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The occurrence of α(1–3)glucan in Cryptococcus, schizosaccharomyces and Polyporus species, and its hydrolysis by a Streptomyces culture filtrate lysing cell walls of Cryptococcus
PRELIMINARY NOTES
313
BBA 21212
The occurrence of =(l-3)glucan in Cryptococcus, Schizosaccharomyces and Polyporus species, and its hydrolysis by a Streptomyces culture filtrate lysing cell walls of Cryptococcus As part of a systematic investigation of the lysis of fungal cell walls by soil micro-organisms we have examined the soil yeasts Cryptococcus albidus and Cryptococcus terreus. When these were grown upon a glucose-peptone medium, the main nitrogenous constituents of the walls were glucosamine, present as chitin, and a variety of amino acids, presumably derived from protein. Only one neutral sugar, glucose, was present in major amounts, and this accounted for some 75 % of the dry matter in C. albidus. fl-Glucans, typified by the glucan of Saccharomyces cerevisiae, occur in the walls of many fungi (cf. ref. I). The main linkage in these glucans is fl(I-3), and many organisms produce enzymes hydrolysing it 2. Two Streptomyces species from soiP and a non-fruiting myxobacterium, Cytophaga johnsonii 4, responded to the presence of walls of Cryptococcus by producing an extracellular fl(I-3)glucanase, but only the Streptomyces species brought about lysis. The products did not include laminaribiose, the disaccharide usually seen amongst the products of ]3(I-3)glucan degradation. Instead a sugar was present with RE value (in n-butanol-acetic acid-water, 4:1:5, v/v/v) intermediate between those of laminaribiose and maltose. Nigerose (Glcp,¢I -+ 3Glc) was subsequently found to run in this position; Rglucose values : maltose, 0.43 ; nigerose, 0.48; laminaribiose, 0.54. The infrared spectrum of Cryptococcus walls suggested that both ~- and flglucans were present. The spectrum of the =-glucan component, distinguished by absorption bands at 11.75/z and 12.15 F, differed from that of glycogen, bacterial dextran, and nigeran 5, but closely resembled that of an ~(I-3)glucan (Fig. I) isolated by JOHNSTON6 from cell walls of Aspergillus niger (Fraction IVR). This type of polysaccharide was first tentatively identified in Polyporus betulinus by D u f f ~, in this institute (more recently found in Polyporus tumulosus by RALPH AND BENDERS), and his preparations obtained by extraction with aqueous alkali proved to have a spectrum identical with that of the A. niger product. The =-glucan was removed from Cryptococcus walls by extraction with 3 % NaOH at 75 ° leaving a fl-glucan residue, resembling but not identical with S. cerevisiae glucan. Some fl-glucan was extracted with
7
8
9
10
11
12
12 14 15 Woveleng th (p)
16
Fig. I. Infrared spectrum of ~(i-3)glucan from walls of A. niger. I mg freeze-dried sample in 12 mm KBr disk.
Biochim. Biophys. Acta, 158 (1968) 313-3I 5
314
PRELIMINARY NOTES
the ~-glucan. A similar situation was found to exist in Schizosaccharomyces pombe, although in this case the insoluble residue from alkali extraction still contained some e-glucan; the alkali-soluble polysaccharide mixture, when treated with a fl-glucanase from Cyt@haga johnsonii yielded a pure a-glucan with an infrared spectrum identical with that of the ~(I-3)glucan from A. niger. The ability of the Streptomyces species to lyse walls of Cryptococcus and Schizosaccharomyces, and the failure of C. johnsonii to do so, is explained by the presence of an ~(I-3)glucanase in the former. There is an increase in reducing power when the culture fluid is incubated at 30 ° and p H 5.0 with a suspension of ~(i-3)glucan from P. betulinus; paper chromatography shows the presence of a series of oligosaccharides with RF values corresponding to those produced by partial acid hydrolysis of ~(I-3)glucan under the conditions of JOHNSTON6, but no glucose. We can find no previous reference to enzymes capable of attacking glucans in which the majority of linkages are ~(I-3), but it seems possible that "mycodextranase"9 is similar. The latter enzyme was capable of hydrolysing ~-glucopyranoside linkages in nigeran, a glucan having both ~(I-3) and ~(I-4) linkages, and was suspected to be specific for the glycosidic linkage of a glucose residue substituted at position 3. Our observations raise the question whether the alkali-soluble glucan fraction described by KREGER1 is an ~(I-3)glucan. It was found by him in walls of several fungi (Schizosaccharomyces, Endomyces, Penicillium, Agaricus) and by WESSELS1° in Schizophyllum commune ("S-glucan"), but has so far been characterised only by the X-ray diffraction pattern. No numerical values for the spacings of the diffraction maxima were published by KREGER1, but he has kindly provided them for us (personal communication, I968), and W. A. MITCHELL of the Pedology Department of this Institute finds ahnost identical values for the P. betulinus and A. niger glucans, namely 9.4, 4.9, 4-17, 3.20 (weak), 2.69 (weak) A (measured on a Philips X-ray diffractometer). Dr. D. R. KREGER has further informed us of infrared spectra which identify the S-glucans from S. pombe and Schizosaccharomyces versatilis, Polyporus sulphureus, and Boletus edulis as ~(I-3)glucans. In a survey of the infrared spectra of other cell walls we could not detect bands of ~(I-3)glucan in Lipomyces starkeyi, Pythium debaryanum, Rhizoctonia sp., Pythomyces chartarum, or Sclerotinia sclerotiorum. These bands were clearly present in A. niger, and possibly present in Penicillium sp., Polystictus versicolor, and Lentinus sp. These observations suggest that c~(I-3)glucans are more widely distributed in cell walls of yeasts and fungi than has been suspected, and that ~(I-3)glucanases must be present among the enzymes produced by micro-organisms capable of lysing these walls. That the Cytophaga fl(I-3)glucanases, which can attack the fl-glucan component in a mixture of a- and fl-glucans extracted from S. pombe walls, should be unable to attack the/3-glucan in the intact wall is presumably due to the more intimate association between the two glucans in the architecture of the wall. Only a simultaneous attack on both ~- and fl-glucans, such as is achieved by the Streptomyces species, enables the enzymes to penetrate the wall. We are very grateful to Dr. L. J. WICKERHAM for identifying the Cryptococcus species; to Dr. I. R. JOHNSTON,of University College, London, for a generous gift of authentic e(I-3)glucan, which considerably simplified the problem of identifying the ~-glucan component; to him and to Dr, S. A. BARKER for samples of nigeran; to Dr. D. R. KREGER for permission to refer to his unpublished results; to Mr. W. A. Biochim. Biophys. Acta, 158 (1968) 313-315
PRELIMINARY NOTES
315
MITCHELL for the X-ray analysis; and to Miss J. I. NORMINGTON and Mrs. E. F. CRUICKSHANKfor valuable technical assistance.
Macaulay Institute for Soil Research, Craigiebuckler, Aberdeen (GreatBritain)
J. s. D. BACON D. JONES V. C. F A R M E R
D. M. WEBLEY I 2 3 4 5 6 7 8 9 IO
D. R. KREGER, Biochim. Biophys. Acta, 13 (1954) 1. A. T. BULL AND C. G. C. CHESTERS, Advan. Enzymol., 28 (1966) 325 . D. JONES AND n . M. WEBLEY, Ptant Soil, 28 (1968) 147. E. A. C. FOLLETT AND D. IV[. WEBLEY, Antonie van Leeuwenhoek, J. Microbiol. Serol., 31 (1965) 361. S. A. BARKER, E. J. BOURNE, M. STACEY AND D. H. WHIFFEN, J. Chem. Soc., (1954) 171. I. R. JOHNSTON, Biochem. J., 96 (1965) 659. R. B. DUFF, J. Chem. Soe., (1952) 2592. B. J. RALPH AND V. J. BENDER, Chem. Ind. London, (1965) 1181. E. T. REESE AND M. MANDELS, Can. J. Microbiol., IO (1964) IO3. J. G. H. WESSELS, Wentia, 13 (1965) i.
Received February i2th, i968 Biochim. Biophys. Acta, 158 (1968) 313-315
313
BBA 21212
The occurrence of =(l-3)glucan in Cryptococcus, Schizosaccharomyces and Polyporus species, and its hydrolysis by a Streptomyces culture filtrate lysing cell walls of Cryptococcus As part of a systematic investigation of the lysis of fungal cell walls by soil micro-organisms we have examined the soil yeasts Cryptococcus albidus and Cryptococcus terreus. When these were grown upon a glucose-peptone medium, the main nitrogenous constituents of the walls were glucosamine, present as chitin, and a variety of amino acids, presumably derived from protein. Only one neutral sugar, glucose, was present in major amounts, and this accounted for some 75 % of the dry matter in C. albidus. fl-Glucans, typified by the glucan of Saccharomyces cerevisiae, occur in the walls of many fungi (cf. ref. I). The main linkage in these glucans is fl(I-3), and many organisms produce enzymes hydrolysing it 2. Two Streptomyces species from soiP and a non-fruiting myxobacterium, Cytophaga johnsonii 4, responded to the presence of walls of Cryptococcus by producing an extracellular fl(I-3)glucanase, but only the Streptomyces species brought about lysis. The products did not include laminaribiose, the disaccharide usually seen amongst the products of ]3(I-3)glucan degradation. Instead a sugar was present with RE value (in n-butanol-acetic acid-water, 4:1:5, v/v/v) intermediate between those of laminaribiose and maltose. Nigerose (Glcp,¢I -+ 3Glc) was subsequently found to run in this position; Rglucose values : maltose, 0.43 ; nigerose, 0.48; laminaribiose, 0.54. The infrared spectrum of Cryptococcus walls suggested that both ~- and flglucans were present. The spectrum of the =-glucan component, distinguished by absorption bands at 11.75/z and 12.15 F, differed from that of glycogen, bacterial dextran, and nigeran 5, but closely resembled that of an ~(I-3)glucan (Fig. I) isolated by JOHNSTON6 from cell walls of Aspergillus niger (Fraction IVR). This type of polysaccharide was first tentatively identified in Polyporus betulinus by D u f f ~, in this institute (more recently found in Polyporus tumulosus by RALPH AND BENDERS), and his preparations obtained by extraction with aqueous alkali proved to have a spectrum identical with that of the A. niger product. The =-glucan was removed from Cryptococcus walls by extraction with 3 % NaOH at 75 ° leaving a fl-glucan residue, resembling but not identical with S. cerevisiae glucan. Some fl-glucan was extracted with
7
8
9
10
11
12
12 14 15 Woveleng th (p)
16
Fig. I. Infrared spectrum of ~(i-3)glucan from walls of A. niger. I mg freeze-dried sample in 12 mm KBr disk.
Biochim. Biophys. Acta, 158 (1968) 313-3I 5
314
PRELIMINARY NOTES
the ~-glucan. A similar situation was found to exist in Schizosaccharomyces pombe, although in this case the insoluble residue from alkali extraction still contained some e-glucan; the alkali-soluble polysaccharide mixture, when treated with a fl-glucanase from Cyt@haga johnsonii yielded a pure a-glucan with an infrared spectrum identical with that of the ~(I-3)glucan from A. niger. The ability of the Streptomyces species to lyse walls of Cryptococcus and Schizosaccharomyces, and the failure of C. johnsonii to do so, is explained by the presence of an ~(I-3)glucanase in the former. There is an increase in reducing power when the culture fluid is incubated at 30 ° and p H 5.0 with a suspension of ~(i-3)glucan from P. betulinus; paper chromatography shows the presence of a series of oligosaccharides with RF values corresponding to those produced by partial acid hydrolysis of ~(I-3)glucan under the conditions of JOHNSTON6, but no glucose. We can find no previous reference to enzymes capable of attacking glucans in which the majority of linkages are ~(I-3), but it seems possible that "mycodextranase"9 is similar. The latter enzyme was capable of hydrolysing ~-glucopyranoside linkages in nigeran, a glucan having both ~(I-3) and ~(I-4) linkages, and was suspected to be specific for the glycosidic linkage of a glucose residue substituted at position 3. Our observations raise the question whether the alkali-soluble glucan fraction described by KREGER1 is an ~(I-3)glucan. It was found by him in walls of several fungi (Schizosaccharomyces, Endomyces, Penicillium, Agaricus) and by WESSELS1° in Schizophyllum commune ("S-glucan"), but has so far been characterised only by the X-ray diffraction pattern. No numerical values for the spacings of the diffraction maxima were published by KREGER1, but he has kindly provided them for us (personal communication, I968), and W. A. MITCHELL of the Pedology Department of this Institute finds ahnost identical values for the P. betulinus and A. niger glucans, namely 9.4, 4.9, 4-17, 3.20 (weak), 2.69 (weak) A (measured on a Philips X-ray diffractometer). Dr. D. R. KREGER has further informed us of infrared spectra which identify the S-glucans from S. pombe and Schizosaccharomyces versatilis, Polyporus sulphureus, and Boletus edulis as ~(I-3)glucans. In a survey of the infrared spectra of other cell walls we could not detect bands of ~(I-3)glucan in Lipomyces starkeyi, Pythium debaryanum, Rhizoctonia sp., Pythomyces chartarum, or Sclerotinia sclerotiorum. These bands were clearly present in A. niger, and possibly present in Penicillium sp., Polystictus versicolor, and Lentinus sp. These observations suggest that c~(I-3)glucans are more widely distributed in cell walls of yeasts and fungi than has been suspected, and that ~(I-3)glucanases must be present among the enzymes produced by micro-organisms capable of lysing these walls. That the Cytophaga fl(I-3)glucanases, which can attack the fl-glucan component in a mixture of a- and fl-glucans extracted from S. pombe walls, should be unable to attack the/3-glucan in the intact wall is presumably due to the more intimate association between the two glucans in the architecture of the wall. Only a simultaneous attack on both ~- and fl-glucans, such as is achieved by the Streptomyces species, enables the enzymes to penetrate the wall. We are very grateful to Dr. L. J. WICKERHAM for identifying the Cryptococcus species; to Dr. I. R. JOHNSTON,of University College, London, for a generous gift of authentic e(I-3)glucan, which considerably simplified the problem of identifying the ~-glucan component; to him and to Dr, S. A. BARKER for samples of nigeran; to Dr. D. R. KREGER for permission to refer to his unpublished results; to Mr. W. A. Biochim. Biophys. Acta, 158 (1968) 313-315
PRELIMINARY NOTES
315
MITCHELL for the X-ray analysis; and to Miss J. I. NORMINGTON and Mrs. E. F. CRUICKSHANKfor valuable technical assistance.
Macaulay Institute for Soil Research, Craigiebuckler, Aberdeen (GreatBritain)
J. s. D. BACON D. JONES V. C. F A R M E R
D. M. WEBLEY I 2 3 4 5 6 7 8 9 IO
D. R. KREGER, Biochim. Biophys. Acta, 13 (1954) 1. A. T. BULL AND C. G. C. CHESTERS, Advan. Enzymol., 28 (1966) 325 . D. JONES AND n . M. WEBLEY, Ptant Soil, 28 (1968) 147. E. A. C. FOLLETT AND D. IV[. WEBLEY, Antonie van Leeuwenhoek, J. Microbiol. Serol., 31 (1965) 361. S. A. BARKER, E. J. BOURNE, M. STACEY AND D. H. WHIFFEN, J. Chem. Soc., (1954) 171. I. R. JOHNSTON, Biochem. J., 96 (1965) 659. R. B. DUFF, J. Chem. Soe., (1952) 2592. B. J. RALPH AND V. J. BENDER, Chem. Ind. London, (1965) 1181. E. T. REESE AND M. MANDELS, Can. J. Microbiol., IO (1964) IO3. J. G. H. WESSELS, Wentia, 13 (1965) i.
Received February i2th, i968 Biochim. Biophys. Acta, 158 (1968) 313-315