INTRODUCTION

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In general, hydrostatic pressure affects almost all physiological activities in living cells. Bett and Cappi (3) studied the viscosity of water as a function of pressure up to 10,000 kg/cm². They found that relative and absolute viscosities decrease with pressure increases from 0 to 2,000 kg/cm² at ambient temperature (3). Decreased viscosity due to high pressure results in the destruction of hydrogen bonding, and this has been reported to be a consequence of increased temperature (3). Thus, the effects of high temperature and high hydrostatic pressure can be analogous for organisms.

To understand the effect of high temperature and high hydrostatic pressure, we have been studying the analogy between hydrostatic pressure and temperature by using Saccharomyces cerevisiae as a model system (11). We have shown that the molecular chaperone Hsp104, as well as the nonreducing disaccharide trehalose, plays important role in barotolerance and thermotolerance (8, 9). However, thermotolerance and barotolerance are essentially different (8). Hsp104 has an optimum temperature for its role in barotolerance, and the temperature for hydrostatic pressure treatment is below this optimum. This lower temperature decreases the importance of Hsp104 to barotolerance (9). Several lines of evidence suggest that Hsp104 (12, 18) and trehalose (4) are important factors in thermotolerance.

Recent studies have shown that neutral trehalase I encoded by NTH1, as well as its homolog, NTH2 (encoding putative neutral trehalase II), is important for thermotolerance, especially for recovery of cells after severe heat shock (14, 15). Neutral trehalase is responsible for breaking down trehalose in the cell. Apparently, it is difficult to understand the mechanism of the contribution of neutral trehalase to thermotolerance, because this enzyme breaks down trehalose, which contributes to thermotolerance. Two models have been proposed to explain the role of neutral trehalase in heat shock recovery (14, 15, 19). The latest model states that trehalose protects cellular proteins against denaturation and subsequent aggregation, but inhibits the solubilization of protein aggregates and the refolding of the partially denatured proteins during recovery from heat shock (19). The inhibition of refolding of protein by trehalose can cause a delay in recovery from heat denaturation. Thus, neutral trehalase can contribute to thermotolerance, especially during recovery.

In this report, we show that neutral trehalase contributes to barotolerance. The deletion of NTH1 decreased barotolerance, and reintroduction of NTH1 by transformation increased barotolerance. Furthermore, induction of neutral trehalase activity under recovery conditions significantly increased barotolerance. Thus, neutral trehalase contributed to barotolerance, especially during recovery conditions.

	MATERIALS AND METHODS

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Strains and growth conditions. The strains and plasmids used for this study are shown in Table 1. Information about some of the parental strains may be seen in references 13, 15 and 16. For precise experiments, the control strains were constructed by introducing URA3, LEU2, or plasmids without the NTH1 gene into the corresponding parent strains (Tables 2 to 4) so that the effect of artificial factors would be minimal. The strains were constructed according to general methods (5, 17) and were grown in YPD medium (2% polypeptone, 1% yeast extract, 2% glucose) or SD medium (0.67% yeast nitrogen base without amino acids) (7) containing 2% glucose or galactose. A preculture grown for 2 days was used to inoculate experimental cultures at a dilution rate of 20 to 5,000. The cells were grown overnight at 30°C in a shaker until the stationary or logarithmic phase of growth (A₆₆₀ of 1.0).

Barotolerance, neutral trehalase activity, and trehalose content. Hydrostatic pressure treatment of cells was performed at 180 MPa (2 h at 25°C). Yeast cells in the growth medium were poured into 3 ml of a glass syringe, the syringe was transferred to a stainless steel vessel (KT-0422; High Pressure Chemical Co., Ltd., Hiroshima, Japan), and the vessel was pressurized with a pressure-generating pump (wp3000; High Pressure Equipment Co., Ltd., Erie, Pa.). Barotolerance was expressed as a percentage of the CFU of the high-pressure-treated cells relative to the untreated control. Neutral trehalase activity in the crude extract (10) was measured in the reaction mixture containing 34 mM imidazole-HCl (pH 7.0) and 0.11 M trehalose (1) as described previously (10). One unit of neutral trehalase is the amount of enzyme that hydrolyzes 1 µmol of trehalose in 1 min. Trehalose content was measured as glucose after hydrolysis with acid trehalase (6). The data shown are mean values of three independent and reproducible experiments.

	RESULTS AND DISCUSSION

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Deletion of NTH1 decreased barotolerance. As a first step in studying the contribution of neutral trehalases to barotolerance, we measured the barotolerance of the NTH1 and/or NTH2 deletion mutants by using logarithmic-phase cells and stationary-phase cells. In Table 2, the trehalose content and barotolerance of the YS18-URALEU (wild type), YSN1-URA (nth1), YSN-LEU (nth2), and YSN1-01 (nth1 nth2) strains are shown. As expected, the strains that had the deletion in the NTH1 gene showed a higher trehalose content in logarithmic-phase cells and stationary-phase cells than the strains that had the NTH1 gene (see references 14 and 15). This is a consequence of a decrease or absence of neutral trehalase activity due to deletion of the NTH1 gene. The NTH2 gene did not affect the accumulation of trehalose. This result also agrees with the result shown by Nwaka et al. (15). They found that although the NTH2 gene is a homolog of NTH1, it shows no neutral trehalase activity.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Growth curve of the strains constructed. YS18-URALEU (wild type; ), YSN1-URA (nth1::LEU2; ), YSN-LEU (nth2::URA3; ), and YSN1-01 (nth1::LEU2/nth2::URA3; ) were grown in YPD medium. The preculture was diluted at a dilution rate of 100, and growth was monitored as the A650.

Reintroduction of neutral trehalase to the NTH1 deletion strain increased barotolerance. To confirm the contribution of neutral trehalase I to barotolerance, we transformed the neutral trehalase I deletion strain (nth1) with plasmid pSEY8 containing the NTH1 gene and its promoter region (pSEY8-NTH1) (13, 16). The resulting strain, called YSN1-pSEY8-NTH1 (nth1 mutant expressing NTH1 from the plasmid), had restored neutral trehalase activity compared to the control strain YSN1-pSEY8 (nth1 mutant containing pSEY8 plasmid alone) (Table 3). Interestingly, YSN1-pSEY8-NTH1 cells, which accumulated less trehalose, showed higher barotolerance than the control strain. This result corresponds with the data in Table 2. It should be mentioned that the barotolerance values shown in Table 3 are lower than those in Table 2. This is possibly because of the strains, the differences in the medium, and the selective pressure of the plasmids used in Table 3.

Neutral trehalase activity contributes to the recovery of cells after pressure treatment. Although our data suggest that neutral trehalase I plays a role in barotolerance, it remains unclear how neutral trehalase performs this function. The finding that trehalose may prevent refolding of heat-denatured proteins (after heat shock) implies that trehalose hydrolysis catalyzed by neutral trehalase is essential for recovery (15, 19). However, there is no direct evidence to support this. Generally, thermotolerance or barotolerance means total tolerance during stress and recovery. Therefore, we tried to estimate the contribution of neutral trehalase during and/or after pressure treatment.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Neutral trehalase activity in cells grown with glucose and galactose. Strain YSN1A-p2.253 grown with glucose or galactose was transferred to glucose or galactose medium. Neutral trehalase activities were measured at the indicated time, as described in the text. , transfer from glucose to glucose; , transfer from glucose to galactose; , transfer from galactose to glucose; , transfer from galactose to galactose.