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Physical and biochemical properties of freeze-tolerant mutants of a yeast Saccharomyces cerevisiae
JOURNAL OF FERMENTATIONAND BIOENGINEERING Vo1. 70, NO. 4, 275-276. 1990
Physical and Biochemical Properties of Freeze-Tolerant Mutants of a Yeast Saccharomyces cerevisiae KEIKO MATSUTANI, l YASUKI FUKUDA, l* KOUSAKU MURATA, 2 AKIRA KIMURA, 2 ICHIRO NAKAMURA ,l A~D NORIO YAJIMA 3
Chukyo Community College, 2216 Toki-cho, Mizunami, Gifu 509-61, J Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, 2 and Asama Kasei Co., Ltd., Chuo-ku, Tokyo 103, 3 Japan Received 2 May 1990/Accepted 10 August 1990
Freeze-tolerant mutants of a yeast Saccharomyces cerevisiae were obtained through repeated mutations. The freeze-tolerance of the yeast cells was thought to be partially induced by the increasing rigidity of the cell surface, although the tolerance altered the susceptibility of the yeast calls to some toxic chemicals.
Freeze-tolerant yeasts have been sought for use in breadmaking by frozen dough methods, and several types of such strains have been screened from natural environment (1-3). We have obtained freeze-tolerant strains of a yeast Saccharomyces cerevisiae by mutation, although not by screening from nature. A comparison of mutant and wildtype cells indicated that the freeze-tolerant mutants were structurally rigid, although phenotypically variegated. For the isolation of freeze-tolerant mutants, cultures (108 cells/ml) of S. cerevisiae IFO 2375 after incubation at 30°C for 16 h in 3.0 ml of YPD nutrient medium (2.0~ glucose, 0 . 5 ~ yeast extract, 1.0~ bactopeptone; pH 5.0) were transferred to a sterilized Schale (10 cm in diameter) and exposed to ultraviolet (UV) light at a distance giving approximately a 980//00 killing rate. Yeast extract and bactopeptone were purchased from Difco Laboratories (Detroit, MI, USA). The cells were collected, suspended in 3.0 ml of fresh YPD, incubated at 30°C for 16 h to grow mutagenized cells and then kept at - 2 0 ° C for 16 h. The frozen culture was thawed at room temperature and treated again with UV as above. The processes of mutation, incubation in fresh YPD and subsequent freezing and thawing were repeated five times and then the cells were spread on YPD-agar (1.5°//00) plates. The large colonies were selected after incubation of the plates at 30°C for 2 d. The cells in each colony were suspended in 3.0 ml of YPD (105 cells/ml) and kept at - 2 0 ° C . Freezing (--20°C) and thawing (room temperature) of the suspensions were then repeated five times at an interval of 16 h without incubation at 30°C and final viable cells in each suspension were counted on YPD-agar plates. Two cultures that showed approximately 750//00survival were chosen and mutants in the cultures were designated FTM-1 and FTM-2. To verify freeze-tolerance, FTM-1 and -2 cells were grown in 3.0 ml of YPD at 30°C and then the cultures (3 × 107 cells/ml) were kept at - 2 0 ° C for 16 h. Freezing (--20°C) and thawing (room temperature) of the cultures were repeated six times at intervals of 16 h without incubation for growth and 0.1 ml of the culture was periodically taken to determine viable cells. The changes in viable cells in FTM-1 and -2 cultures were compared with those of wild-type culture treated similarly (Fig. 1). The viable cells in the wild-type culture apparently decreased after repeated freezing and thawing. On the other hand, in case of
the mutant cultures, no marked decrease in viable cells was found and approximately 7 5 ~ of the initial viable cells survived even after six repetitions of freezing and thawing. This result indicates that the mutants FTM-1 and -2 were freeze-tolerant. Other than the freeze-tolerance, mutant FTM-1 cells were different from wild-type cells with respect to physical and phenotypic properties. The cell size of the mutant FTM-1 (Fig. 2B-top) was almost the same as that of the wild-type (Fig. 2A-top). However, when the mutant FTM1 and wild-type cells growing logarithmically on 3.0 ml of YPD were placed in vacuo, wild-type cells were completely
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64 Time (h)
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FIG. 1. Changes in viable cells of freeze-tolerant mutants during repeated freezing and thawing. The mutants FTM-1 (A), FTM-2 (A) and wild-type ( ~ ) cells were grown on 3.0ml of YPD to 3 × l07 cells/ml at 30°C and then the cultures were subjected to freezing and thawing six times at intervals of 16 h as shown in text. The survival rates are shown relative to the original viable cells.
* Corresponding author. 275
276
MATSUTANI ET AL.
A
J. FERMENT. BIOENG.,
CuCI2 COCI2 CdCI2 ZnCI2 NiCI2 PhG MG NEM 8-HQ IAA
I I 0 -e
I 0 -7
I 0 -6
I 0 -s
Concentration
I 0 -4
10 -3
I I0
2
(M)
FIG. 3. Susceptibility of freeze-tolerant mutant to various chemicals. Mutant FTM-I ( • ) and wild type ( [] ) cells were grown on SDagar plates supplemented with various chemicals. The plates were incubated at 30°C for 2 d and the appearance of colonies was checked. PhG, Phenylglyoxal; MG, methylglyoxal; NEM, N-ethylmaleimde; 8-HQ, 8-hydroxyquinoline; IAA, iodoacetamide. FIG. 2. Structural rigidity of freeze-tolerant mutant in vacuo. Wild-type (A) and mutant FTM-1 (B) cells were grown on YPD and phase contrast micrographs were taken before (top) and after (below) treatment in vacuo. Bars represent 5/~m in length. ruptured (Fig. 2A-below), while almost all o f the mutant cells retained their original cell shapes (Fig. 2B-below). This suggests that the freeze-tolerant mutant cells were structurally rigid, and that the freeze-tolerance was partially induced by an increase in the rigidity o f the cell surface. Structural changes in the cell surface thought to alter the susceptibility o f yeast cells to various chemicals. The freeze4olerant mutant FTM-1 and wild-type cells were grown on SD minimal agar (1.5°/00) plates [2.00/00 glucose, 0.67% yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI, USA); pH 5.0] supplemented with various toxic chemicals (4) (Fig. 3). The mutant cells o f FTM-1 were f o u n d to become more sensitive or resistant to certain toxic chemicals than the wild-type cells. For example, the growth o f the mutant cells was arrested when N-ethylmaleimide (NEM) was included in the plates at 10 5 M, although the wild-type cells could grow even in the presence o f 10 4M o f N E M . On the other hand, in case o f 8-hydroxyquinoline (8-HQ), phenylglyoxal (PhG) or NiC12, mutant cells were more resistant than wild-type cells. Thus, the sensitivities of wild-type and mutant cells varied depending on the types o f chemicals used. This was
presumably due to the altered permeability of these chemicals through cell membranes and the alteration was thought to be caused by the repeated UV irradiations. Thus, the freeze-tolerant mutants o f S. cerevisiae obtained by mutations indicated that the freeze-tolerance could be partially induced by increasing cell rigidity, although the tolerance rendered the yeast cells susceptible to some toxic chemicals. We thank Ms. Tamiko Fukuda for her assistance with the experiments and preparation of the manuscript.
REFERENCES 1. Hino, A., Takano, H., and Tanaka, Y.: New freeze-tolerant yeast for frozen dough preparation. Cereal Chem., 64, 269-275 (1987). 2. Oda, Y., Uno, K., and Ohta, S.: Selection of yeasts for bread making by the frozen-dough method. Appl. Environ. Microbiol., 52, 941-943 (1986). 3. Hahn, Y.-S. and Kawai, H.: Isolation and characterization of freeze-tolerant yeasts from nature available for the frozen-dough method. Agric. Biol. Chem., 54, 829-831 (1990). 4. Murata, K., Fukuda, Y., Shimosaka, M., Watanabe, K., Saikusa, T., and Kimura, A.:Phenotypic character of the methylglyoxal resistance gene in Saccharomyces cerevisiae: expression in Escherichia coli and its application to breeding of wild-type yeast strains. Appl. Environ. Microbiol., 50, 1200-1207 (1985).
Physical and Biochemical Properties of Freeze-Tolerant Mutants of a Yeast Saccharomyces cerevisiae KEIKO MATSUTANI, l YASUKI FUKUDA, l* KOUSAKU MURATA, 2 AKIRA KIMURA, 2 ICHIRO NAKAMURA ,l A~D NORIO YAJIMA 3
Chukyo Community College, 2216 Toki-cho, Mizunami, Gifu 509-61, J Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, 2 and Asama Kasei Co., Ltd., Chuo-ku, Tokyo 103, 3 Japan Received 2 May 1990/Accepted 10 August 1990
Freeze-tolerant mutants of a yeast Saccharomyces cerevisiae were obtained through repeated mutations. The freeze-tolerance of the yeast cells was thought to be partially induced by the increasing rigidity of the cell surface, although the tolerance altered the susceptibility of the yeast calls to some toxic chemicals.
Freeze-tolerant yeasts have been sought for use in breadmaking by frozen dough methods, and several types of such strains have been screened from natural environment (1-3). We have obtained freeze-tolerant strains of a yeast Saccharomyces cerevisiae by mutation, although not by screening from nature. A comparison of mutant and wildtype cells indicated that the freeze-tolerant mutants were structurally rigid, although phenotypically variegated. For the isolation of freeze-tolerant mutants, cultures (108 cells/ml) of S. cerevisiae IFO 2375 after incubation at 30°C for 16 h in 3.0 ml of YPD nutrient medium (2.0~ glucose, 0 . 5 ~ yeast extract, 1.0~ bactopeptone; pH 5.0) were transferred to a sterilized Schale (10 cm in diameter) and exposed to ultraviolet (UV) light at a distance giving approximately a 980//00 killing rate. Yeast extract and bactopeptone were purchased from Difco Laboratories (Detroit, MI, USA). The cells were collected, suspended in 3.0 ml of fresh YPD, incubated at 30°C for 16 h to grow mutagenized cells and then kept at - 2 0 ° C for 16 h. The frozen culture was thawed at room temperature and treated again with UV as above. The processes of mutation, incubation in fresh YPD and subsequent freezing and thawing were repeated five times and then the cells were spread on YPD-agar (1.5°//00) plates. The large colonies were selected after incubation of the plates at 30°C for 2 d. The cells in each colony were suspended in 3.0 ml of YPD (105 cells/ml) and kept at - 2 0 ° C . Freezing (--20°C) and thawing (room temperature) of the suspensions were then repeated five times at an interval of 16 h without incubation at 30°C and final viable cells in each suspension were counted on YPD-agar plates. Two cultures that showed approximately 750//00survival were chosen and mutants in the cultures were designated FTM-1 and FTM-2. To verify freeze-tolerance, FTM-1 and -2 cells were grown in 3.0 ml of YPD at 30°C and then the cultures (3 × 107 cells/ml) were kept at - 2 0 ° C for 16 h. Freezing (--20°C) and thawing (room temperature) of the cultures were repeated six times at intervals of 16 h without incubation for growth and 0.1 ml of the culture was periodically taken to determine viable cells. The changes in viable cells in FTM-1 and -2 cultures were compared with those of wild-type culture treated similarly (Fig. 1). The viable cells in the wild-type culture apparently decreased after repeated freezing and thawing. On the other hand, in case of
the mutant cultures, no marked decrease in viable cells was found and approximately 7 5 ~ of the initial viable cells survived even after six repetitions of freezing and thawing. This result indicates that the mutants FTM-1 and -2 were freeze-tolerant. Other than the freeze-tolerance, mutant FTM-1 cells were different from wild-type cells with respect to physical and phenotypic properties. The cell size of the mutant FTM-1 (Fig. 2B-top) was almost the same as that of the wild-type (Fig. 2A-top). However, when the mutant FTM1 and wild-type cells growing logarithmically on 3.0 ml of YPD were placed in vacuo, wild-type cells were completely
IOO
7' O L.
";=,.5
0
~
~o
0
I
32
I
64 Time (h)
I
96
FIG. 1. Changes in viable cells of freeze-tolerant mutants during repeated freezing and thawing. The mutants FTM-1 (A), FTM-2 (A) and wild-type ( ~ ) cells were grown on 3.0ml of YPD to 3 × l07 cells/ml at 30°C and then the cultures were subjected to freezing and thawing six times at intervals of 16 h as shown in text. The survival rates are shown relative to the original viable cells.
* Corresponding author. 275
276
MATSUTANI ET AL.
A
J. FERMENT. BIOENG.,
CuCI2 COCI2 CdCI2 ZnCI2 NiCI2 PhG MG NEM 8-HQ IAA
I I 0 -e
I 0 -7
I 0 -6
I 0 -s
Concentration
I 0 -4
10 -3
I I0
2
(M)
FIG. 3. Susceptibility of freeze-tolerant mutant to various chemicals. Mutant FTM-I ( • ) and wild type ( [] ) cells were grown on SDagar plates supplemented with various chemicals. The plates were incubated at 30°C for 2 d and the appearance of colonies was checked. PhG, Phenylglyoxal; MG, methylglyoxal; NEM, N-ethylmaleimde; 8-HQ, 8-hydroxyquinoline; IAA, iodoacetamide. FIG. 2. Structural rigidity of freeze-tolerant mutant in vacuo. Wild-type (A) and mutant FTM-1 (B) cells were grown on YPD and phase contrast micrographs were taken before (top) and after (below) treatment in vacuo. Bars represent 5/~m in length. ruptured (Fig. 2A-below), while almost all o f the mutant cells retained their original cell shapes (Fig. 2B-below). This suggests that the freeze-tolerant mutant cells were structurally rigid, and that the freeze-tolerance was partially induced by an increase in the rigidity o f the cell surface. Structural changes in the cell surface thought to alter the susceptibility o f yeast cells to various chemicals. The freeze4olerant mutant FTM-1 and wild-type cells were grown on SD minimal agar (1.5°/00) plates [2.00/00 glucose, 0.67% yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI, USA); pH 5.0] supplemented with various toxic chemicals (4) (Fig. 3). The mutant cells o f FTM-1 were f o u n d to become more sensitive or resistant to certain toxic chemicals than the wild-type cells. For example, the growth o f the mutant cells was arrested when N-ethylmaleimide (NEM) was included in the plates at 10 5 M, although the wild-type cells could grow even in the presence o f 10 4M o f N E M . On the other hand, in case o f 8-hydroxyquinoline (8-HQ), phenylglyoxal (PhG) or NiC12, mutant cells were more resistant than wild-type cells. Thus, the sensitivities of wild-type and mutant cells varied depending on the types o f chemicals used. This was
presumably due to the altered permeability of these chemicals through cell membranes and the alteration was thought to be caused by the repeated UV irradiations. Thus, the freeze-tolerant mutants o f S. cerevisiae obtained by mutations indicated that the freeze-tolerance could be partially induced by increasing cell rigidity, although the tolerance rendered the yeast cells susceptible to some toxic chemicals. We thank Ms. Tamiko Fukuda for her assistance with the experiments and preparation of the manuscript.
REFERENCES 1. Hino, A., Takano, H., and Tanaka, Y.: New freeze-tolerant yeast for frozen dough preparation. Cereal Chem., 64, 269-275 (1987). 2. Oda, Y., Uno, K., and Ohta, S.: Selection of yeasts for bread making by the frozen-dough method. Appl. Environ. Microbiol., 52, 941-943 (1986). 3. Hahn, Y.-S. and Kawai, H.: Isolation and characterization of freeze-tolerant yeasts from nature available for the frozen-dough method. Agric. Biol. Chem., 54, 829-831 (1990). 4. Murata, K., Fukuda, Y., Shimosaka, M., Watanabe, K., Saikusa, T., and Kimura, A.:Phenotypic character of the methylglyoxal resistance gene in Saccharomyces cerevisiae: expression in Escherichia coli and its application to breeding of wild-type yeast strains. Appl. Environ. Microbiol., 50, 1200-1207 (1985).