FEMS MicrobiologyLetters 94 (1002)271-276 ,~t31992 Federation of European MicrobiologicalSocieties 0378-1007/02/$t15.00 Published by Elsevier
271
FEMSLE IM961
Construction of Saccharomyces cerevisiae strains that accumulate relatively low concentrations of trehalose, and their application in testing the contribution of the disaccharide to stress tolerance P a u l V. A t t f i e l d , A n a R a m a n a n d C a r o l y n J. N o r t h c o t t Yeast Physiology.' Group. Burns Philo Technolo,~" and Research ('emre, North Rvth'. New S~mth Ii~h's. ,4ustralia
Received 3 April 1092 Revision received 6 May It;02 Accepted 6 May 1002 Key words: Trehalose; Stress response; Thermotolerance: Freeze-thaw tolerance; Saccharon~yces ceret'isiae
1. S U M M A R Y
2. I N T R O D U C T I O N
Genetically related diploid strains of Saccharomyces ceret'isiae that accumulate varied amounts
T r e h a l o s e (a-D-glucopyranosyl a-D-glucopyranoside) accumulates in vegetative Saccharomyces cerevisiae under conditions of reduced growth rate. nutrient limitation, or exposure to physicochemical stresses. Cells starved specifically of carbon, nitrogen, phosphorous or sulphur accumulate large quantities of the sugar [1]. Various workers have reported the accumulation of trehalose by yeast exposed to heat, ethanol, oxidants and heavy metals [2-7]. The disaccharide is also found in ascospores and is apparently essential for germination [8]. It is evident from these studies that trehalose accumulates in cells undergoing physiological transition, differentiation or stress. Trehalose offers a potential energy reserve for cells and may even function in moderating glycolytic flux under certain circumstances [1,9,10]. There is increasing evidence for the additional and perhaps primary role of trehalose as a cellular protectant during stress. It can protect artificial m e m b r a n e s and isolated enzymes during
of trehalose during starvation for nitrogen have been constructed. Strains that produced > 5% trehalose (dry cell weight) were more tolerant of thermal, or freeze-thaw stresses than strains that produced < 4% trehalose. Thus trehalose appears to play a role in stress tolerance of yeast. The significance of these results is that, for the first time, a series of related, unmutated strains have been used to test the effect of trehalose on thermotolerance. Previous studies employed either heat shock treatment, or mutated strains to provide trehalose variations, and as such the contribution of the disaccharide to stress tolerance could not necessarily be separated from other factors such as heat shock proteins.
Correspondence to: P.V. Attfield, Yeast Physiology Group,
Burns Philo Technology and Research Centre, PO Box 210, North Ryde, NSW 2113, Australia.
272 freezing or dehydration [11,12]. Yeast storing high levels of trehalose, or with the disaccharide added exogenously, show high tolerance to freezing and desiccation [13-16]. it has been reported that yeast cells that accumulate trehalose during heat shock acquire thermotolerance [4,5,14,17]. However, experiments in which yeast with varying capacities for storage of trehalose were heat stressed in the absence of previous heat shock treatment, failed to prove whether the disaccharide is responsible for acquisition of thermotolerance [6,7]. Studies on the possible role of trchalose in thermotolerance have employed either heat shock treatment to force trehalose accumulation, or diverse mutant strains that are deficient in control of nutaerous activities including trehalose metabolising enzymes [4-7,14]. A problem with the former approach is that it is very difficult to distinguish between the possible roles of trehalose and heat shock proteins, or other thermally induced factors, in stress tolerance when a thermal stress has already been used as a means of inducing trehalose accumulation. A problem with applying diverse, mutated strains in comparative studies is that stress tolerance may be affected by altered strain properties that are unknown and therefore uncontrolled. In an attempt to overcome these problems we have adopted the approach of generating a series of strains that are related in terms of their genetic origin and that produce various levels of trehalose u n d e r conditions of nitrogen starvation. This p a p e r reports the construction of these strains and their application to the study of the role of trehalose in stress tolerance.
3. M A T E R I A L S A N D M E T H O D S 3.1. Y e a s t strains, m e d i a a n d c u l t u r e c o n d i t i o n s
Table 1 lists the strains used in this work. Standard genetic methods were employed in strain construction [18]. Complete minimal medium comprised 2% ( w / v ) glucose, Difco yeast nitrogen base without amino acids at the concentration recommended by the manufacturer, and 50 m g / l adenine sulphate. Amino acids were
Table I Saccharomyces cerevisiae strains used in this work, and tre-
halose accumulated during starvation for nitrogen Strain
Genotype or derivation Haploids AH22 a leu2 his4 malO DE6.1D a ade2 lys2 MAL6 YNI38 aade21en2his4MAL6 a ade2 leu2 his4 malO YNI41 a ade2 leu2 malO YNI43 YNI45 a leu2 his4 MALO YNI46 a leu2 his4 malO YNI48 a ade2 malO YN149 a l y s 2 M A L 6 Diploids YNI50 AH22× DE6.1D YN154 YNI45 × DE6.1D YNI46× DE6.1D YNI55 YNI56 AH22 × YN 148 YNI57 AH22 × YN 149 YNI58 YN138 X DE6.1D ade2/ade2
YNI59 YNI60
4.52 (I).78) I).46 (0.21) 0.49(0.19) 11.66({}.23) 1.69 (0. I I ) 5.17 (0.52) 5.33 ( I. 10) 5.99 ({).76) 3.95 (0.21)
[2] [19] This work This work This work This work This work This work This work
6.45(11.19) 5.53(I).43) S.58 (2.41) 9.{)3(1.73) 6.78 (0.78)
This work This work This work This work This work
3.8(I (1.02)
This work
2.Sl (0.90)
This work
2.41 (0.22) 8.38 (0.92) 7.17(0.10) 7.18(2.01) 8.28 (2.25)
This work This work This work This work This work
YNI41 × DE6.1D ade2/ade2
YNI43 X DE6.1D ade2/ade2
YNI61 YNI62 YNI63 YNI64
% Trehalose Sourceor (drycell wt) reference
YN 145 x YN 148 YNI46× Y N I 4 8 YNI45 × Y N 1 4 9 YN 146 x YN 149
Trehalose levels shown are means of 3 to 5 replicates. Figures in brackets are +_standard deviations.
added at 100 m g / I where required. Nitrogenstarvation medium consisted of 0.3% ( w / v ) glucose, Difco yeast nitrogen base without amino acids or ammonium sulphate at the recomm e n d e d concentration, 50 m g / I adenine sulphate, and 20 mM K H 2 P O 4 adjusted to pH 6.0 with NaOH. Liquid cultures were incubated at 30°C and 180 rpm with medium to conical flask volume ratio of 1:5. Solid medium for testing survivors of stress treatments was 1% ( w / v ) glucose, 0.5% ( w / v ) yeast extract, 1% ( w / v ) bacteriological peptone, 50 m g / I adenine sulphate, and 1.5% ( w / v ) bacteriological agar. Plates were placed at 30°C for 3 to 5 days prior to counting colonies.
273
3.2. Nitrogen-start'ation protocol Single colony inocula were grown in 10 ml complete minimal medium for 6 h. These cultures were diluted to 2 x 105/ml into 50 ml prewarmcd and aerated complete minimal medium and incubated to a density of 5-10 x 107/ml. They were then harvested by centrifugation at 3000 × g and room temperature (25°C), washed twice by centrifugation in sterile distilled water at room temperature and resuspended in 50 ml prewarmed and aerated nitrogen-starvation medium. Nitrogen-starvation cultures were incubated for 20 h.
3.3. Heat stress protocol Cells were heat stressed following removal of 4 ml culture from incubation medium and transfer to 22-mm diameter glass test tubes. Samples were heated rapidly to 50°C in a 65°C water bath before immediate transfer to a 52°C water bath for 5 min. Samples were shaken occasionally during this incubation to keep cells suspended. Stress was relieved by removal of tubes to an ice-water bath and cooling to 25°C. Sample temperature was monitored throughout these procedures by thermometer. Followitt?, alleviation of heat stress, cells were diluted in one-quarter strength Ringer's solution at room temperature and plated immediately on solid medium. Survival was determined by comparison with viable counts made of unstressed culture samples.
3.4. Freeze-thaw stress protocol Cells were subjected to freeze-thaw stress by removal of 1 ml cult,ire from incubation medium and transfer to sterile Eppendorf tubes, which were then plunged into liquid nitrogen after having caps pierced once using a sterile syringe needle. Tubes were removed after 5 min and placed in a 30°C water bath for 10 min. The process was then repeated. After the second cycle of freezing and thawing, cells were diluted and plated as described in 3.3. Analysis of cells by light microscopy revealed no evidence of cell clumping either before or after heat or freezethaw stress treatments.
3.5. Assay of trehalose Trehalose was extracted with trichloroacetic
acid and assayed by the anthrone method as described previously [2].
4. RESULTS AND DISCUSSION Capacities of the various strains for storage of trehalose during starvation for nitrogen are shown in Table 1. The haploid strains AH22 and DE6.1D showed strong and weak trehalose accumulation phenotypes, respectively. Failure of DE6.1D to store a large amount of trehalose under conditions of nonproliferation has been reported previously [19]. Strains AH22 and DE6.1D were mated to give diploid strain YNI50, which was subsequently sporulated. Asci were pooled, disrupted, the spores plated and haploid yeast isolated. Several of these were picked and screened using the. nitrogen-starvation protocol to identify high or low trehalose storage phenotypes (Table 1). Haploids with defined trehalose accumulation properties were then mated to give the series of genetically related diploid strains also listed in Table 1. The nature of the low trehalose accumulation phenotype associated with DE6.1D is uncertain. This strain produces low levels of trehalose-6phosphate synthase under conditions normally expected to produce high levels of this activity ([19] and unpublished results of this laboratory). Trehalose concentration is dependent upon the balance between trehalose-6-phosphate synthase and trehalases [10,20-22]. Neutral trehalase is activated by cAMP-dependent phosphorylation [23-26] and trehalose-6-phosphate synthase is apparently less active when phosphorylated [22,27]. There is evidence that the original mutant strain from which the low trehalose accumulation phenotype associated with DE6.1D was derived has unusually high levels of enzymes in their phosphorylated states [28], producing high trehalase but low trehalose-6-phosphate synthase activities. Thus the reduced capacities for trehalose accumulation in DE6.1D and its derivatives (Table 1) may be due to impaired post-translational control of trehalose synthetic and degradative enzymes, rather than mutations in the genes for these enzymes. Trehalose levels of diploid strains derived from two haploids exhibiting strong accu-
274
mulation were not significantly greater than those in diploids derived from one haploid with strong and one with weak trehalose accumulation phenotypes (Table 1). Moreover the low trehalose storage characteristic appears to be recessive since, in all crosses involving weak and strong trehalose-accumulating haploids, the strong accumulation phenotype was expressed in resultant diploids (Table 1). Furthermore, combinations of two weak trehalose-accumulating haploids gave rise to diploids that produced relatively low but significant levels of the disaccharide, implying that the low storage characteristic is 'leaky'. These observations could be expected if DE6.1D does
Iot
I i
i
i
i
Fig. 2. Survival of Saceharon~ycescerecisiae strains exposed to freeze-thaw stress. Strains were starved fl~r nitrogen, exposed to two cycles of freezing in liquid nitrogen and thawing at 30°C, and subsequently plated as described in MATERIAI.SAND METIIODS. Other details are as described in the legend to Fig.
I,
)-
)-
)..
)-
)-
)..
)..
~.
~
)-
Fig. I. Survival of Saeeharomyces eerecisiae strains exposed to thermal stress. Strains were starved for nitrogen, exposed to 52°C for 5 min and subsequently plated for survivors as described in MATERIAI.S AND METIIODS. Values are means of three replicated experiments+_standard deviations. 100q~- viable counts of unstressed, control cultures varied between 4 × 1 0 7 and 1 . 2 x l 0 s c f u / m l . See Table I for relative trehalose accumulation by these strains.
have impaired enzyme-phosphorylation control of trehalose levels. Construction of a series of strains derived from the same gene pool but exhibiting varied trehalose storage capacities, opens the way for specific testing of the rolc of trehalose in s:ress tolerance. The diploid strains shown in Table 1 were tested for their abilities to survive either a 52°C heat stress for 5 min (Fig. 1), or cycles of freezing in liquid nitrogen and thawing at 30°C (Fig. 2). Strains YN158, YNI59 and YN160, which were derived from crosses between haploids of low trehalose accumulation type, and which stored relatively low levels of the disaccharide under conditions of nitrogen-starvation (Table 1), showed greater sensitivities than other strains to both
275 tivcly scnsitivc to these stresses (Figs. i-31. T h e s e results imply that trehalose plays a role in survival of yeast exposed to stress, but that for these strains at least, its c o n c e n t r a t i o n is not critical above a certain threshold.
D
O
ACKNOWLEDGEMENT
D
D
T h e a u t h o r s thank the M a n a g e m e n t of B u r n s Philp R & D Pty. Ltd. for their kind permission to publish this work.
REFERENCES [1] Lillic. S.II. and Pringle. J,R. (1980) J. Bacteriol, 143.
1384-13q4.
A
[2] Attficld. P.V. 11987) FEBS Lett. 225. 259-263.
& A
A
&
D ~A
IA
•
t
Trehalose (70 Dry Cell Weight) Fig. 3. Relationship between trehalose concentration and post-stress survival. Data taken from Table I and Figs. 1 and 2. D. thermal stress; • , frecze-thav, stress.
stress types (Figs. 1 and 2). T h e s e results suggest that t r c h a l o s e can e n h a n c e thermal and freezethaw tolerances. Moreover, it is possible to be r e a s o n a b l y confident a b o u t such a role for the disaccharide w h e n using the described strains, b e c a u s e potential effects of heat shock p r o t e i n s and u n k n o w n variable factors o c c u r r i n g in less closely related and m u t a t e d strains are reduced. T h i s c a n n o t be said of previous studies w h e r e heat shock t r e a t m e n t s and various m u t a n t s of m o r e diverse genetic b a c k g r o u n d were e m p l o y e d [4-7,14,17]. Notably no strict correlation was observed b e t w e e n survival and a m o u n t of t r e h a l o s c stored, b e y o n d a certain level. T h o s e strains able to a c c u m u l a t e trehalose at > 5% (dry cell weight) w e r e comparatively resistant to the t h e r m a l or f r e e z e - t h a w stresses, w h e r e a s those strains producing the disaccharide at < 4% were c o m p a r a -
[3] Grba. S.. Oura. E. md Suomalainen. tl. (19751 Eur. J. Appl. Microbiol. 2. 29-37. [41 Ilottiger. T.. Schmutz, P. and Wiemken, A. (19~71 J. Bacteriol. 169, 5518-5522. [5] Hottigcr, T.. Bollel. T. and Wiemkcn, A. 119891 FEBS Left. 255, 431-434. [6] Panek. A.D.. Ferrcira, R. and Panel A.C. 11989) Biochimie 71,313-318. [7] Panck. A.C.. Mansure-Vania. J.J.. Paschoalin, M.F. and Panek, A.D. (199111 Biochimic 72, 77-79. [81 Panek, A.D. and Bernades. E.J. (19831 ('urr. Oenct. 7, 393-397. [9] Panek. A.D. and Mattoon, J.R. 11977) Arch. Biochem. Bit)phys. 183. 3116-316. [10] Panek, A.D. 11985) J. Biotechnol. 3, 121-1311. [I I] Rudolph, A. and Crowe, J.H. 119851 Cu, obiol. 22, 367377. [12] ('rowe. J.II.. ('rowe, L.M., Carpenter. J.F. and Aur¢llWistrom, C. (19871 Biochem. J. 242. I-111. [13] Gadd, G.M., ('hailers. K. and Reed, R.H. (1987) FEMS Micr~biol. Lclt. 48, 249-254. [14] tloltiger. T., Boiler, T. and Wiemken, A. 119871 FEBS Lett. 220, 113-115. [15] Gclinas. P.. Fiset. G.. keDuy, A, and Goulet, J. (1989) Appl. Environ. Microbiol. 55. 2453-2459. [16] Oda, Y.. Uno, K. and Ohta, S. (198G) AOOI. Environ. Microbiol. 52. 941-943. [17] DeVirgilio. C., Piper. P., Boiler. T. and Wiemken. A. (1991) FEBS Lett. 288, 86-90. [18] Sherman, F.. Fink. G.R. and tlick, J.B. 11981) Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spriag Harbor NY. [19] Operti. M.S.. Olivcira. D.E., Freitas-Valle, A.B.. Oestreicher, E.G.. Mattoon. J.R. and Panel A.D. 119821 Curr. Genet. 5. 6,9-76.
276 [20] Thevelein, J.M. (1984) Microbiol. Rev. 48, 42-59, [21] Wiemken. A. 0990) Antonie Van Leeuwenhoek 58, 209217. [22] Panek. A.D. and Panek. A.C. 0990) J. Biotechnol. 14. 229-238. [23] Uno. 1., Matsumoto. K., Adachi. K. and ]shikawa, T. (1983) J. Biol. Chem. 258, 10867-10872. [24] Thevelein. J.M. and Beullens. M. (1985) J. Gen. Microbiol. 131. 3199-3209.
[25] Wiemken, A. and Schellenberg, M. 0982) FEBS Left. 150. 329-331. [26] Francois, J., Eraso, P. and Gancedo, C. (1987) Eur. J. Biochem. 164, 369-373. [27] Panek, A.C.. DeAraujo, P.S.. Moara-Neto, V. and Panek, A.D. (1987) Curr. Genet. I1. 459-465. [28] Panek, A.C., Francois. J. and Panek, A.D. (1988) Curt. Genel. 13, 15-2(I.
271
FEMSLE IM961
Construction of Saccharomyces cerevisiae strains that accumulate relatively low concentrations of trehalose, and their application in testing the contribution of the disaccharide to stress tolerance P a u l V. A t t f i e l d , A n a R a m a n a n d C a r o l y n J. N o r t h c o t t Yeast Physiology.' Group. Burns Philo Technolo,~" and Research ('emre, North Rvth'. New S~mth Ii~h's. ,4ustralia
Received 3 April 1092 Revision received 6 May It;02 Accepted 6 May 1002 Key words: Trehalose; Stress response; Thermotolerance: Freeze-thaw tolerance; Saccharon~yces ceret'isiae
1. S U M M A R Y
2. I N T R O D U C T I O N
Genetically related diploid strains of Saccharomyces ceret'isiae that accumulate varied amounts
T r e h a l o s e (a-D-glucopyranosyl a-D-glucopyranoside) accumulates in vegetative Saccharomyces cerevisiae under conditions of reduced growth rate. nutrient limitation, or exposure to physicochemical stresses. Cells starved specifically of carbon, nitrogen, phosphorous or sulphur accumulate large quantities of the sugar [1]. Various workers have reported the accumulation of trehalose by yeast exposed to heat, ethanol, oxidants and heavy metals [2-7]. The disaccharide is also found in ascospores and is apparently essential for germination [8]. It is evident from these studies that trehalose accumulates in cells undergoing physiological transition, differentiation or stress. Trehalose offers a potential energy reserve for cells and may even function in moderating glycolytic flux under certain circumstances [1,9,10]. There is increasing evidence for the additional and perhaps primary role of trehalose as a cellular protectant during stress. It can protect artificial m e m b r a n e s and isolated enzymes during
of trehalose during starvation for nitrogen have been constructed. Strains that produced > 5% trehalose (dry cell weight) were more tolerant of thermal, or freeze-thaw stresses than strains that produced < 4% trehalose. Thus trehalose appears to play a role in stress tolerance of yeast. The significance of these results is that, for the first time, a series of related, unmutated strains have been used to test the effect of trehalose on thermotolerance. Previous studies employed either heat shock treatment, or mutated strains to provide trehalose variations, and as such the contribution of the disaccharide to stress tolerance could not necessarily be separated from other factors such as heat shock proteins.
Correspondence to: P.V. Attfield, Yeast Physiology Group,
Burns Philo Technology and Research Centre, PO Box 210, North Ryde, NSW 2113, Australia.
272 freezing or dehydration [11,12]. Yeast storing high levels of trehalose, or with the disaccharide added exogenously, show high tolerance to freezing and desiccation [13-16]. it has been reported that yeast cells that accumulate trehalose during heat shock acquire thermotolerance [4,5,14,17]. However, experiments in which yeast with varying capacities for storage of trehalose were heat stressed in the absence of previous heat shock treatment, failed to prove whether the disaccharide is responsible for acquisition of thermotolerance [6,7]. Studies on the possible role of trchalose in thermotolerance have employed either heat shock treatment to force trehalose accumulation, or diverse mutant strains that are deficient in control of nutaerous activities including trehalose metabolising enzymes [4-7,14]. A problem with the former approach is that it is very difficult to distinguish between the possible roles of trehalose and heat shock proteins, or other thermally induced factors, in stress tolerance when a thermal stress has already been used as a means of inducing trehalose accumulation. A problem with applying diverse, mutated strains in comparative studies is that stress tolerance may be affected by altered strain properties that are unknown and therefore uncontrolled. In an attempt to overcome these problems we have adopted the approach of generating a series of strains that are related in terms of their genetic origin and that produce various levels of trehalose u n d e r conditions of nitrogen starvation. This p a p e r reports the construction of these strains and their application to the study of the role of trehalose in stress tolerance.
3. M A T E R I A L S A N D M E T H O D S 3.1. Y e a s t strains, m e d i a a n d c u l t u r e c o n d i t i o n s
Table 1 lists the strains used in this work. Standard genetic methods were employed in strain construction [18]. Complete minimal medium comprised 2% ( w / v ) glucose, Difco yeast nitrogen base without amino acids at the concentration recommended by the manufacturer, and 50 m g / l adenine sulphate. Amino acids were
Table I Saccharomyces cerevisiae strains used in this work, and tre-
halose accumulated during starvation for nitrogen Strain
Genotype or derivation Haploids AH22 a leu2 his4 malO DE6.1D a ade2 lys2 MAL6 YNI38 aade21en2his4MAL6 a ade2 leu2 his4 malO YNI41 a ade2 leu2 malO YNI43 YNI45 a leu2 his4 MALO YNI46 a leu2 his4 malO YNI48 a ade2 malO YN149 a l y s 2 M A L 6 Diploids YNI50 AH22× DE6.1D YN154 YNI45 × DE6.1D YNI46× DE6.1D YNI55 YNI56 AH22 × YN 148 YNI57 AH22 × YN 149 YNI58 YN138 X DE6.1D ade2/ade2
YNI59 YNI60
4.52 (I).78) I).46 (0.21) 0.49(0.19) 11.66({}.23) 1.69 (0. I I ) 5.17 (0.52) 5.33 ( I. 10) 5.99 ({).76) 3.95 (0.21)
[2] [19] This work This work This work This work This work This work This work
6.45(11.19) 5.53(I).43) S.58 (2.41) 9.{)3(1.73) 6.78 (0.78)
This work This work This work This work This work
3.8(I (1.02)
This work
2.Sl (0.90)
This work
2.41 (0.22) 8.38 (0.92) 7.17(0.10) 7.18(2.01) 8.28 (2.25)
This work This work This work This work This work
YNI41 × DE6.1D ade2/ade2
YNI43 X DE6.1D ade2/ade2
YNI61 YNI62 YNI63 YNI64
% Trehalose Sourceor (drycell wt) reference
YN 145 x YN 148 YNI46× Y N I 4 8 YNI45 × Y N 1 4 9 YN 146 x YN 149
Trehalose levels shown are means of 3 to 5 replicates. Figures in brackets are +_standard deviations.
added at 100 m g / I where required. Nitrogenstarvation medium consisted of 0.3% ( w / v ) glucose, Difco yeast nitrogen base without amino acids or ammonium sulphate at the recomm e n d e d concentration, 50 m g / I adenine sulphate, and 20 mM K H 2 P O 4 adjusted to pH 6.0 with NaOH. Liquid cultures were incubated at 30°C and 180 rpm with medium to conical flask volume ratio of 1:5. Solid medium for testing survivors of stress treatments was 1% ( w / v ) glucose, 0.5% ( w / v ) yeast extract, 1% ( w / v ) bacteriological peptone, 50 m g / I adenine sulphate, and 1.5% ( w / v ) bacteriological agar. Plates were placed at 30°C for 3 to 5 days prior to counting colonies.
273
3.2. Nitrogen-start'ation protocol Single colony inocula were grown in 10 ml complete minimal medium for 6 h. These cultures were diluted to 2 x 105/ml into 50 ml prewarmcd and aerated complete minimal medium and incubated to a density of 5-10 x 107/ml. They were then harvested by centrifugation at 3000 × g and room temperature (25°C), washed twice by centrifugation in sterile distilled water at room temperature and resuspended in 50 ml prewarmed and aerated nitrogen-starvation medium. Nitrogen-starvation cultures were incubated for 20 h.
3.3. Heat stress protocol Cells were heat stressed following removal of 4 ml culture from incubation medium and transfer to 22-mm diameter glass test tubes. Samples were heated rapidly to 50°C in a 65°C water bath before immediate transfer to a 52°C water bath for 5 min. Samples were shaken occasionally during this incubation to keep cells suspended. Stress was relieved by removal of tubes to an ice-water bath and cooling to 25°C. Sample temperature was monitored throughout these procedures by thermometer. Followitt?, alleviation of heat stress, cells were diluted in one-quarter strength Ringer's solution at room temperature and plated immediately on solid medium. Survival was determined by comparison with viable counts made of unstressed culture samples.
3.4. Freeze-thaw stress protocol Cells were subjected to freeze-thaw stress by removal of 1 ml cult,ire from incubation medium and transfer to sterile Eppendorf tubes, which were then plunged into liquid nitrogen after having caps pierced once using a sterile syringe needle. Tubes were removed after 5 min and placed in a 30°C water bath for 10 min. The process was then repeated. After the second cycle of freezing and thawing, cells were diluted and plated as described in 3.3. Analysis of cells by light microscopy revealed no evidence of cell clumping either before or after heat or freezethaw stress treatments.
3.5. Assay of trehalose Trehalose was extracted with trichloroacetic
acid and assayed by the anthrone method as described previously [2].
4. RESULTS AND DISCUSSION Capacities of the various strains for storage of trehalose during starvation for nitrogen are shown in Table 1. The haploid strains AH22 and DE6.1D showed strong and weak trehalose accumulation phenotypes, respectively. Failure of DE6.1D to store a large amount of trehalose under conditions of nonproliferation has been reported previously [19]. Strains AH22 and DE6.1D were mated to give diploid strain YNI50, which was subsequently sporulated. Asci were pooled, disrupted, the spores plated and haploid yeast isolated. Several of these were picked and screened using the. nitrogen-starvation protocol to identify high or low trehalose storage phenotypes (Table 1). Haploids with defined trehalose accumulation properties were then mated to give the series of genetically related diploid strains also listed in Table 1. The nature of the low trehalose accumulation phenotype associated with DE6.1D is uncertain. This strain produces low levels of trehalose-6phosphate synthase under conditions normally expected to produce high levels of this activity ([19] and unpublished results of this laboratory). Trehalose concentration is dependent upon the balance between trehalose-6-phosphate synthase and trehalases [10,20-22]. Neutral trehalase is activated by cAMP-dependent phosphorylation [23-26] and trehalose-6-phosphate synthase is apparently less active when phosphorylated [22,27]. There is evidence that the original mutant strain from which the low trehalose accumulation phenotype associated with DE6.1D was derived has unusually high levels of enzymes in their phosphorylated states [28], producing high trehalase but low trehalose-6-phosphate synthase activities. Thus the reduced capacities for trehalose accumulation in DE6.1D and its derivatives (Table 1) may be due to impaired post-translational control of trehalose synthetic and degradative enzymes, rather than mutations in the genes for these enzymes. Trehalose levels of diploid strains derived from two haploids exhibiting strong accu-
274
mulation were not significantly greater than those in diploids derived from one haploid with strong and one with weak trehalose accumulation phenotypes (Table 1). Moreover the low trehalose storage characteristic appears to be recessive since, in all crosses involving weak and strong trehalose-accumulating haploids, the strong accumulation phenotype was expressed in resultant diploids (Table 1). Furthermore, combinations of two weak trehalose-accumulating haploids gave rise to diploids that produced relatively low but significant levels of the disaccharide, implying that the low storage characteristic is 'leaky'. These observations could be expected if DE6.1D does
Iot
I i
i
i
i
Fig. 2. Survival of Saceharon~ycescerecisiae strains exposed to freeze-thaw stress. Strains were starved fl~r nitrogen, exposed to two cycles of freezing in liquid nitrogen and thawing at 30°C, and subsequently plated as described in MATERIAI.SAND METIIODS. Other details are as described in the legend to Fig.
I,
)-
)-
)..
)-
)-
)..
)..
~.
~
)-
Fig. I. Survival of Saeeharomyces eerecisiae strains exposed to thermal stress. Strains were starved for nitrogen, exposed to 52°C for 5 min and subsequently plated for survivors as described in MATERIAI.S AND METIIODS. Values are means of three replicated experiments+_standard deviations. 100q~- viable counts of unstressed, control cultures varied between 4 × 1 0 7 and 1 . 2 x l 0 s c f u / m l . See Table I for relative trehalose accumulation by these strains.
have impaired enzyme-phosphorylation control of trehalose levels. Construction of a series of strains derived from the same gene pool but exhibiting varied trehalose storage capacities, opens the way for specific testing of the rolc of trehalose in s:ress tolerance. The diploid strains shown in Table 1 were tested for their abilities to survive either a 52°C heat stress for 5 min (Fig. 1), or cycles of freezing in liquid nitrogen and thawing at 30°C (Fig. 2). Strains YN158, YNI59 and YN160, which were derived from crosses between haploids of low trehalose accumulation type, and which stored relatively low levels of the disaccharide under conditions of nitrogen-starvation (Table 1), showed greater sensitivities than other strains to both
275 tivcly scnsitivc to these stresses (Figs. i-31. T h e s e results imply that trehalose plays a role in survival of yeast exposed to stress, but that for these strains at least, its c o n c e n t r a t i o n is not critical above a certain threshold.
D
O
ACKNOWLEDGEMENT
D
D
T h e a u t h o r s thank the M a n a g e m e n t of B u r n s Philp R & D Pty. Ltd. for their kind permission to publish this work.
REFERENCES [1] Lillic. S.II. and Pringle. J,R. (1980) J. Bacteriol, 143.
1384-13q4.
A
[2] Attficld. P.V. 11987) FEBS Lett. 225. 259-263.
& A
A
&
D ~A
IA
•
t
Trehalose (70 Dry Cell Weight) Fig. 3. Relationship between trehalose concentration and post-stress survival. Data taken from Table I and Figs. 1 and 2. D. thermal stress; • , frecze-thav, stress.
stress types (Figs. 1 and 2). T h e s e results suggest that t r c h a l o s e can e n h a n c e thermal and freezethaw tolerances. Moreover, it is possible to be r e a s o n a b l y confident a b o u t such a role for the disaccharide w h e n using the described strains, b e c a u s e potential effects of heat shock p r o t e i n s and u n k n o w n variable factors o c c u r r i n g in less closely related and m u t a t e d strains are reduced. T h i s c a n n o t be said of previous studies w h e r e heat shock t r e a t m e n t s and various m u t a n t s of m o r e diverse genetic b a c k g r o u n d were e m p l o y e d [4-7,14,17]. Notably no strict correlation was observed b e t w e e n survival and a m o u n t of t r e h a l o s c stored, b e y o n d a certain level. T h o s e strains able to a c c u m u l a t e trehalose at > 5% (dry cell weight) w e r e comparatively resistant to the t h e r m a l or f r e e z e - t h a w stresses, w h e r e a s those strains producing the disaccharide at < 4% were c o m p a r a -
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