Compatible Solutes Protect against Chaotrope (Ethanol)-Induced, Nonosmotic Water Stress

Water stress is one of the major stresses experienced by cellularsystems and can take a number of distinct forms. In responseto turgor-related osmotic stress, cells produce compatible solutesthat are macromolecule protectants and also counteract the outflowof water from stressed cells. In this report we show that thegermination of conidia of Aspergillus nidulans, a sensitiveindicator of water stress, in the presence of ethanol is correlatedwith the intracellular concentration of the compatible solutesglycerol and erythritol, which protect against both osmoticand nonturgor forms of water stress.

INTRODUCTION

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Introduction

Cellular systems consist of up to 97% water, so thermodynamic changesin water availability cause fundamental biological stress that canseverely impact vital metabolic processes (3). Water stresscan be induced by a net loss of water from cells, e.g., dueto high levels of extracellular solutes such as NaCl that reducecell turgor and induce osmotic stress. Cells exposed to suchagents are subjected to both reduced turgor and stabilizingeffects on macromolecules, which can lead to membrane rigidityand impairment of protein structure (3, 16, 19, 25). Intracellularwater activity also may be reduced by chaotropic compounds,such as ethanol, that decrease the strength of hydrogen bondingand other electrostatic interactions and thereby perturb thestructure and function of hydrated macromolecules, includingnucleic acids, proteins, and lipids.

Cells generally respond to osmotic stress by acquiring and/orproducing compatible solutes that protect macromolecule structureand at the same time increase the osmotic pressure of the cytoplasmand thereby counteract water loss from cells (1, 3). Our workinghypothesis is that these compatible solutes also protect againstintracellular water stress due to alterations in the hydrationof cellular macromolecules caused by ethanol, a chaotropic solutethat does not cause osmotic stress.

The ascomycete Aspergillus (Emericella) nidulans is a modelmicroorganism whose genetics and metabolism, including thoserelated to stress and toxicology, have been widely studied.A. nidulans, like most yeast and fungal cells, synthesizes low-molecular-weightpolyols, such as glycerol, in response to osmotic stress (3, 21).Our objective in this study was to determine whether increasedintracellular levels of glycerol and erythritol affect conidialgermination in A. nidulans when the organism is cultured underconditions that reduce cell turgor and induce osmotic stress(e.g., in a high-NaCl environment), as well as those that increaseintracellular water stress by reducing the strength of water-macromoleculeinteractions (e.g., at high ethanol concentrations).

MATERIALS AND METHODS

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Materials AND METHODS

Organism, media, and production of conidia.
A culture of A. nidulans (IFO 4342) was obtained from the Institutefor Fermentation Osaka (Osaka, Japan) and maintained on potatodextrose agar (PDA; Nissui, Tokyo, Japan) at 25°C. All mediawere buffered with 21.3 g of 2-(N-morpholino)ethanesulfonicacid (MES) liter^-1, and the pH was adjusted to 5.8 with 2.5M NaOH. The water activity (a_w) values of the media were determinedby using a Humidat-IC II apparatus (Novasina, Talstrasse, Switzerland)fitted with a conventional humidity sensor and a protectiveeVALC alcohol filter (Novasina) (13). Three measurements ofmedium a_w were taken at 25°C for one sample from each independentexperiment, and the a_w variation was within ±0.003.

The media used were PDA (control), PDA plus 180 g of glycerolliter^-1, and PDA plus 115 g of KCl liter^-1 (Table 1). The glyceroland KCl concentrations employed were based on those used inprevious studies (7, 8). Petri plates were inoculated with 2-mm-diameterplugs of agar taken from the periphery of an exponential-phaseculture growing on medium of the same composition. Aerial conidiawere obtained from these cultures by incubation for 4 days (forcultures on PDA) or 8 days (for cultures on PDA plus glycerol andon PDA plus KCl) at 25°C. Plates of each medium were kept togetherin a sealed bag of low-density polyethylene to maintain a constantrelative humidity and the medium a_w while allowing gaseous exchange(17, 20). Conidia from each medium were harvested into sterileAnalaR water (Merck, Poole, United Kingdom), the suspensionwas filtered through glass wool to remove any hyphal fragments,and the conidia were washed and freeze-dried as described previously (7).

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TABLE 1. Intracellular polyols from conidia produced on different media

Extraction and analysis of compatible solutes.
Freeze-dried conidia (10 mg) were resuspended in 1 ml of AnalaRwater in a 2-ml microcentrifuge tube, sonicated (for 120 s atan amplitude of 28 µm), placed in a boiling water bath(5.5 min), and filtered (10). Polyol-containing filtrates wereinjected onto a series 4500 high-pressure liquid chromatograph(Dionex Corp., Sunnyvale, Calif.) fitted with a CarboPac PA1column (Dionex) and quantified by pulsed electrochemical detection (10).Analyses were performed in triplicate with conidia harvestedfrom three separate experiments that had been carried out atdifferent times.

Assessment of germination.
Germination on PDA (control; 0.998 a_w), PDA plus NaCl, and PDAplus ethanol was assessed over a range of concentrations insix-vented disposable petri plates (90 by 15 mm). At 25°C,the a_w values of PDA plus ethanol were (percentages signifyweight per volume) 0.977 (4%), 0.975 (4.5%), 0.973 (5%), 0.971 (5.5%),0.969 (6%), 0.967 (6.5%), 0.961 (7%), 0.957 (7.5%), and 0.955(8%), and the a_w values of PDA plus NaCl were 0.975 (8%), 0.968 (10%),0.960 (12%), 0.953 (14%), 0.947 (16%), 0.940 (18%), 0.934 (20%),and 0.930 (22%). Ethanol was incorporated into media that hadcooled to <50°C after sterilization to avoid losses through evaporation.

Conidia were harvested into sterile distilled water (if germinationwas assessed on PDA) or into sterile distilled water containingethanol or NaCl at the concentration corresponding to that inthe germination medium used, centrifuged, washed, and resuspendedin the wash solution, as described previously (7). Petri platesof each medium were inoculated with 200 µl of the conidialsuspension (which was spread with a glass rod, giving approximately5,000 conidia cm^-2), sealed in polyethylene bags, and incubatedat 25°C. Two 9-mm-diameter disks were removed from eachplate at intervals of approximately 6 h and stained with lactophenol cottonblue, and the germination status of 100 conidia from each treatment(three replicates) was assessed by light microscopy. Conidia withgerm tubes longer than their diameter were considered to have germinated.Thirty well-separated germinated conidia from each treatment(three replicates) were evaluated for germ tube length, numberof germ tubes per conidium, and diameter of imbibed spores.

RESULTS

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Results

Compatible-solute contents of conidia.
Conidia contained three main polyols, glycerol, erythritol,and mannitol (Table 1), as well as trace amounts of arabitol(<1 mg g of dry conidia^-1). The culture medium used did notaffect the concentration of mannitol in conidia (44 to 48 mg g^-1)but did affect the glycerol and erythritol contents of conidia(Table 1). Conidia from A. nidulans cultures grown on PDA (low-compatible-soluteconidia) contained negligible amounts of glycerol and erythritol,whereas those produced on PDA plus glycerol (high-compatible-soluteconidia) contained 90 mg of glycerol plus erythritol g^-1 (dry weight).Conidia obtained from A. nidulans grown on PDA plus KCl containedglycerol and erythritol concentrations that were low but higherthan control concentrations (Table 1).

Germination and germ tube growth of conidia in relation to polyol content.
Germination on PDA without stressors was rapid; by 6.5 h, themajority of control (low-compatible-solute) conidia had imbibedwater and swollen, and 5% had already germinated (data not shown).By 12 h, almost 90% had germinated, and the mean length of theprimary germ tubes had reached 34 µm (Table 2). In contrast,by 48 h, only 4% of low-compatible-solute conidia had germinatedon media containing 6.5% (wt/vol) ethanol, and even fewer hadgerminated on media containing $>=$ 7% (wt/vol) ethanol,despite extensive conidial swelling. For the conidia that didgerminate, the mean lengths of the germ tubes and the proportionof conidia with multiple germ tubes decreased with increasingethanol concentration in the medium. Low-compatible-solute conidiaalso failed to germinate on medium containing $>=$ 16%(wt/vol) NaCl (Table 2).

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TABLE 2. Influence of water stressors on germination parameters of conidia containing different levels of compatible solutes^a

Conidia containing large amounts of glycerol and erythritolgerminated only marginally more quickly on PDA than did controlconidia (Table 2). However, these high-compatible-solute conidiagerminated rapidly on media containing ethanol or NaCl; by 12h, they had begun to germinate on media containing 4% (wt/vol)ethanol or 8% (wt/vol) NaCl, and by 34 h, the majority had germinatedon media containing 5.5% (wt/vol) ethanol or 14% (wt/vol) NaCl(data not shown) and germ tube growth was proportionally advanced.By 48 h, most conidia had germinated on media containing allbut the highest concentrations of ethanol or NaCl, and eventhe conidia at these highest concentrations had imbibed waterand were apparently preparing to germinate (Table 2). Conidiafrom cultures grown on PDA plus KCl, the medium with the lowesta_w, were slightly more resistant to ethanol and salt stressthan those grown on unmodified PDA but substantially less resistantto both stressors than high-compatible-solute conidia producedon PDA plus glycerol (Table 2), eliminating a_w of the culturemedium as a possible variable affecting the observed differencesin germination.

DISCUSSION

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Discussion

Studies of water stress in microorganisms have focused mainlyon turgor-related (osmotic) stress caused by kosmotropic solutes (3)such as polyethylene glycol 600 and NaCl. In such cases, microbialcells synthesize and/or accumulate low-molecular-weight compatiblesolutes (3). Glycerol and erythritol enhance conidial germinationof several fungal species under NaCl-induced osmotic stress (8)and also have this effect on A. nidulans (Table 2). In contrast,mannitol is relatively ineffective as an osmoprotectant (2, 4, 7, 8).

In response to chaotropic solutes (e.g., LiCl, urea, and ethanol),microbial cells also up-regulate proteins involved in proteinstabilization, lipid metabolism and membrane structure, proteinsynthesis, and energy metabolism (14). Ethanol and other chaotropesreadily traverse lipid bilayers (5, 6, 15, 23) but nevertheless reducewater activity and decrease electrostatic interactions in biologicalmacromolecules (see Materials and Methods) (14, 16, 19, 25).We found that compatible solutes protect against ethanol (Table 2),which is consistent with the hypothesis that ethanol's primaryeffects on microbial metabolism are through perturbation ofwater-macromolecule relations. Glycerol and erythritol alsoreduce the adverse effects of ethanol on enzymes and membranesin vitro (18, 22). There is some evidence that the amino acidproline may play the same role in some microbial cells, suchas those of Saccharomyces cerevisiae (3, 11, 24).

In conclusion, the osmoprotectants glycerol and erythritol protectagainst ethanol-induced water stress in A. nidulans. These resultsare consistent with the hypothesis that compatible solutes aregeneral protective agents against different forms of water stressand extend our previous observations that microbial growth andmetabolism are inhibited by chaotrope-induced water stress (11, 12, 14).These results also raise the question of whether the principalstress mediated by both chaotropic and kosmotropic solutes isthe same, namely, a perturbation of water-macromolecule relations.

ACKNOWLEDGMENTS

This research was funded by the Ministry of Education, Science,Sports and Culture of Japan; the Sagawa Foundation for Promotionof Frontier Science, Kumamoto, Japan; and MIFRIEND (EU). J.E. Hallsworth gratefully acknowledges the receipt of a postdoctoralfellowship from the Japan Society for the Promotion of Science,Tokyo, Japan.

We thank K. Kayata and K. Kawashita (Sojo University, Kumamoto,Japan) for providing technical assistance and P. Nicholls andT. J. McGenity (University of Essex, Essex, United Kingdom)and S. Rainieri (Suntory Limited, Osaka, Japan) for helpful discussions.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom. Phone: 44-1206 872535. Fax: 44-1206 872592. E-mail: jehalls{at}essex.ac.uk.

REFERENCES

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