Germination and Outgrowth of Single Spores of Saccharomyces cerevisiae Viewed by Scanning

Transcription

1 JOURNAL OF BACTrIOLOoY, Mar. 1972, p Copyright 0) 1972 American Society for Microbiology Vol. 109, No. 3 Printed in U.S.A. Germination and Outgrowth of Single Spores of Saccharomyces cerevisiae Viewed by Scanning Electron and Phase-Contrast Microscopy PAUL ROUSSEAU, HARLYN 0. HALVORSON,' LEE A. BULLA, JR., AND GRANT ST. JULIAN Laboratory of Molecular Biology and Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706, and Northern Regional Research Laboratory,2 Peoria, Illinois Received for publication 4 October 1971 Single spores of Saccharomyces cerevisiae were examined during germination and outgrowth by scanning electron and phase-contrast microscopy. Also determined were changes in cell weight and light absorbance, trehalose utilization, and synthesis of protein and KOH-soluble carbohydrates. These studies reveal that development of the vegetative cell from a spore follows a definite sequence of events involving dramatic physical and chemical modifications. These changes are: initial rapid loss in cellular absorbance followed later by an abrupt gain in absorbance; reduction in cell weight and a subsequent progressive increase; modification of the spore surface with concomitant diminution in refractility; elongation of the cell and augmentation of surface irregularities; rapid decline in trehalose content of the cell accompanied by extensive formation of KOH-soluble carbohydrates; and bud formation. Germination and outgrowth are developmental stages in the transition of a spore to a vegetative cell (4, 5). Both stages consist of simultaneous chemical and physical alterations that are reflected in the appearance of the spore. In this report, germination is regarded as a change from a light-refractile spore to a nonrefractile cell. Outgrowth designates the sequence of development after germination that culminates with cell division. Previously, germination and outgrowth of bacterial spores have been examined with a scanning electron microscope (16). The three-dimensional images produced by the scanning microscope revealed distinctive changes in spore surface and overall anatomy during the developmental process. In this investigation, we examined germination and outgrowth of single spores of Saccharomyces cerevisiae by scanning electron and phase-contrast microscopy. The only other work involving microscopy reported on the germination of this organism includes thin sections of resting and germinated spores contained within an ascus (7) and hand-drawn representations of different morphological ' Present address: Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Mass A laboratory of the Northem Marketing and Nutrition Research Division, Agricultural Research Service, U.S. Department of Agriculture. stages observed with a light microscope (6, 12). In an effort to distinguish morphological features as well as certain chemical and physical modifications that occur during the developmental phases, we also determined changes in cell weight and light absorbance, trehalose utilization, and synthesis of protein and KOHsoluble carbohydrates during germination and outgrowth. MATERIALS AND METHODS Organism and cultural conditions. A diploid S. cerevisiae Y-55 (20) was used in this study. Details concerning its growth and sporulation and the techniques for preparation of single spores were described earlier (15). Spores were germinated in a 0.2% succinic acid synthetic medium designated SSM (17); glucose and Tween 80 (15) were added aseptically before inoculation to give a final concentration of 1% for each component. Inoculated flasks were aerated by rotary agitation at 250 rev/min :30 C, in an incubator shaker. Samples were removed from the flasks at 30-min intervals and quickly cooled. Cells then were separated from the culture medium by centrifugation (70 x g) at 5 C, washed three times with cold sterile phosphate-citrate buffer (0.02 M, ph 7.0), and finally suspended in this buffer. Measurement of germination and outgrowth. Germination and outgrowth were monitored by measuring spectrophotometrically the change in absorbance at 600 nm. To obtain the number of various morphological forms, six separate differential counts 1232

2 VOL. 109, 1972 S. CEREVISIAE GERMINATION 1233 were made by phase-contrast microscopy. The percentage of each cell form in the population was calculated as the average number of that specific cell form (germinated or outgrown) divided by the total number of cells per microscopic field x 100. Analytical measurements. Dry weight determinations were done with cells collected on individual preweighed membrane filters. Proteins were extracted by the procedure of Ogur and Rosen (11), and their concentration was determined by the method of Lowry et al. (9). The anthrone method (1) was used to measure carbohydrates; cells were shaken for 40 min at room temperature in flasks containing 0.5 M trichloroacetic acid. This procedure was repeated until the acid extract contained no anthrone-positive material. The extracted cell pellet was resuspended in 30% KOH for 3 hr in a boiling-water bath, and then KOH-soluble carbohydrates (glycogen and mannan) were determined; trehalose was measured in pooled acid extracts and was confirmed by paper chromatography (2, 14, 19, 21). All carbohydrate determinations are expressed as glucose units. Microscopy. The techniques for observing specimens by phase-contrast and scanning electron microscopy were modified from those described earlier (3) Ḟor phase-contrast microscopy, mounting slides were prepared by spreading a thin film of 1% Noble agar evenly over the surface of glass microscope slides. A small amount (about 0.05 ml) of a cell suspension was placed on the solidified agar surface and covered with a cover slip. Cells were photographed on Panatomic-X film through Neofluar phase optics of a Zeiss WL microscope. Squares (10 by 10 mm) cut from glass microscope slides were placed on aluminum specimen stubs for scanning electron microscopy. About 0.05 ml of a diluted cell suspension was spread over the mounting surface, dried, and coated with aluminum to a thickness of 15 nm. Specimens were examined in a Cambridge Stereoscan Mark II scanning electron microscope at an accelerating voltage of 20 kv; the final aperture was 200,um, and the beam specimen angle was 450. RESULTS The distribution of different cell forms (germinated and outgrown) of S. cerevisiae was determined and used as an index for synchrony of germination and outgrowth. A normal spore preparation contained about 95% refractile spores. After 30 min of incubation (designated T30) in the SSM medium, only 45% of the population xemained refractile, with the other cells appearing semirefractile or completely phase-dark. The percentage of germinated cells, indicated by semirefractility and phase-darkness, increased from 55% at T30 to approximately 85% at T60 and 95% at TY0. By T.20, nearly 100% of the population was germinated. All germinated cells by this time were swollen and slightly elongated; swelling of individual cells began at T., Upon initial bud formation at T,50, the cells clumped; thereafter, it was difficult to determine accurately the percentage of various morphological cell forms. During germination (To to T,,), there was a rapid decrease in light absorbance by the cell population; total loss in absorbance was approximately 15% (Fig. 1). At T,20, the cells began to increase in absorbance and continued to T240. Cell weight, expressed as per cent dry weight, decreased about 10% during the first 60 min of incubation, after which it steadily increased throughout the remaining incubation period. Interestingly, trehalose content of the spores (Fig. 2) diminished concomitantly with the loss in cellular absorbance (To to T9O; see also Fig. 1), whereas KOH-soluble carbohydrates (glycogen mannan; 10, 13, 21) increased simultaneously with the gain in absorbance (T120 to T240). Net protein synthesis began after the first 45 min of incubation. Figure 3 presents scanning electron micrographs of S. cerevisiae with phase-contrast photographs inserted. The photographs disclose the sequence of morphological change from refractile spore to bud formation. Figure 3A is an ascus containing four ungerminated ascospores arranged in the shape of a u U.uO Time, Minutes FIG. 1. Light absorbance (-) and cell dry weight (0) during germination and outgrowth of S. cerevisiae. Spores were incubated in succinic acid synthetic medium (17), and absorbance readings were taken at 600 nm. Dry weight measurements are an average of three separate experiments done in triplicate.

3 1234 ROUSSEAU ET AL. J. BACTERIOL. Time, Minutes FIG. 2. Trehalose utilization and synthesis of protein and of KOH-soluble carbohydrates during germination and outgrowth of S. cerevisiae. All measurements are an average of two separate experiments. Carbohydrates are expressed as micrograms of glucose determined by the anthrone reagent (1). tetrahedron. This arrangement appears to be typical because approximately 90% of the asci examined in the scanning electron microscope exhibited a tetrahedral form. In phase-contrast microscopy, most of the asci appeared to be planar and to contain only three highly refractile ascospores (Fig. 3A, insert); however, the fourth spore can be readily detected in a lower plane. The outer surface of the intact ascospores appeared smooth in the scanning microscope. However, an individual spore which appeared free of both the ascus wall and outer spore wall (To) exhibited some surface irregularity (Fig. 3B). The corresponding phase-contrast photograph shows a spherical refractile spore. Figure 3C is a germinated spore (T30) that has lost some refractility. The spore wall surface also appears more uneven than that of the To spore. A greater loss in refractility and more intense modification of the spore surface occurred between T30 and T60. The spore at T60 (Fig. 3D) is rough with continuous ridges over the entire surface. At T90 (Fig. 3E, insert), maximum phasedarkness was reached; also, the spore swelled and became pear-shaped. The scanning electron micrograph in Fig. 3E depicts heightened surface irregularity and the beginning of outgrowth (arrow). Further outgrowth (T120) is seen in Fig. 3F as an extension of one end of the cell (arrow). This elongation continued up to T1,0, by which time initial bud formation had occurred (Fig. 3G, arrow). The surface of the outgrowth is smoother than that of the older, opposing portion of the cell. This contrast in topography was not discernible by phase-contrast. microscopy (Fig. 3G insert). Figure 3H is an intermediate stage of' bud formation that shows an enlarged bud (arrow). Figure 3I reveals a mature bud (T2,0, arrow) whose surface is smooth and similar to that of' the outgrown portion of the mother cell. This appearance again is compared to the rough surface of the older portion of the mother cell. By T240, the majority of cells were conjugated (Fig. 3J). DISCUSSION The modification in surface topography of S. cerevisiae ascospores during germination and outgrowth presents an interesting aspect of' cellular differentiation. Our study with the scanning electron microscope reveals that the ascospore wall undergoes marked alteration during the developmental process. Skinner et al. (18) and Hashimoto et al. (7) contend that the inner spore wall serves as the cell wall of' the new vegetative cell. Two distinct phases compose the sequence of events from spore to vegetative cell by S. cerevisiae. The first stage involves several dramatic changes during the initial 90 min of incubation: (i) rapid loss in light absorbance; (ii) reduction in cell weight; (iii) modification of the spore surface and concomitant diminution in refractility; (iv) increase in cell size and altered shape; and (v) decrease in trehalose content. The second stage (T90 to T240) includes: (i) abrupt gain in cellular absorbance; (ii) progressive increase in cell weight; (iii) elongation of cell and augmentated surface irregularity; (iv) bud formation; and (v) formation of KOH-soluble carbohvdrates. Loss in light absorbance and ref'ractilitv along with the changes in surface structure may reflect internal chemical modification of' the spore wall. If trehalose is a precursor of' glucan, as Leloir suggests (8), the rapid decrease in trehalose content of the cell f'ollowed by extensive formation of KOH-soluble carbohydrates may indicate glycogen and manrnan biosynthesis. Net protein synthesis occurs early during germination. Whether the newly synthesized protein becomes incorporated into cell wall of the vegetative cell has yet to be determined. Further studies are now in progress on the biochemical and physiological events of germination and outgrowth in this organism.

4 VOL. 109, 1972 S. CEREVISIAE GERMINATION I 1235 FIG. 3A-D. Scanning electron micrographs (x 10,000 or x20,000) of S. cerevisiae with corresponding phasecontrast micrographs (x2,375) inserted. A, ascus containing four ungerminated ascospores; B, T, ungerminated, separated single spore; C, T,, germinated spore; D, T60, germinated spore. Ellipses with designated numbers represent either 0.5 or 0.1 fnm.

5 1236 ROUSSEAU ET AL. J. BACTERIOL. FIG. 3E-G. Scanning electron micrographs (x 10,000 or x20,000) of S. cerevisiae with corresponding phasecontrast micrographs (x2,375) inserted. E, T,0, germinated spore; F, T120, outgrown spore; G, T,50. initial bud formation (arrow). Ellipses with designated numbers represent either 0.5 or 0.1 fim.

6 VOL. 109, 1972 S. CEREVISIAE GERMINATION 1237 v F. I I.0 '.,v Downloaded from FIG. 3H-J. Scanning electron micrographs (x 10,000 or x20,000) of S. cerevisiae with corresponding phasecontrast micrographs (x2,375) inserted. H, T,80, intermediate bud formation (arrow); I, T210, mature bud (arrow) and mother cell; J, T240, conjugation. Ellipses with designated numbers represent either 0.5 or 0.1 Mm. on December 9, 2018 by guest

7 1238 ROUSSEAU ET AL. J. BACTERIOL. ACKNOWLEDGMENTS We thank F. L. Baker and G. L. Adams for their able technical assistance. This investigation was supported by Public Health Service research grant AI-1459 from the National Institute of Allergy and Infectious Diseases and by research grant B from the National Science Foundation. H. 0. Halvorson was the recipient of a Research Career Professorship from the National Institutes of Health. LITERATURE CITED 1. Ashwell, G Colorimetric analysis of sugars, p In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press Inc., New York. 2. Avigad, G Accumulation of trehalose and sucrose in relation to the metabolism of a-glucosides in yeast of defined genotype. Biochim. Biophys. Acta 40: Bulla, L. A., G. St. Julian, R. A. Rhodes, and C. W. Hesseltine Scanning electron and phase-contrast microscopy of bacterial spores. Appl. Microbiol. 18: Gould, G. W Germination, p In G. W. Gould and A. Hurst (ed.), The bacterial spore. Academic Press Inc., New York. 5. Halvorson, H. O., J. C. Vary, and W. Steinberg Developmental changes during the formation and breaking of the dormant state in bacteria. Annu. Rev. Microbiol. 20: Hansen, E. C Recherches sur la physiologie et la morphologie des ferments alcooliques. VIII. Sur la germination des spores chez les Saccharomyces. C. R. Trav. Lab. Carlsberg 3: Hashimoto, T., S. F. Conti, and H. B. Naylor Fine structure of microorganisms. Ill. Electron microscopy of resting and germinating ascospores of Saccharomyces cerevisiae. J. Bacteriol. 76: Leloir, L. F The uridine coenzymes, p In C. Liebecq (ed.), Third International Congress of Biochemistry, Brussels. Academic Press Inc., New York. 9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Northcote, D. H., and R. W. Horne The chemical composition and structure of yeast cell wall. B3iochem. J. 51: Ogur, M., and G. Rosen The nucleic acids of plant tissues. I. The extraction and estimation of' deoxypentose nucleic acid and pentose nucleic acid. Arch. Biochem. 25: Palleroni, N. J The nutritional requiremenits for the germination of yeast spores. Phyton Ann. Sci. Bot. 16: Pazonyi, B., and L. Murkus The fractionation and measurement of carbohydrates in relation to the lif'e cycle of yeast (in Hungarian). Agrokem. Talajtan 4: Roth, R Carbohydrate accumulation during the sporulation of yeast. J. Bacteriol. 101: Rousseau, P., and H. 0. Halvorson Preparation and storage of single spores of Saccharomvces cerelisiae. J. Bacteriol. 100: St. Julian, G., L. A. Bulla, Jr., and C. W. Hesseltine Germination and outgrowth of Bacillus thuringiensis and Bacillus alvei spores viewed by scanning electron and phase-contrast microscopy. Can. J. Microbiol. 17: Sebastian, J., B. L. A. Carter, and H. 0. Halvorson Use of yeast populations fractionated by zonal centrifugation to study the cell cycle. J. Bacteriol. 108: Skinner, C. E., C. H. Emmons, and H. M. TI'suchiva Morphology and classification of the yeasts ancd yeast-like fungi, p In Henrici's molds, yeast. and actinomycetes, 2nd ed. John Wiley & Sons. Illc.. New York. 19. Suomalainen, H., and S. Pfaffli Changes in the carbohydrate reserves of baker's yeast during growth and on standing. J. Inst. Brew. 67: Tauro, P., and H. 0. Halvorson Effect of' gene position on the timing of enzyme synthesis in synchronous cultures of yeast. J. Bacteriol. 92: Trevelyan, W. E., and J. S. Harrison Studies on yeast metabolism. VII. Yeast carbohydrate f'ractions. Separation from nucleic acid, analysis, and behavior during anaerobic fermentation. Biochem. J. 63:2>-232:.