Mycologia
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Managbanag, J. R.
Right arrow Articles by Torzilli, A. P.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Managbanag, J. R.
Right arrow Articles by Torzilli, A. P.
Agricola
Right arrow Articles by Managbanag, J. R.
Right arrow Articles by Torzilli, A. P.
Mycologia, 94(3), 2002, pp. 384-391.
© 2002 by The Mycological Society of America

An analysis of trehalose, glycerol, and mannitol accumulation during heat and salt stress in a salt marsh isolate of Aureobasidium pullulans


Jim Ray Managbanag

     American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia, 20110-2209

Albert P. Torzilli 1

     Department of Biology, George Mason University, Fairfax, Virginia, 22030-4444

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

Polyhydroxy compounds from Aureobasidium pullulans exposed to stress treatments of heat, salt, and simultaneous heat and salt were isolated, identified, and quantified. Results from both thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) showed that concentrations of trehalose, mannitol, and glycerol increased under stress conditions that induce osmotic- and thermotolerance in A. pullulans. The cellular concentration of trehalose increased in heat-stressed and in simultaneously heat- and salt-stressed cells but not in cells subjected to salt stress alone. Mannitol increased under all stress conditions examined, while an increase in intracellular glycerol was apparent only in salt-stressed cells. The significance of these findings in relation to stress tolerance in salt marsh environments is discussed.

Key words: Aureobasidium, glycerol, HPLC, mannitol, stress response, TLC, trehalose


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
In salt marsh environments, the microbial community is subjected to variations in temperature and salinity depending on the time of day and season. The combination of summer low tides with increasing temperature and evaporation during the day can produce simultaneous conditions of high temperature and osmotic stress. To this end, certain marine bacteria and fungi have been shown to increase their salinity optimum for growth when exposed to elevated temperatures, a phenomenon described as the "Phoma" pattern (Molina and Hughes 1982Citation ). Aureobasidium pullulans (Deuteromycota) is frequently isolated from marine environments including coastal sands, seawater, sediments, and marine animals (Kohlmeyer and Kohlmeyer 1979Citation ). When the salt marsh isolate of A. pullulans used in this study is exposed to increasing temperatures it exhibits an upward shift in its salinity optimum for growth (Torzilli et al 1985Citation ). Torzilli (1997)Citation also has shown that high temperatures and salinities induce the synthesis of stress proteins in this fungus which in turn is correlated with the acquisition of thermo- and osmotolerance. Such physiological adjustments are well suited for surviving conditions characteristic of salt marsh ecosystems.

Under conditions where temperatures exceed the normal growth range, cells experience stress due to the damaging effect of heat on intracellular macromolecules, such as heat-sensitive enzymes, and to cell membranes (Henle et al 1982Citation ). In the budding yeast Saccharomyces cerevisiae, two major cellular responses have been shown to counteract the lethal physiological effects of heat: the induction of heat shock proteins and an increase in the intracellular production of the dissacharide, trehalose (Attfield 1987Citation ). Both prokaryotic and eukaryotic cells are capable of responding to increased but sublethal temperatures (heat shock) by undergoing rapid and massive synthesis of a subset of cellular proteins commonly referred to as heat shock proteins, HSP (Lindquist 1986Citation ). Heat shock proteins have been shown to confer thermotolerance to subsequent lethal exposures at elevated temperature (Lindquist 1986Citation ) and may even induce cellular resistance to rapid freezing (Komatsu et al 1990Citation ). In contrast, an investigation by Hall (1983)Citation showed the absence of thermotolerance in S. cerevisiae even when HSPs were induced. A report by Hottiger et al (1989)Citation indicated that trehalose is more important in the cellular induction of thermotolerance in S. cerevisiae than HSP. This is consistent with work by Watson et al (1984)Citation who reported only a decrease in thermotolerance instead of total loss after cycloheximide was added to inhibit protein synthesis in S. cerevisiae. This reinforced the idea that HSP are not unique in the induction of thermotolerance and that other stress response mechanisms also are involved (Panek et al 1990Citation ).

In saline environments, cells encounter stress due to increased electrolyte concentrations that tend to inhibit metabolic functions (Adler et al 1982Citation ). In S. cerevisiae, hyperosmotic stress induces the synthesis of stress proteins and an increase in the intracellular production of glycerol (Lewis et al 1995Citation , Nass and Rao 1999Citation ). Data from sodium dodecyl sulfate-polyacrylamide gel elecrophoresis (SDS-PAGE) showed that some of these proteins overlapped those synthesized during heat shock (Lewis et al 1995Citation ). Furthermore, Lewis et al reported that the induction of salt tolerance was decreased by approximately 50% but not eliminated in cells that were salt shocked in the presence of cycloheximide compared to untreated cells. Since glycerol also was observed to increase during the treatment, it was implicated as a factor contributing to the acquisition of salt tolerance. As is the case for S. cerevisiae, stress tolerance in A. pullulans also may involve solute accumulation as well as stress protein synthesis. Therefore, we investigated solute accumulation by A. pullulans under conditions of heat and salt stress known to induce osmotic and thermotolerance in this salt marsh isolate.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Strain isolation and propagation – The Aureobasidium pullulans strain employed in this investigation was originally isolated from surface sediment detritus in a Spartina salt marsh in Mathews County, Virginia, and has been deposited in the American Type Culture Collection (ATCC # MY-MYA-115). Stock cultures were maintained on potato dextrose agar (PDA). The composition of the experimental medium was as follows: glucose, 10 g; KH2PO4, 0.5 g; K2HPO4, 0.5 g; MgSO4·7H2O, 0.5 g; NaNO3, 0.83 g; and deionized water to 1 L.

Heat and salt tolerance experiments – Inoculum for experiments was prepared according to methods described by Torzilli (1997)Citation . Experiments were initiated by adding 1 x 108 cells of inoculum to 1 L Erlenmeyer flasks containing 500 mL of experimental medium. These cultures were incubated at 25 C with agitation (200 rpm) for 40 h (exponential phase) before being exposed, in triplicate, to the stress treatments described below. The temperature and salt treatments employed in these experiments have been shown to induce thermotolerance and osmotic tolerance in the isolate of A. pullulans studied here (Torzilli 1997Citation ).

Heat treatment – Forty-hour cultures designated for heat shock were immersed in a water bath (Lab-Line shaker bath) oscillating at 200 RPM at 35 C for 45 min, while 40 h control cultures were left at 25 C and 200 RPM for 45 min.

Salt treatment – The addition of NaCl to 40 h cultures at a final concentration of 4.5% constituted the salt treatment. The NaCl was sterilized by drying at 180 C overnight. Cultures (including controls lacking salt) were maintained at 25 C and 200 RPM for 45 min.

Heat and salt treatment – The addition of NaCl to 40 h cultures at a final concentration of 4.5% with immersion at 35 C at 200 RPM for 45 min constituted the heat and salt treatment. Forty-hour control cultures were left at 25 C and 200 RPM for the same time period.

Homogenization of cells – Following the 45 min treatments, cell suspensions were collected in 250 mL sterile centrifuge bottles and centrifuged for 20 min at 6555 x g. This fungus produces a polysaccharide, pullulan, which moves with cells during centrifugation forming white "halos" around the cell pellets. Supernatants were decanted and the pullulan was carefully removed with a Pasteur pipette. The pellets then were combined and resuspended with 5 mL of sterile deionized H2O, transferred to sterile 50 mL polyethylene tubes (Corning), and subjected to vortexing with equal volumes of sterile glass beads (0.25–0.30 mm) for 15 rounds, each round comprised of 3 cycles of 30 s each. The tubes were immersed in an ice bath for approximately 15 s between cycles. This resulted in approximately 90% cell breakage. Homogenates were centrifuged for 8 min at 1475 x g. Supernatants were removed and saved. Four mL of sterile deoinized H2O were added to the remaining pellets, and the pellets were subjected again to at least two more rounds of vortexing. These supernatants were collected, pooled with the previously collected supernatants, and stored at -70 C.

Thin-layer chromatography (TLC) – Standards (2%) which consisted of trehalose (Sigma, St. Louis, Missouri), glucose (Difco, Detroit, Michigan), mannitol (Beckton Dickinson, Franklin Lakes, New Jersey), and glycerol (Sigma) were spotted along with cell extracts onto Silica Gel 60 plates (Merck, Darmstadt, Germany) and developed with butanol-pyridine-water (15:30:20, vol/vol) as the mobile phase. The spots were detected by spraying with 0.5% KMnO4 in 1 N NaOH.

Bio-Rad protein assay – The protein concentration of cell extracts was determined in duplicate with the Bio-Rad protein reagent according to the macro procedure provided by the manufacturer (Bio-Rad, Hercules, California). The absorbances were read at 595 nm in a Spectronic 20D+ (Spectronic Instruments Inc., Rochester, New York).

High performance liquid chromatography (HPLC) – Quantitative analysis was carried out by HPLC (Waters, Milford, Massachusetts) with a refractive index detector (410, Waters), a Pb++ form cation-exchange column (Hamilton HC-75, 305 x 7.8 mm), and double-distilled H2O for the mobile phase. A column heater (Eppendorf, Westbury, New York) was appended to the system to maintain column temperature at 85 C. Fifty-µL samples or standards were injected and chromatographed at a constant flow rate of 1 mL/min (mobile phase). Peak integrations were carried out with the Millenium HPLC software package (Waters). Prior to HPLC, the samples were boiled for approximately 10 min to denature proteins and centrifuged for 10 min at 8160 x g. The supernatants were transferred to HPLC vials (12 x 32 mm, Waters) and stored at -20 C until further use. Standards of trehalose, glucose, mannitol, and glycerol ranging from 0.1% to 1% were utilized.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
When A. pullulans cells grown at 25 C for 40 h were exposed to sublethal stress conditions (35 C, 4.5% NaCl, or concurrent treatments of both), the intracellular levels of certain polyhyroxy compounds increased compared to the controls as shown by both TLC and HPLC. Thin-layer chromatograms of cell extracts that were heat shocked at 35 C for 45 min showed a marked increase in trehalose levels compared with controls (data not shown). This represented a near doubling of the mean cell concentration of trehalose compared with untreated controls (TABLE I ) as determined by HPLC (compare the results for control cells in Fig. 1 with those for heat-shocked cells in Fig. 2 ). Although changes in mannitol were not obvious by TLC analysis (due to the overlapping migration of glucose and mannitol and perhaps other sugars in the extracts) HPLC clearly showed a nearly two-fold increase in mannitol as a result of heat shock (Fig. 2 , Table I ). The low level of glycerol in untreated cells did not increase in heat-shocked cells (Fig. 2 , Table I ) and glycerol was not detected by TLC. The peak with a retention time of approximately 5 min that appeared in all the HPLC chromatograms appears to be a product of the polysaccharide pullulan which is produced by this fungus. The pullulan "halos" that form around cell pellets during centrifugation produce peaks with a retention time of approximately 5 min when subjected to HPLC.


View this table:
[in this window]
[in a new window]
  		TABLE I. Stress treatments and resulting solute concentrations as determined by HPLC

 


View larger version (14K):
[in this window]
[in a new window]
  		 FIG. 1. HPLC chromatogram of polyol accumulations in control cells.

 


View larger version (14K):
[in this window]
[in a new window]
  		 FIG. 2. HPLC chromatogram of polyol accumulations in heat-shocked cells.

 
An exposure to a sublethal concentration of 4.5% NaCl for 45 min elicited an increase in the internal concentration of glycerol from trace levels in controls to a mean concentration of 1.89 mg/mg protein in treated cells (Table 1 , Fig. 3 ). The mean mannitol concentration doubled in these cells. Various attempts to separate the trehalose peaks from the glucose peaks in salt-treated cells by HPLC (Fig. 3 ) were unsuccessful and precluded a quantitative determination of trehalose concentrations when salt was present. However, qualitative analysis employing TLC (Fig. 4 ) showed that the internal level of trehalose in the salt-shocked cells was the same as in control cells.



View larger version (13K):
[in this window]
[in a new window]
  		 FIG. 3. HPLC chromatogram of polyol accumulations in salt-stressed cells.

 


View larger version (116K):
[in this window]
[in a new window]
  		 FIG. 4. TLC of cellular extracts from salt-stressed and control cells. 1. trehalose. 2. glucose. 3. mannitol. 4. glycerol. 5. salt treatment. 6. control

 
Cells treated simultaneous with NaCl (4.5%, 45 min) and heat (35 C, 45 min), exhibited solute production similar to that of cells treated with heat alone. As was the case for the salt-shocked cells, the difficulty in resolving the HPLC trehalose/glucose peaks precluded quantification (Fig. 5 ). But TLC chromatograms (Fig. 6 ) indicated an increase in intracellular trehalose levels in the cells subjected to both heat and salt compared with the controls. This increase is probably due to the heat component only since high salt did not elicit an increase in trehalose. The intracellular mannitol level nearly doubled in treated compared to untreated cells (Table I , Fig. 5 ). The intracellular glycerol concentration was the same in the heat-/salt-stressed cells and in the controls (Table I , Fig. 5 ).



View larger version (14K):
[in this window]
[in a new window]
  		 FIG. 5. HPLC chromatogram of polyol accumulations in cells exposed to a simultaneous treatment of heat and salt.

 


View larger version (103K):
[in this window]
[in a new window]
  		 FIG. 6. TLC of cellular extracts from simultaneously heat-/salt-treated and control cells. 1. trehalose. 2. glucose. 3. mannitol. 4. glycerol. 5. heat and salt treatment. 6. control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Trehalose has been proposed to play an important role in the ability of organisms to withstand environmental stresses (Van Laere 1989Citation ). For example, a correlation between intracellular trehalose concentration and thermotolerance has been reported in S. cerevisiae (Lewis et al 1995Citation ; Hottiger et al 1987Citation ). Reports have shown that trehalose is also an important compound involved in the survival of yeasts such as S. cerevisiae, Torulaspora delbrueckii, and Zygosaccharomyces rouxii when exposed to severe osmotic stress conditions (Hounsa et al 1998Citation , D'Amore et al 1991Citation ).

Glycerol also has been shown to be a compatible solute for cells exposed to lowered water potential. Studies involving more than twenty yeasts and filamentous fungi indicate that glycerol is the primary osmoticum produced by many fungal cells subjected to NaCl stress when glucose is the carbon source (Blomberg and Adler 1993Citation ).

The role of mannitol in stress response has been investigated by Chatuverdi et al (1997)Citation who created a new mannitol biosynthetic pathway in an osmosensitive, glycerol-defective mutant of S. cerevisiae by transforming this yeast with multi-copy plasmids that encoded the mannitol -1- phosphate dehydrogenase gene obtained from Escherichia coli. Using this system, the investigators showed that the transformation restored the ability of the mutant strain to grow in the presence of a high NaCl concentration. Therefore, mannitol is capable of substituting for glycerol as the primary intracellular osmolyte in S. cerevisiae.

Torzilli (1997)Citation showed that exponentially-growing A. pullulans cells exposed to a sublethal treatment of heat, salt, or a simultaneous exposure to both, responded by synthesizing a set of stress proteins as demonstrated by SDS-PAGE. The ability of A. pullulans to withstand subsequent lethal treatments of heat and salt was correlated with the enhanced synthesis of these stress proteins. Furthermore, preliminary data revealed that inhibition of protein synthesis reduced, but did not eliminate, the ability of cells to survive lethal heat stress, indicating that other mechanisms are involved in the acquisition of stress tolerance. As reported here, the enhanced production of compatible solutes under the same conditions that induce stress tolerance in A. pullulans (Torzilli 1997Citation ) suggests that increases in the cellular concentrations of trehalose, glycerol, and mannitol also contribute to the acquisition of stress tolerance.

Torzilli (1997)Citation also reported that the stress pretreatments exhibited reciprocity with respect to the induction of stress tolerance. That is, not only did sublethal treatments of heat and salt induce thermotolerance and osmotolerance respectively, but the heat pretreatment protected against a lethal osmotic stress and the salt pretreatment protected against a lethal temperature treatment. Results presented here suggest that mannitol may contribute to this reciprocity since its concentration increased in both heat- and salt- treated cells. Although trehalose and glycerol levels increased only under heat and osmotic stress, respectively, they each have been implicated in both thermotolerance and osmotolerance in S. cerevisiae (Lewis et al 1995Citation ), and therefore may contribute to reciprocity in A. pullulans also.

Fungi inhabiting live or dead salt marsh plant shoots are subjected to periodic wetting and drying by salt water. This mycoflora can experience a gradual and simultaneous elevation in temperature and salinity due to evaporation during the day as the tide recedes, followed by a decline with the next high tide. The results reported here, together with earlier studies, are indicative of A. pullulans' ability to adapt to fluctuating environmental stresses characteristic of salt marsh ecosystems. Such physiological flexibility is consistent with the well-documented, cosmopolitan distribution of this fungus (Kolhmeyer and Kohlmeyer 1979Citation , Cooke 1959Citation ).


   FOOTNOTES
 
1 Corresponding author, atorzill{at}gmu.edu Back

Accepted for publication September 25, 2001.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Adler L, Pedersen A, Tunblad-Johansson I., 1982 Polyol accumulation by two filamentous fungi grown at different concentrations of NaCl Physiol Plant 56:139-142

Attfield PV., 1987 Trehalose accumulates in Saccharomyces cerevisiae during exposure to agents that induce heat shock response FEBS Lett 225:259-263[Medline]

Blomberg A, Adler L., 1993 Tolerance of fungi to NaCl In: Jennings DH, ed. Stress tolerance of fungi. New York: Marcel Dekker. p 209–231

Chatuverdi V, Bartiss A, Wong B., 1997 Expression of bacterial mtlD in Saccharomyces cerevisiae results in mannitol synthesis and protects a glycerol-defective mutant from high-salt and oxidative stress J Bac 179:157-162

Cooke WB., 1959 An ecological history of Aureobasidium pullulans (deBary) Arnaud Mycopathol Mycol Appl 12:1-45

D'Amore T, Crumplen R, Stewart GG., 1991 The involvement of trehalose in yeast stress tolerance J In Microbiol 7:191-196

Hall BG., 1983 Yeast thermotolerance does not require protein synthesis J Bac 156:1363-1365

Henle KJ, Nagle WA, Moss AJ, Herman TS., 1982 Polyhydroxy compounds and thermotolerance: a proposed concatenation Rad Res 92:445-451[Medline]

Hottiger T, Boller T, Wiemken A., 1987 Rapid changes of heat and desiccation tolerance with changes of trehalose content in Saccharomyces cerevisiae cells subjected to temperature shifts FEBS Lett 220:113-115[Medline]

Hottiger T, 1989 Correlation of trehalose content and heat resistance in yeast mutants altered in the RAS / adenylate cyclase pathway: Is trehalose a thermoprotectant? FEBS Lett 255:431-434[Medline]

Hounsa C, Brandt EV, Thevelein J, Hohmann S, Prior BA., 1998 Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress Microbiology 144:671-680[Medline]

Kohlmeyer J, Kohlmeyer E., 1979 Marine mycology: the higher fungi New York: Academic Press. 690 p

Komatsu Y, Kaul SC, Iwahashi H, Obuchi K., 1990 Do heat shock proteins provide protection against freezing? FEMS Microbiol Lett 72:159-162

Lewis JG, Learmonth RP, Watson K., 1995 Induction of heat, freezing and salt tolerance by heat and salt shock in Saccaromyces cerevisiae Microbiology 141:687-694[Medline]

Lindquist S., 1986 The heat shock response Annu Rev Biochem 55:1151-1191[Medline]

Molina FI, Hughes GC., 1982 The Growth of Zalerion maritimum (Linder) Anastasiou in response to variation in salinity and temperature J Exp Mar Biol Ecol 61:147-166

Nass R, Rao R., 1999 The yeast endosomal Na+/H+ exchanger, Hhx 1, confers osmotolerance following acute hypertonic shock Microbiology 145:3221-3228[Medline]

Panek AC, Vania JJM, Paschoalin MF, Panek D., 1990 Regulation of trehalose metabolism in Saccharomyces cerevisiae during temperature shifts Biochimie 72:77-79[Medline]

Torzilli AP., 1997 Tolerance to high temperature and salt stress by a salt marsh isolate of Aureobasidium pullulans Mycologia 98:786-792

Torzilli AP., Vinroot S, West C., 1985 Interactive effect of temperature and salinity on growth and activity of a salt marsh isolate of Aureobasidium pullulans Mycologia 77:278-284

Van Laere A., 1989 Trehalose, reserve and/or stress metabolite? FEMS Microbiol Rev 63:201-210

Watson K, Dunlop G, Cavicchioli R., 1984 Mitochondrial and cytoplasmic protein synthesis are not required for heat shock acquisition of ethanol and thermotolerance in yeast FEBS Lett 172:299-302[Medline]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Managbanag, J. R.
Right arrow Articles by Torzilli, A. P.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Managbanag, J. R.
Right arrow Articles by Torzilli, A. P.
Agricola
Right arrow Articles by Managbanag, J. R.
Right arrow Articles by Torzilli, A. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS