Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae

Abstract

We examined the role of intracellular proline under freezing and desiccation stress conditions in Saccharomyces cerevisiae. When cultured in liquid minimal medium, the proline-nonutilizing mutant containing the put1 mutation (proline oxidase-deficient) produced more intracellular proline, and increased the cell survival rate as compared to the wild-type strain after freezing and desiccation. We also constructed two PUT1 gene disruptants. PUT1-disrupted mutants in minimal medium supplemented with external proline at 0.1% accumulated higher proline levels than those of the control strains (17–22-fold). These disruptants also had a 2–5-fold increase in cell viability compared to the control strains after freezing and desiccation stresses. These results indicate that proline has a stress-protective function in yeast.

Gene disruption, Proline oxidase, Stress resistance, Saccharomyces cerevisiae

1 Introduction

Cellular freezing injury is of two general types [1,2]. In the first, slower cooling rates cause osmotic shrinkage of a cell and freeze extracellular water. In the second, dehydration occurs and biological macromolecules and/or membrane components denature. More rapid freezing does not permit the transport of intracellular water through the membrane, and impairs membrane structure or function as ice crystals form in the cells. Therefore, these observations suggest that freezing, desiccation and osmotic stresses, so-called water stresses, provide common deleterious damages to the cell membrane and functional proteins [3].

We have investigated the cryoprotective effect of amino acids on freezing stress in Saccharomyces cerevisiae, and found that proline, known as an osmoprotectant, has cryoprotective activity nearly equal to that of glycerol or trehalose. We also isolated freeze-tolerant mutants with higher levels of intracellular proline from proline analogue-resistant mutants [4]. S. cerevisiae can grow on proline as the sole source of nitrogen. Proline utilization requires two enzymes, proline oxidase and Δ¹-pyrroline-5-carboxylate (P5C) dehydrogenase, to convert proline into glutamate [5–7]. Therefore, we expect that the put1 mutation, which inactivates proline oxidase, or the disruption of the PUT1 gene would result in intracellular proline accumulation. This paper examines the role of proline in the freezing or desiccation resistance of S. cerevisiae by comparing intracellular proline level and cell viability after exposure to stresses of the put1 mutant and the PUT1 gene disruptants.

2 Materials and methods

2.1 Strains and plasmids

The S. cerevisiae strains used in this study are described in Table 1. Strains MB1433 and MB329-17C were derived from a cross between strains S288C and Σ1278b [8]. Strain CKY2 was derived from S288C. Strain XU-1 is a haploid derived from a sake yeast strain K-9, which is a diploid prototroph [9]. Escherichia coli strain JM109 [recA1 Δ(lac-proAB) endA1 gyrA96 thi-1 hsdR17 relA1 supE44/(F ′traD36 proAB⁺lacI^qZ ΔM15)], and the plasmid vector pBluescript II SK+ (Toyobo Biochemicals, Osaka, Japan) were used to subclone the PUT1 gene. Two plasmids YEp24 [10] and pRS406 [11] (Stratagene, La Jolla, CA, USA), which contain the URA3 gene, were used to construct the plasmid for PUT1 gene disruption and for the integration of the URA3 gene into the host strain, respectively.

1

Genotypes and sources of S. cerevisiae strains used in this study

Strain	Genotype	Source
MB1433	αura3-52 trp1 PUT1	M.C. Brandriss
MB329-17C	αura3-52 trp1 put1-54	M.C. Brandriss
CKY2	a ura3-52 his4-619	C. Kaiser
CKY2U	a his4-619 URA3a	This study
HKCKY2U	a ura3-52 his4-619 put1::URA3	This study
XU-1	a ura3	Y. Kubo
XU-1U	a URA3b	This study
KHXU-1U	a ura3 put1::URA3	This study
a,bStrains CKY2U and XU-1U were obtained by integrating the URA3 gene into strains CKY2 and XU-1, respectively.

2.2 Media

The media used for growth of S. cerevisiae were SD (2% glucose, 0.67% Bacto-yeast nitrogen base without amino acids [Difco Laboratories, Detroit, MI, USA]), and YPD (2% glucose, 1% Bacto-yeast extract, 1% Bacto-peptone). To examine the phenotype of the PUT1 disruptants, 0.1% monosodium glutamate or proline was used instead of ammonium sulfate as the sole source of nitrogen; required supplements were added to the media for auxotrophic strains. E. coli strains were grown in Luria-Bertani (LB) medium [12] containing ampicillin (50 μg ml⁻¹). If necessary, 2% agar was added to solidify the medium.

2.3 DNA manipulation and transformation

The enzymes used for DNA manipulation were obtained from Takara Shuzo (Kyoto, Japan). One-step gene replacement by homologous recombination in S. cerevisiae was performed according to the method of Rothstein [13]. Conventional techniques [14] were used for other manipulations such as S. cerevisiae genomic DNA preparation and transformation. Southern blot analysis was carried out using ECL direct nucleic acid labelling and detection systems based on enhanced chemiluminescence (Amersham Pharmacia Biotech, Buckinghamshire, UK).

2.4 Cloning of the PUT1 gene and plasmid construction for PUT1 disruption

Genomic DNA was prepared from S. cerevisiae MB1433, and forward and reverse primers for PUT1 were designed based on the available nucleotide sequences [15]. The forward primer was 5′-GAGGATCCGAACACAAACTCCA-3′ (the underlined sequence shows the position of a BamHI site), and the reverse primer was 5′-GCGGTACCCCAAAATCCTTACA-3′ (the underlined sequence shows the position of a KpnI site). 50 ng of genomic DNA was added as a template to a solution containing 10 μl of 10×PCR buffer (100 mM Tris-HCl [pH 8.3], 500 mM KCl, 15 mM MgCl₂), 8 μl of each deoxyribonucleotide triphosphate at a concentration of 2.5 mM, 1 μl of a preparation containing each of the two primers at a concentration of 1 mM, 2.5 U of Ex Taq DNA polymerase, and enough distilled water to bring the total volume to 100 μl. Twenty-five PCR cycles (94°C for 1 min, 50°C for 1 min, 72°C for 2 min) were carried out with a Gene Amp PCR system 2400 (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). The unique amplified band of 1976 bp was digested with BamHI and KpnI and then ligated to the BamHI and KpnI sites of pBluescript II SK+. The nucleotide sequence of the cloned DNA was confirmed with a Model 377 DNA sequencer from Perkin-Elmer Applied Biosystems using the dideoxy chain termination method. A plasmid harboring the PUT1 gene was designated pPUT1. The plasmid pPUT1U was then constructed by deleting the 970-bp PstI-BglII fragment in the PUT1 gene from plasmid pPUT1 and inserting the 1.2-kb HindIII fragment containing the URA3 gene isolated from plasmid YEp24 by blunt-end ligation.

2.5 Assay of proline oxidase activity

Yeast cells were grown in 500-ml flasks containing 50 ml of SD medium at 30°C for 48 h with shaking. Cell extracts were prepared from the culture samples and proline oxidase (EC 1.4.3.2) activity was assayed by monitoring the amount of P5C-o-aminobenzaldehyde complex as previously described [5]. The millimolar extinction coefficient of the P5C-o-aminobenzaldehyde complex was 2.71 [16]. One unit of specific activity was expressed as 1 nmol of P5C formed per min per mg of protein. Protein concentrations were determined using a Bio-Rad Protein Assay kit (Hercules, CA, USA) using bovine serum albumin as the standard protein.

2.6 Freezing and desiccation resistance tests

In a 500-ml flask, yeast cells were grown in 50 ml of SD medium at 30°C for 48 h with shaking to the stationary phase. Culture samples were diluted with SD medium to an absorbance of 7 at 600 nm. In the case of PUT1 gene disruptants and the control strains, 0.1% proline was added into SD medium.

For freezing stress, approximately 1×10⁸ cells were harvested from 1 ml of diluted suspension by centrifugation (1500×g, 5 min), washed with SD medium, and suspended in 1 ml of the same medium. Aliquots (0.1 ml) of cells were transferred into 1.5-ml microcentrifuge tubes and immediately stored at −20°C. In this condition, it took about 1 h until cells were frozen, assuming that cooling rate was slow (approximately 0.5–1°C min⁻¹). Samples of frozen cells were thawed at room temperature for 20 min, serial dilutions were made in 0.9% NaCl, and aliquots plated immediately on YPD plates. To induce desiccation stress, approximately 5×10⁸ cells in 5 ml of diluted suspension were collected on sterilized- membrane filter (0.45 μm mixed cellulose ester; Toyo Roshi, Tokyo, Japan), and washed with 0.9% NaCl. The filter was placed in a sterilized dish and stored at 20°C for various days. Samples of dried cells were rehydrated in 5 ml of SD, serial dilutions were made in 0.9% NaCl, and aliquots were plated immediately on YPD plates. Survival rates are expressed as percentages of the number of colonies after incubation at 30°C for 2 days relative to the number of colonies before freezing or desiccation.

2.7 Intracellular contents of proline

After cultivation in 50 ml of SD medium supplemented with or without 0.1% proline at 30°C for 48 h with shaking to the stationary phase in a 500-ml flask, the culture samples were diluted with SD medium to an absorbance of 7 at 600 nm. 5 ml of cell suspension (approximately 5×10⁸ cells) was removed, the cells were washed twice with 0.9% NaCl and suspended in 0.5 ml of distilled water. The 1.5-ml microcentrifuge tube containing cells was transferred to a boiling water bath and intracellular amino acids were extracted by boiling for 10 min. After centrifugation (5 min at 15 000×g), each supernatant was subsequently quantitated with an amino acid analyzer (L-8500A, Hitachi Co., Tokyo, Japan). Proline content was expressed as a percentage of dry weight.

3 Results

3.1 Resistance to freezing and desiccation stresses

When cultured in liquid SD medium, the proline level in the put1 mutant (MB329-17C) was approximately 2% of its dry weight, while no proline was detected in the wild-type strain (MB1433). Proline oxidase activity of the put1 mutant was 0.3 U mg⁻¹ (only 25% of the wild-type). Freezing (Fig. 1A) and desiccation (Fig. 1B) resistances were higher in the put1 mutant strain than in the wild-type strain, although the effect was observed only after each stress had been imposed for 12 h. During exposure to stresses, the proline values decreased in MB329-17C cells probably due to the severe cellular damage (Fig. 1). These results indicate a positive correlation between proline content and water stress resistance.

1

Changes of cell viability and intracellular proline content during freezing (A) and desiccation (B) of S. cerevisiae. Wild-type (MB1433) and put1 mutant (MB329-17C) strains were grown in SD medium for 48 h, and the cell density was adjusted. Aliquots of the cells were removed and frozen at −20°C or desiccated at 20°C for the time indicated. Cell viability of wild-type (○) and put1 mutant (●) strains was expressed as a percentage of the number of colonies after freezing or desiccation relative to the number of colonies prior to freezing or desiccation. Additional aliquots of wild-type (△) and put1 mutant cells (▲) were removed and measured for intracellular proline content as described in Materials and methods. The data shown are means±S.D. from three independent experiments.

3.2 Disruption of the PUT1 gene in S. cerevisiae

The put1 mutant (MB329-17C) was derived from a cross between strains S288C and Σ1278b. To examine the general role of intracellular proline in the stress resistance, we constructed the PUT1 disruptants from two strains (CKY2 and XU-1) with different genetic backgrounds. For the PUT1 gene disruption, the plasmid pPUT1U was digested with BamHI and KpnI, and the linear 2.2-kb fragment containing put1::URA3 with homologous ends was integrated into the PUT1 locus in ura3-52 PUT1 yeast strains, CKY2 and XU-1, by transformation. To remove the influence of uracil auxotrophy on this study, plasmid pRS406 was cut with StuI in the URA3 gene and the linearized plasmid was then introduced for the integration of the URA3 gene to obtain the control strains (CKY2U and XU-1U). Yeast transformants with the put1::URA3 construct were selected on SD plates supplemented with all the auxotrophic requirements except for uracil. The resultant putative disruptants, KHCKY2U and KHXU-1U, from several uracil prototrophic transformants were selected and used for genomic Southern blot analysis to confirm the PUT1 disruption (Fig. 2). Genomic DNAs prepared from transformants (KHCKY2U and KHXU-1U) and the control strains (CKY2U and XU-1U) were completely digested with EcoRV, and the fragments were separated onto a Hybond-N⁺ nylon membrane (Amersham Pharmacia Biotech). When the 1.5-kb PstI-KpnI fragment from plasmid pPUT1 was used as a probe, 4.7-, 4.4-, and 0.7-kb fragments from the intact PUT1 gene were detected in the control strains (lanes 1 and 3). Meanwhile, two bands corresponding to the 4.7- and 1.0-kb fragments were observed for two disruptants with put1::URA3 (lanes 2 and 4). Therefore, in KHCKY2U and KHXU-1U strains, the intact PUT1 gene was replaced by the URA3-disrupted fragment by homologous recombination.

2

Southern blot analysis of genomic DNAs from the wild-type and the PUT1-disrupted S. cerevisiae strains. A: The construction used to disrupt the PUT1 gene. The locations of the BglII (Bg), EcoRV (EV), HindIII (H), KpnI (K) and PstI (P) sites are marked. The PUT1 and URA3 genes are indicated by the open and shaded bars, respectively. The arrows on the gene show the direction of transcription. B: Southern blotting. 5 μg of yeast genomic DNA from each strain was digested with EcoRV, electrophoresed 0.8% agarose gel, transferred onto a nylon membrane, and hybridized with a 1483-bp PstI and KpnI fragment bearing most of the PUT1 gene labelled using the ECL direct nucleic acid labelling system. Lane 1, CKY2U; lane 2, KHCKY2U; lane 3, XU-1U; lane 4, KHXU-1U. The 4683-, 4393-, and 718-bp fragments indicate native PUT1 loci (lanes 1 and 3). The insertion of the URA3 gene into the PUT1 region causes the EcoRV fragment to increase in size by 968 bp (lanes 2 and 4). A HindIII digest of λDNA was used as the size standard.

3.3 Phenotype and proline oxidase activity of PUT1 disruptants

We examined the growth of PUT1 disruptants on minimal medium containing 0.1% monosodium glutamate or proline, instead of ammonium sulfate, as the sole source of nitrogen (Table 2). As expected, both disruptants failed to grow on proline-containing plates, while the wild-type strains could utilize proline as their nitrogen source.

2

Proline utilization and proline oxidase activities of the wild-type and the PUT1-disrupted S. cerevisiae strains

Strain	Genotype	Nitrogen sourcea		Specific activity of proline oxidase (U mg−1)b
		Glutamate	Proline
CKY2U	PUT1 URA3	+	+	2.7
HKCKY2U	put1::URA3	+	−	0.55
XU-1	PUT1 URA3	+	+	17
HKXU-1	put1::URA3	+	−	0.58

^aMonosodium glutamate or proline was supplied at 0.1%. The carbon source was 2% glucose. Growth on the media was scored after 2 days of incubation at 30°C. Plus and minus signs indicate growth and absence of growth, respectively.

^bVariations in the values were below 5%.

Disruption of the PUT1 gene was also confirmed by measurement of proline oxidase activity in cell extracts (Table 2). Proline oxidase activities of the parent strains, CKY2U and XU-1U, were detected at significant levels, while KHCKY2U and KHXU-1U strains exhibited weakened activity (approximately 0.6 U mg⁻¹).

3.4 Intracellular proline and resistance to freezing and desiccation

We examined the relationship between cellular proline levels and resistance to water stress conditions in the PUT1 disruptants (Table 3). No detectable proline had accumulated in the PUT1 disruptants after cultivation in SD medium in the absence of proline (data not shown). To increase the intracellular level of proline, 0.1% proline was added to the liquid medium. The proline level in the PUT1 disruptants was approximately 17–22-fold that of the control strains. When the cell suspensions were frozen at −20°C for 3 days, the PUT1 disruptants (KHCKY2U and KHXU-1U) showed an approximately 2-fold increase in survival rates after freezing as compared with that of the control strains. The cell viability in the two disruptants was 1.4–1.6-fold that of the control strains. The significant cryoprotective effect in the PUT1 disruptants still remained after freezing at a much lower temperature (−80°C) for 3 days. However, the effect of proline was not significantly observed after freezing at −20°C for 2 days and prolonged storage of the cells for up to 1 week caused a gradual loss of freeze resistance ability in the disruptants (data not shown). A similar tendency was observed in the disruptants exposed to desiccation stress (Table 3). With desiccated cells the survival percentage was lower than with frozen cells. The survival rate of the PUT1 disruptants was 2–5 times higher than the control strains following desiccation at 20°C for 4 days. Therefore, the PUT1 disruptants exhibited a substantial increase in survival relative to the wild-type strain under freezing and desiccation stress conditions, and this increased stress resistance relatively correlated with the elevated cellular proline content.

3

Intracellular proline content before stresses and cell viability after stresses in the wild-type and the PUT1-disrupted S. cerevisiae strains

Strain	Genotype	Proline content (% of dry weight)	Viability (%)a
			Freezing	Desiccation
CKY2U	PUT1 URA3	0.13±0.012	32±8.7	0.83±0.14
HKCKY2U	put1::URA3	2.9±0.29	58±13	1.4±0.16
XU-1	PUT1 URA3	0.50±0.082	59±2.2	1.3±0.42
HKXU-1	put1::URA3	8.7±1.4	82±6.7	6.3±1.7
The data shown are means±S.D. from three independent experiments.

^aCell viability was the same as given in Fig. 1 after freezing at −20°C for 3 days after desiccation at 20°C for 4 days. Total number of cells corresponding to 100% was 1–5×10⁷.

4 Discussion

In this work, we first focused on intracellular proline's role during freezing and desiccation stresses, which constitute the common dehydration process in the cells. As expected, the accumulation of proline in a proline oxidase-deficient mutant yeast grown in minimal medium conferred a better survival rate than that of the wild-type strain under both freezing and desiccation stress conditions (Fig. 1). To increase or minimize the production of a specific metabolite in yeast cells, including some organic acids, trehalose or urea, each gene encoding the related enzyme, fumarase [17], acid trehalase [18], or arginase [9], has been successfully disrupted by a one-step gene replacement method. We attempted to construct a yeast strain which accumulates proline after cultivation in minimal medium. In S. cerevisiae, proline oxidase, the product of the PUT1 gene, is a mitochondrially localized enzyme and increases approximately 30-fold when proline is substituted for ammonia as the nitrogen source [8,15]. It is known that S. cerevisiae can use either glutamate or proline as its sole source of nitrogen, but that the put1 mutant can no longer utilize proline [5]. To suppress the induction of the PUT1 gene by intracellular proline, we disrupted the PUT1 gene from two yeast strains. The proline oxidase activities of the disruptants were not completely abolished (Table 2). The reason for the residual activities remains unclear; however, the weak activity of another oxidase for proline or some unexpected blank reaction may be conceivable.

When the PUT1 disruptants were grown in the medium without added proline, no detectable proline accumulated in the cells. It is probable that the genetic backgrounds of three strains used (S288C, Σ1278b, and a sake yeast strain) would result in the differences of proline metabolism and of proline contents in the cells. Therefore, by supplementing 0.1% proline in a liquid medium, we expected to elevate the intracellular proline content by enhancing the transport system that brings proline into the cell and by decreasing proline utilization while growing the cells. In S. cerevisiae, it is known that the two proline transport systems, the proline-specific permease (PUT4) and the general amino acid permease (GAP1), are regulated not by proline induction but by nitrogen catabolite repression [19]. In two PUT1 disruptants, there was an approximately 20-fold increase in cellular proline content compared with that of the control strains having proline-degrading enzyme (Table 3). At the same time, the elevated levels of proline in these disruptants correlated with increased resistance to freezing and desiccation, whereas the degree of resistance varied between the strains or the stress conditions. One of the PUT1 disruptants (KHXU-1U) accumulated nearly 9% of its dry weight, and cell viability showed a better survival rate than that of the other disruptant (KHCKY2U) with 3% of its dry weight after freezing and desiccation stresses.

In this study, the effect of proline on water stress resistance was relatively small in contrast to the differences in cellular proline levels between the wild-type and PUT1 disruptants. Our results could suggest that the freezing and desiccation resistances of S. cerevisiae may be influenced by some factors in addition to proline. Recent works revealed that trehalose is a critical membrane-protecting agent, and also confers increased cell viability under environmental stress conditions [20–22]. Accordingly, the combination of cryoprotectants, including trehalose, glycerol, and proline, would further contribute to the enhancement of cellular stress resistance.

Generally speaking, it is relatively difficult to breed industrial baker's yeast strain with a higher freeze resistance compared to that of a laboratory strain. The process that proline is added externally to the cell or the dough would be somewhat troublesome for practical application. However, the proline analogue-resistant mutants with higher amounts of proline in the cells [4], which probably increase flux in the proline metabolic pathway, might overcome the problem. It may be possible to create a freeze-resistant baker's diploid strain or an active dry yeast strain by the disruption of both copies of the PUT1 gene. The investigation of this is currently in progress.

Acknowledgements

We thank Marjorie C. Brandriss (University of Medicine and Dentistry of New Jersey, Newark, NJ, USA), Chris Kaiser (Massachusetts Institute of Technology, Boston, MA, USA), and Yoshito Kubo (Fukui Food Processing Research Institute, Fukui, Japan) for providing yeast strains, and Kazunori Hirai for assistance in the preparation of figures. This work was supported by a grant from Fukui Prefectural Scientific Research Foundation (H.T.).