Enzymic lysis of three species of yeasts

[ 233 ] Trans. Br. mycol. Soc. 59 (2), 233- 240 (1972) Printed in Great Britain

ENZYMIC LYSIS OF THREE SPECIES OF YEASTS ByJ.P.G.BALLESTA Instituto de Biologia Celular, Velazquez 144, Madrid, Spain (With

1

Text-figure)

The cell wall structures of three species of yeasts - Torulopsis aeris Marcilla & Feduchy, Geotrichum lactis Fres. and Oospora suaueolens (Linder) Lindau - have been studied using lytic enzymes. Extensive lysis was only achieved by mixtures of enzymes or by complex lytic systems derived from soil organisms. The 'structural' components of the walls, mainly chitin and glucan, seem to be protected from the lytic action of the enzymes by other 'cementing' polysaccharides. The glucan is sensitive to P(I-3) and P(I4J) glucanases. Those polysaccharides containing sugars other than glucose are released from the wall after lysis without complete degradation to monomers.

The cell wall of Saccharomyces cerevisiae Hansen has been studied in great detail and the models available of the yeast wall structure are based mostly on data obtained from that species. The most recent one (Lampen, 1968) proposes that the outer layer of the wall is made of phosphomannan molecules covering an inner layer of glucan, both polysaccharides being linked together by protein molecules. It is hard to tell how far this model is valid for other species of yeast, some of which differ significantly from S. cerevisiae as far as chemical composition of the wall is concerned (Kreger, 1954; Crook & Johnston, 1962; Ballesta & Villanueva, 1971); however, it seems reasonable to suppose that the general idea of a cell wall having one structural component responsible for the rigidity of the wall and one, or several, cementing polymers may be valid for all species of yeast and even fungi (Aronson , 1965). Reported data seem to indicate that in some species chitin might totally or partially replace the glucan as structural polysaccharide (Kreger, 1954); and the fact that other monosaccharides in addition to mannose are found in some yeast cell walls (Ballesta, Uruburu & Villanueva, 1969, Crook & Johnston, 1962) points to the existence of other cementing polymers besides mannan. The results that we have obtained studying the structure and composition of the cell wall of three species of yeasts seem to confirm these statements (Ballesta & Villanueva, 197 I). The use of enzymes which specifically cleave certain components of the wall has been of great help for the study of cell structure in bacteria (Ghuysen, 1968) as well as in yeast and fungi (Skujins, Potgieter & Alexander, 1965; McLellan & Lampen, 1968; Pengra, Cole & Alexander, 1969; Ballesta et al. 1969; Eddy, 1958). In this communication we present some data from studies in which lytic enzymes have been used as tools for the elucidation of cell wall structures. 15

MYC

59

Transactions British Mycological Society

234

MATERIALS AND METHODS

Organisms andgrowth conditions The three species used were obtained from Coleccion Espanola de Cultivos Tipo: Torulopsis aeris Marcilla & Feduchy, 133I, Oospora suaveolens (Linder) Lindau, 1335 and Geotrichum lactis Fres., I028. Growth conditions and methods for the preparation of cell walls have been described recently (Ballesta & Villanueva, 197I). Lyticpreparations The organisms used for preparing lytic systems have been described previously (Villanueva, 1966). Micromonospora chalcea (Foulerton) 0rskov (Gascon, Ochoa & Villanueva, 1965), Streptomyces RA (Aguirre, GarciaAcha & Villanueva, 1963) as well as Streptomycesflavovirens Waksman 128 were isolated from soil samples, and lytic preparations precipitated from the cultures by ammonium sulphate as described elsewhere (Ballesta et al. 1969). The gut juice of the snail Helix pomatia was obtained from L'Industrie Biologique Francaise. Lysis of intact cells Cells harvested during exponential growth were washed with phosphate buffer 0·05 MpH 6'7 and then resuspended in I M (NH4hS04 at a concentration of 5 mg dry weight/mi. This suspension was mixed with 2 vol. of the lytic preparation and incubated at 37°. The final mixture was made up to 0·6 M with mannitol to make the medium osmotically stable for protoplasts. Enzymic tests fJ(I-3) and fJ(I-6) glucanase actrvity was tested on fJ(I-3) glucan (laminarine) and fJ(I-6) glucan (pustulan) respectively at 37° with a 2 h incubation period in 0·05 Macetate buffer pH 5.0. Chitin in 0.025 M phosphate buffer, pH 6'3, was used for the chitinase test. The reaction mixture contained IOO flg of enzyme preparation and I mg of substrate per ml of buffer. When lytic preparations were used, 1'0 ml of preparation was incubated with 5.0 mg of walls for 6 hat 37°. The extent of the reaction was measured by the release of reducing sugars determined by the method of Somogyi with glucose as standard. One unit of enzyme was defined as the amount needed to release 0·5 mg of reducing sugars N-acetylglucosamine was measured by the technique of in I h at Reissig, Strominger & Leloire (1955). Chitinase was obtained from the Worthington Biochemical Corp. fJ(I-6) glucanase from Penicillium brefeldianum Dodge was a gift from Dr E. T. Reese. fJ(I-3) glucanase was purified as reported elsewhere from Rhizopus arrhizus Fischer (Ballesta, 197I).

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Lysis ofyeasts.]. P. G. Ballesta

235

Fractionation ofcell walls and analysis ofits components The method used for fractionation of the cell walls has been described in detail elsewhere (Ballesta & Villanueva, 1971). It involves treatment of wall samples with 1 M KOH; the alkali-insoluble residue forms the fraction C-I. From the soluble part, fraction D-II is precipitated by (NH4)2S04 at saturation. Fraction C-III is formed by the material still soluble after the above mentioned precipitation. Acid hydrolysis and chromatography of the components of the different fractions were carried out as previously reported (Ballesta & Alexander, 1971a). RESULTS

Lysis ofintact cells by lytic preparations Cells from exponentially growing cultures of T. aeris, O. suaveolens and G. lactis were treated with lytic preparations obtained from different organisms as described in Methods. The sensitivity to lysis was tested by checking the formation of protoplasts in medium osmotically stabilized with mannitol. In Table 1 the results of this type of test are shown together with the enzymic activities of the lytic preparations used. Several lytic systems were obtained by growing Streptomyces RA under the conditions described in Methods, using cell walls of different species of yeasts as unique carbon sources. In this way we tried to induce lytic enzymes specific for the walls used as nutrients. It can be seen, however, that no significant improvement could be obtained in the activity of the lytic preparation against the wall used. T. aeris displayed a strong resistance to lysis, as previously reported (Ballesta et al. 1969), O. suaveolens and G. lactis were attacked to a greater or lesser extent by all the preparations used, the latter organism being more sensitive. It is noteworthy that although the structural components of the cell wall of these species are chitin and glucan (Ballesta & Villanueva, 1971), there is no direct relationship between the chitinase and glucanase activity of the lytic preparations and their capacity for lysing cells.

Enzymic Irydrolysis ofpurified cell walls Lysis by lytic preparations

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Samples of purified cell walls were incubated at with lytic preparations and the extent of digestion was checked by the loss of dry weight of the samples and by chromatography of the products of hydrolysis. When the cell walls were treated with lytic preparation from the Streptomyces 128 for 5 h the residue resistant to hydrolysis amounted to 80 %, 26 % and less than 10 % of the dry weight of the original sample in T. aeris, O. suaveolens and G. lactis respectively. Analysis of the hydrolysis-resistant residues made by chromatography of the HCI hydrolysates revealed the presence of glucose and glucosamine as the only monosaccharides in the three cases studied. 15-2

Transactions British Mycological Society Table

I.

Actioity

of various lyticpreparations Activity on polysaccharides (units/rnl)

Activity on cell walls Lytic preparation from:

Miaomonospora Helix pomatia Streptomyces RA (t)* Streptomyces RA (0)* Streptomyces RA (g)* Streptomyces RS Streptomyces 128

A

(

T. aeris O. suaveolens

P(I-3) glucan

P(I-6) glucan

o-oq 0'45 0'34 0'43 0'23 0'55

I'g 1'5 0'78 1'10 0'47 1'0

I3 0'7 06 1'1 0'55 0,8

G. lactis

± ± +

± ± ++

++ ++ ++++

++ +++ +++++

+

Chitin

+

Activity on cells was tested by checking the formation of protoplast. The relative amount of protoplast is indicated by the number of plus signs. (-) No protoplasts, (±) Occasional protoplasts. * (t), (0), (g) lytic systems from Streptomyces RA grown in T, aeris, O. suaveolens, and G. lactis respectively.

100 90

...

Vi'

A+B+C

80

C
OIl

~ '" 70 OIl

t::

'u ~

60

'" -="0

50

"0

'" '" C
<;

'"'"0

u

~

B+C 40 30

"Ell OIl

::t

20 10

2 3 4 Time of incubation (h) Fig.

I.

5

Release of reducing sugars from G. lactis cell walls by purified enzymes. A, Chitinase; B, p(r-6) glucanase; C, P(I-3) glucanase.

In the supernatant, after centrifugation of the reaction mixture, glucose was the major spot detected by chromatography in the three cases; N-acetylglucosamine was also present but in much lower proportional amounts. Galactose was found in the case of O. suaoeolens and G. lactis. Several unidentified slow-moving spots were also detected, corresponding probably to oligosaccharides. It is interesting to note that despite the presence of considerable amounts of mannose in the cell wall of O. suaoeolens and G. lactis (Ballesta &

Lysis of yeasts. J. P. G. Ballesta Table

2.

Wall pretreatment None P (I-3) glucanase P(I-o) glucanase

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Activi0' ofchitinase on cell walls ofyeasts T. aeris

O. suaoeolens

G. lactis

o 5,6 1'3

0'3 15'7 3'4

0'2 23'4 6'3

Data given as p.g of N-acetylglucosamine released per mg of walls.

Table 3. Activi0' of a mixture

ofglucanases and chitinase on cell walls

Sample

Resistant residue (% )

T, aeris O. suaueolens G. lactis

45 40

79

Reducing sugars released (%)

N-acetylglucosamine released (%)

15 23 31

0'3 6 15

Data given as percentage of cell wall dry weight. Cell walls were incubated with a mixture of P(I-3), (J(I-o) glucanases and chitinase. The loss of dry weight and the release of reducing sugars and N-acetylglucosamine after 5 h of treatment was checked.

Villanueva, 1971) this sugar was not detected in the chromatograms. This probably indicates that mannose had been released into the medium in the form of a polysaccharide during hydrolysis.

Lysis by purified eneymes Cell walls of the three species differed in sensitivity to the various purified enzymes used. Fig. 1 shows the enzymic release of reducing sugars from cell walls of G. lactis. O. suaueolens gave closely similar results both qualitatively and quantitatively whereas walls of T. aeris, although giving qualitatively similar results, were much more resistant to attack by these enzymes. The synergistic action of the three enzymes used is obvious from these results . Specially noticeable is the case of chitinase, which alone shows practically no activity but which strongly stimulates the activity of the glucanases. This suggests a protective role for the glucanasesensitive compounds on chitin, a role which is also clearly indicated by the results presented in Table 2. In these experiments cell walls of G. lactis, previously treated with glucanases, were exposed to the action of chitinase for 2 h and the release of N-acetylglucosamine was checked. As shown in Table 2, prior treatment of these walls with glucanases, especially {J(1-3) glucanase, makes possible the subsequent action of the chitinase. The sensitivity of the three species of yeast to the mixture of enzymes is shown in Table 3, After 5 h of treatment with a mixture of fJ(1-3), fJ(1-6) glucanases and chitinase, T. aeris cell walls only lost 21 % of their dry weight compared with 55 % in the case of O. suaveolens and 60 % with G. lactis. It is interesting to note that with T. aeris the reducing sugars recovered in the supernatant account for almost the total loss of dry weight detected whereas with the other two species only 50 % of the material released had reducing activity.

Transactions British Mycological Society Table 4. Activiry oj purified eneymes on Geotrichum lactis cell wallfractions Enzyme P(I-3) glucanase P(I4i) glucanase Chitinase

Fraction C-I 198'1

23'4

46-6

Fraction C-II

Fraction C-III

58'2 97'0 3'4

36'6 117'9 0

Data given as pg of glucose released per mg of fraction,

Analysis of the residues resistant to the action of the enzymes showed the presence of the same sugars that compose the intact walls (Ballesta & Villanueva, 1971), namely glucose and glucosamine in T. aeris, and in addition galactose and mannose in O. suaueolens and G. lactis, although the latter two sugars were present in higher proportions than in intact walls.

Lysis oj cell wallfractions As summarized in Methods, it is possible by means of chemical treatments to fractionate yeast cell walls into three portions (Ballesta & Villanueva, 1971) of different chemical composition. These fractions were incubated for 5 h with the purified enzymes and the results in the case of G. lactis are expressed in Table 4. Fraction C-I is more sensitive to the action of jJ(I-3) glucanase and chitinase. Fractions C-II and C-III are insensitive to chitinase and more sensitive to jJ(1-6) than jJ(1-3) glucanase. When the products of hydrolysis by jJ(1-6) glucanase of the wall fractions were chromatogrammed using similar products from pustulan (a polymer of glucose with jJ(I-6) bonds) as standards it was possible to detect the spots corresponding to dimers and trimers (gentobiose and gentotriose) as well as glucose monomers. Similarly, when cell wall fractions and laminarine (a polymer of glucose with jJ(I-3) bonds) were hydrolysed by jJ(I-3) glucanase the spots of the corresponding dimers and trimers (laminaribiose and laminaritriose) were detected. However, in this case other unidentified spots were also detected. DISCUSSION

The results reported in this paper give a complementary picture to that previously obtained by means of pure chemical studies (Ballesta & Villanueva, 1971) of the wall structure of the three yeasts studied. It is confirmed here that in these species, T. aeris, G. lactis and O. suaveolens, the glucan is at least partially jJ(I-3) and jJ(I-6) glucose linked. This type of gluean has been demonstrated in S. cereoisiae and other species of yeasts (Duff, 1952; Bishop, Blanck & Gardner, 1960; Wallen, Rhodes & Shulke, 1965; Manners & Patterson, 1966), It is interesting to note that whereas two cell wall fractions enriched in glucan have been isolated and have been shown to be enriched in jJ(1-3) and fJ(I-6) bonds respectively, the chitin seems to be associated almost exclusively with the former glucan. The glucosamine existing in the other fractions (Ballesta & Villanueva, 197 I) is either not in the form of chitin

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or else it is very well protected from the action of the chitinase. Nonchitin forming glucosamine has been found as bridges between the protein and polysaccharide moieties of glucopeptide molecules in yeast cell walls (Sentandreu & Northcote, 1968). According to the latest model for yeast cell wall structure (Lampen, 1968), polysaccharides containing sugars other than glucose play a cementing role and in some way protect the so-called structural ones. This protective action is indicated by our results obtained by lysing intact cells. It is seen how enzymes which attack the structural macromolecules are not sufficient by themselves to lyse the cells, other enzymes also being required. In fact a phosphomannanase has been reported to play an important role in the process of lysis (McLellan & Lampen, 1968), and a series of highly specific glucanases have been reported in cultures of Cytophagajohnsonii Stanier (Bacon et at. 1970). The specific mechanism whereby certain species of yeast and fungi resist lysis has been studied in detail in several cases. In most instances, resistance is conferred upon the wall by a non-structural component which protects the sensitive ones from the action of lytic enzymes (Bloomfield & Alexander, 1967; Ballesta et at. 1969; Ballesta & Alexander, 1971). In other instances, however, the mechanism of resistance is not so well defined (Pengra et at. 1969). The resistance of T. aeris to lysis has been studied in some detail elsewhere (Ballesta et at. 1969). It was shown that a xylose-containing polysaccharide, easily removable from the wall by mechanical means, possibly plays an important role in the mechanism of resistance of this yeast. The data reported here, obtained with T. aeris cell wall which lacks that polysaccharide, seem to indicate that other cell wall components are also important in determining the resistance of this species. On the other hand the complete breakdown of all the cell wall components is not necessary in order to achieve lysis since, as shown above (Table 3), only the 50 % of the released wall material has reducing power and no free mannose is detected in the hydrolysates during lysis. The synergistic action of the enzymes also confirms the protecting function of the wall components. In agreement with the results discussed above, the chitin seems closely associated with a glucan having mainly P( 1-3) bonds, and it is necessary to remove this by the action of glucanase in order to expose the chitin to lytic enzymes (Table 3). Similar results were reported for Rhizoctonia solani Kuhn by Potgieter & Alexander (1966). Part of this work was carried out while the author was at the Department of Agronomy, Cornell University, Ithaca, N.Y. I wish to thank Professor M. Alexander for reading and commenting on this manuscript, and Professor J. R. Villanueva for help and encouragement. REFERENCES

J. R., GARCIA.AcHA, I. G. & VILLANUEVA, J. R. (1963). An enzyme(s) from a Streptomyces sp, to prepare mold 'protoplasts'. Experientia 19, 82-85. ARONSON,J. M. (1965). The cell wall. In The Fungi, vol. I (ed. G. C. Ainsworth and A. Sussman). New York: Academic Press.

AGUIRRE, M.

Transactions British Mycological Society BACON, j. S. D., GORDON, A. H., JONES, D., TAYLOR, I. F. & WEBLEY, D. M. (1970). The separation of p-glucanases produced by Cytophaga johnsonii and their role in the lysis of yeast cell walls. Biochemical Journal 120, 67-68. BALLESTA,j. P. G. (1971). Purificacion y propiedades de la P(I-3) glucanasa de Rhizopus arrhizus, Microbiologia Espanola 24, 257-269. BALLESTA, J. P. G. & ALEXANDER, M. (1971). Resistance of Z,ygorhynchus sp. to lysis. Journal of Bacteriology 106, 938-945. BALLESTA,J. P. G. & ALEXANDER, M. (1972). Susceptibility of several basidiomycetes to microbial lysis. Transactions of the British Mycological Society 58, 482-487. BALLESTA,J. P. G. & VILLANUEVA,J. R. (1971). The cell wall components of various species of yeasts. Transactions of the British Mycological Society 56, 403-410. BALLESTA, J. P. G., URUBURU, F. & VILLANUEVA, J. R. (1969). Resistance of Torulopsis aeris cells to enzymatic lysis. Transactions of the British Mycological Society 52, 1-7. BISHOP, C. T., BLANCK, F. & GARDNER, P. E. (1960). The cell wall polysaccharides of Candida albicans: glucan, mannan, and chitin. Canadian Journal of Chemistry 38, 869-88 1. BLOOMFIELD, B. ]. & ALEXANDER, M. (1967). Melanins and resistance of fungi to lysis. Journal of Bacteriology 93, 1276-1280. CROOK, E. M. & JOHNSTON, I. R. (1962). Analysis offungal cell wall. BiochemicalJournal 83, 325-33 I. DUFF, R. B. (1952). The constitution of a glucosan from the fungus Polyporus betulinus. Journal of Chemical Society, pp. 2529-2594. EDDY, A. A. (1958). The structure of the yeast cell wall. II. Degradative studies with enzymes. Proceedings of the Royal Society (B) 149, 425-440. GASCON, S., OCHOA, A. G. & VILLANUEVA, J. R. (1965). Production of yeast and mold protoplasts by treatment with the strepzyme of Micromonospora. Canadian Journal of Microbiology II, 573-580. GHUYSEN, J. M. (1968). Use of bacteriolytic enzymes in determination of wall structure and their role in cell metabolism. Bacteriological Reviews 33, 425-464. KREGER, D. R. (1954). Observations on cell walls of yeasts and some other fungi by X-ray diffraction and solubility tests. Biochimica et Biophysica Acta 13, 1-9. !.AMPEN,]. O. (1968). External enzymes of yeast: their nature and formation. Antonie van Leeuwenhoek 34, I - I 8. McLELLAN, W. L. & LAMPEN,J. O. (1968). Phosphomannase (PR-factor), an enzyme required for the formation of yeast protoplasts. Journal of Bacteriology 95, 967-974. MANNERS, D.J. & PATIERSON,j. C. (1966). A re-examination of the molecular structure of yeast glucan. Biochemical Journal I9c-20C. PENGRA, R. M., COLE, M. A. & ALEXANDER, M. (1969). Cell wall and lysis of Mortierella paroispora hyphae. Journal of Bacteriology 97, 1056-1061. POTGIETER, H. H. & ALEXANDER, M. (1966). Susceptibility and resistance of several fungi to microbial lysis. Journal of Bacteriology 91, 1056-1061. REISSIG, J. L., STROMINGER, J. L. & LELOIR, L. F. (1955). A modified calorimetric method for the estimation of N-acetylamino sugars. Journal of Biological Chemistry 2 1 7, 959-966. SENTANDREU, R. & NORTHCOTE, D. H. (1968). The structure of a glycopeptide isolated from yeast cell wall. Biochemical Journal 109, 419-432. SKUJINS,J.j., POTGIETER, H.J. & ALEXANDER, M. (1965). Dissolution offungal cell wall by a streptomycete chitinase and P( 1-3) glucanase, Archives of Biochemistry and Biophysics III, 358-364. VILLANUEVA,]. R. (1966). Protoplasts offungi. In Thefungi, ed. Ainsworth and Sussman. New York: Academic Press. WALLEN, L. L., RHODES, R. A. & SHULKE, H. R. (1965). Physical properties and chemical composition of p-glucans from fleshy fungi. Applied Microbiology 13, 272-278.

(Accepted for publication 26 March 1972)