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Temperature is one of the most important parameters affecting the growth and survival of microorganisms (6, 24). Most microorganisms are mesophiles and occupy temperature niches that are not regarded as extreme. Psychrophilic microbes, including psychrophilic yeasts (24), are capable of growth at temperatures below 0°C and have a maximum growth temperature of 20°C (15). It has been proposed that a thermophilic yeast should be defined as a yeast which has a minimum temperature for growth of 20°C and no restriction on the maximum temperature for growth (24). If this definition is used, all of the species in this category are enteric yeasts isolated from digestive tracts of various animals (20). These yeasts, which have growth temperature limits between 20 and 46°C (20, 26), include the respiratory-deficient organisms Candida slooffii and Torulopsis pintolopesii and the respiratory-competent organisms Saccharomyces telluris and Torulopsis bovina. All of these yeasts have been reclassified as Arxiozyma telluris (4, 22), are facultative anaerobes (27), and either occur as respiratory-deficient organisms or are capable of giving rise to stable respiratory-deficient mutants either spontaneously or by ethidium bromide induction (2, 26).

The heat shock response, whereby exposure to a mild, nonlethal heat shock renders cells resistant to a subsequent challenge at a higher, normally lethal temperature, appears to be a universal response in all organisms (5, 9, 19). In the mesophilic yeast Saccharomyces cerevisiae a mild heat shock at 37°C induces tolerance to a normally lethal temperature, generally 48 to 55°C. Thermotolerant cells synthesize the disaccharide trehalose (3, 7) and a specific set of proteins, termed the heat shock proteins (9, 18, 25). In heat-stressed yeasts, trehalose appears to maintain the structure and integrity of cell membranes and proteins (7, 18).

In this paper we describe the heat shock response of two strains of the thermophilic enteric yeast A. telluris and compare this response to the response reported previously for the mesophilic yeast S. cerevisiae.

Yeast strains and culture conditions. A. telluris CBS 1787 (respiratory deficient) and CBS 2760 (respiratory competent) were used in this study. Cultures were grown at 35°C on a rotary shaker (180 rpm) in YEP medium [0.5% yeast extract, 0.5% bacteriological peptone, 0.3% (NH₄)₂SO₄, 0.3% KH₂PO₄, 2% glucose]. Experimental cultures were grown to optical densities at 600 nm of 0.2 to 0.4, corresponding to logarithmic-phase cells at concentrations of approximately 2 × 10⁶ to 6 × 10⁶ cells ml¹. All experiments were repeated a minimum of three times and produced consistent results.

Stress tolerance assays. Preliminary experiments were undertaken to determine the optimal temperatures required to give appropriate stress tolerance kinetics for a heat shock response. Intrinsic thermotolerance was measured by rapidly heating cells grown at 35 to 47°C in a 70°C water bath and transferring them to a 47°C oscillating water bath for the duration of the 60-min time course. Induced thermotolerance was measured by exposing cultures to a 30-min 40°C heat shock prior to heat stress at 47°C. Subsamples (0.5 ml) were taken at various times and diluted in YEP medium. Duplicate YEP agar spread plates were prepared and incubated at 35°C for 1 to 2 days. Thermotolerance was assessed by determining the percentage of CFU after heat treatment compared to the CFU in an unstressed control (100% survivors).

Trehalose determination. Trehalose was extracted in triplicate from 80 ml (5 to 7 mg [dry weight] of washed cells. Control and heat-shocked cells were extracted with 0.5 M trichloroacetic acid at 4°C, and the trehalose concentrations were estimated by a modified anthrone method (8).

[³⁵S]methionine labelling, protein extraction, and electrophoresis. Prior to [³⁵S]methionine labelling, 40 ml of culture was washed and resuspended in 2 ml of YNB medium (0.67% yeast nitrogen base, 0.3% KH₂PO₄, 2% glucose) without amino acids. [³⁵S]methionine (100 µCi; specific activity, 1,150 Ci mmol¹) was added to control and heat shock samples, and the preparations were incubated at the appropriate temperatures for 30 min. Cells were pelleted and protein was extracted as previously described (14). Protein concentrations were determined by using a Coomassie blue microassay procedure (Pierce). Proteins (10 µg) and low-range molecular weight standards (Bio-Rad) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using a 10% polyacrylamide separating gel and a 4% polyacrylamide stacking gel. The gels were silver stained, dried, and exposed to Hyperfilm-MP (Amersham) at 70°C for 5 to 7 days before they were developed.

Western immunoblot analysis. Following extraction and electrophoresis as described above, proteins were transferred to Hybond-C super nitrocellulose membranes (Amersham). Western immunoblotting was carried out by using an Amersham ECL Western blot detection kit according to the manufacturer's instructions. The final membranes, washes prior to detection were modified as follows: three washes (5 min each in phosphate-buffered saline-0.3% Tween 20, followed by three washes (5 min each in phosphate-buffered saline-0.1% Tween 20. Anti-hsp 104 polyclonal antibody (Affinity BioReagents), anti-hsp 90 monoclonal antibody (a kind gift from P. Piper, University College London), anti-hsp 70 monoclonal antibody (Affinity BioReagents), and anti-hsp 60 monoclonal antibody (StressGen) were used at dilutions of 1:1,000, 1:750, 1:5,000, and 1:1,000, respectively. The appropriate secondary antibody was used at a dilution of 1:1,000. Membranes were exposed to Hyperfilm-MP for times ranging from a few seconds to a few minutes before they were developed.

Intrinsic thermotolerance and induced thermotolerance. Intrinsic thermotolerance and induced thermotolerance to a 47°C heat stress over a 60-min time course were measured in respiratory-competent strain 2760 and respiratory-deficient strain 1787 of A. telluris. A 40°C heat shock for 30 min prior to a 47°C heat stress conferred tolerance to both strains, and the levels of viability for the 60-min experiment were close to 100% (Fig. 1). The levels of induction for intrinsic thermotolerance and induced thermotolerance were similar in both strains.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Intrinsic thermotolerance and induced thermotolerance in mid-logarithmic-phase cultures of A. telluris 2760 (respiratory competent) (circles) and A. telluris 1787 (respiratory-deficient) (triangles). Intrinsic tolerance ( and ) was measured by transferring cells directly to 47°C. Induced thermotolerance was monitored at 47°C following a 40°C heat shock for 30 min ( and ). Levels of thermotolerance are expressed as percentages of survivors after the appropriate treatment compared with the number of survivors in a 35°C control sample.

Trehalose. A 40°C heat shock for 30 min resulted in marked increases in trehalose accumulation; the amounts of trehalose increased approximately 12- and 17-fold in strains 2760 and 1787, respectively. The absolute levels of trehalose were less for both control and heat shock samples in strain 1787 (control, 0.63% [wt/vol]; heat shock, 10.7% [wt/vol]) than in strain 2760 (control, 1.2% [wt/vol]; heat shock, 13.9% [wt/vol]).

Stress proteins. As shown by [³⁵S]methionine-labelled de novo protein synthesis, a 40°C heat shock for 30 min induced synthesis of proteins whose molecular masses corresponded to the molecular masses of hsp 104, hsp 90, and members of the hsp 70 family in both strain 2760 and strain 1787 (Fig. 2). Furthermore, a ca. 35-kDa heat shock-inducible protein was identified in both strains (Fig. 2).

View larger version (52K): [in this window] [in a new window]   FIG. 2.   Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiogram of [35S]methionine-labelled protein extracts from A. telluris 2760 (respiratory competent) (a) and A. telluris 1787 (respiratory deficient) (b). The conditions used were as follows: 35°C for the control (lanes 1 and 3) and 40°C for the heat shock (lanes 2 and 4). The arrows indicate new or increased heat shock protein synthesis. The positions of molecular mass standards are indicated on the left and right.

View larger version (58K): [in this window] [in a new window]   FIG. 3.   Western blot analysis of A. telluris 2760 (respiratory competent) (a) and A. telluris 1787 (respiratory deficient) (b). Proteins from the 35°C control treatment (lanes 1 and 3) and the 40°C heat shock treatment (lanes 2 and 4) were probed with anti-hsp 104 (1:1,000), anti-hsp 90 (1:750), anti-hsp 70 (1:5,000), and anti-hsp 60 (1:1,000).

Conclusions. The organisms which we examined are found in the digestive tracts of warm-blooded domestic and wild animals and are obligate or facultative saprophytes, although there is a report of isolation of A. telluris from soil (reviewed in reference 20). Despite their relatively narrow temperature range for growth, these organisms were capable of a heat shock response similar to that of S. cerevisiae. Key heat shock proteins with molecular masses of 104, 90, 70, and 60 kDa, as well as the disaccharide trehalose, were synthesized upon mild heat shock. In addition, a ca. 35-kDa heat shock-inducible protein, which may correspond to glyceraldehyde-3-phosphate dehydrogenase (17), was prominent in the enteric yeast.