase 60000 (100,al at concentrations of 1 mg, 0-2 mg and 0-06 mg ml- 1) . Cells were incubated for 1 h at 37°C and the lytic reagents removed by washing the spheroplasts with uptake medium. The treatment of washed cells with pullulanase 16-2
0007-1536/81/2828-7450 $01.00 © 1981 The British Mycological Society
452
Pullulan elaboration by spheroplasts of Aureobasidium pullulans
(pullulan-6-glucanohydrolase, E.C. 3.2.1.41) was accomplished by suspending 50 mg dry weight of cells in 0'1 M citrate-phosphate buffer (4'0 ml), pH 5'4 made up to 0'7 M with KCl. Pullulanase (o-oj zekatals) was added and the cells were incubated and shaken at 37° for 1 h before being washed and prepared for electron microscopy. Similarly, a comparable concentration of washed cells was incubated with a mixture of z-mercaptoethanol (0'05 M) and EDTA (4Na)-ethylenediamine tetraacetate (0'05 M) at 22° for 1 h.
Polymer production and cellular incorporation of glucose The uptake medium used was identical to the growth medium except that NH 4Cl and yeast extract were omitted, the concentration of glucose was 10 mM, the pH adjusted to 5'5 and KCI added to a final concentration of 0'7 M. [14C-U]-nglucose, 0'2 ,umoles containing 9'2 x 104Bq of 14C isotope were introduced to the uptake medium just prior to the addition of cells, and the suspension was agitated in a shaking water bath at 27°. Excreted polymer was measured by transferring aliquots (200,u1) of cell-free medium on to 2 x 2 em squares of filter paper (Whatman, 3 mm). These were extracted three times in 67 % aqueous ethanol, then squares were removed and dried with infra-red illumination. Counts were made in a scintillant comprising 0'5 % PPO (2,5-diphenyloxazole) and 0'01 % POPOP (1,4-bis[2-phenyloxazolyl] benzene) in toluene. The polysaccharide product was identified as pull ulan by co-precipitation with unlabelled pullulan, treatment with pullulanase and identification of the released [14C]-maltotriose using paper chromatography (Catley, 1971a). The concentration of glucose present in the uptake medium was measured using the glucose oxidase reagent (Lloyd & Whelan, 1969). Electron microscopy Cells were suspended in 2 % aqueous KMnO 4 for at least 2 h at room temperature, then washed in distilled water until all traces of colour had been removed from the washings. Dehydration in a series of ethanol washes (15 min in each of 50 %, 70 %, 90 %-twice and 100 %-twice, vIv) was completed by rinses in epoxy propane (10 min twice), then acetone (10 min) before suspension in 50 % resin (Epon Brz-acetone). After 1 h this was replaced by undiluted resin and the preparation heated at 100°. Samples were sectioned with an LKB microtome and examined using an AEI 802 transmission electron microscope. Photographs were taken using Ilford SP 353 film.
Chemicals All chemicals used were Analar or of the highest purity available commercially. Zymolyase 60000 was purchased from the Kirin Brewery Co. Ltd, Takasaka, Gumma Pref., Japan and pullulanase was supplied by Boehringer Corporation (London) Ltd. RESUL TS
The extent to which components of the cell wall have been removed by a range of treatments is revealed in Fig. 1. Comparison of Fig. 1 a with Fig. 1 b shows that little, if any, encroachment has been made by exposure of the cells to pullulanase. The effect of z-mercaptoethanol/Bfr'I'A on the integrity of the cell wall is shown in Fig. 1c. The sulphydryl reagent complements the enzymic treatment and probably acts by reducing the disulphide bonds of constituent cell-wall proteins (Larnpen, 1968). Some dissolution of the wall is seen but it is not extensive. Nevertheless the presence of mercaptoethanol in the enzymic digest must aid the removal of polysaccharide components by increasing their accessibility. Fig. 1 d illustrates the ability of Zymolyase to remove most of the cell wall. Preparations of spheroplasts using Zymolyase at these concentrations (10,ug ml- 1) afforded cells that were incapable of polymer production or glucose utilization (Fig. 2). Reduction of the carbohydrase concentrations to 2'0 and 0'6,ug ml" produced spheroplasts that fully maintained their ability to assimilate glucose but were impaired in their rate of pull ulan production to some 45 % of the untreated cells. Cross-sections of these cells revealed walls (Fig. 1 e, f) that had been damaged, but to a lesser extent than those (Fig. 1 d) subjected to the higher concentration of enzyme. It therefore appears that a controlled removal of the cell wall is possible. DISCUSSION
The polymorphic forms of A. pullulans, and the influence of the environment on them, have been described as a number of cycles (Ramos & GardaAcha, 1975a). Studies of the chemical composition of cell walls (Brown & Nickerson, 1965; Brown & Lindberg, 1967a, b; Brown, Hanic & Hsiao, 1973; Dominguez, GoiH & Uruburu, 1978) have been reported, and a comparison of extracellular polysaccharides with those in the cell wall has been published (Kikuchi et al., 1973). Recent examinations of cell ultrastructure have revealed the budding process in both single-cell and mycelial forms (Ramos, Garcia-Acha & Peberdy,
B. J. Gatley and Anne Hutchison
453
(a ) ...._ _
,f
(c)
.....- - - - - - - 1 1 •
.
,
. . . ... .
. ',
.'
Fig. 1, Ultra-thin sections of blastospores of A. pullulans, (a) Untreated control. (b) Pullulanase treatment. (c) 2-mercaptoethanoljEDTA treatment. Zymolyase treatment at final concentrations of (d) 10 /lg ml" l ) (II) 2 /lg ml-\ (f) 0·6 /kgml", Bar marker represents 1 /lm.
454
Pullulan elaboration by spheroplasts of Aureobasidium pullulans
1975); the hyphae have been studied using highvoltage electron microscopy and reconstruction techniques (Crang & Pechak, 1978) ; and scanning electron microscopy has been used to visualize the sequence of developmental stages (Passmore, 1973; Pechak & Crang, 1977). Freeze-etching and negative staining have revealed details of the yeast cell-wall architecture and the relationship between the external face of the membrane and the innermost layer of the cell wall (Ramos & Garcia-Acha, 1975 b). The preparation of spheroplasts and protoplasts from A . pullulans has been achieved using a number of lytic preparations. Hydrolases used in these digestions have been isolated from Streptomyces venezuelae Ehrlich, Gottlieb, Burkholder, Anderson & Pridham RA and Micromonospora chalcea (Foulerton) 0rskov (Ramos & Garcia-Acha, 1972), Oerskovia xanthineolytica and a Basidiomycete QM 806 (Jeffries et al., 1977). The lytic agent used in the present investigation is Zymolyase, a carbohydrase preparation obtained from the culture filtrate of Arthrobacter luteus (Kitamura, Kaneko & Yamamoto, 1971; Kaneko, Kitamura & Yamamoto, 1973; Kuo & Yamamoto, 1975) and containing endo P-(1 ..... 3) glucanase activity which is active against the majority of P-(1 ..... 3) glucans (Kitamura & Yamamoto, 1972). The action of pullulanase was also investigated, for it had been suggested that pull ulan is not only an extracellular polysaccharide that is elaborated into the medium but is also a minor component of the cell wall (Kikuchi et al., 1973; Dominguez et al., 1978). However, whilst it has been observed that whole cell walls are susceptible to a-amylase (Dominguez et al., 1978) and extracted cell-wall polymers are hydrolysed by pullulanase (Kikuchi et al., 1973) there is no unequivocal evidence that these enzymes catalyse the hydrolysis of glucosidic bonds in pullulan originally located in the cell wall. Under the conditions of the present investigation no destruction of the cell wall by pullulanase could be detected (Fig. 1 b). Digestion with Zymolyase yielded cells that were contained by a variable residuum of cell wall (Fig. 1 d-f). In examining spheroplast formation in Saccharomyces cerevisiae it has been observed that a small proportion of the remaining cell wall is sufficient to maintain the original cell shape (Darling, Theilade & Birch-Andersen, 1969; Pringle, Forsdyke & Rose, 1979). The intermediate stage, en route to the spheroplast and protoplast, has been termed the prospheroplast. Both spheroplasts and prospheroplasts derived from S. cerevisiae are osmotically sensitive. Contrasting with this fragility, the spheroplasts of A. pullulans did not lyse when suspended in
10
12
-
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10
~
'J
6
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4
x 8
o
60
120
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Fig. 2. Consumption of glucose C-, e, A, 'f' ) and elaboration of pullulan CD, 0, 6, 17) by cells treated with Zymolyase at final concentrations of 10 pg ml'! C_, D ); 2/~g rnl" ce, 0) and Q'6pg ml " ! CA, 6 ), and by untreated cells C'f', 17 ).
hypotonic medium. However, despite their resistance to lysis those prepared using the highest concentration of Zymolyase (Fig. 1 d) did not incorporate labelled glucose, even in a stabilizing medium of 0'7 M-KCl, and it is possible that protein complexes in the plasmalemma, responsible for the binding and assimilation of glucose into the cell (Burger, Hejmova & Kleinzeller, 1959 ; de la Fuente & Sols, 1962), may have been perturbed or lost by the remo val of neighbouring cell-wall structures. The concern of the present investigation was to examine the ability of the yeast cell to elaborate pullulan subsequent to the removal of part or all of the cell wall. Clearly any modification to the peripheral architecture must still allow assimilation and metabolism of the glucose carbon source. Accordingly a series of cell-wall digestions was designed wherein partial removal of the cell wall was achieved. The extent of digestion is revealed in Fig. 1 e, f and the corresponding changes in glucose utilization and glucan excretion are presented in Fig. 2. It is apparently possible to remove ceJl wall to the extent where glucose absorption remains undiminished but polymer elaboration is appreciably attenuated. It must be concluded that components of the cell wall playa role in the elaboration of pullulan and that the Zymolyase preparation can modify or destroy them. There is no evidence to indicate what this loss may be attributed to. It could be explained as a loss or inhibition of peri plasmically located
B.J. Catley and Anne Hutchison polymerase. Some evidence suggests that lipidphosphoglucose intermediates may be involved in the biosynthesis of pullulan (Taguchi et al., 1973). The location of polysaccharide synthetases, or enzymes modifying existing polysaccharides, on the external face of some microbial plasma membranes is established (Larsen & Haug, 1970, 1971; Brown, Wilson & Richardson, 1976; Zaar, 1979), and if lipid intermediates are involved in pullulan biosynthesis it is possible that polysaccharide assembly may be located in the periplasmic region of the cell.
REFERENCES
BARTNICKI-GARCIA, S. (1973). Fundamental aspects of hyphal morphogenesis. In Microbial Differentiation, pp. 245-267. Cambridge University Press. BROWN, R. G. & NICKERSON, W. J. (1965). Cornposittion and structure of cell walls of filamentous and yeast-like forms of Aureobasidium (Pullularia) pullulans, Proceedings of the American Society for Microbiology, p. 26. BROWN, R. G. & LINDBERG, B. (1967 a). Polysaccharides from cell walls of Aureobasidium (Pullularia) pullulans. Part I. Glucans. Acta Chemica Scandinavica zt, 2379-2382. BROWN, R. G. & LINDBERG, B. (1967 b). Polysaccharides from cell walls of Aureobasidium (Pullularia) pullulans. Part II. Heteropolysaccharide. Acta Chemica Scandinavica 21, 2383-2389. BROWN, R. G., HANIC, L. A. & HSIAO, M. (1973). Structure and chemical composition of yeast chlamydospores of Aureobasidium pullulans, Canadian Journal of Microbiology 19, 163-168. BROWN, R. M., WILSON, J. H. M. & RICHARDSON, C. L. (1976). Cellulose biosynthesis in Acetobacter xylinum, Visualization of the site of synthesis and direct measurement of the in vivo process. Proceedings of the National Academy of Sciences U.S.A. 73, 4565-45 69. BURGER, M., HEJMovA, L. & KLEINZELLER, A. (1959). Transport of some mono- and di-saccharides into yeast cells. Biochemical Journal 71, 233-242. CATLEY, B. J. (1971 a). Utilization of carbon sources by Pullularia pullulans for the elaboration of extracellular polysaccharides. Applied Microbiology zz, 64 1-649. CATLEY, B. J. (1971 b). Role of pH and nitrogen limitation in the elaboration of the extracellular polysaccharide pullulan by Pullularia pullulans. Applied Microbiology zz, 650-654. CATLEY, B. J. (1979). Pullulan synthesis by Aureobasidium pullulans. In Microbial Polysaccharides and Polysaccharidases, pp. 69-84. Academic Press. CRANG, R. E. & PECHAK, D. G. (1978). Serial section reconstruction of the black yeast, Aureobasidium pullulans by means of high voltage electron microscopy. Protoplasma 96, 225-234. DARLING, S., THEILADE, J. & BIRCH-ANDERSEN, A. (1969). Kinetic and morphological observations on
455
Saccharomyces cereoisiae during spheroplast formation. Journal of Bacteriology 98, 797-810. DE LA FUENTE, G. & SOLS, A. (1962). Transport of sugars in yeasts. II. Mechanisms of utilization of disaccharides and related glycosides. Biochimica et Biophysica Acta 56, 49-62. DOMINGUEZ, J. B., GONI, F. M. & URUBURU, F. (1978). The transition from yeast-like to chlamydospore cells in Pullularia pullulans. Journal of General Microbiology 108, 111-117. DuRAN, A., BOWERS, B. & CABIB, E. (1975). Chitin synthetase zymogen is attached to the yeast plasma membrane. Proceedings of the National Academy of Sciences, U.S.A. 71,,3952-3955. FLEET, G. H. & PHAFF, H. J. (1974). Glucanases in Schizosaccharomyces. Isolation and properties of the cell-wall associated jf-(l -+ 3)-glucanases. Journal of Biological Chemistry 249, 1717-1728. JEFFRIES, T. W., EVELEIGH, D. E., MAcMILLAN, J. D., PARRISH, F. W. & REESE, E. T. (1977). Enzymatic hydrolysis of the walls of yeast cells and germinated fungal spores. Biochimica et Biophysica Acta 499, 10-23. KANEKO, T., KITAMURA, K. & YAMAMOTO, Y. (1973). Susceptibilities of yeasts to yeast cell wall lytic enzymes of Arthrobacter luteus. Agricultural and Biological Chemistry 37, 2295-2302. KELLY, P. J. & CATLEY, B. J. (1977). The effect of ethidium bromide mutagenesis on dimorphism, extracellular metabolism and cytochrome levels in Aureobasidium pullulans. Journal of General Microbiology 101" 249-254. KIKUCHI, Y., TAGUCHl, R., SAKANO, Y. & KOBAYASHI, T. (1973). Comparison of extracellular polysaccharide produced by Pullularia pullulans with polysaccharides in the cells and cell wall. Agricultural and Biological Chemistry 37, 1751-1753. KITAMURA, K., KANEKO, T. & YAMAMOTO, Y. (1971). Lysis of viable yeast cells by enzymes of Arthrobacter luteus. Archives of Biochemistry and Biophysics 145, 402-404. KITAMURA, K. & YAMAMOTO, Y. (1972). Purification and properties of an enzyme, Zymolyase, which lyses viable yeast cells. Archives of Biochemistry and Biophysics 153, 403-406. Kuo, S.-C. & YAMAMOTO, S. (1975). Preparation and growth of protoplasts, Methods in Cell Biology 11, 169- 183. LAMPEN, J. O. (1968). External enzymes of yeast: their nature and formation. Antonie van Leeuwenhoek 34, 1-18. LARSEN, B. & HAUG, A. (1970). Biosynthesis of alginate. Part 1. Composition and structure of alginate produced by Azotobacter vinelandii (Lipman). Carbohydrate Research 17, 287-296. LARSEN, B. & HAUG, A. (1971). Biosynthesis of alginate. Part III. Tritium incorporation with polymannuronic acid 5-epimerase from Azotobacter oinelandii. Carbohydrate Research 1,0, 225-232. LLOYD, J. B. & WHELAN, W. J. (1969). An improved method for enzymic determination of glucose in the presence of maltose. Analytical Biochemistry 30, 467-470.
456
Pullulan elaboration by spheroplasts of Aureobasidium pullulans S. M. (1973). Scanning electron microSANTOS, E., VILLANUEVA, J. R. &
PASSMORE, scopy of yeast colonies. Journal of The Institute of Brewing 79, 237-243. PECHAK, D. G. & CRANG, R E. (1977). An analysis of Aureobasidium pullulans developmental stages by means of scanning electron microscopy. Mycologia 69, 7 83-792. PRINGLE, A. T., FORSDYKE, J. & ROSE, A. H. (1979). Scanning electron microscopy study of Saccharomyces cerevisiae spheroplast formation. Journal of Bacteriology 140, 289-293. RAMos, S. & GARciA-AcHA, I. (1972). Production and regeneration of protoplasts of Pullularia pullulans, In 3rd International Symposium on Yeast Protoplasts-« Salamanca, p. 57. RAMOS, S. & GARciA-AcHA, I. (1975a). A vegetative cycle of Pullularia pullulans. Transactions of the British Mycological Society 64, 129-135. RAMos, S. & GARcfA-AcHA, I. (1975b). Cell wall enzymatic lysis of the yeast form of Pullularia pullulans and wall regeneration by protoplasts. Archives of Microbiology 1°4,271-277. RAMos, S., GARciA-ACHA, I. & PEBERDY, J. F. (1975). Wall structure and the budding process in Pullularia pullulans. Transactions of the British Mycological Society 64, 283-288.
SENTANDREU, R (1978). The plasma membrane of Saccharomyces cerevisiae, isolation and properties. Biochimica et Biophysica Acta 508, 39-54. SANTOS, T., SANCHEZ, M., VILLANUEVA, J. R & NOMBELA, C. (1979). Derepression of P-1,3glucanases in Penicillium italicum: localization of the various enzymes and correlation with cell wall glucan mobilization and autolysis. Journal of Bacteriology 137,6-12. TAGUCHI, R, SAKANO, Y., KIKUCHI, Y., SAKUMA, M. & KOBAYASHI, T. (1973). Synthesis of pullulan by acetone-dried cells and cell-free enzyme from Pullularia pullulans, and the participation of lipid intermediate. Agricultural and Biological Chemistry 37, 1635- 1641. WALLENFELS, K., KEILlCH, G., BECHTLER, G. & FREUDENBERGER, D. (1965). Untersuchungen an Pullulan. IV. Die Klarung des Strukturproblems mit physikalischen, chemischen und enzymatischen Methoden. Biochemische Zeitschrift 341, 433-450. ZAAR, K. (1979). Visualization of pores (export sites) correlated with cellulose production in the envelope of the gram negative bacterium Acetobacter xylinum, Journal of Cell Biology 80, 773-777.
(Received for publication 29 May 1980)