Folia Microbiol. 28, 452--457 (1983)

Regeneration of the Cell Wall of Protoplasts of the Fission Yeast Schizosaccharomyces versatilis in Liquid Media and Their Reversion to Cells M. GABRIEL and M. KoPEcxX Department of Biology, Medical Faculty, J.E. Purkyn~ University, 662 43 Brno, Czechoslovakia

Received February 14, 1983

ABSTRACT. Regeneration of the cell wall a n d reversion of protoplasts with a completely regenerated cell wall to cells were studied b y light a n d electron microscopy in protoplasts of the fission yeasts Schizosaccharomyces versatilis. On their surface the protoplasts regenerated a complete new wall even in liquid media. The wall regeneration began with the f o r m a t i o n of a t h i n irregular n e t of fiat bundles of long microfibrils a n d the n e t was gradually" filled w i t h aggregates of short straight microfibrils a n d small piles of a m o r p h o u s material. Osmotically resistant org a n i s m s with regenerated walls were detected after a 4 - - 6 - h cultivation. D e p e n d i n g on the n u t r i e n t m e d i u m u s e d 10--80 ~o of protoplasts with the regenerated wall were obtained t h a t reverted s u b s e q u e n t l y to cells. The high percentage of the wall regeneration a n d reversion to cells was reached b y combining cultivation in a poor m e d i u m with t h a t in a rich m e d i u m . Reversion to cells could only occur after the protoplasts h a d regenerated rigid cell walls. These walled protoplasts u n d e r w e n t septation, and, b y polar growth, produced cylindrical cells, f u r t h e r dividing b y fission.

Protoplasts of budding yeasts of the genus Saccharomyce8 can produce a complete new cell wall and revert to normal cells only during cultivation in a solid nutrient medium (gelatin, agar) (Ne6as 1961; Svoboda 1966). On the other hand, protoplasts prepared from fission yeast Schizosaccharomyces pombe can regenerate the complete wall and revert to normal cells even in liquid media (Svoboda 1967). This ability was attributed to the formation of an extensive fibrillar network, which, according to Ne6as et al. (1968), prevents diffusion of the wall polymers of the matrix by mechanisms similar to those of gels. The complete new wall is formed beneath the network and cell division follows (Ne6as et al. 1968). However, the ability of S. pombe protoplasts to regenerate cell walls in liquid media may also be due to the synthesis of wall polymers of different physico-ehemical properties from those found in budding yeasts. While during the first 2 h of cultivation (Ne6as et al. 1968) the fibrillar network is similar to the network of S. cerevisiae protoplasts in both ultrastructure (KopeckA et al. 1967) and chemical composition, being composed of highly crystalline ~-(1-+3)-glucan (Kreger and Kopeck~ 1973, 1976, 1978), in S. pombe protoplasts

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the synthesis of ~-(1-+3)-glucan is followed upon prolonged cultivation by the synthesis of another insoluble polysaccharide, ~-(1-~3)-glucan, which accumulates in the form of short microfibrils (Kreger and Kopeck~ 1978), particularly when the wall regeneration is blocked b y snail enzymes (Havelkovs 1972). Protoplasts of another strain of fission yeasts, S. versatilis, are also a suitable model for studying the wall biogenesis (Gabriel 1982). They can also regenerate in liquid media (Kopecks 1975) and their nuclei can be observed in the phase-contrast microscope (Kopecks and Gabriel 1978). In addition to the so-called yeast ~-glucan (Kreger 1954) and galactomannan (Gorin and Spencer 1968), cell walls of S. versatilis contain ~-(1-+3)-glucan, which was demonstrated b y chemical methods (Bacon et al. 1968, Kreger 1954). However, the ultrastructure of the cell wall in this species has not yet been investigated. The regeneration of the complete cell wall of protoplasts of S. versatilis during cultivation in liquid media and their reversion to cells dividing by fission are described in the present communication. MATERIAL AND M E T H O D S

Microorganism. The cells of Schizosacharomyces japonicus var. versatilis CC 60-255, kindly supplied b y Prof. H.J. Phaff (University of California, Davis, USA) were cultivated on 2 ~ wort agar (pH 5.5). Stock cultures were maintained at 4 ~ Preparation of protoplasts. For the preparation of protoplasts the cells were transferred to l0 mL of wort medium, cultivated for 12 h at room temperature; 1 mL of the suspension was then added to a fresh wort medium and the culture was shaken for 12 h at 27 ~ Cells from the exponential phase of growth were taken and washed with 0.3 ~ mannitol. The crude gastric juice of Helix pomatia, diluted 2 : 1 with citrate--phosphate buffer (pH 5.5) and osmotically stabilized by mannitol at a final concentration of 0.7 M with traces of MgSOa was then added. Protoplasts were washed with 0.7 ~ mannitol (pH 5.5) by repeated centrifugation (10 min, 670 g). The centrifugation also facilitated the mechanical release of the protoplasts from the residues of partially digested cell walls. Cultivation of protoplasts. Fresh protoplasts were t~ransferred in the liquid medium, i.e. in wort medium osmoticallv stabilized with mannitol or KC1 and in the N1 synthetic medium (Kelletl et aI. 1954), in a thin layer of the liquid medium on the surface of osmotically stabilized wort agar and on Difco mycological agar (composition: Bacto Soytone, 10 g; Bacto Dextrose, 10 g; Bacto Agar, 15 g; water 1 L). The percentages of protoplasts with regenered walls and those reverting to cells were determined microscopically. Electron microscopy of wall structures. Protoplasts inoculated in liquid media were osmotically lyzed in distilled water at various time intervals, their cytoplasm was dissolved in 0.5 ~ sodium dodecylsulphate (50 ~ 10 rain) and the residual wall structures were washed b y repeated centrifugation ( 3 X 1 0 m i n , 1 500g). Wall structures were applied to electron microscopic grids and after drying coated with platinum at an angle of 15 ~ and observed in the T E S L A BS 500 electron microscope.

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RESULTS

Formation of protoplasts When the crude snail enzyme osmotically stabilized by mannitol was added to the culture of cells from the exponential growth phase the cell walls of most cells in the population were digested during 30--60 min and spherical protoplasts were formed (Plate 1). Whereas in the original yeasts nuclei with nucleoli could be well seen under the phase-contrast microscope practically in all cells (Kopecks and Gabriel 1978), after the conversion of the cells to protoplasts the nuclei were less distinct.

Regeneration of the cell wall and reversion of walled protoplasts to cells During the first 3 h of cultivation in liquid media the protoplasts remained osmotically sensitive. Only after 4--6 h the protoplasts became osmotically resistant, indicating t h a t a firm cell wall was formed on their surface. In this phase the cell wall was visible in the phase-contrast microscope (Plate 2). Protoplasts with regenerated cell walls were mostly spherical. The first revertants were found after 4--6 h of cultivation. T h e y arose from the walled protoplasts by polar growth as cylindrical cells which later divided by septation. More frequently, however, the walled protoplasts were first divided by septation into two compartments (Plate 3), often unequal in size, which then gave rise to cylindrical cells by polar growth (Plate 4). These revertants, identical in shape with the initial culture, divided further b y fission. Whereas in a wort medium osmotically stabilized with 0.4 M KC1 (Kopecks 1975) only about 10 % of the protoplasts regenerated their cell walls, in a wort medium osmotically stabilized with mannitol as m a n y as 50 ~/o of the protoplasts produced complete cell walls. Most of them reverted to cells. During cultivation in a thin liquid layer on agar surface the percentage of wall regeneration and reversion to cells slightly increased (to 65 %). The other protoplasts only grew and perished. In the N1 medium used for the regeneration of budding yests, about 50 ~ of the protoplasts regenerated their cell walls. The other protoplasts only grew and perished. However, only 15--20 ~ of the protoplasts produced septa and reversion to cells occurred in about 10 % of the protoplasts. During cultivation on the Difco mycological agar more t h a n 80 ~ protoplasts regenerated their cell wall, but both septation and reversion to cells were rare. By transferring the protoplasts with the regenerated cell walls from the mycological agar where regeneration proceeded for 6 h, to wort agar and after a subsequent 2--3-h cultivation it was possible to reach over 80 % of revertants.

Electron microscopy of cell wall regeneration No wall structures were isolated from the surfaces of fresh protoplasts. During the first hour of cultivation in liquid media thin fibrillar networks were formed on protoplast surfaces. They grew in number for another hour of incubation (Plate 5). The basic structural units of the nets were long parallel microfibrils arranged in flat bundles that were crossed and interwoven. After 2 h of incubation the interfibrillar spaces gradually became

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filled with short microfibrils and some amorphous material (Plate 6) a n d after 5 h of incubation protoplasts were covered with a complex surface structure consisting of microfibrils and amorphous matrix (Plate 7). In this culture even reverting protoplasts could be detected (Plate 8) from which typical cylindrical cells grew. Their smooth cell walls contrasted with t h e rough fibrillar surfaces of the reverting protoplasts. No differences in the ultrastructure of cell walls were found between protoplasts grown in different regenerating media (N1 medium, wort medium, mycological agar). DISCUSSION

Protoplasts of the fission yeast Schizosaccharomyces versatilis, prepared b y treatment with snail enzymes, can regenerate complete cell walls and revert to cells morphologically identical with those of the initial cell culture both in liquid media and on the surface of solid media. In this respect they resemble protoplasts of th fission yeast Schizosaccharomyces pombe (Svoboda 1967). However, in contrast with these and other yeast protoplasts so far studied (cf. NeSas 1971), the onset of wall regeneration occurred in Schizosaccharomyces versatilis protoplasts 2,--3 times faster, the first regenerated walls being produced by protoplasts as early as after 5 h of cultivation in liquid media. Quantitative differences in the composition of wall polymers m a y be one of the reasons for the faster wall regeneration in S. versatilis as compared with S. pombe. In S. pombe it is known t h a t about 80 % of the cell wall is formed b y insoluble polysaccharides, of which insoluble ~-glucan, which consists of branched ~-(1->3)-gluean and branched ~-(1-+6)-glucan (Bush et al. 1974; Manners et al. 1974), makes up about 50 ~o of the wall, while a b o u t 30 ~o of the wall is built from ~-(1->3)-glucan (Kreger 1954; Bush et al. 1974). In S. versatilis only the qualitative composition of polysaccharides is k n o w n - - t h e y contain galactomannan (Gorin and Spencer 1968), ~-(1-->3)-glucan (Kreger 1954; Bacon et al. 1968) and ~-glucan (Kreger 1954). The rate of synthesis of matrix polysaccharides, which is different in S. pombe and in S. versatilis, m a y be another factor playing a role in the regeneration of cell walls in protoplasts. Whereas no interfibrillar material can be found in the network of S. pombe protoplasts after a 2-h cultivation in liquid media (NeSas et al. 1968; Kreger and Kopecks 1978), the nets o f S. versatilis are distinctly masked b y the interfibrillar material after 2-h cultivation and after 5 h complete cell walls enabling reversion were formed. In S. pombe, osmotically resistant walls are formed as late as after 15 h a n d reversion occurs after about 20 h of incubation (NeSas et al. 1968). The reason for the higher rate of synthesis of some wall polymers (including the amorphous matrix) are not known. It can hardly be accounted for b y a faster supply of components for the biosynthesis of one polymer as compared with the others. Possible explanations m a y be that either biosynthesis of one protein (which is later glycosylated) is preferred to t h a t of the others or a competition of various nucleotide carriers for the substrate m a y be involved, or the rate of glycosylation m a y v a r y in different proteins, etc. Some evidence for the differences in polysaccharide composition of the cell walls between S. pombe and S. versatilis can also be derived from the

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fact that to obtain protoplasts S. versatilis cells had to be treated with 30 ~o Helix pomatia gastric juice solution (as reported in this paper), as compared with only 1.5 % solution used for S. pombe cells (Svoboda 1967). In the regenerating walls of S. pombe protoplasts two structural components-fibrils and amorphous matrix--could be distinguished (NeSas etal. 1968, and others). As shown in this study, in S. versatilis, in addition to long straight microfibrils arranged in flat bundles, short straight microfibrils were present. The chemical nature of the microfibrils was not studied. The long microfibrils seen in the regenerating walls of both S. pombe and Saccharomyces cerevisiae protoplasts were identified as ~-(1-->3)-glucan and the short microfibrils found in S. pombe protoplasts were proved to be ~-(1-->3)-glucan (Kreger and Kopeck~ 1973, 1976, 1978). The chemical nature of the amorphous component of regenerated cell walls is not known; galactomannan or amorphous glucan(s) might be involved. The protoplasts of S. versatilis can regenerate complete cell walls also in liquid media but the yield is 10--80 % depending on the type of cultivation medium. The significance of nutritional conditions for cell wall regeneration has been known since the first papers on yeast protoplast regeneration (Ne5as 1961). The N1 medium, which is relatively poor in nutrients, proved to be suitable for wall regeneration in budding yeast protoplasts. On the other hand, richer media of wort type support growth and thus ofter~ decrease the percentage of wall regeneration in protoplast populations. In our experiments a high yield of revertants in a liquid medium was achieved by combining cultivation on Difco mycological agar, a poor medium, with that in nutritionally rich wort medium. While the former medium facilitated the formation of rigid cell walls in most of the protoplasts, the latter medium provided conditions for reversion of walled protoplasts to cells. After a complete cell wall had been formed, a septum could be produced which divided the walled protoplast into two compartments. Septation in S. versatilis resembled that in S. pombe protoplasts (NeSas et al. 1968; Havelkovs 1969; Gabriel 1982), except that in the latter species it began much later. The mechanism of septation and reversion of the walled protoplasts appears to be identical in both organisms. REFERENCES BAco~ J.D.S., Jo~r~s D., FARMER V.C., WEBr,EY D.M.: The oceurence of ~-(1-->3)-glucan in Cryptoccocus, Schizosaccharomyces and Polyporus species and its hydrolysis b y a Streptomyces culture filtrate lysing cell walls of Cryptoccocus. Biochim.Biophys.Acta 158, 313 (1968). BUSH D.A., HORIS~E~GE~ M., HOR~A~ I., WU~SCH P.: The wall structure of Schizosaccharomyces pombe. J.Gen.Microbiol. 81, 199 (1974). GABRIEL M.: Relation of the karyokinesis to cytokinesis in regenerating and reverting protoplasts of Schizosaccharomyces versatilis, as studied by time-lapse microcinematography. (In Czech) Kvaen~ pr~mysl 28, 114 (1982). GoR~N P.A.J., SPEZ~CEe. J.F.T.: Galactomannans of Trichosporon fermc~tants and other yeasts: proton magnetic resonance and chemical studies. Can.J.Chem. 5~i, 299 (1968). HAVELKOV~ M.: Electron microucopy study of cell structures and their changes during growth and regeneration of Schizosacch(Iromyces pombe protoplasts. Folia J]/licrobiol. 1~, 155 (1969). HAVELKOV~- M.: Experimental inhibition of cell wall formation and of reversion in Nadsonia elongata and Schizosctcchctromyces pombe protoplasts. Protoplasmct 75, 405 (1972). KOPECrZ~-M.: The isolation of protoplasts of the fission yeast Schizoscwcharomyces by Trichoderma viride and snail enzymes. Folia Microbiol. 20, 273 (1975). KOPECK.~ ~i., ~TVRT:N'i(~EK O., NE~AS O.: Formation and properties of fibrillar network formed in yeast protoplasts as the first step of biosynthesis of cell wall, p. 73 in 1%. Miiller (Ed.): Symposium i~ber Hcfeprotoplasten. Akademie Verlag, Berlin 1967.

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KoPEcE]~ M., G,~BRIEL M.: Staining the nuclei in cells and protoplasts of living yeasts, moulds a n d green algae with the antibiotic lomofungin. Arch.Microbiol. 119, 305 (1978). K~z~o~ D.R.: Observations on cell walls of yeasts and some other fungi b y X - r a y diffraction and solubility tests. Biochim.Biophys.Acta 13, 1 (1954). ] K ~ o E ~ D.R., K o P ~ : c ~ M.: On the nature of the fibrillar nets formed by protoplasts of Saccharomyces cerevisiae in liquid media, p. 117 in J.R. Villanueva et al. (Eds.): Yeast, Mould and Plant Protoplasts. Academic Press, New Y o r k - - L o n d o n 1973. KlU~G~.~ D.R., KOPECK.~ 1~I.: On the nature a n d formation of the fibrlUar nets produced b y protoplasts of Saccharomyces cerevisiae in liquid media. An electronmicroseopic, X - r a y diffraction and chemical study. J.Gen.Microbiol. 9"2, 207 (1976). KR~.OER D.R., KOPECK~ M.: The nature of the nets produced b y protoplasts of Schizosaccharomyces pombe during the first stage of wall regeneration in liquid media. J.Gen.Microbiol. 1@8, 269 (1978). KELLETI T., SZABOLZI G., LENVAI A., GARZO T.: Untersuchungen fiber die lebensi~ahigen EiweisskSrper yon Saccharomyces cerevisiae. Die Regeneration in sterilen F i l t r a t yon zerst6rten Hefezellen. Acta Sci.Hung. 5, 213 (1954). MA~rN~EgSD.J., MAsseur A.J., PATTERSON J.C. : The heterogenity of glucan preparations from the walls of various yeasts. J.Gen.Microbiol. 80, 411 (1974). NE~AS O. : Physical conditions as i m p o r t a n t factors for the regeneration of n a k e d yeast protoplasts. Nature 192, 580 (1961). NE~AS O.: Cell wall synthesis in yeast protoplasts. Bacteriol.Rev. 35, 149 (1971). NE~AS O., SVOBOI)AA., HAV~.LXOVA,M. : Mechanism of regeneration of yeast protoplasts. V. Formation of the cell wall in Schizosaccharomyces pombe. Folia Biol. (Prague) 14, 80 (1968). SVOBOI)A A.: Regeneration of yeast protoplasts in agar gels. Exp.CcU Re~. 4~, 640 (19{}6). SVOBODA A.: Regeneration ability of protoplasts of different yeast species, p. 31 in R. Mfiller (Ed.): Symposium i~ber Hefeprotoplasten. Akademie Verlag, Berlin 1967. The plates will be found at the end of the issue.

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PLATE 1. F r e s h p r o t o p l a s t s of S. versatilis w a s h e d w i t h t h e o s m o t i c stabilizer a n d inoculat e d in t h e n u t r i e n t m e d i u m . P h a s e c o n t r a s t . PLATE 2. P r o t o p l a s t r e g e n e r a t i n g 4.5 h on t h e Mycological a g a r h a v e a solid cell wall t h a t does n o t lyze in a h y p o t o n i c m e d i u m . P h a s e c o n t r a s t . PLATE 3. P r o t o p l a s t s r e g e n e r a t e d in t h e N1 m e d i u m . P h a s e c o n t r a s t . PLAT]: 4. P r o t o p l a s t s r e g e n e r a t i n g a n d r e v e r t i n g in t h e w o r t h m e d i u m . P h a s e c o n t r a s t .

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P L A T E 5. P a r t i a l l y filled fibrilar n e t w o r k after a 2-h c u l t i v a t i o n of t h e p r o t o p l a s t s in t h e liquid medium. PLATE 6. I d e n t i c a l s t r u c t u r e s a t a h i g h e r m a g n i f i c a t i o n . S h o r t fibrils a n d piles of a m o r p h o u s filling m a t e r i a l c a n well be seen in m e s h e s of t h e n e t w o r k .

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PLATE 7. D~tail of the regenerated cell wall of the protoplast showing prominent bundles of mierofibrils, densely filled with other wall components. PLATE 8. Reversion of the protoplast to the cell.