Tolerance mechanism of the ethanol-tolerant mutant of sake yeast

JOURNAL OF BIOSCIENCEAND BIOENGINEERING Vol. 90, No. 3, 313-320. 2000

Tolerance Mechanism of the Ethanol-Tolerant Mutant of Sake Yeast YOSHIAKI

OGAWA, l* ASAKO NITTA, l HIROFUMI UCHIYAMA, l THE LATE TAKESHI IMAMURA, ’ HITOSHI SHIMO12 AND KIYOSHI ITO Tatsuuma-honke Brewing Co. Ltd., 2-6 Tateishi-cho, Nishinomiya 662-0943’ and National Research Institute of Brewing, 3-7-l Kagamiyama, Higashi-Hiroshima 739-0046,2 Japan Received 24 April 2OOO/Accepted 26 June 2000

Several ethanol-tolerant mutants have been bred from industrial sake yeasts, but the mechanism of ethanol tolerance in these mutants has not been elucidated. After the determination of the entire genome sequence of Saccharomyces cerevisiae, various methods to monitor the whole-gene expression of the yeast have been developed. In this study, we used a commercially available nylon membrane on which virtually every gene of S. cerevisiae was spotted to compare expression profiles between the ethanol-tolerant mutant and its parent sake yeast to investigate the mechanism of ethanol tolerance in this mutant. As a result, we found that several genes were highly expressedonly in the ethanol-tolerant mutant but not in the parent strain. These genes were known to be induced in cellsthat were exposed to various stresses,such as ethanol, heat, and high osmolarlty, or at the stationary-phase but not at the log-phase. In the ethanol-tolerant mutant, the expression level of these stressresponsive genes was further increased after exposure to ethanol. We also found that substances such as catalase, glycerol and trehalose that may have protective roles under stressful conditions were accumulated in high amounts in the ethanol-tolerant mutant. The ethanol-tolerant mutant also exhibited resistance to other stresses including heat, high osmolarity and oxldative stressin addition to ethanol tolerance. These results indicate that the mutant exhibits multiple stresstolerance because of elevated expression of stress-responsivegenes, resulting in accumulation of stressprotective substances. [Key words: Saccharomycescerevisiae,sake yeast, ethanol-tolerant

There is no microorganism that has been closely related to human life from ancient times more than yeasts. In particular, Saccharomyces cerevisiae has been widely used for industrial purposes, e.g., brewing of alcoholic beverages and manufacturing of bread. Sake is a Japanese traditional alcoholic beverage produced by fermentation of steamed rice using Aspergillus oryzae, which is the source of saccharification enzymes, and sake yeasts. Sake yeasts are classified as S. cerevisiae and are known to produce higher concentrations of ethanol in sake brewing than other yeasts such as wine or beer yeasts, and the final ethanol concentration reaches about 20% in sake mash. However, a high concentration of ethanol produced in the late stage of sake mash causes yeast cell death which degrades the quality of sake. Accordingly, tolerance to high concentration of ethanol is one of the most desirable characteristics in industrial sake yeasts. Several ethanol-tolerant mutants such as Kll (1) and SR4-3 (2), which can survive even at the final stage of sake fermentation at which a high ethanol concentration is present, have been bred from traditional sake yeasts and are used in sake brewing. These yeasts can produce high concentrations of ethanol even in the late stage of brewing. In addition to ethanol tolerance, these mutants exhibit resistance to the Kl killer toxin and a cell wall lysis enzyme, zymolyase (1, 3). These characteristics suggest that the cell wall structure is changed in these yeasts because the primary targets of the Kl killer toxin and zymolyase are B-1,6-glucan (4, 5) and ,B-1,3-glucan (6) of the cell wall, respectively. On the other hand, it has been reported that the cell membrane is the primary target of ethanol toxicity and that there is a relationship between membrane fluidity and ethanol * Corresponding

mutant, GeneFilters”]

tolerance (7-10). Thus, it is considered that the structural change in the cell wall or plasma membrane might be related to ethanol tolerance; however, the general mechanism of ethanol tolerance in yeasts is still unclear. The reason for the increased ethanol tolerance in ethanoltolerant mutants is also unknown. S. cerevisiae is widely used as a model organism in molecular and cellular biology because highly useful classical and molecular genetic methods, applicable to industrial yeasts including sake yeast, are available. Generally, sake yeasts have good characteristics for brewing sake but have undesirable properties for breeding and genetic analysis similar to other industrial yeasts. Although sake yeasts are usually diploid, they hardly sporulate, and even if spores are formed, they hardly germinate (11). In addition to these properties, sake yeasts usually do not possess auxotrophic markers commonly used for transformation experiments in laboratory yeasts. Therefore, genetic analyses of ethanol-tolerant mutants by classical and molecular genetic methods have been very difficult. Recently, the genomic DNA sequencing of S. cerevisiae has been accomplished (12). As a result, various methods for monitoring the gene expression of the entire yeast genome have been developed. GeneFilters@ is a commercially available microarray system and consists of two sheets of nylon membrane filters on which PCR products corresponding to over 6200 genes of yeast are spotted (13). Using GeneFiltersO, we screened genes that were differently expressed in the ethanol-tolerant mutant and its parent strain. In this analysis, several genes whose expressions were very weak in the parent strain but highly expressed in the ethanol-tolerant mutant were found. We discuss the involvement of these genes in ethanol tolerance of the ethanol-tolerant mutant.

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Yeast strains, medium, and growth condition S. cerevisiae K7 (MAT a/a), K701 (MAT a/a), and Kll (MAT a/u) obtained from the Brewing Society of Japan, SR4-3 (MAT a/a) (2), and YPHSOO (MAT a ura3-52 1~~2-801 ade2-101 trpl-A63 his34200 leu2-Al) were used in this study. Yeast cells were cultured in YPAD (1% yeast extract, 2% bactopeptone, 2% glucose, 40mg/f adenine) and YNB (2% glucose, 0.67% yeast nitrogen base w/o amino acids) media at 20°C. RNA extraction Yeast cells were cultured in YPAD medium at 20°C under static condition and harvested at log-phase (OD660= 1.0). Total RNA was extracted from yeast cells by the hot phenol extraction method (14). Poly(A)+ RNA was purified by affinity chromatography using an oligo (dT) spun column (Pharmacia, Sweden). Hybridization with GeneFilters@ The cDNA probes were synthesized with [a-33P] dCTP (Amersham, Little Chalfont, England) using a cDNA labeling kit (GenHunter, Nashville, TN, USA) with 250 ng of poly(A)+ RNA, and purified with a Sephadex G-50 column (Pharmacia). GeneFilters@ (Research Genetics, Huntsville, AL, USA) were hybridized with the probe for 16 h at 50°C in a buffer containing 50% formamide, 6 x SSC [l X SSC: 180 mM NaCl, 10 mM sodium phosphate, and 1 mM EDTA (pH 7.0)], 5 x Denhardt’s solution, and 0.5 pg/ml polydeoxyadenosine. After washing twice with 2 X SSC-1% SDS at 50°C for 20min and once with 0.5 x SSC-1% SDS at 25”C, the radioactivity of the membrane was analyzed with the BAS-1000MAC phosphor imaging system (Fujifilm, Tokyo). Northern blot analysis Ten /*g of total RNA was denatured at 65°C for 5 min in a mixture containing 50% formamide, MOPS [20 mM 3-(IV-morpholino) propanesulfonic acid, 5 mM sodium acetate, 1 mM EDTA (pH 7.0)], and 0.16 volume of formaldehyde (37% v/v). The samples were electrophoresed in a gel of 1% agarose, MOPS, and 0.17 volume of formaldehyde (37% v/v), then the fractionated products were blotted onto a nylon membrane (Hybond-N+; Amersham) using 0.05 M NaOH. DNA probes were synthesized by polymerase chain reaction using primers listed in Table 1 and the genomic DNA of K701 as a template. The probes were labeled with [@PI dCTP (Amersham) using Ready-ToTABLE

Primer GPOI-I Gf’ilf -2 (‘TT-I CT? I-2 HSf’f2.I HSP12-2 SPf- / Sf’f-2 HOR7.I HOR7-2 TPSI-I r’sl-2 TPS2./ TPS2.2 MSN2-I MSN2-2 AC‘TJ-I AU-2

1. PCR primers used in this study Sequence S-AAGAAAGCCAAGCGTGTAGAC-3’ 5’.TCTTCGTCGTGGGGTGATAAG3‘ 5’-AGAGAATGGTTATCACTACCG-3’ 5’dSAGACAAGAGAAGGATTTTTT.3’ 5’-TTAATACAACCCACAAACACA-3’ S-GAAATAGAACAATACGCACAC-3’ 5’-TTTATGCTAAAAATCCAGAAG-3’ 5’-TCGTTAACATAGTGCTCAATA-3’ S’XTACGGMAAACCATAAAGAG-3’ 5’-ATATTGCACACCGAGAGAAAA-3’ S-CTCXrTGGAGACGCTTGATTTG-3’ 5’-AAAAAGGGCCATGGGATAGAA-3’ S-AGAGCAAGTACAGGAAGCATA-3’ 5’-TCTTTTACCTACCGCTGTTTC-3’ 5’.CGGTT’lYXXATCCTACTTCAT-3’ S-CCCTCTTCATCCTTAGTCCTG-3’ 5’-A(;GTT(;CTGCTTTGGTTATT-3’ 5’-TAGAAACACTTGTGGTGAA-3’

Go DNA labeling beads (Pharmacia). Blots in the membrane were hybridized with the DNA probes for 16 h at 50°C in a buffer containing 50% formamide, 2 X SSPE [I x SSPE: 180 mM NaCl, 10 mM sodium phosphate, and 1 mM EDTA (PH 7.0)], 5 x Denhardt’s solution, and 20 pg/ml denatured salmon sperm DNA. These were followed by washing twice with 2 X SSPE-0.1% SDS for 20 min at 50°C. Radioactivity of the membrane was analyzed with the BAS-1000MAC phosphor imaging system. Cells were harAnalysis of glycerol and trehalose vested, washed with ice-cold water, resuspended in 5 ml of water, and boiled for 10min. After the cell suspension was centrifuged at 10,000 x g for 5 min, the amounts of glycerol and trehalose were determined by HPLC analysis on a CarboPac MA-l column using the DX500 gradient chromatography system equipped with an electrochemical detector ED-40 (Dionex, Sunnyvale, CA, USA). Before injection, the column was equilibrated with 60 mM NaOH for at least 15 min. The column was eluted with 60mM NaOH for 4min, followed by a NaOH gradient of 60 to 700mM over 4 to 34min at a flow rate of 0.4ml/min. Under these conditions, retention times of glycerol and trehalose were 9.8 min and 28.9 min, respectively. Analysis of catalase activity Cells were harvested, washed twice with ice-cold distilled water, resuspended in an extraction buffer [25 mM Tris-HCI (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 10 mM /3-mercaptoethanol, and 1 mM PMSF], and disrupted using a mini-bead beater (Biospec Products, Bartlesville, OK, USA) at 4°C in the presence of an equal volume of 0.5-mm-diameter glass beads. The resulting suspension was then centrifuged at 5000 X g for 5 min at 4”C, and the supernatant was used for enzyme assay. Catalase activity of the cell extract was assayed by measuring the decrease in absorbance at 240nm using hydrogen peroxide as a substrate in a 0.05 M phosphate buffer (pH 7.0) at 25°C. One unit of catalase activity is defined as the amount of enzyme that catalyzes the conversion of 1 pmol hydrogen peroxide/min at 25°C. The amount of protein was determined by the Bradford method (15) using a protein assay kit (Bio-Rad, Richmond, CA, USA) with bovine serum albumin as a standard. Ethanol tolerance test Cell culture in YPAD medium incubated for 2 d at 15°C under static condition were washed with ice-cold sterilized water and resuspended in a 0.1 M acetate buffer (pH 4.2) containing 1% glucose and 20% (v/v) ethanol. After a 7-d incubation at 15”C, aliquots of the cell suspensions were spread onto a YPAD agar plate and the colony number was determined after a 2-d incubation at 30°C. Thermotolerance test Exponentially growing cells were transferred to glass tubes and incubated at 50°C. Every 3 min, aliquots of cell cultures were transferred into microtubes, cooled in ice, diluted serially (lo-fold at each step) in microtubes, and spotted onto a YPAD agar plate. After a 3-d incubation at 2O”C, colonies were photographed. Oxidative stress resistance test Exponentially growing cells were diluted serially (lo-fold at each step) in microtubes, and spotted onto a YPAD agar plate containing 3 mM hydrogen peroxide. After a 3-d incubation at 2O”C, colonies were photographed. High osmolarlty resistance test Exponentially growing cells were washed once with a 0.1 M phosphate buffer (pH 5.9), resuspended in YNB medium containing 3

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M NaCl, and incubated at 20°C. Every 6 h, aliquots of the cell suspension were spread on a YPAD agar plate after serial dilution. After a 3-d incubation at 2O”C, the colony number was counted and then the survival rate was calculated. GFP fluorescence microscopy A haploid segregant of SR4-3 was separated by random spore analysis. The haploid cells were spread onto a YNB agar plate containing 2mg/ml 5fluoroorotidic acid and 40mg/ml uracil in order to obtain uracil auxotroph mutants (16). After a one-week incubation at 2O”C, a mutant exhibiting both uracil auxotrophy and ethanol tolerance (u-5) was screened for further experiments. The u-5 cells were transformed using a pMsnZGFP, which contained a gene coding for the Msn2-GFP fusion protein and URA3 as a selection marker (17), and fluorescence of the cells was observed under a fluorescence microscope (Nikon ECLIPSE E600 Tokyo). Images were scanned with a chilled CCD camera (Hamamatsu Photonix) and analyzed with Adobe Photoshop 4.0 software. RESULTS Identification of genes highly expressed in the ethanoltolerant mutant by expression profiling Several ethanol-tolerant mutants have been isolated from industrial sake yeasts, e.g., Kll and SR4-3 are mutants of K7 and K701, respectively (1, 2). Since SR4-3 exhibited much more tolerance to ethanol than Kll (Fig. l), SR43 and K701 were used for the expression profiling analysis. SR4-3 has specific characteristics, such as Kl-killertoxin and zymolyase resistance, that are not observed in the parent strain at the exponential growth phase (2). Therefore, we analyzed the expression profile of the two strains at the exponential growth phase. Messenger RNA was extracted from exponentially growing cells of SR4-3 and K701 cultured under static condition, because sake brewing is carried out anaerobically. Messenger RNA was then converted to 33P-labeled single-stranded cDNA, and the resultant probe was hybridized with GeneFilters@. After autoradiography, the expression profile of every gene was compared between SR4-3 and K701 by visual observation. Although most genes were expressed at similar levels, there were several genes whose expression levels were very low or not detectable in K701 but were highly expressed in SR4-3. Typical profiles are shown

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in Fig. 2. From this result, we have noted that several stress-responsive genes such as GPDI, CTTI, CYC7, HSPI2, HOR7 and SPII were highly expressed in SR43. GPDl encodes glycerol-3-phosphate dehydrogenase, which is necessary for the adjustment of the intracellular osmolarity under high osmotic stress condition (18). CTTl encodes cytosolic catalase T, which is important for resistance to oxidative stress (19). CYC7 encodes iso2-cytochrome c, which transfers electrons to cytochrome c oxidase, and is induced by osmotic stress (20). HSPl2 encodes the protein induced by heat shock (21), and HOR7 is a gene induced under high osmolarity, but the functions of its protein product have not been clarified (22). SPII encodes a putative cell wall protein and is known to be induced at the stationary phase (23, 24). In addition to these genes, several others whose protein products’ functions have not been determined yet were also highly expressed in SR4-3 (data not shown). On the contrary, there were few genes whose expressions were very high in K701 but very low or not detectable in SR43 (data not shown). Northern blot analysis of the stress-responsive genes To confirm whether these stress-responsive genes are indeed highly expressed in SR4-3, transcription levels of

Filter I

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SR4-3 K701 SR4-3 Kll K7 FIG. 1. Ethanol tolerance of the mutant strains and their parent strains. Cells were cultured in YPAD medium for 2d, harvested, washed with sterilized water, and transferred to a buffer solution containing 20% (v/v) ethanol as described in Materials and Methods. Cell viability was determined after incubation for 7 d at 15°C.

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FIG. 2. Expression profiles of a ethanol-tolerant mutant, SR4-3, and its parent strain using GeneFilters” analysis. (A) Representative expression profile of SR4-3. (B) The section of the microarray indicated by boxes in Fig. 2A. Arrows indicate genes that were highly expressed in SR4-3 as compared to K701. Detailed information on GeneFilters” can be obtained from the homepage of the supplier (http://www.resgen.com/).

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GPDI CTTl HSPl2 SPII ACT1

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FIG. 3. Northern blot analysis of genes that were identified as highly expressed ones in SR4-3 using GeneFilters”. Yeast cells were cultured in YPAD medium at 20°C to the exponential phase. Cells were harvested and immediately analyzed (Lanes l-4) or further incubated for 2 h in YPAD medium containing 10% ethanol (Lanes 5-8). Total RNA was extracted from the cells, and gene expression was analyzed by Northern blotting. ACT1 mRNA was used as the control. Lanes 1 and 5, Strain SR4-3; lanes 2 and 6, strain Kll; lanes 3 and 7, strain K7; and lanes 4 and 8, strain K701.

GPDI, CTTI, HSPIZ, and SPII in exponentially growing cells were analyzed by Northern hybridization. Figure 3 shows that the expression levels of these genes in SR4-3 (Lane 1) were higher than those in the parent strain K701 (Lane 4). It has been reported that these genes were not normally expressed in exponentially growing cells but were expressed in the cells that were exposed to various stresses such as ethanol, heat, and high osmolarity, or nutrient starvation (22, 25, 26). Moreover, these genes have multiple STREs (stress-responsive elements; consensus CCCCT or AGGGG) in their promoter regions (27, 28), which are believed to be involved in general stress responses. Therefore, we examined whether or not the expression of these genes was induced by ethanol in the strains used in this study. The expression levels of these genes were increased in parent strains when they were cultured for 2 h in the medium containing 10% (v/v) ethanol (Lanes 7, S), indicating that these genes are indeed stress-induced in the sake yeasts. Surprisingly, the expression levels of these genes in SR4-3 were much higher (Lane 5) than those in the parent strain (Lane 8) under ethanol-stress condition. These results indicate that not only SR4-3 expresses several stress-responsive genes even at the vegetative phase but the expression levels of these genes are further increased under the stress conditions. As regards the other ethanol-tolerant mutant, Kll (Lanes 2, 6), the expression levels of some genes were slightly increased as compared with K7 (Lanes 3, 7); however, the increase in expression levels was not very significant as that observed in SR4-3 and K701. These results suggest that increased expression of the stress-responsive genes affects ethanol tolerance of SR4-3 and Kll corresponding to the extent of the increased expression. Accumulation of glycerol and catalase in SR4-3 Based on the increased expression of stress-responsive genes, we hypothesized that SR4-3 accumulates high amounts of stress-inducible substances even in the absence of stress. Because GPDI and CTTI are highly expressed in SR4-3, we analyzed the intracellular glycerol concentration and catalase activity in exponentially grow-

SR4-3 K11 K7 K701 FIG. 4. Intracellular glycerol contents and catalase activities in log-phase cells. (A) Intracellular glycerol contents. Exponentially growing cells were harvested, washed with water, resuspended in water, and boiled for 10 min. After centrifugation, supernatants were subjected to HPLC analysis. (B) Catalase activity. Exponentially growing cells were harvested, washed with water, resuspended in extraction buffer, and disrupted using glass beads. Catalase activity of cell extracts were assayed by measuring the decrease in the level of peroxide. Data represent mean values of at least three independent experiments and bars indicate SD.

ing cells. Figure 4 shows that the amount of intracellular glycerol in SR4-3 was 2.4-fold higher than that in K701, and a very high catalase activity was observed in SR4-3, while a low activity was observed in K701. As regards Kll, although glycerol accumulation and catalase activity were less than those in SR4-3, the amount of intracellular glycerol was 1.6-fold higher, and catalase activity of Kll was significantly higher than those of the parent strain, K7. These results indicate that the expression of stress-responsive genes results in the production of stress-inducible substances, e.g., glycerol and catalase, in ethanol-tolerant mutants even at the log phase like under stressful conditions. Trehalose is also accumulated in SR4-3 It has been suggested that trehalose functions not only as a carbohydrate reserve but also as a protectant that contributes to survival of yeasts under various stressful conditions (29). It has been reported that there is a close correlation between accumulation of trehalose and acquisition of thermotolerance in yeast (30). In trehalose biosynthesis, the catalytic subunit of the trehalose-6-phosphate synthase encoded by TPSI (31) and that of the trehalose-6-phosphate phosphatase encoded by TPS2 (32) play central roles. It has been reported that these two genes contain multiple STREs in their promoter regions (27, 33) and expression of these genes is induced by various stresses. Considering that many stress-responsive genes were induced in SR4-3, we expected that TPSI and TPS2 were

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SR4-3 Kli K7 K701 FIG. 5. (A) Northern blot analysis of TPSl and TPS2. Yeast cells were cultured in YPAD medium at 20°C. Exponentially growing cells (Lanes l-4) or cells which were transferred to YPAD medium containing 10% ethanol and further incubated for 2 h (Lanes 5-8) were harvested. Total RNA was extracted from the cells, and gene expression was analyzed by Northern blotting. ACT1 mRNA was used as the control (see Fig. 3). Lanes 1 and 5, Strain SR4-3; lanes 2 and 6, strain Kll; lanes 3 and 7, strain K7; and lanes 4 and 8, strain K701. (B) Intracellular trehalose content, Exponentially growing cells were harvested, washed with water, resuspended in water, and boiled for 10 min. After centrifugation, supernatants were subjected to HPLC analysis. Data represent mean values of at least three independent experiments and bars indicate SD.

also highly express,ed in SR4-3. However, the expression of TPSI and TPS2 in SR4-3 and K701 was similarly at a low level as analyzed using GeneFilters@. Therefore, we carried out Northern blot analysis of TPSl and TPS2 to reinvestigate whether TPSl and TPS2 were highly expressed in SR4-3. Figure 5A shows that both TPSl and TPS2 were expressed only in SR4-3 particularly in exponentially growing cells (Lane 1). The addition of ethanol to the medium resulted in a further increase in TPSl and TPS2 expression in all strains tested, indicating that these genes are indeed stress inducible. Expression levels of these genes in SR4-3 in the presence of ethanol were much higher than those in the parent strain. We expected that the increased expression of TPSl and TPS2 resulted in higher production of trehalose in SR4-3; therefore, we analyzed the intracellular trehalose amount. Figure 5B shows that the amount of intracellular trehalose in exponentially growing cells in SR4-3 was 4-fold higher than that in K701. However, no difference in the intracellular trehalose amount was observed between Kll and K7. These results suggest that trehalose accumulation is one of the reasons for ethanol tolerance of SR4-3. SR4-3 exhibits multiple stress resistance It is well known that yeast cells exposed to mild stress acquire multiple stress resistance (34-36). This phenomenon is called cross-protection in terms of stress responses and is caused by expression of stress-responsive genes under mild stressful conditions. SR4-3 showed higher ethanol tolerance than its parent strain, and substances related

Time (h)

FIG. 6. Multiple stress tolerance of ethanol-tolerant mutants. Cells were cultured in the YPAD medium at 20°C. (A) Thermotolerance. Exponentially growing yeast cultures were further incubated for 3, 6, 9, 12 min at 50°C diluted serially (IO-fold at each step), spotted onto a YPAD agar plate, and inoculated at 2O’C for 3 d. (B) Cell viability under high osmolarity stress condition. Exponentially growing cells were harvested and resuspended in YNB medium containing 3 M NaCI. Aliquots of cell suspension were spread onto a YPAD agar plate and incubated for 3 d. Closed squares, Strain SR4-3; closed circles, strain Kll; open circles, strain K7; and open squares, strain K701. (C) Growth under oxidative stress. Exponentially growing yeast cultures were diluted serially (lo-fold at each step), spotted onto a YPAD agar plate containing 3 mM hydrogen peroxide, and grown at 20°C for 5 d.

to stress tolerance were highly produced. Therefore, it is likely that SR4-3 exhibits resistance to other stresses in addition to ethanol tolerance. We measured tolerance of the ethanol-tolerant mutants to high temperature, osmotic stress and oxidative stress. First, thermotolerance was determined by measuring viability of exponentially growing cells after heat shock at 50°C. As shown in Fig. 6A, almost all cells of SR4-3 survived after heat treatment for 12mir-1, while viability of K701 and K7 was greatly decreased by the same treatment. Next, the survival after exposure to osmotic stress was analyzed. As shown in Fig. 6B, significant decrease in viability was observed when K701 was transferred to a medium containing 3 M NaCl, whereas no decrease in viability was observed in SR4-3 after exposure to the same osmotic stress. Finally, oxidative stress tolerance was analyzed by culturing the strains on the medium containing hydrogen peroxide. As shown in Fig. 6C, SR4-3 could grow on the

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medium containing 3 mM hydrogen peroxide, whereas K701 could not grow on the same medium. Kll also exhibited tolerance to heat and osmotic stress, but the degree of tolerance was much less than that of SR4-3. These results indicate that increased expression of the stress-responsive genes renders SR4-3 multiple stress resistant to heat, osmotic stress, oxidative stress, and ethanol. MsN2 expression and Msntp localization are normal in SR4-3 It has been reported that the zinc finger pro-

teins Msn2p and Msn4p are required for induction of the expression of a number of stress-responsive genes that have STREs in their promoter regions (28, 37). Under stressful conditions, Msn2p migrates from the cytosol to the nucleus and binds to STREs, which activate the transcription of stress-responsive genes (37, 38). Therefore, we hypothesized that increased expression of M&V2 or constitutive localization of Msn2p to the nucleus results in increased expression of many stress-responsive genes in SR4-3. To confirm this hypothesis, the transcription level of MSN2 in SR4-3 was compared with that in K701 at the exponential phase by Northern blot analysis, however, no difference was observed (Fig. 7A). Next, to investigate the localization of Msn2p, a haploid segregated from SR4-3 (u-5) was transformed with a plasmid encoding Msn2p tagged with green fluorescent protein (38) and the fluorescence of cells was observed (Fig. 7B). We have confirmed that the transformant exhibited the same characteristics as those of SR4-3, such as ethanol tolerance, and Kl-killer-toxin and zymolyase resistance (data not shown). In the exponentially growing cells, the fluorescence was observed to be diffused throughout the cytoplasm. After addition of ethanol to the medium, the fluorescence rapidly concentrated in the nucleus as reported in a wild-type laboratory strain (17). These results indicate that changes in A4SN2 expression or the localization of its product to the nucleus is not the reason for multiple stress tolerance of SR4-3 and that the strain must have an unknown mutation which causes increased expression of the stress-responA MSN2

No stress

10% Ethanol

FIG. 7. (A) Northern blot analysis of MSN2. Yeast cells were cultured in YPAD medium at 20°C. Total RNA was extracted from exponentially growing cells, and gene expression was analyzed by Northern blotting. ACTI mRNA was used as the control (see Fig. 3). Lane 1, Strain SR4-3; lane 2, strain Kll; lane 3, strain K7; and lane 4, strain K701. (B) Localization of Msn2p. The u-5 cells carrying pMsnZp-GFP were cultured in YPAD medium at 20°C. Msn2p-GFP fluorescence of exponentially growing cells or cells that were resuspended in YPAD medium containing 10% ethanol and further incubated for 2 h was observed under fluorescent microscope.

sive genes. DISCUSSION

Comparison of the expression profiles of every gene between SR4-3 and K701 and Northern blot analysis revealed that the stress-responsive genes, GPDI, CTTI, HSP12, SPII, TPSI and TPS2 were highly expressed in SR4-3 even at the log phase. The expression of these genes is usually induced only under stressful conditions or at the stationary phase. Expression of these genes further increased after addition of ethanol to the medium. Because of relatively low sensitivity of expression profiling method we have employed, which also does not allow quantification of expression levels, we may not have detected genes whose expression level was low in SR4-3 but significantly higher than that in K701. However, although we could not detect TPSI and TPS2 by the expression profiling method, we could detect the increased expression of these genes in SR4-3 by Northern blot analysis. Therefore, it is likely that other genes involved in stress responses are highly expressed in SR4-3. As a consequence of higher expression of these genes, stress-inducible substances, such as catalase, glycerol and trehalose were highly accumulated in SR4-3. Although the detailed mechanism of ethanol tolerance of SR4-3 is still unclear, we believe that the increased expression of the stress-responsive genes is a reason for ethanol tolerance of SR4-3. In particular, the accumulation of trehalose seems to contribute to ethanol tolerance of SR4-3, because it has been suggested that trehalose functions as a protectant against various stresses in yeasts (29). Increased expression of stress-responsive genes and accumulation of stress protectants suggest that SR4-3 may exhibit multiple stress resistance in addition to ethanol tolerance. Actually, SR4-3 showed resistance to other stresses such as heat shock, high osmolarity and oxidative stress. Another ethanol-tolerant mutant, Kll, also exhibited similar but less prominent resistance and tolerance than SR4-3, e.g., resistance to the Kl killer toxin and zymolyase, and ethanol tolerance (2). As expected, Kll showed increased expression of the stress-responsive genes and increased production of the stress-inducible substances compared with K7, which is the parent strain of Kll, however, the expression levels of these genes in Kll were significantly lower than those in SR4-3. It is likely that this lower induction of the stress-responsive genes is a reason for lower stress tolerance of Kll than that of SR4-3. It is noteworthy that an increased trehalose accumulation was observed only in SR4-3, but not in Kll. These results indicate that the expression levels of the stress-responsive genes agree well with the extent of ethanol and other stress tolerance. The common characteristic among ethanol-tolerant mutants is resistance to the Kl killer toxin and zymolyase (2). This characteristic is related to the cell wall structure because primary targets of these substances are cell wall components. The Kl killer toxin is adsorbed to the cell wall p-1, 6-glucan, followed by its internalization and action as a toxin (38). Zymolyase is a cell-walldegrading enzyme and mainly comprised ,8-l, 3-glucannase and protease (39). Among the genes whose expression is increased in SR4-3, SPIl encodes a cell wall protein whose expression is induced by various stresses (24). As a result of increased expression of SPII, the cell wall

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layer may be covered with this protein which is anchored to the cell wall by binding to P-1,6-glucan and ,3-1,3-glucan. Therefore, it is possible that a higher content of the cell wall protein renders ethanol-tolerant mutants resistant to the Kl killer toxin and zymolyase. It is well known that the expression of genes encoding some cell wall proteins and j-1,3-glucan-synthesizing enzymes is induced by stresses (40, 41). Therefore, the possibility that other genes related to cell wall components are highly expressed in the ethanol-tolerant mutant cannot be eliminated. What gene mutation is responsible for the ethanol tolerance in SR4-3? Although we have not yet identified such mutation by conventional gene cloning methods, some possible explanations can be inferred. Yeast cells under stressful conditions display two types of adaptive responses. One is a stress-specific response, and the other is a general stress response. It is well known that yeast cells exposed to one kind of mild stress can acquire multiple stress resistance (cross protection) in addition to acquisition of resistance to a subsequent more severe stress of the same kind (34-36). This cross-protection phenomenon is linked to the general stress response (42), in which the Ras-CAMP-PKA signaling pathway is involved (43). It has been reported that inactivation of protein kinase A (PKA) induces the expression of stress-responsive genes similar to that observed in SR4-3. These stress-responsive genes have c&element called STRE in its promoter region (27). Following inactivation of PKA, the CzHz zinc finger protein Msn2p is transferred from the cytoplasm to the nucleus, binds to STRE and activates transcription of the stress-responsive genes (28, 37). In fact, the genes highly expressed in SR4-3 contain multiple STREs in their promoter regions; therefore, it is conceivable that the STRE-dependent transcription pathway was activated in SR4-3. A mutation in ras2 or overexpression of PDE2 can cause low PKA activity and induce the expression of stress-responsive genes because of the resulting low concentration of CAMP (43). If this were the case in SR4-3, constitutive localization of Msn2p should be observed in SR4-3. However, the result shown in Fig. 7 revealed that localization of Msn2p was normal. Therefore, we conclude that the Ras-cAMPPKA pathway is normal in SR4-3. Although a higher basal expression of stress-responsive genes could be caused by other unknown factors, a further increase in expression of these genes by ethanol in SR4-3 is probably mediated by the Ras-CAMP-PKA pathway. Recently, it has been reported that DBF2 is associated with the control of stress-responsive genes (44). Overexpression of DBF2 causes increased expression of stressresponsive genes, resulting in acquisition of multiple stress tolerance, as observed in SR4-3. However, all the phenotypes observed in SR4-3 cannot be explained by overexpression of DBF2. DBF2 expression is not induced by oxidative stress, and tolerance to this stress is not increased by overexpression of DBF2 (44), whereas SR4-3 exhibits oxidative stress tolerance. Besides, it has been reported that Sok2p acts downstream of PKA and regulates stress-responsive genes negatively. Therefore, the sok2 disruptant shows increased expression of some stress-responsive genes (45). Preliminary genetic analysis of SR4-3 reveals that its Kl-killer-toxin resistant phenotype is recessive. A heterozygote obtained by crossing the haploid segregated from SR4-3 (u-5) with Kl-killertoxin sensitive laboratory strain (YPH500) showed Kl-

OF ETHANOL-TOLERANT

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OF SAKE YEAST

3 I!,

killer-toxin sensitive phenotype (data not shown). However, further genetic analysis was impossible because of poor sporulation and germination of the zygote. Based on the finding that Kl-killer-toxin resistance phenotype of SR4-3 was not complemented by the wild-type SOK2 (data not shown), a mutation in SOK2 was, therefore, not the reason for the phenotypes of SR4-3. Constitutive expression of the stress-responsive genes in the ethanol-tolerant mutants may have disadvantages for their growth, because accumulation of stress protectants is unnecessary under growth conditions without stresses. The ethanol-tolerant mutants showed slightly slow growth as compared with parent strains (1, 2). They also produced higher amounts of organic acid by unknown mechanisms. We could not determine the mutation in SR4-3 that confers on it multiple stress tolerance. However, we believe that identification of a gene responsible for the multiple stress tolerance of SR4-3 will provide further insight into stress response mechanisms in yeasts and will be very helpful in developing more sophisticated stress-tolerant industrial yeast strains. ACKNOWLEDGMENT We are grateful to Dr. Christoph Schuller (Vienna University) for kindly providing the fusion plasmid pMsn2-GFP. REFERENCES 1. Hara, S., Sasaki, M., Obata, T., and Noshiro, K.: Sot. Brew. Japan, 71, 301-304 (1976). (in Japanese) 2. Nitta, A., Uchiyama, H., and Imamura, T.: Breeding of ethanol tolerant sake yeasts from Kl killer-resistant mutants. Seibutsukogaku, 78, 77-81 (2000). 3. Hara, S., Yamamoto, N., Fukada, Y., Obata, T., and Noshiro, K.: Sot. Brew. Japan, 71, 564-568 (1976). (in Japanese) 4. Hutchins, K. and Bussey, H.: Cell wall receptor for yeast killer toxin: involvement of (I-6)-,‘i-D-glucan. J. Bacterial., 154, 161169 (1983). 5. Boone, C., Sommer, S. S., Hensel, A., and Bussey, H.: Yeast KRE genes provide evidence for a pathway of cell wall ;j-glucan assembly. J. Cell Biol., 110, 1833-1843 (1990). 6. Kitamura, K. and Yamamoto, Y.: Lysis of yeast cells showing low susceptibility to zymolyase. Agric. Biol. Chem., 45, 17611766 (1981). 7. Ingram, L.O. and Buttke, T. M,: Effects of alcohols on microorganisms. Adv. Microbial. Physiol., 4, 40-44 (1984). 8. Thomas, D. S., Hossack, J. A., and Rose, A. H.: Plasmamembrane lipid composition and ethanol tolerance. Arch. Microbial., 117, 239-245 (1978). 9. D’Amore, T. and Stewart, G. G.: Ethanol tolerance of yeast. Enzyme Microb. Technol., 9, 322-330 (1987). 10. Agudo, L.C.: Lipid content of Saccharomyces cerevisiae strains with different degrees of ethanol tolerance. Appl. Microbiol. Biotechnol., 37, 647-651 (1992). 11. Nakazawa, N., Harashima, S., and Oshima, Y.: Sot. Brew. Japan, 88, 354-356 (1993). (in Japanese) 12. Mewes, H. W., Albermann, K., Bahr, M., Frishman, D., Gleissner, A., Hani, J., Heumann, K., Kleine, K., Maierl, A., Oliver, S.G., Preiffer, F., and Zoller, A.: Overview of the yeast genome. Nature, 387, 7-73 (1997). 13. Cox, K. H., Pinchak, A. B., and Cooper, T. G.: Genome-wide transcriptional analysis in S. cerevisiae by mini-array membrane hybridization. Yeast, 15, 708-713 (1999). 14. Kohrer, K. and Domdey, H.: Preparation of high molecular weight RNA. Methods Enzymol., 194, 398-405 (1991). 15. Bradford, M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254 (1976).

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