Antonie van Leeuwenhoek 61: 317-322, 1992. 9 1992 Kluwer Academic Publishers. Printed in the Netherlands.
The flocculation of wine yeasts: biochemical and morphological characteristics in Zygosaccharomyces flocculation in Zygosaccharomyces
Giovanna Suzzi,* Patrizia Romano & Marzia Benevelli
Dipartimento di Protezione e Valorizzazione Agroalimentare, Sezione Microbiologica, Universit~ di Bologna, Coviolo, 42100 Reggio Emilia, Italy Received 11 September 1991; accepted in revised form 6 February 1992
Key words: flocculation, proteinases, SEM, Zygosaccharomyces bailii, Zygosaccharomyces fermenmti Abstract
The floc-forming ability of flocculent strains of Zygosaccharomyces bailii and Z. fermentati, isolated from musts, was tested for susceptibility to proteinase and sugar treatments. Z. fermentati was found highly resistant to the proteolytic enzymes tested, whereas Z. baili was only trypsin-resistant. The inhibition of flocculation by sugars distinguished two types: in Z. fermentati flocculation was completely inhibited by mannose, in Z. bailli by various sugars. By SEM observation, the cell surface of Zygosaccharomyces revealed the presence of a column structure, resulting from fusion of vesicles present on the cell surface.
Introduction
Flocculation of yeast cells is a particular case of cell adhesion which can be defined as the aggregation of cells to form fairly stable multicellular associations under physiological conditions (Calleja et al. 1981). This phenomenon is well known in brewer's yeast for its technological interest (for a review see Calleja 1987), in particular in Saccharornyces cerevisiae and S. uvarum (formerly classified as S. carlsbergensis). In yeasts sexual and non-sexual flocculation systems are known. Sexual co-flocculation is predominantly mediated by protein-protein interactions between cells, as shown in Hansenula (Crandall et al. 1974), Schizosaccharomyces pombe (Calleja 1974; Johnson et al. 1988) and Saccharomyces (Yanagishima & Yoshida 1981). Asexual self-flocculation of brewing strains of S. cere-
visiae is always mediated by protein situated on the cell surface upon activation by Ca 2+, the surface protein interacts with carbohydrate to form a protein-carbohydrate complex (Miki et al. 1982; Kihn et al. 1988). In the genus Zygosaccharomyces flocculent growth or growth in agglutinated masses can occur (Lodder 1970). This characteristic seems to be fairly common in Zygosaccharomyces and in this genus flocculation is also linked to calcium ions (Suzzi & Romano 1990). In this paper, different aspects of Zygosaccharomyces flocculation are investigated by studying the influence of proteolytic enzyme digestion and the inhibition by various saccharides with the aim to know the pattern of cell-cell interaction. Therefore, the surface of the flocculent cells was examined by scanning electron microscopy (SEM).
318 Materials and methods
Evaluation of flocculation
Organisms and culture conditions
Flocculation, scored by eye and compared with appropriate controls, was graded on a scale from 0 (non-flocculent) to 4 (very flocculent).
Flocculent yeast strains of the genus Zygosaccharomyces, belonging to the collection of our Department and isolated from musts (Romano & Suzzi 1991), were used. The strains F50, F51 and F59 were identified as Z. fermentati and the strains F32, F34 and F40 as Z. bailii. The yeasts were cultured in Sabouraud (DIFCO) liquid medium, at 25~ without shaking.
Scanning electron microscopy (SEM) Two preparatory methods for SEM visualization were used: splash-freeze lyophilization as proposed by Sowden & Walker (1988) and the procedure reported by Rodriguez & Dominguez (1984). Samples were viewed using a Hitachi model 510.
Proteinase treatments Flocculent cells were treated with proteinases according to the method of Hodgson et al. (1985). The following enzymes were used: pronase, proteinase k and trypsin, obtained from Boehringer Mannheim, and chymotrypsin from Sigma.
Sugar treatments Flocculated cells from 4 days cultures were washed twice with water and resuspended in 50 mM-sodium phosphate, 50mM-EDTA buffer, pH7.5, to disperse the flocs. The resulting suspension was dispensed in six 1-ml portions into centrifuge tubes and centrifuged. The supernatant was removed and the cells were resuspended in 3 ml of 1-M solutions of the following sugars: fructose, galactose, glucose, maltose and mannose. After shaking for a few seconds, the flocculation was determined immediately and after 20 min, and compared with the control (flocculent cells resuspended in water).
Results
Phenotypic expression of flocculation Flocculent cells of Zygosaccharomyces showed to possess intense cell-cell interactions, resulting in the formation of a coherent layer on the bottom of the recipient. After shaking the layer broke up into large aggregates, which could be swirled up into suspension and then clumped again. Two different flocculation phenotypes were discriminated by protease digestion (Table 1). One phenotype was characterized by high resistance to the proteolytic enzymes and pertained to Z. fermentati. In the other phenotype, belonging to Z. bailii the flocforming ability was irreversibly lost during incubation with pronase, proteinase K and chymotrypsin. Trypsin did not affect any strains.
Table 1. Flocculation phenotypes in Zygosaccharomyces. Yeast
Initial degree of flocculence
Degree of flocculation after treatment ~ Proteinase K
Trypsin
Pronase
Chymotrypsin
4 4 3
4 4 3
4 3 2
4 4 3
4 4 3
4 4 3
0 0 0
0 0 0
4 4 3
0 0 0
Zygosacch. fermentati: F50 F51 F59
Zygosacch. bailii: F32 F34 F40
t0 - non-flocculent; 1 - poorly flocculent; 2 - weakly flocculent; 3 - moderately flocculent; 4 - very flocculent.
319 2
Figs. 1-2. SEM of Zygosacch. fermentati F59 (Fig. 1) and Zygosacch. bailii F34 (Fig. 2). Micrographs of flocculent cells, dried by splash-freeze lyophilization, show surface details. Note the presence of a heavy coat, covering the cells and jointing them into a continuous floc. The coat is more consistent in Zygosacch. bailii.
Inhibition of flocculation by sugars
were differences in surface structure, which might correlate with differences in the biochemical tests. Two preparative techniques were used because the appearance of flocculating cells depends upon the procedure used. SEM examination of flocs prepared by the two methods showed obvious differences. Flocs dried by splash-freeze lyophilization (Figs. 1 and 2) showed a smooth coat that was thick enough to obscure details of the scars. The coat was much more consistent in Z. bailii. Flocs prepared by the other technique showed a coarse but so thin surface layer that bud scars and other details were not obscured (Figs. 3 and 4). Adjacent cells were connected by column structures (Figs. 5 and 6), that could be single or occasion-
Two definite patterns of inhibition were obvious (Table 2). Z. fermentati strains flocculated normally, except in presence of mannose, where there was a complete irreversible inhibition. Flocculation in strain of Z. bailii was completely inhibited by 1-M fructose, galactose, maltose and mannose. The other strains flocculated normally only in presence of fructose. Inhibition of flocculation by sugars was reversible by removal of sugars.
Surface features of flocculent Zygosaccharomyces Zygosaccharomyces strains were examined by scanning electron microscopy to determine if there
Table 2. Inhibitory effect of various sugars on flocculation in Zygosaccharomyces. Yeast
Control
Fructose
Galactose
Glucose
Maltose
Mannose
Zygosacch. fermentati: F50 F51 F59
Zygosacch. bailii: F32 F34 F40
-
+
4-
+
+
+ -
+ +
+ +
+ 4-
44-
+, inhibitory effect on flocculation; - , no inhibitory effect.
320 3
~ii ~II
:
i~i
Figs. 3~6. SEM of flocculent cells prepared with the method of Rodriguez & Dominguez (1984): Zygosacch.fermentatistrain F59 (Figs. 4-5), Zygosacch. bailiistrain F34 (Figs. 3-6). Cell surfaces appear to be highly detailed so that bud scars and vesiclescan be identified (Figs. 3--4). Flocculent cells are connected by a column structure, which is more distinguishable in the Figs. 5 and 6. Cell surfaces present numerous vesicles (Fig. 6).
ally joint together (Fig. 3). T h e cell surface showed numerous vesicles, the presence of which could be related to the column structure formation. Figure 7 shows two vesicles that are linking together to form a column. In contrast to the biochemical tests, SEM did not reveal significant differences between the flocculent cells of Z. bailii and Z. fermentati.
Conclusions Flocculation depends on intense interaction between yeast cells which is modulated by genetic,
environmental and metabolic factors. The flocculation in Zygosaccharomyces has various features in c o m m o n with that in other yeasts, such as S. cerevisiae and S. pombe, with respect to the mechanisms of cell-cell interaction and the involvement of cell wall protein and carbohydrate (Miki et al. 1982; Johnson et al. 1988). Proteinase treatments of flocculent cells have indicated an important role for protein components of the cell wall, and have discriminated four different flocculation phenotypes in S. cerevisiae wine yeasts (Suzzi & R o m a n o 1991). By proteolytic digestion Zygosaccharomyces can be distinguished into two phenotypes, i.e. 1) proteinase- and trypsin-resistant, pertaining to Z.
321
fermentati, and 2) Z. bailii, only resistant to pronase. The tests on inhibition of flocculation by sugars confirm the occurrence of two distinct types. In the first type, including Z. fermentati, flocculation is completely inhibited by mannose only, in the second type, including Z. bailii, by various sugars. These mechanisms probably involve a mannosespecific protein, lectin-like, in Z. fermentati and a broad-specificity lectin in Z. bailii, analogous to what Stratford (1989) reported in S. cerevisiae. The SEM images of flocculent cells do not relate to the two different phenotypes, comparable rather than contrasting SEM micrographs having been found in Z. fermentati and Z. bailii. Only the surface layer of protein which mediates flocculation is more smooth in the latter. A heavy coat of proteinaceous material has been found as a typical SEM appearance of flocculent fission-yeast cells (Johnson et al. 1989). Threads and 'fimbriae' have been seen in S. cerevisiae (Day et al. 1975) and 'sex-hairs' (Calleja et al. 1977), hairs and hairlike threads in S. pombe (Johnson et al. 1989). The surface of the fiocculent cells of Zygosaccharomyces, as seen by SEM, does not show any hairlike threads. It is still possible that hairs beyond the resolution of our scanning electron microscope occur. However, flocculent strains of Zygosaccharomyces produce a different structure, a column structure, which appears to originate from fusion of vesicles present on the cell surface. These vesicles, extruded through the cell wall, could be considered as a synaptic receptor for flocculation, probably composed of one or more mannanprotein complexes. They might be involved both in protein secretion and in flocculation, such as Day et al. (1975) suggested for the fimbriae. The obvious difference in extent of coat between Z. fermentati and Z. bailii suggests differences in the secretion of protein to their cell surface. It remains to be done to investigate the multifunctional role of vesicles and to establish if the aggregation in Zygosaccharomyces is a prerequisite to sex or has some parasexual implication.
Fig. 7. SEM of vesicles of flocculent strain F59. Two vesicles appear to fuse together to form a column.
Acknowledgement Research was supported by National Research Council of Italy, special project RAISA, subproject 4, paper n ~ 313.
References Calleja GB (1974) On the nature of the forces involved in the sex-directed flocculation of a fission yeast. Can. J. Microbiol. 20:797-803 - - (1987) Cell aggregation. In: Rose AH & Harrison JS (Eds) The Yeasts, vol. 2 (pp 165-238) Academic Press, London Calleja GB, Yoo BY & Johnson BF (1977) Fusion and erosion of cell walls during conjugation in the fission yeasts (Schizosaccharornyces pombe). J. Cell Sci. 25:139-155 - - (1981) The cell wall as sex organelle in fission yeast. In: O'Day DH & Horgen PA (Eds) Sexual Interactions in Eukaryotic Microbes (pp 225-259). Academic Press, New York Crandall M, Lawrence LM & Saunders RM (1974) Molecular complementarity of yeast glycoprotein mating factors. Proc. Natl. Acad. Sci. 71:26--29 Day AW, Poon NH & Stewart GG (1975) Fungal fimbriae. IlI. The effect on flocculation in Saccharomyces. Can J. Microbiol. 21:558--564 Hodgson JA, Berry DR & Johnston JR (1985) Discrimination by heat and proteinase treatments between flocculent phenotypes conferred on Saccharornyces cerevisiae by the genes FL01 and FL05. J. Gen. Microbiol. 131:3219-3227 Johnson BF, Walker T, Calleja GB & Seligy VL (1988) Sexual co-flocculation and asexual self-flocculation in budding and fission yeasts: experimental establishment of a fundamental difference. Can. J. Microbiol. 34:1105-1107
322 Johnson BF, Sowden LC, Walker T, Yoo BY & Calleja GB (1989) Use of electron microscopy to characterize the surfaces of flocculent and nonflocculent yeast cells. Can. J. Microbiol. 35:1081-1086 Kihn JC, Masy CL & Mestdagh MM (1988) Yeast flocculation: competition between nonspeciflc repulsion and specific bonding in cell adhesion. Can. J. Microbiol. 34:773-778 Lodder J (1970) The Yeasts: a Taxonomic Study. Amsterdam, North Holland Publ. Comp Miki BLA, Poon NH, James AP & Seligy VL (1982) Possible mechanism for flocculation interactions governed by gene FL01 in Saccharomyces cerevisiae. J. Bacteriol. 150:878-889 Rodriguez C & Dominguez A (1984) The growth characteristics of Saccharornycopsis lipolytica: morphology and induction of mycelium formation. Can. J. Microbiol. 30:605-612 Romano P & Suzzi G (1991) Preliminary characterization of Zygosaccharomyces for oenological purpose. Proc. Int.
Syrup. Biotechnology and wine quality, Frascati (Roma), Italy Sowden LC & Walker T (1988) A procedure for the study by scanning electron microscopy of flocculating cells of the fission yeast Schizosaccharomyces pombe. Can. J. Microbiol. 34:577-582 Stratford M (1989) Evidence for two mechanisms of flocculation in Saccharomyces cerevisiae. Yeast 5:$441-$445 Suzzi G & Romano P (1990) Flocculazione in Zygosaccharomyces. Ind. Bevande 19:306-308 - - (1991) Flocculent phenotypes in wine yeasts. Lett. Appl. Microbiol. 13:7-10 Yanagishima N & Yoshida K (1981) Sexual interactions in Saccharomyces cerevisiaewith special reference to the regulation of sexual agglutinability. In: O'Day DH & Horgen PA (Eds) Sexual Interactions in Eukaryotic Microbes (pp 261-295). Academic Press, New York
The flocculation of wine yeasts: biochemical and morphological characteristics in Zygosaccharomyces flocculation in Zygosaccharomyces
Giovanna Suzzi,* Patrizia Romano & Marzia Benevelli
Dipartimento di Protezione e Valorizzazione Agroalimentare, Sezione Microbiologica, Universit~ di Bologna, Coviolo, 42100 Reggio Emilia, Italy Received 11 September 1991; accepted in revised form 6 February 1992
Key words: flocculation, proteinases, SEM, Zygosaccharomyces bailii, Zygosaccharomyces fermenmti Abstract
The floc-forming ability of flocculent strains of Zygosaccharomyces bailii and Z. fermentati, isolated from musts, was tested for susceptibility to proteinase and sugar treatments. Z. fermentati was found highly resistant to the proteolytic enzymes tested, whereas Z. baili was only trypsin-resistant. The inhibition of flocculation by sugars distinguished two types: in Z. fermentati flocculation was completely inhibited by mannose, in Z. bailli by various sugars. By SEM observation, the cell surface of Zygosaccharomyces revealed the presence of a column structure, resulting from fusion of vesicles present on the cell surface.
Introduction
Flocculation of yeast cells is a particular case of cell adhesion which can be defined as the aggregation of cells to form fairly stable multicellular associations under physiological conditions (Calleja et al. 1981). This phenomenon is well known in brewer's yeast for its technological interest (for a review see Calleja 1987), in particular in Saccharornyces cerevisiae and S. uvarum (formerly classified as S. carlsbergensis). In yeasts sexual and non-sexual flocculation systems are known. Sexual co-flocculation is predominantly mediated by protein-protein interactions between cells, as shown in Hansenula (Crandall et al. 1974), Schizosaccharomyces pombe (Calleja 1974; Johnson et al. 1988) and Saccharomyces (Yanagishima & Yoshida 1981). Asexual self-flocculation of brewing strains of S. cere-
visiae is always mediated by protein situated on the cell surface upon activation by Ca 2+, the surface protein interacts with carbohydrate to form a protein-carbohydrate complex (Miki et al. 1982; Kihn et al. 1988). In the genus Zygosaccharomyces flocculent growth or growth in agglutinated masses can occur (Lodder 1970). This characteristic seems to be fairly common in Zygosaccharomyces and in this genus flocculation is also linked to calcium ions (Suzzi & Romano 1990). In this paper, different aspects of Zygosaccharomyces flocculation are investigated by studying the influence of proteolytic enzyme digestion and the inhibition by various saccharides with the aim to know the pattern of cell-cell interaction. Therefore, the surface of the flocculent cells was examined by scanning electron microscopy (SEM).
318 Materials and methods
Evaluation of flocculation
Organisms and culture conditions
Flocculation, scored by eye and compared with appropriate controls, was graded on a scale from 0 (non-flocculent) to 4 (very flocculent).
Flocculent yeast strains of the genus Zygosaccharomyces, belonging to the collection of our Department and isolated from musts (Romano & Suzzi 1991), were used. The strains F50, F51 and F59 were identified as Z. fermentati and the strains F32, F34 and F40 as Z. bailii. The yeasts were cultured in Sabouraud (DIFCO) liquid medium, at 25~ without shaking.
Scanning electron microscopy (SEM) Two preparatory methods for SEM visualization were used: splash-freeze lyophilization as proposed by Sowden & Walker (1988) and the procedure reported by Rodriguez & Dominguez (1984). Samples were viewed using a Hitachi model 510.
Proteinase treatments Flocculent cells were treated with proteinases according to the method of Hodgson et al. (1985). The following enzymes were used: pronase, proteinase k and trypsin, obtained from Boehringer Mannheim, and chymotrypsin from Sigma.
Sugar treatments Flocculated cells from 4 days cultures were washed twice with water and resuspended in 50 mM-sodium phosphate, 50mM-EDTA buffer, pH7.5, to disperse the flocs. The resulting suspension was dispensed in six 1-ml portions into centrifuge tubes and centrifuged. The supernatant was removed and the cells were resuspended in 3 ml of 1-M solutions of the following sugars: fructose, galactose, glucose, maltose and mannose. After shaking for a few seconds, the flocculation was determined immediately and after 20 min, and compared with the control (flocculent cells resuspended in water).
Results
Phenotypic expression of flocculation Flocculent cells of Zygosaccharomyces showed to possess intense cell-cell interactions, resulting in the formation of a coherent layer on the bottom of the recipient. After shaking the layer broke up into large aggregates, which could be swirled up into suspension and then clumped again. Two different flocculation phenotypes were discriminated by protease digestion (Table 1). One phenotype was characterized by high resistance to the proteolytic enzymes and pertained to Z. fermentati. In the other phenotype, belonging to Z. bailii the flocforming ability was irreversibly lost during incubation with pronase, proteinase K and chymotrypsin. Trypsin did not affect any strains.
Table 1. Flocculation phenotypes in Zygosaccharomyces. Yeast
Initial degree of flocculence
Degree of flocculation after treatment ~ Proteinase K
Trypsin
Pronase
Chymotrypsin
4 4 3
4 4 3
4 3 2
4 4 3
4 4 3
4 4 3
0 0 0
0 0 0
4 4 3
0 0 0
Zygosacch. fermentati: F50 F51 F59
Zygosacch. bailii: F32 F34 F40
t0 - non-flocculent; 1 - poorly flocculent; 2 - weakly flocculent; 3 - moderately flocculent; 4 - very flocculent.
319 2
Figs. 1-2. SEM of Zygosacch. fermentati F59 (Fig. 1) and Zygosacch. bailii F34 (Fig. 2). Micrographs of flocculent cells, dried by splash-freeze lyophilization, show surface details. Note the presence of a heavy coat, covering the cells and jointing them into a continuous floc. The coat is more consistent in Zygosacch. bailii.
Inhibition of flocculation by sugars
were differences in surface structure, which might correlate with differences in the biochemical tests. Two preparative techniques were used because the appearance of flocculating cells depends upon the procedure used. SEM examination of flocs prepared by the two methods showed obvious differences. Flocs dried by splash-freeze lyophilization (Figs. 1 and 2) showed a smooth coat that was thick enough to obscure details of the scars. The coat was much more consistent in Z. bailii. Flocs prepared by the other technique showed a coarse but so thin surface layer that bud scars and other details were not obscured (Figs. 3 and 4). Adjacent cells were connected by column structures (Figs. 5 and 6), that could be single or occasion-
Two definite patterns of inhibition were obvious (Table 2). Z. fermentati strains flocculated normally, except in presence of mannose, where there was a complete irreversible inhibition. Flocculation in strain of Z. bailii was completely inhibited by 1-M fructose, galactose, maltose and mannose. The other strains flocculated normally only in presence of fructose. Inhibition of flocculation by sugars was reversible by removal of sugars.
Surface features of flocculent Zygosaccharomyces Zygosaccharomyces strains were examined by scanning electron microscopy to determine if there
Table 2. Inhibitory effect of various sugars on flocculation in Zygosaccharomyces. Yeast
Control
Fructose
Galactose
Glucose
Maltose
Mannose
Zygosacch. fermentati: F50 F51 F59
Zygosacch. bailii: F32 F34 F40
-
+
4-
+
+
+ -
+ +
+ +
+ 4-
44-
+, inhibitory effect on flocculation; - , no inhibitory effect.
320 3
~ii ~II
:
i~i
Figs. 3~6. SEM of flocculent cells prepared with the method of Rodriguez & Dominguez (1984): Zygosacch.fermentatistrain F59 (Figs. 4-5), Zygosacch. bailiistrain F34 (Figs. 3-6). Cell surfaces appear to be highly detailed so that bud scars and vesiclescan be identified (Figs. 3--4). Flocculent cells are connected by a column structure, which is more distinguishable in the Figs. 5 and 6. Cell surfaces present numerous vesicles (Fig. 6).
ally joint together (Fig. 3). T h e cell surface showed numerous vesicles, the presence of which could be related to the column structure formation. Figure 7 shows two vesicles that are linking together to form a column. In contrast to the biochemical tests, SEM did not reveal significant differences between the flocculent cells of Z. bailii and Z. fermentati.
Conclusions Flocculation depends on intense interaction between yeast cells which is modulated by genetic,
environmental and metabolic factors. The flocculation in Zygosaccharomyces has various features in c o m m o n with that in other yeasts, such as S. cerevisiae and S. pombe, with respect to the mechanisms of cell-cell interaction and the involvement of cell wall protein and carbohydrate (Miki et al. 1982; Johnson et al. 1988). Proteinase treatments of flocculent cells have indicated an important role for protein components of the cell wall, and have discriminated four different flocculation phenotypes in S. cerevisiae wine yeasts (Suzzi & R o m a n o 1991). By proteolytic digestion Zygosaccharomyces can be distinguished into two phenotypes, i.e. 1) proteinase- and trypsin-resistant, pertaining to Z.
321
fermentati, and 2) Z. bailii, only resistant to pronase. The tests on inhibition of flocculation by sugars confirm the occurrence of two distinct types. In the first type, including Z. fermentati, flocculation is completely inhibited by mannose only, in the second type, including Z. bailii, by various sugars. These mechanisms probably involve a mannosespecific protein, lectin-like, in Z. fermentati and a broad-specificity lectin in Z. bailii, analogous to what Stratford (1989) reported in S. cerevisiae. The SEM images of flocculent cells do not relate to the two different phenotypes, comparable rather than contrasting SEM micrographs having been found in Z. fermentati and Z. bailii. Only the surface layer of protein which mediates flocculation is more smooth in the latter. A heavy coat of proteinaceous material has been found as a typical SEM appearance of flocculent fission-yeast cells (Johnson et al. 1989). Threads and 'fimbriae' have been seen in S. cerevisiae (Day et al. 1975) and 'sex-hairs' (Calleja et al. 1977), hairs and hairlike threads in S. pombe (Johnson et al. 1989). The surface of the fiocculent cells of Zygosaccharomyces, as seen by SEM, does not show any hairlike threads. It is still possible that hairs beyond the resolution of our scanning electron microscope occur. However, flocculent strains of Zygosaccharomyces produce a different structure, a column structure, which appears to originate from fusion of vesicles present on the cell surface. These vesicles, extruded through the cell wall, could be considered as a synaptic receptor for flocculation, probably composed of one or more mannanprotein complexes. They might be involved both in protein secretion and in flocculation, such as Day et al. (1975) suggested for the fimbriae. The obvious difference in extent of coat between Z. fermentati and Z. bailii suggests differences in the secretion of protein to their cell surface. It remains to be done to investigate the multifunctional role of vesicles and to establish if the aggregation in Zygosaccharomyces is a prerequisite to sex or has some parasexual implication.
Fig. 7. SEM of vesicles of flocculent strain F59. Two vesicles appear to fuse together to form a column.
Acknowledgement Research was supported by National Research Council of Italy, special project RAISA, subproject 4, paper n ~ 313.
References Calleja GB (1974) On the nature of the forces involved in the sex-directed flocculation of a fission yeast. Can. J. Microbiol. 20:797-803 - - (1987) Cell aggregation. In: Rose AH & Harrison JS (Eds) The Yeasts, vol. 2 (pp 165-238) Academic Press, London Calleja GB, Yoo BY & Johnson BF (1977) Fusion and erosion of cell walls during conjugation in the fission yeasts (Schizosaccharornyces pombe). J. Cell Sci. 25:139-155 - - (1981) The cell wall as sex organelle in fission yeast. In: O'Day DH & Horgen PA (Eds) Sexual Interactions in Eukaryotic Microbes (pp 225-259). Academic Press, New York Crandall M, Lawrence LM & Saunders RM (1974) Molecular complementarity of yeast glycoprotein mating factors. Proc. Natl. Acad. Sci. 71:26--29 Day AW, Poon NH & Stewart GG (1975) Fungal fimbriae. IlI. The effect on flocculation in Saccharomyces. Can J. Microbiol. 21:558--564 Hodgson JA, Berry DR & Johnston JR (1985) Discrimination by heat and proteinase treatments between flocculent phenotypes conferred on Saccharornyces cerevisiae by the genes FL01 and FL05. J. Gen. Microbiol. 131:3219-3227 Johnson BF, Walker T, Calleja GB & Seligy VL (1988) Sexual co-flocculation and asexual self-flocculation in budding and fission yeasts: experimental establishment of a fundamental difference. Can. J. Microbiol. 34:1105-1107
322 Johnson BF, Sowden LC, Walker T, Yoo BY & Calleja GB (1989) Use of electron microscopy to characterize the surfaces of flocculent and nonflocculent yeast cells. Can. J. Microbiol. 35:1081-1086 Kihn JC, Masy CL & Mestdagh MM (1988) Yeast flocculation: competition between nonspeciflc repulsion and specific bonding in cell adhesion. Can. J. Microbiol. 34:773-778 Lodder J (1970) The Yeasts: a Taxonomic Study. Amsterdam, North Holland Publ. Comp Miki BLA, Poon NH, James AP & Seligy VL (1982) Possible mechanism for flocculation interactions governed by gene FL01 in Saccharomyces cerevisiae. J. Bacteriol. 150:878-889 Rodriguez C & Dominguez A (1984) The growth characteristics of Saccharornycopsis lipolytica: morphology and induction of mycelium formation. Can. J. Microbiol. 30:605-612 Romano P & Suzzi G (1991) Preliminary characterization of Zygosaccharomyces for oenological purpose. Proc. Int.
Syrup. Biotechnology and wine quality, Frascati (Roma), Italy Sowden LC & Walker T (1988) A procedure for the study by scanning electron microscopy of flocculating cells of the fission yeast Schizosaccharomyces pombe. Can. J. Microbiol. 34:577-582 Stratford M (1989) Evidence for two mechanisms of flocculation in Saccharomyces cerevisiae. Yeast 5:$441-$445 Suzzi G & Romano P (1990) Flocculazione in Zygosaccharomyces. Ind. Bevande 19:306-308 - - (1991) Flocculent phenotypes in wine yeasts. Lett. Appl. Microbiol. 13:7-10 Yanagishima N & Yoshida K (1981) Sexual interactions in Saccharomyces cerevisiaewith special reference to the regulation of sexual agglutinability. In: O'Day DH & Horgen PA (Eds) Sexual Interactions in Eukaryotic Microbes (pp 261-295). Academic Press, New York
