JOURNAL OF BACTERIOLOGY, May 1969, p. 797-810 Vol. 98, No. 2Copyright © 1969 American Society for Microbiology Printed in U.S.A.

Kinetic and Morphological Observations onSaccharomyces cerevisiae During

Spheroplast FormationSVEN DARLING, J0RGEN THEILADE, AND AKSEL BIRCH-ANDERSEN

Departments of Biochemistry and Electron Microscopy, Royal Dental College, DK-8000 Aarhus C,Denmark and Department of Biophysics, Statens Seruminstitut, DK-2300 Copenhagen S, Denmark

Received for publication 29 January 1969

A strain of Saccharomyces cerevisiae which produced elongated cells under ourgrowth conditions was investigated. By digestion of the cell walls with snail enzyme,the cells became spheroplasts after a transient state which we termed "prosphero-plast." The prospheroplast could be lysed like the spheroplast, but it retained theshape of the original yeast cell if osmotically protected. Prospheroplasts and sphero-plasts were prepared, and thin sections of samples taken throughout the process ofwall removal were studied in the electron microscope, at regular intervals up to thetime of complete conversion to spheroplasts. In addition, cell wall remnants re-covered from spheroplast preparations were shadow cast for electron microscopy.This material revealed structures resembling bud scars with attached membranousmatter. The kinetic studies showed that after a certain period of time all cells weretransformed into prospheroplasts, whereas spheroplast formation started later,depending on the enzyme concentration. In sections, the prospheroplasts appearedto be formed by detachment of the cell walls. Both the prospheroplasts and thespheroplasts showed asymmetric cytoplasmic membranes in which the outer leafletsappeared coated with a dense fibrillar layer. The experiments suggest that, afterenzyme digestion, the cytoplasmic membrane retains a coating which is rigid in theprospheroplast but which loses rigidity when the cell is transformed into a sphero-plast.

The formation of protoplasts from yeasts hasbeen studied by several authors. Observations onprotoplast formation from a strain of Saccharo-myces carlsbergensis, by digestion of the cell wallwith snail enzyme, were reported by Eddy andWilliamson (8). The cells in this strain areelongated, and it was observed, on examinationunder phase contrast, that at an early stage of di-gestion the protoplast retracted from one or bothpoles of the cell. Later the protoplast was ex-truded through a hole in the cell wall, and thishole was frequently found at the site of a budscar. The protoplasts derived from these cellsimmediatelyattaineda sphericalform. Holter andOttolenghi (11) studied protoplast formationfrom strains of S. carlsbergensis and Schizo-saccharomyces pombe and made similar observa-tions. Svihla, Schlenk, and Dainko (24) studieda strain of Candida utilis, the cells of which areoval, and observed the same stages in protoplastformation as those described by all of the previ-ously mentioned authors. They noticed, however,

that extrusion of the protoplasts occurred in theequatorial zone, with a frequency almost equalto that at the poles where bud scars normally arefound. Mendoza and Villanueva (15) studied theformation of protoplasts from C. utilis by use oflytic enzymes from Streptomyces GM or snailenzyme. They found that the cell wall seems to beattacked most readily in the equatorial zone wherethe protoplast emerged from the elongated cell.The liberation of protoplasts has very rarely beenobserved at the poles where buds normally arefound (26). The same holds for Saccharomycesfragilis studied by Muller (17). Liberation ofprotoplasts from cells of Endomyces magnusiitreated with snail enzyme took place at one of thepoles, and immediately the protoplast attained thespherical shape (28).A protoplast, by definition, retains no rem-

nants of the original cell wall (5, 25), otherwiseit should be called a spheroplast (23). Whetherthe spherical entities liberated from yeast cellscould be considered real protoplasts was ex-

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amined by Ottolenghi (18); in many cases, hefound that so-called protoplasts obtained from astrain of Sacccharomyces cerevisiae, when trans-ferred to a suitable hypotonic solution, could swellwithout bursting. By the sudden change in size, avery thin membrane which apparently had sur-rounded the cell was now shed in the suspendingmedium. The membrane, to which annular struc-tures similar to bud scars were attached, wasvisible under phase contrast.

In this paper, preparations of the type describedby Ottolenghi (18) were studied in the electronmicroscope. Our results indicate that such ma-terial retained on spheroplasts, formed during anextended period of snail enzyme digestion, con-stitutes the cell wall remnants, and throughoutthe present paper we use this term only for thistype of material.The formation of spheroplasts from a strain of

S. cerevisiae, the cells of which are elongatedunder our conditions of culture, was also studied.The cells form spheroplasts by digestion with snailenzyme through a transient state we call a pro-spheroplast. Thus, we have reserved the termspheroplast for spherical and osmotically sensi-tive cells; the term prospheroplast is used forosmotically sensitive cells from which the cell wallis detached without simultaneous loss of theoriginal shape of the cell.

Experiments designed to elucidate the kineticsof prospheroplast and spheroplast formation,together with ultrastructural investigations ofdifferent stages in this process, are reported.

MATERIALS AND METHODSOrganism and culture conditions. S. cerevisiae

Hansen no. 983 was obtained from the CarlsbergLaboratory, Copenhagen, Denmark. The yeast wasmaintained on agar slopes containing, per liter ofdeionized water: KH2PO4, 2.0 g; MgSO4*7H20, 1.0 g;(NH4)2SO4, 1.0 g; glucose, 20 g; Difco yeast extract,3.0 g; Difco Neopeptone, 3.5 g; Difco Special Agar(Noble), 30 g. The yeast was grown for 18 hr at 30 Cin test tubes containing 10 ml of the same mediumwithout agar. This culture was used as the inoculumfor production of yeast cells.The cultures used in the experiments were grown in

1,000-ml triple baffled shake flasks with stainless-steel caps (Bellco Glass Inc., Vineland, N.J.). Eachflask contained 250 ml of medium, having the follow-ing composition per liter of deionized water: KH2PO4,0.2 g; (NH4)2HP04, 3.50 g; MgSO4-7H20, 0.25 g;sodium citrate (NA3C6H507 *53%H20), 1.0 g; yeastextract (Difco), 3.0 g; glucose, 1.0 g; sodium lactate(70 to 72%, w/w), 10 ml. The pH was adjusted to 5.0with 3 M H3PO4 before autoclaving at 120 C for 20min. The flasks were inoculated with 0.5 ml of the cul-ture described above and were shaken for 18 hr at 30 Con a rotatory shaker (125 rev/min).The yeast was harvested by centrifugation at 4 C in a

Servall RC2 centrifuge with an SS-34 rotor and wassubsequently washed three times in the centrifuge tubewith 0.6 M KCI (3,000 X g for 10 min). After thethird washing, the yeast was spun down at 35,000 X gfor 30 min. The yield was approximately 6 g (wetweight) per liter of medium.

The growth medium described was selected fromamong several media having different carbon sources.Good yields were obtained with glucose in differentconcentrations as the sole carbon source. However, thecells formed sheroplasts less readily, and the sphero-plasts were rather unstable in the digestion medium.With lactate as the carbon source, the yield variedconsiderably, but the cells were highly susceptible todigestion and formed stable spheroplasts. Addition ofa small amount of glucose to this medium secured aconstant yield of yeast and had no influence on theability to form stable spheroplasts.

Preparation of spheroplasts. The cell wall was di-gested at 30 C (unless otherwise stated) with a snailenzyme ("Suc digestif d'Helix pomatia stabilise", lotno. 7800, Nov. 3, 1966) obtained from l'IndustrieBiologique Francaise, Paris, France. Before use, theenzyme was diluted with an equal volume of 1.2 MKCI containing 1 mg of cysteine hydrochloride per ml(14). The yeast was preincubated with ,1-mercapto-ethanol (6, 7) for 30 min at 30 C at pH 7.5 in a buffercontaining 0.05 M tris(hydroxymethyl)aminomethane(Tris), 0.60 M KCI, 0.03 M f3-mercaptoethanol, and0.4 mm ethylenediaminetetraacetic acid (EDTA). A2-ml amount of buffer per g (wet weight) of yeast wasused. To this amount of yeast, 0.8 ml of enzyme solu-tion was added. The suspension, containing approxi-mately 3 X I01 cells per ml, was shaken gently on arotatory shaker during preincubation and digestion.After digestion for 1 hr, the conversion of the yeastcells into spheroplasts was completed. Based on cellcounts the yield was 90 to 100% and remained un-changed after 2 additional hr of digestion. Finally,the spheroplasts were spun down and washed threetimes with 0.6 M KCI in the centrifuge (1,500 X g for5 min).

Preparation of prospheroplasts. The method is es-sentially the same as for the preparation of sphero-plasts, except that the amount of enzyme added wasreduced to 0.2 ml per g of yeast. Under these condi-tions, osmotically resistant cells disappeared after30 min, and the cells were transformed into pros-pheroplasts and a few per cent of spheroplasts. In thenext period (60 to 75 min), only a slight increase inthe number of spheroplasts occurred (at maximum10%). The prospheroplasts were harvested in thesame way as the spheroplasts.

Cell counts. The progress of spheroplast formationwas followed by counting in a hemocytometer. Thetotal number of cells (NT) and the number of sphero-plasts (Nsp) were counted in a sample of the digestionmixture diluted 400 times with 0.6 M KCI. The num-ber of intact yeast cells (NY) was determined in asample diluted 400 times with water. The number ofprospheroplasts (Npsp) was calculated from the equa-tion: Npsp = NT-NY- Nsp Well-developed budswere counted as individual cells. Ghosts were notcounted.

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Isolation of cell wall remnants from spheroplasts.The method used for preparation of membranous ma-

terial from yeast spheroplasts is a modification ofthat described by Ottolenghi (18). Spheroplasts pre-pared from 10 g of yeast were suspended in 100 mlof 8 M urea and left overnight at room temperature.The suspension was then diluted with 100 ml ofwater and centrifuged at 35,000 X g for 20 min. Theprecipitate was washed with 40 ml of water andcentrifuged. The sediment was then treated with 40 mlof urea for 20 min and washed twice with 40 ml ofwater (35,000 X g for 20 min). Subsequently, thesediment was extracted with 40 ml of 0.1 M potassiumacetate in absolute ethyl alcohol and centrifuged at17,000 X g for 10 min. This extraction procedure wasrepeated. The remaining material was now washedthree times with 40 ml of water with subsequentcentrifugations (3,000 X g for 10 min), suspended in10 ml of 3% NaOH, and heated in a boiling-waterbath for 3 hr. After this treatment, the preparationwas centrifuged and washed three times with water(3,000 X g for 10 min). From this material, suspen-sions in redistilled water were used for electronmicroscopy.

Electron microscopic technique. The cells were sus-pended in 0.6 M KCl to yield a final count of 3 X 108to 5 X 108 cells per ml. To 10-ml samples of this sus-pension, we added 1 ml of 3% glutaraldehyde inVeronal acetate buffer, pH 6.1 (21), in which thecells were prefixed for 10 min. Subsequently, the sam-ples were centrifuged for 10 min at 5,000 X g; thecells were suspended in 10 ml of 3% glutaraldehydein Veronal acetate buffer (pH 6.1) and fixed for 1 hrat room temperature. After centrifugation, the pelletwas embedded in warm (45 C) melted agar (1.5%agar in Veronal acetate buffer, pH 6.1), and smallagar blocks with the cells were fixed overnight at roomtemperature in 1% OS04 in the same acetate bufferby the method of Ryter and Kellenberger (20).Finally, the blocks were dehydrated in increasingconcentrations of acetone and embedded in VestopalW (M. Jaeger, Vesenaz, Geneva, Switzerland).

Thin sections were prepared on an LKB ultrotomeI microtome with glass knives and were counter-stained with uranyl magnesium acetate (9) and leadcitrate (19).

The cell wall remnants prepared from spheroplastswere shadow cast with palladium or with a platinum-palladium alloy at an angle of 18 degrees.

Electron microscopy was carried out with aPhilips EM 200 electron microscope. Negatives wereobtained at primary magnifications of 1,400, 2,900,and 9,500 times, and prints were made by photo-graphic enlargement as desired. Approximately 650recordings were studied.

RESULTS

Formation of prospheroplasts and spheroplasts.The formation of prospheroplasts and sphero-plasts as related to time is shown in Fig. 1. Twophases in spher.oplast formation are easily recog-nized. In the first phase, the yeast cells weretransformed into prospheroplasts and a few per

cent of spheroplasts (Fig. 1 at 30 to 45 min). Inthe second phase, the prospheroplasts wererapidly transformed into spheroplasts (Fig. 1 at160 to 200 min). Spheroplast formation with dif-ferent amounts of enzyme, 0.8, 0.4, and 0.2 mlper g of yeast, is shown in Fig. 2. In these threeexperiments, osmotically resistant cells disap-peared after 10, 15, and 40 min, respectively, andonly prospheroplasts and spheroplasts were thenpresent. The length of the first phase in sphero-plast formation varied with the amount of en-zyme in the digestion mixture. The rate of the

-80-

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0 30 60 120 150 180 210 Mm.

FIG. 1. Percentage of osmotically sensitive cells(calculatedfrom cell counts at time zero) plotted againsttime. The s olid line represents the percentage of os-

motically sensitive spheroplasts, and the dotted linerepresents the percentage of prospheroplasts. Thiscurve was obtained by calculation with the equationgiven in the text. Two phases of spheroplast formationare recognized. During the first phase, the yeast cellsare transformed into prospheroplasts and afew per centofspheroplasts. During the secondphase, the prosphero-plasts are rapidly transformed into spheroplasts. Thearrow indicates the time at which osmotically resistantcells were no longer present.

10

0 61

c

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0 30 60 90 120 Min.

FIG. 2. Percentage of osmotically sensitive sphero-plasts, calculatedfrom cell counts at time zero, plottedagainst time in minutes in three experiments withvarious enzyme concentrations. In curves A, B, and C,the enzyme concentrations were 0.8, 0.4, and 0.2 ml perg of yeast, respectively. Although the first phase of

spheroplas formation varies with the amount ofenzymethe rate of transformation of prospheroplasts intospheroplasts appears to be independent of the enzymeconcentration.

10 A B C

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transformation of prospheroplasts into sphero-plasts, however, appeared to be independent ofthe enzyme concentration. This was confirmed inother experiments. The rate of transformationexpressed in percentage per minute was, however,found to vary considerably (3 to 6% per min).

Stability of prospheroplasts. This experimentwas performed at 25 C. The formation of pro-spheroplasts and spheroplasts was followed andsamples were withdrawn at suitable intervals. Atthe same time, samples were examined in theelectron microscope to verify that the cell wallwas detached.The cells were harvested by centrifugation and

washed three times with 0.6 M KCI. Then the cellswere suspended in 0.6 M KCl, and the volume wasadjusted to that of the sample. The suspensionwas shaken gently, and the transformation ofprospheroplasts into spheroplasts was followed(Fig. 3). The values correspond to the number ofspheroplasts expressed in percentage of the origi-nal number of yeast cells. The value at zero-timecorresponds to that of the sample at the time ofwithdrawal. Curve A shows the stability of pro-spheroplasts formed after treatment of yeast cellswith snail enzyme for 60 min, and curve B showsthe stability of those formed after 140 min ofdigestion. Similar experiments were performedwith 0.8 M KCI, 0.05 M Tris buffer (pH 7.5) with0.6 M KCI and 0.4 mm EDTA, or with Tris-KClbuffer containing 0.01 M magnesium sulfate. Theresults were essentially the same as those obtainedwith 0.6 M KCI. Thus, the stability of prosphero-plasts is dependent on the time of digestion. Thelater they are harvested, the greater is the pro-portion which is transformed into spheroplasts.

60 BB

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In

O 1 2 3 HRSFIG. 3. Stability of the prospheroplasts in the experi-

ment shown in Fig. I after 60 min (curve A) and 140min (curveB) of entzyme digestioni. The percentage ofspheroplasts of the original number of yeast cells isplotted against time after withdrawal of the sample. Thevalue at zero-time corresponds to that of the sample atthe time of withdrawal, when the cells were washed. Thestability ofprospheroplasts is dependent on the durationof digestion.

Light microscopic observations. The two phasesin spheroplast formation were observed underphase contrast and are shown diagrammaticallyin Fig. 4. The cell wall loosened from the pro-spheroplast at an early stage. Then the prosphero-plast was extruded through one end of the partlydigested cell wall (Fig. 4, 1-4), retaining its shapedespite the apparent loss of a supporting wall.Occasionally, the extrusion did not take place,but the cell wall loosened and dissolved gradually(Fig. 7). In the second phase, the prospheroplastwas transformed into a spheroplast through inter-mediate steps (Fig. 4, 6a, and 6b). Conversion tothe spherical form did not necessarily involve thewhole surface at once (Fig. 4, 6b). The direct for-mation (Fig. 1, 4) was rarely observed, except indigestion mixtures with a high enzyme concen-tration.

Electron microscopic observations. In general,our observations on the ultrastructure of the un-treated cells of S. cerevisiae confirmed those ofprevious reports (13, 27). The cytoplasm of thecell (Fig. 5 and 6a) was surrounded by a triple-layered cytoplasmic membrane showing many in-vaginations. The cytoplasm was densly packedwith ribosomes. The nucleus was usually more orless centrally located and often exhibited an ir-regular outline (Fig. 5). Mitochondria wereobserved peripherally in the cytoplasm and hadthe well-established internal organization ofcristae, with a matrix of varying density in be-tween. A great number of the sectioned cells re-vealed membrane-bound vacuoles of varioussizes (Fig. 5 and 6a), often containing some in-

1 3

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FIG. 4. Schematic illustration of the two phases insperoplast formation as observed in the light micro-scope. (1) The intact yeast cell with a bud scar. (2)Partial detachment of the cell wall. (3) Extrusion of theprospheroplast from the cell wall. (4) The prosphero-plast. (5a) The prospheroplast is losing its elongatedshape and is transformed into a spheroplast (6). Con-version to the sphericalform does not necessarily involvethe whole surface at once (5b). The direct formation ofthe spheroplast from the intact cell (la to 6) was rarelyobserved.

l la

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I

_w_ -_-w.,_c;_

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FIG. 5. Electron micrograph of sectioned untreated yeast cells. Each cell is surrounded by a cell wall (cw) ofrather even thickness. The elongated cell form is apparent in longitudinally sectioned cells. In the cytoplasm, thenuclei (n) are distinguishable as are various organelles and vesicular inclusions. The bar in this and all of the follow-ing electron micrographs represents I jum, unless otherwise stated. X 7,000.

clusions of more or less digested cytoplasmicmaterial. Many membranous profiles werescattered in the cytoplasm. They were particu-larly prevalent adjacent to the surrounding cyto-plasmic membrane (Fig. 6a), where they oftendisplaced the ribosomes. Several layers of thesemembranes, arranged parallel to the cytoplasmicmembrane, were frequently noted.The cell wall was observed to be rather even in

thickness except for the areas of bud scars. It ap-peared to consist of three zones of differentelectron densities, with the middle portionexhibiting the lowest electron density (Fig. 5

and 6a). The inner boundary of the cell wallappeared to be in intimate contact with the cyto-plasmic membrane, which it followed into allindentations. The cell wall and the cytoplasmicmembrane of cells pretreated with mercapto-ethanol did not differ in ultrastructure from thesestructures in untreated cells (Fig. 6b); the samewas true for the nucleus and the cytoplasmiccontent.We observed some changes in the cell walls of

cells fixed for sectioning immediately after theaddition of snail enzyme. It must be remembered,however, that the centrifugation procedure for

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inv cm.

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FIG. 6. (a) Part of an untreated yeast cell. The cell wall (cw) appears to consist of three zones of differentelectron densities. The inner boundary of the cell wall seems to be in intimate contact with the cytoplasmic membrane(cm). The cytoplasm is surrounded by a triple-layered cytoplasmic membrane showing many invaginations (inv).Membranous profiles are seen adjacent to the cytoplasmic membrane in the cytoplasm (arrows), which is denselypacked with ribosomes. Part ofa large membrane-bound vacuole (v) is seen. X 118,000. (b) Yeast cell treated withmercaptoethanol. No distinct differences are noted as compared with the untreated cell (Fig. 5a). Mitochondria(m) are seen in the cytoplasm. X 104,000. (c) Yeast cell fixed after the addition of snail enzyme (time zero). Thecell wall (cw) is partly detached (arrow) from the cytoplasmic membrane (cm). This is probably due to enzymaticaction during the 20 mim before the cell was washed. The differentiation of the cell wall into three zones is lessobvious. The cell wall material in the upper left corner isfrom an adjacent cell. X 112,000.

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washing out the enzyme took approximately 20min and, consequently, some effect of the enzymewas to be expected. The cell wall was partly de-tached from the cytoplasmic membrane of thecell (Fig. 6c). Furthermore, the differentiation ofthe cell wall into zones became less obvious as themost intermediate electron-lucent zone acquiredapproximately the same density as the adjacentzones (Fig. 6c).

Prospheroplasts were examined at varioustimes (20, 40, 60, 80, and 100 min) during theenzymatic digestion of the yeast cells at 30 C. Asall cells in the culture were not in the samephysiological state, the individual cells exhibitedvarious degrees of change after enzymatic treat-ment for a certain period of time. Certain altera-tions, however, seemed to be definitely related tothe length of enzymatic digestion. In Fig. 7, someprospheroplasts obtained by treating yeast cellswith snail enzyme for 20 min are shown as atypical example of cells after this length of treat-ment. The cells have lost most of their walls.Partly digested cell wall material was occasionallyretained in localized regions, most frequently inrelation to the bud scars. Apart from this theyappeared unaffected, even retaining their elon-gated shape (Fig. 5). The cell wall was detachedfrom the cytoplasmic membrane, and often awide gap was observed between this and theloosened cell wall (Fig. 7). Several extrusions ofmembrane-bound cytoplasm were frequentlyfound in the gap between the cell and the detachedcell wall (Fig. 7 and 8). It was generally observedthat the cytoplasmic membrane was asymmetric,the outer leaflet of the membrane being moredense as if coated with a dense fibrillar layer(Fig. 8). After further digestion, more cell wallmaterial was removed, but the asymmetric ap-pearance of the cytoplasmic membrane was stillobserved (Fig. 8). This figure also shows that thecytoplasmic extrusions still persisted.

Spheroplasts appeared devoid of cell wall ma-terial (Fig. 9), and the limiting cytoplasmicmembrane retained its undulating course andasymmetric appearance (Fig. 10). The numerousindentations of the cytoplasmic membraneshowed up particularly well in tangential sectionsof the spheroplast (Fig. 10).The shadowed preparations of the cell wall

remnants from spheroplasts (Fig. 11) revealedstructures resembling bud scars in form and size.The bud scars were seen in clusters held togetherby a thin layer of granular material. This granularlayer was also present in the central regions of thebud scars in preparations from spheroplaststreated for 60 min with snail enzyme (Fig. 11).In preparations of spheroplasts from cells treatedfor 120 min, some of the granular material could

still be found within the bud scars (Fig. 11),whereas it was almost absent in bud scars fromcells treated for 180 min (Fig. 11).

DISCUSSIONUnder the culture conditions employed, the

yeast strain used for the present investigationhad elongated cells. This made it possible to dif-ferentiate between two phases during spheroplastformation, which occurs when the intact yeastcells are subjected to enzymatic digestion of theircell walls. During the first phase, the cells losttheir cell wall, but retained their characteristicelongated form if they are osmotically protected.We term the cells in this phase prospheroplasts.During the second phase, the cells rapidly attaineda spherical form and were thus transformed intospheroplasts. Attempts were therefore made tocorrelate the kinetics with the ultrastructure ofthe cells after various periods of time during theformation of the spheroplasts.Our observations on the ultrastructure of intact

cells of S. cerevisiae generally conform with thoseof other investigators examining thin sections ofthese cells in the electron microscope (1, 2, 10,13, 27).With respect to the cell wall, as seen in thin

sections, our observations are in agreement withthose of Agar and Douglas (1) and of Vitols,North, and Linnane (27). There is no evidenceeither from these investigators or from our studiesthat the cell wall consists of two separate "mem-branes," as was claimed by Bartholomew andLevin (4). The cell wall of intact yeast has threezones of different electron densities, with the in-termediate zone having a lower electron densitythan the outer and inner zones (Fig. 6).

Furthermore, our results are in accordancewith the observations reported by investigatorsusing the freeze-etching technique for the studyof the ultrastructure of yeast cells (16, 22). Theundulating course of the cytoplasmic membrane,as described by these authors, is also evident inour electron micrographs of thin sections.The ultrastructure of the spheroplasts reveals

no apparent difference from that of. the intactyeast cell except for the loss of shape and cellwall. Our observations on the cytoplasmic mem-brane of the spheroplasts demonstrate the cleft-like invaginations (Fig. 10a and b) that havebeen observed in frozen-etched preparations ofintact yeast cells and spheroplasts (16, 22). Theasymmetry of the cytoplasmic membrane asobserved in intact cells as well as in prosphero-plasts and spheroplasts, apparently produced bya fibrillar layer attached to the outer layer of thecytoplasmic membrane, may well represent thefine fibrils described by Streiblova (22). Accord-

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cw

q%

-Cw

FIG. 7. Prospheroplasts produced by treating yeast cells with snail enzyme for 20 min before washing. Cellwall material (cw) is left in certain regions. The cell wall is generally detachedfrom the cells, as illustrated in thecell in the upper right corner. Abbreviations: n, nuclei; m, mitochondria; v, vacuoles; ce, cytoplasmic extrusions.X 18,000.

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a -QJ4

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FIG. 8. (a) Part of a prospheroplast obtained after 20 min of enzyme digestion. Almost completely digested cellwall material (cw) canl be distingutished. Tlte triple-layered cytoplasmic membrane (cm) can be distinguished. Thetriple-layered cytoplasmic membrane (cm) is asymmetric, the outer leaflet of the membrane being more dense asif coated with a fibrillar layer. The cytoplasm is densely packed with ribosomes except for the zone adjacent to thecytoplasmic membrane where membra1nouis structuires predominate, often arranged as mesosomes (me). X 105,000.(b) Part of a prospheroplast obtained after 40 miii of enzynme cligestion. Almost completely digested cell wallmaterial (cw) cani be seen. In the gap between the cell anid the cell wall, cytoplasmic extrusions (ce) are found.These are also limited by an asymmetric cytoplasmic membrane and contain ribosomes. X 105,000.

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ce

FIG. 9. Spheroplasts obtained after enzymatic treatment for 60 min. (The cells are slightly oval due to compres-sion produced during sectioning.) No cell wall material is seen. Several cytoplasmic extrusions (ce) are present.x 10,000.

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0.5u

cm-

b 'v 'i r>; nFIG. 10. (a) Spheroplast obtained after enzymatic digestion for 120 min. (The cell is slightly oval due to com-

pression during sectioning.) The cytoplasmic membrane (cm) has retained its asymmetric appearance and has anundulating course with small invaginations (arrows). The cytoplasm is packed with ribosomes except for the regionadjacent to the cytoplasmic membrane, in which zone membranous structures running parallel to the cytoplasmicmembrane dominate. Abbreviations: n, nucleus; m, mitochondria; v, vacuoles. X 51,000. (b) Tangential sectionofa spheroplast obtained after enzymatic digestion for 120 min, demonstrating the cleft-like invaginations (arrows)of the cytoplasmic membrane (cm) into the cytoplasm. The asymmetric appearance of the cytoplasmic membrane isevident. X 65,000.

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L

FIG. 11. (a) Shadowed preparation of cell wall remnants from spheroplasts obtained after 60 min of enzymedigestion. Ringlike structures resembling bud scars are seen. The cluster of bud scars is held together by a thinlayer of granular material, which is also present in the central region of the bud scar. X 19,000. (b) Shadowedpreparation of cell wall remnants from spheroplasts obtained after 120 miii of enzyme digestion. Some of the budscars show less granular material in the central region. X 19,000. (c) Shadowed preparation of cell wall remnantsfrom spheroplasts obtained after 180 min ofenzyme digestion. In this preparation, almost all bud scars are devoid ofgranular material in the central region. X 18,000.

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ing to Streiblovai (22), these fibrils, representingremnants of the innermost cell wall layer, con-

nect the cell wall to the cytoplasmic membrane.These fibrils were found in intact cells as well as

in spheroplasts (22).We attempted to find a possible difference in

ultrastructure between prospheroplasts andspheroplasts by examining samples of cells con-sisting mainly of prospheroplasts and othersamples containing spheroplasts only. However,no distinct differences were found which couldexplain why the prospheroplasts retain theirelongated form. It was characteristic of prospher-oplasts, as well as of spheroplasts, that the outerleaflet of the cytoplasmic membrane was thick-ened as if a fibrillar layer was attached to it.From a protoplast preparation of S. cerevisiae,

Ottolenghi (18) isolated ringlike structures at-tached to membranous matter visible in phase-contrast microscopy. With essentially the sameisolation method as Ottolenghi (18), we were

able to demonstrate, in shadow cast preparations,ringlike structures resembling bud scars to whicha membranous material was attached (Fig. 11).It is most likely that the cell wall remnants il-lustrated in our shadowed preparations fromspheroplasts are part of the innermost cell walllayer described by Streiblova (22). Moreover,this material seemed to be morphologicallyidentical to that isolated by Bacon et al. (3) andby Houwink and Kreger (12) from intact cellsof S. cerevisiae. In addition these authors em-

ployed physical and chemical techniques toanalyze the composition of this material andfound it to consist primarily of chitin. Pre-liminary results of chemical analysis on our

cell wall remnants prepared from spheroplastsessentially support the chemical findings of Baconet al. (3).

Additional experiments in our laboratoryyielded the bud scars and membranous materialdescribed by Ottolenghi (18) when the followingpreparation technique was used. Spheroplastswere treated with a hypertonic solution of potas-sium chloride and were centrifuged in a suitablegradient. Cell wall remnants with the charac-teristic bud scars were obtained in the super-natant fluid. Under the optical microscope we

were able to follow the shedding of the budscars. This seems to indicate that the cell wallremnants which are still present on the sphero-plasts form a rather rigid structure, which isdetached when the cytoplasm of the spheroplastis shrunk in the hypertonic medium.To elucidate the kinetics of the formation of

spheroplasts from intact yeast cells through theintermediate prospheroplast stage, the stabilityof the prospheroplasts was determined early in

the prospheroplast stage and towards the endof this period (Fig. 1). The stability of theprospheroplasts in a 0.6 M potassium chloridesolution or in Tris-KCI buffer with and withoutthe addition of magnesium ions decreased duringthis transformation period. The same changesoccurred in a 0.8 M potassium chloride solution.Therefore, it is unlikely that the transformationfrom the prospheroplast into the spheroplast isdue to an osmotic effect of the medium. It ismore likely, however, that the enzymatic actionon the remaining cell wall material of the pro-spheroplast takes place gradually during the wholeprospheroplast stage, although the obviousvisible result, the actual formation of the roundspheroplast, occurs quickly. Apparently, thechanges in the cell wall material resulting in thetransformation from prospheroplast to sphero-plast must be very subtle, since no differences inthe ultrastructure of the two types of cells couldbe demonstrated.The stability tests also showed that the forma-

tion of spheroplasts from prospheroplasts isindependent of the presence of the enzyme, sincethe transformation also occurs after intensivewashing. The experiments on the effect of theenzyme concentration also seemed to supportthe contention that the second phase of sphero-plast formation is independent of the presence ofthe enzyme, as the spheroplast formation ratedid not change significantly when the enzymeconcentration was altered (Fig. 3). The lengthof the prospheroplast phase, however, is depend-ent on the enzyme concentration, which is indica-tive of an action of the enzyme during the fullprospheroplast period. This suggests that theenzyme is acting on the cell wall material of theprospheroplast without morphologically chang-ing the structures responsible for the rigidity ofthe cell up to a certain point. At this point, thecell wall material has been weakened to such anextent that it loses its rigidity very abruptly, andthe spherical form of the cell is attained. Furtherstudies are under way to elucidate the reactionsinvolved in the transformation process fromprospheroplast to spheroplast, with the aim ofdemonstrating the morphological structures re-sponsible for the rigidity of the enveloping ma-terial of the prospheroplast.

ACKNOWLEDGMENTS

This investigation was supported by a grant from the DanishState Research Foundation.We thank Inge Simonsen, Department of Biochemistry, and

Inge Jochimsen, Department of Electron Microscopy, for skillfulltechnical assistance. The valuable criticism and suggestions offeredduring the preparation of the final manuscript by R. G. E. Murray,Department of Bacteriology, University of Western Ontario,London, Ontario, Canada, are gratefully acknowledged.

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LITERATURE CITED

l. Agar, H. D., and H. C. Douglas. 1955. Studies of budding andcell wall structure of yeast. Electron microscopy of thinsections. J. Bacteriol. 70:427-434.

2. Agar, H. D., and H. C. Douglas. 1957. Studies on the cyto-log:cal structure of yeast: electron microscopy of thinsections. J. Bacteriol. 73:365-375.

3. Bacon, J. S. D., D. Davidson, D. Jones, and I. F. Taylor.1966. The location of chitin in the yeast cell wall. Biochem.J. 101:36c-38c.

4. Bartholomew, J. W., and R. Levin. 1955. The structure ofSaccharomyces carlsbergensis and S. cerevisiae as deter-mined by ultra-thin sectioning methods in electron micros-copy. J. Gen. Microbiol. 12:473-477.

5. Brenner, S., F. A. Dark, P. Gerhardt, M. H. Jeynes. 0.

Kandler, E. Kellenberger, E. Klieneberger-Nobel, K.McQuillen, M. Rubio-Huertos, M. R. J. Salton, R. E.Strange, J. Tomcsik, and C. Weibull. 1958. Bacterialprotoplasts. Nature (London) 181:1713-1715.

6. Davies, R., and P. A. Elvin. 1964. The effect of P-mercapto-ethanol on release of invertase and formation of proto-

plasts of Saccharomyces fragilis. Biochem. J. 93:8p-9p.7. Duell, E. A., S. Inoue, and M. F. Utter. 1964. Isolation and

properties of intact mitochondria from spheroplasts ofyeast. J. Bacteriol. 88:1762-1773.

8. Eddy, A. A., and D. H. Williamson. 1957. A method ofisolating protoplasts from yeast. Nature (London) 179:1252-1253.

9. Frasca, J. M., and V. R. Parks. 1965. A routine technique fordouble-staining ultrathin sections using uranyl and leadsalts. J. Cell Biol. 25:157-161.

10. Hashimoto, T., S. F. Conti, and H. B. Naylor. 1959. Studiesof the fine structure of microorganisms. IV. Observationson budding Saccharomyces cerevisiae by light and electronmicroscopy. J. Bacteriol. 77:344-354.

11. Holter, H., and P. Ottolenghi. 1960. Observations on yeastprotoplasts. C. R. Trav. Lab. Carlsberg 31:409-422.

12. Houwink, A. L., and D. R. Kreger. 1953. Observations on thecell wall of yeasts. An electron microscope and X-raydiffraction study. Antonie van Leewenhoek J. Microbiol.Serol. 19:1-24.

13. Linnane, A. W., E. Vitols, and P. G. Nowland. 1962. Studieson the origin of yeast mitochondria. J. Cell Biol. 13:345-350.

14. Marini, F., P. Arnow, and J. 0. Lampen. 1961. The effect ofmonovalent cations on the inhibition of yeast metabolismby nystatin. J. Gen. Microbiol. 24:51-62.

15. Mendoza, C. G., and J. R. Villanueva. 1964. Stages in theformation of yeast protoplasts with strepzyme. Nature(London) 202:1241-1242.

16. Moor, H., and K. MUhlethaler. 1963. Fine structure infrozen-etched yeast cells. J. Cell Biol. 17:609-628.

17. MUller, R. 1967. Mikroskopische Analyse der Protoplasten-entstehung und -reversion, p. 37-41. In R. MUller (ed.),Symposium uiber Hefe-Protoplasten, Jena, 1965. AkademieVerlag, Berlin.

18. Ottolenghi, P. 1966. Do yeasts form true protoplasts? C. R.Trav. Lab. Carlsberg. 35:363-368.

19. Reynolds, E. S. 1963. The use of lead citrate at high pH as an

electron-opaque stain in electron microscopy. J. Cell Biol.17:208-212.

20. Ryter, A., and E. Kellenberger. 1958. Etude au microscopeelectronique de plasmas contenant de l'acide desoxyri-bonucleique. Z. Naturforsch. 13b:597-605.

21. Sabatini, D. D., K. Bensch, and R. J. Barmett. 1963. Cyto-chemistry and electron microscopy. The preservation ofcellular ultrastructure and enzymatic activity by aldehydefixation. J. Cell Biol. 17:19-58.

22. Streiblova, E. 1968. Surface structure of yeast protoplasts..J. Bacteriol. 95:700-707.

23. Svihla, G., and F. Schlenk. 1967. The influence of cultureconditions on the formation of spheroplasts from Candidautilis, p. 15-20. In R. MUller (ed.), Symposium uber HefeProtoplasten, Jena, 1965. Akademie-Verlag, Berlin.

24. Svihla, G., F. Schlenk, and J. L. Dainko. 1961. Spheroplastsof the yeast Candida utilis. J. Bacteriol. 82:808-814.

25. Tulasne, R., R. Minck, A. Kirn, and J. Krembel. 1960.Delimitation de la notion de formes L des bacteries:protoplastes, spheroplasts et formes L. Ann. Inst. Pasteur99 :859-874.

26. Villanueva, J. R., R. Sentandreu, and C. G. Mendoza. 1967.Protoplast release and its structure in Candida utilis, p.21-25. In R. Muller (ed.), Symposium uber Hefe-Proto-plasten, Jena, 1965. Akademie-Verlag, Berlin.

27. Vitols, E., R. J. North, and A. W. Linnane. 1961. Studies on

the oxidative metabolism of Saccharomyces cerevisiae. I.

Observations on the fine structure of the yeast cell. J.Biophys. Biochem. Cytol. 9:689-699.

28. Zvjagilskaja, R. A., and T. P. Afanasjewa. 1967. Die Iso-

lierung und Franktionierung der Protoplasten aus Hefe-

zellen von Edomyces magnusil, p. 27-29. In R. Muller(ed.), Symposium Uber Hefe-Protoplasten, Jena, 1965.Akademie-Verlag, Berlin.

810 J. BACTERIOL.

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nloaded from