. 13: 529–539 (1997)

The Osmotic Hypersensitivity of the YeastSaccharomyces cerevisiae is Strain and Growth MediaDependent: Quantitative Aspects of the Phenomenon

ANDERS BLOMBERG

Department of General and Marine Microbiology, University of Goteborg, Medicinaregatan 9C, 413 90 Goteborg,Sweden

Received 22 March 1996; accepted 22 October 1996

Osmotic hypersensitivity is manifested as cellular death at magnitudes of osmotic stress that can support growth.Cellular capacity for survival when plated onto high NaCl media was examined for a number of laboratory andindustrial strains of Saccharomyces cerevisiae. During respiro-fermentative growth in rich medium with glucose asenergy and carbon source, the hypersensitivity phenomenon was fairly strain invariant with a threshold value ofabout 1 -NaCl; most strains fell within a 300 m range in LD10 values (lethal dose yielding 10% survival).Furthermore, all but one of the strains displayed similar differential death responses above the threshold value, i.e.ten-fold decreased viability for every 250 m increase in salinity. Addition of small amounts of salt to the growthmedium drastically improved tolerance and shifted the hypersensitivity threshold to higher NaCl concentrations.This salt-instigated tolerance could partly be reversed by washing in water. The washing procedure depleted cells ofthe glycerol that they had accumulated under saline growth, and the contribution from glycerol to the improvedtolerance was about 50% in the two strains examined. Growth on derepressing carbon sources like galactose, ethanolor glycerol gave strain-dependent responses. The laboratory strain X2180–1A drastically improved tolerance whilethe bakers’ yeast strain Y41 did so only marginally. It was concluded that all strains of S. cerevisiae display theosmotic hypersensitivity phenomenon in qualitative terms while the quantitative values differ. It was also proposedthat growth rate does not dictate the level of osmotic hypersensitivity of S. cerevisiae.? 1997 by John Wiley & Sons,Ltd.

Yeast 13: 529–539, 1997.No. of Figures: 6. No. of Tables: 2. No. of References: 37.

— osmotic hypersensitivity; Saccharomyces cerevisiae; strains; NaCl; plating; derepression

INTRODUCTION

Osmotic hypersensitivity is manifested as cellulardeath subsequent to a shift to magnitudes ofosmotic stress that can normally support growth.Thus, despite a potential to adapt and grow atthe post-shift osmolarity, certain cell states areextremely sensitive, i.e. hypersensitive, to the newstress conditions. The yeast Saccharomyces cere-visiae can grow in a range of environmental osmo-larities. However, at extremes of osmotic stress thisyeast grows very slowly, mainly as a consequenceof an disproportional deviation of carbon and

energy into production of the cellular osmolyteglycerol (Blomberg and Adler, 1992; Brown, 1978).If the stress conditions are governed by theaddition of NaCl, the maximal concentration atwhich proliferation can occur is about 2·5 .Survival of S. cerevisiae, however, can be dramati-cally effected at substantially lower concentrationsof salt. This can be observed during growth inbasal medium by plating on high water potentialmedium (basal medium) and low water potentialmedium (high concentration of stress solute;MacKenzie et al., 1986). A dramatic drop incolony-forming units (CFU) is encountered athigher NaCl concentrations in the plates whereonly a minor proportion of the culture forms

Correspondence to: Anders Blomberg.Contract grant sponsor: NFR

CCC 0749-503X/97/060529–11 $17.50? 1997 by John Wiley & Sons Ltd

colonies. This has been attributable to death ratherthan dormancy (MacKenzie et al., 1986). How-ever, yeast placed in high NaCl do not actuallydie until after 2 h (Edgley and Brown, 1983).The osmotic character of the phenomenon hasbeen validated by survival studies conducted withaddition of either salt or sugar to the stress media(MacKenzie et al., 1986). However, nothing isknown about the mechanisms of this irreversibleosmolarity-instigated cell damage. The phenom-enon is physiological and not genetic because cellsthat do survive and form colonies under conditionsof high stress will exhibit the same growth-relatedosmotic hypersensitivity if inoculated into freshbasal medium.It was initially assumed that osmotic hypersen-

sitivity was a feature specific for a limited numberof yeasts. Among 11 yeast species tested, it wasonly observed for S. cerevisiae and Candida krusei(MacKenzie et al., 1986). The phenomenon isconditional, however, since it is only encounteredabove certain threshold values of stress solute;no drop in viability was recorded for S. cerevisiaebelow 30% (w/v) of glucose (MacKenzie et al.,1986). Thus, the initial observation that Debaryo-myces hansenii did not display the phenomenonwas later shown to be a consequence of the stressrange applied, and at higher concentrations ofNaCl this extremely salt-tolerant species also re-vealed hypersensitivity during exponential growth(Larsson and Gustafsson, 1993). Osmotic hyper-sensitivity was also recorded for exponentiallygrowing cultures of Candida utilis if plated on highglucose media (Pardo et al., 1991). The phenom-enon might thus be valid for many if not all speciesof yeast and not restricted to specific examples.Strains of S. cerevisiae have been shown to differ

in growth properties relating to the osmolarity ofthe medium, with some strains deviating substan-tially from others (Anand and Brown, 1968).There are few quantitative studies evaluating thestrain dependence of osmotic hypersensitivity.Lewis et al. (1995) reported strain variation insurvival at 1·5 -NaCl. However, they did notinvestigate quantitatively the cause of this strain-dependent survival, e.g. shift in hypersensitivitythresholds or response characteristics.The major gene pool of most strains of S.

cerevisiae currently in use for molecular biologystudies originates from the strain S288C, or strainsclosely related to it (Mortimer and Johnston,1986). However, genotypic and phenotypic differ-ences, even among laboratory strains, are apparent

for features closely linked to the salt response(Wieland et al., 1995) or to other phenomena likeglucose derepression (Brown and Trumpower,1995). It was thus important to ascertain thegenerality of the osmotic hypersensitivity responseby performing studies on a number of strains ofdiverse origin, prior to conducting any detailedmolecular studies. Furthermore, a more thoroughquantitative description of the phenomenon wasneeded to facilitate future studies utilizing specificmutants and to distinguish the impact fromspecific genetic alterations.Strain-dependent quantitative differences pre-

sented in this study might prove fundamental inelucidating stress tolerance determinants. Thework presented should thus be regarded as abaseline for future research into a phenomenonwith the potential to contribute to our knowledgeabout the effects of sudden dehydration and thegeneral stress response of yeast cells. It furtherhighlights that care should be used in extrapolatingexperimental data even within one species and,when possible, the same strain(s) should be usedfor interlaboratory comparisons.

MATERIALS AND METHODS

Strains and growth conditions

Strains of S. cerevisiae used in this study areindicated in Table 1. The standard YPD mediumused contained 1% (w/v) yeast extract, 2% (w/v)bacteriological peptone and 2% (w/v) D-glucose.During the experimental period strains were main-tained on YPD slopes (+4)C) or stored long termin 20% glycerol at "85)C. A loopful of yeast cellswas inoculated into 5 ml of YPD medium in 15 mlFalcon tubes and incubated at 30)C overnight(15–20 h) in a rollerdrum. The overnight precul-ture was inoculated to an optical density at 610 nmof 0·1 (which corresponds to a cell density of about1#106 cells/ml) in 10 ml of growth medium in asidearmed flask (125 ml), incubated at 30)C ona rotary shaker (110 rpm) and harvested at anoptical density (610 nm) of 0·3–0·5.When the impact on osmotic hypersensitivity

from growth in saline medium was investigated,the YPD growth medium was supplemented withindicated amounts of NaCl. Depletion of theaccumulated glycerol was performed by transfer-ring 1 ml of the culture to a microcentrifuge tubeand centrifuge (13 000#g; 3 min), after which thecell pellet was resuspended and washed once with

530 .

? 1997 by John Wiley & Sons, Ltd. . 13: 529–539 (1997)

1 ml of water, and finally resuspended in 1 ml ofwater prior to dilution in water.For the starvation experiment, the overnight

preculture was prepared as above, and these cellswere spun down (5000#g; 5 min); the pellet wasresuspended in 5 ml of sterile milliQ water. Avolume corresponding to approximately 5#107

cells/ml was centrifuged (13 000#g; 3 min) andthe pellet resuspended in 10 ml of sterile milliQwater to yield a final optical density (610 nm) of0·5. The cell suspension was incubated in 125 mlflasks on a rotary shaker (110 rpm) at 30)C for24 h prior to the drop plate analysis.

Drop plate analysisThe colony-forming capacity of the culture was

determined on salt series YPD plates with 2% (w/v)agar, containing the indicated amounts of NaCl.The plates were left to dry in piles for at least 2days at room temperature to minimize the risk ofwater droplets forming on the agar surface, whichwould impair the drop plate analysis. At the timeof harvest the cells were diluted in water (milliQ)or, where indicated, in NaCl solutions isotonic tothe growth medium, to 10"1, 10"2, 10"3 and10"4. All procedures were performed at roomtemperature (no difference in the results was foundwhen cells after harvest were kept on ice beforeplating) and 10 ìl drops (duplicates) from each

dilution were placed onto each agar plate in thesalt series (0–12% [w/v] NaCl). The whole platingprocedure was completed within 20 min. Plateswere incubated at 30)C in plastic bags until col-onies were visible (plates were routinely countedafter 2, 7 and 14 days).

Construction of respiratory-deficient mutantsMutants were induced by ethidium bromide

treatment essentially as described before (Slonimskiet al., 1968). An overnight culture of strainX2180–1A and Y41 growing in synthetic medium(YNB) (Norbeck and Blomberg, 1996) was inocu-lated into fresh YNB to a cell density of about2#106 cells per ml. Ethidium bromide was addedto a final concentration of 10 ìg/ml and the cul-tures were incubated in the dark at 30)C on arotary shaker for 24 h. The cultures were subse-quently plated onto YPDG plates (rich platescontaining both 1% [w/v] glucose and 1% [w/v]glycerol), and incubated for 2 days at 30)C. Therespiratory defect of a number of small colonieswas confirmed by plating on both YPD and YPE(2% [v/v] ethanol) or YPGly (2% [w/v] glycerol)plates. Two of the isolates (for each strain)only capable of growth on glucose-containingplates were used for the test on the influence ofrespiration on osmotic hypersensitivity. No respir-atory activity was detected in these petite strains.

Table 1. Description of the isolates/strains of Saccharomyces cerevisiae used in this study.

Strain designation Isolation/genotype Reference/source

Industrial strains and natural isolatesCBS 439 Silvaner grapes; Germany CBSa

CBS 2421 Kefyr grains; Japan CBSCBS 2087 Flowers of lychee; China CBSY41; ATCC 38531 Bakers’ yeast; Australia Anand and Brown (1968)JB-A Bakers’ yeast; Sweden Jastbolaget; SwedenCBS 7764 Trout intestine; Sweden Andlid et al. (1995)CBS 7765 Trout intestine; Sweden Andlid et al. (1995)J52c The sea; Sweden Norkrans (1966)J16c The sea; Sweden Norkrans (1966)

Laboratory strainsS288C MATa, SUC2, mal, mel, gal2, CUP1 YGSCb

X2180–1A MATa, SUC2, mal, mel, gal2, CUP1 YGSCX2180–1B MATá, SUC2, mal, mel, gal2, CUP1 YGSCSKQ MATa/á, ade1/+, +/ade2, +/his1 Bataille et al. (1988)

aCentraalbureau voor Schimmelcultures, Delft, The Netherlands: http://www.cbs.knaw.nl.bYeast Genetic Stock Centre, University of California, U.S.A.cThese strains recently received CBS numbers: J16=CBS 8291 and J52=CBS 8292.

531 .

? 1997 by John Wiley & Sons, Ltd. . 13: 529–539 (1997)

Measurements of glycerol content and therespiratory capacityAnalysis of glycerol was carried out by enzy-

matic methods, as described previously (Blomberget al., 1988).The respirometric measurements were per-

formed in a Cyclobios oxygraph (Haller et al.,1994). Samples to be analysed (2 ml) were with-drawn from the growing cultures and immediatelyapplied to the oxygraph.

RESULTS

Strain-dependent differences in osmotichypersensitivityIn this screen for intraspecies variation in hyper-

sensitivity to high concentrations of NaCl, anumber of genetically defined haploid and diploidlaboratory strains of S. cerevisiae, as well asnatural isolates and industrial strains, of unknowngenetic background and ploidy, were used (Table1). The natural isolates represented a number ofdifferent biotopes regarding the prevailing magni-tude in osmotic stress or type of stress soluteencountered; e.g. sylvaner grapes (CBS 439), flow-ers of lychee (CBS 2087), bakers’ yeast (Y41 andJB-A), fish intestine (CBS 7764 and 7765) andmarine waters (J16 and J52). Thus, strains CBS439 and CBS 2087 would represent variants se-lected for survival and growth in sugar-containingenvironments, while J16 and J52 would probablybe better adapted to NaCl. However, these strainswere not selected a priori for any specific osmo-related property, and are thus unlikely to representthe complete spectrum of S. cerevisiae strains asfar as osmotic hypersensitivity is concerned. Theyshould merely be viewed as an almost random butslightly environmentally biased collection ofstrains.Overnight cultures were inoculated into rich

medium without NaCl and during the subsequentrespiro-fermentative exponential growth dilutedand plated on salt-containing rich media plates.The commonly used haploid laboratory strainX2180–1A, which is isogenic to strain S288C(Mortimer and Johnston, 1986) that is utilized inthe genome sequencing project, clearly displayedosmotic hypersensitivity (Figure 1). For this labo-ratory strain the salinity threshold for the phenom-enon to be manifested was about 1·1 -NaCl,below which no significant decrease in viabilitywas observed. At higher NaCl concentrations an

increasing proportion of the culture was unable toform colonies on the salt plates. The industrialstrain Y41 exhibited a lower threshold value ofapproximately 0·75 -NaCl. For both these strainsthe phenomenon was manifested equally well inthe OD range 0·3 to 0·8 during exponential growth(mid-exponential phase), with only slight varia-tions in relative numbers of survivors (Figure 1).Subsequent analysis of other strains was thereforeconducted at about OD 0·5 after growth for atleast two generations. The technical problems ofaccurately determining the threshold valueprompted use of another criterium for the inter-strain comparison. Thus, the lethal dose of NaClyielding only 10% survivors (LD10), determined bygraphical interpolation, was chosen (dotted line inFigure 1). Strain-specific LD10 values were highlyreproducible. The standard deviation was less than6% in the two strains (X2180–1A and Y41) forwhich LD10 values were determined in at least tenindependent cultures (Table 2).For the strain-specific differences in LD10 values

to represent variation in hypersensitivity thresh-olds, the strains should display an identical differ-ential salt-dependent decrease in viability at highermagnitudes of stress. The CFU values above theNaCl threshold for strains X2180–1A and Y41

Figure 1. Colony-forming capacity on NaCl-containing YPDplates for cells of S. cerevisiae growing exponentially in therespiro-fermentative phase in YPD medium without addition ofsalt. Cultures were tested for osmotic hypersensitivity at differ-ent cell densities, measured as OD values at 610 nm: circles,OD610=0·3; squares, OD610=0·4; triangles, OD610=0·6; dia-monds, OD610=0·8. Cultures were diluted in water prior toplating. Filled symbols represent strain X2180–1A and opensymbols strain Y41. The colony-forming capacity on plateswithout salt was set to 100%. The dotted line indicates 10%survival and the intercepts with the curves the LD10 values(dose of NaCl to yield only 10% viability). Results are from onetypical experiment.

532 .

? 1997 by John Wiley & Sons, Ltd. . 13: 529–539 (1997)

decreased more or less exponentially, i.e. ten-foldfor every 250 m increase in concentration ofNaCl (Figure 1). As a good approximation thisrelationship was true for most strains tested (Fig-ure 2; Table 2). The only exception was CBS 2087,which displayed a less severe response per unit ofNaCl increase (slope "0·8; Table 2). Another oddfeature of this strain was its low maximum NaClconcentration tolerated for growth; no colonieswere observed at 1·8 -NaCl even after prolongedincubation (25 days), and on the 1·6 -NaCl platesthe colonies produced were very small. Biochemi-cal classification of this isolate from flowers oflychee has grouped it as a strain of S. cerevisiae,thus indicating it as a rare variant of this species.Most of the strains tested displayed hypersensi-

tivity threshold values between those obtainedfor X2180–1A and Y41. Even strains isolatedfrom salty environments, like strains J16 and J52,were not more tolerant than the laboratory strains(Figure 2B). The only two extremes were CBS 2087and CBS 439, which both exhibited 100% viabilityup to 1·25 -NaCl in the plates (Figure 2A).Studies on the impact of rate of growth on generalstress tolerance have indicated a relationship; theslower the cells grow the better they withstand thestress applied (Elliot and Futcher, 1993). Most ofthe strains tested for osmotic hypersensitivity in

this study grew with similar generation times.Thus, no correlation was observed between growthrate and the hypersensitivity threshold. Forexample, the two strains Y41 and CBS 439, whichdisplayed a 300 m difference in LD10 values,exhibited no significant difference in growth rate.

Growth in NaCl medium alters the hypersensitivitythresholdIt has been shown qualitatively that adding

minor amounts of NaCl to the growth mediumenhances the tolerance of exponentially growingyeast cells to osmotic stress (MacKenzie et al.,1986). In the present study, this response wasexamined quantitatively for the laboratory strainX2180–1A and the industrial strain Y41, whichdisplay clear differences in hypersensitivity duringgrowth in basal medium (Figure 1, Table 2).Addition of up to 360 m-NaCl to the culturemedium did not hamper growth; the generation

Table 2. Strain-dependent variables during mid-exponential respiro-fermentative growth in basal YPDmedium.

StrainGenerationtime (h)

LD10 (exp. growth)()

Slope ofdeclinea

CBS 439 1·7&0·17 1·38&0·07 (3) "1·9CBS 2421 2·1&0·23 1·26&0·05 (3) "1·6CBS 2087 2·7&0·02 1·71&0 (3) "0·8CBS 7764 1·6&0·01 1·21&0 (2) "1·9CBS 7765 2·2&0·11 1·13&0·03 (2) "1·5Y41 1·8&0·05 1·06&0·06 (10) "1·5JB-A 1·6&0·01 1·20&0·01 (2) "1·7J52 1·5&0·07 1·19&0·02 (2) "1·6J16 1·8&0·19 1·17&0·05 (2) "1·8S288C 1·9&0·11 1·29&0 (2) "1·7X2180-A 1·7&0·01 1·34&0·07 (10) "1·8X2180–1B 1·7&0·19 1·35&0·04 (3) "1·5SKQ2n 1·7&0·01 1·21&0·01 (3) "1·3

aSlope obtained by linear regression including all data pointsat NaCl concentrations above the hypersensitivity threshold.Multiplication with 2·303 yields the NaCl-induced specificdeath constant ("1).

Figure 2. Strain-dependent hypersensitivity to plating onNaCl-containing YPD media during mid-exponential growth inthe respiro-fermentative phase on YPD medium. (A) CBS 2087(filled symbols; CBS 2087 does not grow at 1·8 -NaCl); CBS439 (open symbols); (B) J16 (filled symbols); J52 (open sym-bols); (C) Y41 (filled symbols); JB-A (open symbols); (D) CBS7765 (filled symbols); CBS 7764 (open symbols). Cultures werediluted in water prior to plating. The colony-forming capacityon plates without salt was set to 100%. The dotted line indicates1 -NaCl to facilitate intergraph comparison of thresholdvalues. Results are averages from at least two independentexperiments.

533 .

? 1997 by John Wiley & Sons, Ltd. . 13: 529–539 (1997)

time remained the same as for control cultures.At higher salinities, however, both strains wereslightly retarded, with strain Y41 growing with ageneration time of 2·2 and 2·9 h at 0·54 and0·71 -NaCl, while the corresponding values forX2180–1A were 2·0 and 2·4 h. The salt-instigatedincreased tolerance of strain Y41 was roughlyproportional to the salinity of the growth medium;an increase of 360 m-NaCl enhanced the LD10value by about 580 m (Figures 3 and 4). It isimportant to note that salt addition did not oblit-erate the phenomenon, which was implicated inthe original report (MacKenzie et al., 1986), butrather shifted the hypersensitivity threshold tohigher values. The laboratory strain X2180–1Adisplayed a qualitatively similar response to strainY41 (Figure 4).If the stress magnitude (difference in NaCl con-

centration) solely determines the amount of viablecells, the improved tolerance for salt-grown cul-tures could be a consequence of a decrease in thedelta-NaCl encountered. One likely physiologicalbuffering substance in this respect is the saline-instigated accumulation of glycerol, which athigher extracellular salinities can reach molarintracellular levels (Blomberg and Adler, 1992).However, the cells can easily be depleted oftheir accumulated glycerol by washing in water

(Blomberg and Adler, 1989). Accordingly, the salt-grown cells were washed before NaCl plating. Nointracellular glycerol could be detected in thewater-washed cells, while before washing the intra-cellular content of cells grown on 0·7 -NaCl wasapproximately 2 ìmol glycerol per mg dry weight.This washing treatment resulted in markedlydecreased LD10 values for both strains (Figure 4),consistent with previously reported qualitativedata (MacKenzie et al., 1986), implicating glycerolas one of the factors contributing to the enhancedsalt tolerance.However, even if cultures displayed decreased

tolerance as a result of washing, the salt-grownglycerol-depleted cells were more tolerant thancells grown under non-saline conditions. This indi-cates that factors other than glycerol are involvedin the enhanced NaCl tolerance. At a growthmedium salinity of 360 m, the contribution ofglycerol to the increased NaCl tolerance wascalculated from the decreased LD10 values forwashed cells to 63% and 47% for strain Y41 andX2180–1A, respectively. The procedure of growingcells in salt and subsequently washing these salt-adapted cells, amplified the strain differencesobserved in basal medium, i.e. the difference inLD10 values between these two strains increasedto almost 600 m for cells grown in 0·7 -NaClmedium and washed in water.

Figure 3. Colony-forming capacity on NaCl-containing YPDmedia for cells of strain Y41 of S. cerevisiae during mid-exponential growth in the respiro-fermentative phase in YPDmedium with addition of the following amounts of NaCl in thegrowth medium: 0 (open circles); 0·18 (filled circles); 0·36 (open squares); 0·54 (filled squares); 0·71 (filled triangles).Cultures were diluted in NaCl solutions of the same salinity asthe growth medium prior to plating. The colony-formingcapacity on plates without salt was set to 100%. The dotted lineindicates 10% survival and the intercepts with the curves theLD10 values (dose of NaCl to yield only 10% viability). Resultsare averages from two independent experiments.

Figure 4. LD10 values for strains X2180–1A (circles) and Y41(squares) as a function of growth medium salinity, and theimpact of glycerol depletion. Filled symbols indicate valuesfrom cultures diluted in solutions isotonic to the growthmedium, while open symbols represent values after washingand diluting cells in water. Results are averages from twoindependent experiments.

534 .

? 1997 by John Wiley & Sons, Ltd. . 13: 529–539 (1997)

Growth on derepressing carbon sources influencesosmotic hypersensitivity in a strain-dependentfashion

Saline growth instigated higher tolerance toNaCl plating, a feature that was not solely a conse-quence of the accumulated glycerol. So, what isthe additional determinant of this salinity-inducedresistance? Cells growing exponentially in salinemedia display a number of phenotypic changes,e.g. enzymes in central metabolism have recentlybeen shown to alter in expression (Norbeck andBlomberg, 1996). Osmotic stress will also affect thebalance in flux of carbon between respirationand fermentation (O} lz et al., 1993). Furthermore,growth on derepressed carbon sources improvesheat-shock tolerance (Elliot and Futcher, 1993).These observations prompted an investigation

into the impact of growth under glucose-derepressed conditions on osmotic hypersensitiv-ity. Glucose-derepressed conditions cause majordifferences in central metabolism influencing theexpression of glycolytic enzymes as well as therespiratory capacity. Three carbon sources (galac-tose, ethanol and glycerol) were thus tested inaddition to glucose, since growth on any of theseresults in a derepressed phenotype (Fraenkel,1982). Growth of strain X2180–1A under thesederepressing conditions resulted in a markedlyincreased tolerance to high salinity plating, andover the whole salt range tested only a minorimpact on viability on NaCl plates was observed(Figure 5). Growth on the different carbon sourcesresulted in no significant difference in this strain,all three substrates dramatically improving thetolerance with a roughly 700 m increase in thevalue of LD10. Strain Y41, however, displayed asomewhat less striking response, and growth ongalactose or ethanol only slightly affected thehypersensitivity (Figure 5B). This result is consist-ent with data obtained in an earlier study on thisstrain, where post-diauxic growth on ethanol onlymarginally influenced osmotic hypersensitivity(Blomberg et al., 1988). Glycerol was the carbonsource that exhibited the greatest impact onosmotic hypersensitivity in strain Y41; the LD10value increased by 400 m. Thus, the osmotichypersensitivity of strains X2180–1A and Y41 wasqualitatively similar but quantitatively rather dif-ferently affected by the change in the carbonsource of the growth medium. Growth on thesealternative carbon sources affected the growth rateof the two strains similarly, resulting in generation

times of 3·5, 4·9 and 5·3 h for galactose, ethanoland glycerol, respectively.Glucose represses a wide array of cellular func-

tions. One of the most prominent is respiration,which is diminished about three-fold duringgrowth on glucose compared to growth on ethanol(Blomberg et al., 1988). We tested whether thisindicates that enhanced respiration is the cause ofthe improved tolerance for derepressed X2180–1A,and whether the strain-dependent variation intolerance is a consequence of strain-specific levelsof respiratory activity during glucose growth; thehigher the respiratory capacity the higher the salttolerance. The respiratory capacity was thus testedunder growth in basal rich medium with glucose ascarbon source. The two strains exhibited similarlevels of respiration under these conditions, 2·8

Figure 5. Carbon source-induced changes in osmotic hyper-sensitivity. Colony-forming capacity on NaCl-containing YPDmedia for cells of S. cerevisiae growing exponentially in therespiro-fermentative phase in non-saline YP medium sup-plemented with either glucose (open circles), galactose (filledcircles), ethanol (open squares) or glycerol (filled squares) ascarbon and energy source. (A) X2180–1A; (B) Y41. Cultureswere diluted in water prior to plating. The colony-formingcapacity on plates without salt was set to 100%. The dotted lineindicates 10% survival and the intercepts with the curves theLD10 values (dose of NaCl to yield only 10% viability). Resultsfrom one typical experiment.

535 .

? 1997 by John Wiley & Sons, Ltd. . 13: 529–539 (1997)

and 2·7 ìmol O2/mg dry weight per h for Y41 andX2180–1A, respectively. In addition, respiratory-deficient mutants (petite mutants) were con-structed from strains X2180–1A and Y41. Two ofthe petite mutants from each strain were examinedduring exponential growth in basal medium, andno significant reduction in their hypersensitivitythreshold was observed compared to the corre-sponding respiratory-competent wild type (datanot shown). Apparently, different levels of respir-ation per se are not the cause of the strain-dependent variation in osmotic hypersensitivity;rather, other cellular factors affected by glucoserepression are candidates for cellular determinants.

Starvation drastically affects osmotichypersensitivityCells growing in basal medium were subjected to

a total starvation regime. After 24 h of starvationthe osmotic hypersensitivity was tested. This treat-ment drastically changed the capacity for all strainsto survive plating at high NaCl concentration(Figure 6 and data not shown). No change in vi-ability was observed during the starvation regime.All strains examined improved their tolerance,although they were affected to different extents.After this drastic derepressing treatment, the Y41strain exhibited a LD10 value of about 1·7, whileX2180–1A was much more tolerant and about 20%

of the population was viable at 2·15 -NaCl. Ap-parently some strains like Y41 are inherently moresensitive than others irrespective of the state ofproliferation. The death curves for these starvedcultures were slightly steeper compared to thoseobtained for proliferating cells (compare Figures 5and 6), indicating somewhat different deathmechanisms for the two cell states.

DISCUSSION

Strain-specific stress responsesA large number of strains are currently in use in

the yeast community. Most, but not all, strainsemployed in academic research have a phylo-genetic link to the type strain S288C (Mortimerand Johnston, 1986). It should be remembered thatS288C is the result of ‘scientist selection’ over theyears from progenitor strains originating frominterbreeding of isolates of S. cerevisiae and relatedSaccharomyces species (Mortimer and Johnston,1986; Sherman, 1991). Thus, one should expectphysiological differences between these academicvariants and other isolates of S. cerevisiae. Suchdifferences have been demonstrated in this studyfor the bakers’ yeast Y41 and the S288C progenyX2180–1A. Strain-specific properties are wellknown within biotechnology, where special meta-bolic features of individual isolates have beeninstrumental to the industrial utilization of yeast(Kielland-Brandt, 1994). The results of the presentstudy thus highlight in general terms the need for astrict standardization of the use of strains if resultsare to be compatible between laboratories.During respiro-fermentative exponential growth

in rich medium with glucose as energy and carbonsource, the qualitative as well as the quantitativeaspects of the hypersensitivity phenomenon werefairly strain invariant. This constancy was mani-fested as a phenomenological threshold value formost strains of about 1 -NaCl, below which moreor less 100% viability was observed, and a subse-quent exponential decline in viability of a factor often for every 250 m increase in NaCl in theplates. This implies that the osmotic hypersensitiv-ity phenomenon is widespread among isolates of S.cerevisiae, from trout intestine to grapes, and thusappears closely linked to the general physiology ofthis species. However, different treatment of cellsdrastically enhanced minor strain differences, andin certain conditions the LD10 values of the twostrains most extensively studied, Y41 and X2180–1A, differed by almost 700 m.

Figure 6. Starvation-induced changes in osmotic hypersensi-tivity of S. cerevisiae strains. Colony-forming capacity on YPDplates containing indicated amounts of NaCl for culturesstarved for 24 h: CBS 439 (open circles); Y41 (filled circles);X2180–1A (open squares); SKQ2n (filled squares). Cultureswere diluted in water prior to plating. The colony-formingcapacity on plates without salt was set to 100%. The dotted lineindicates 10% survival and the intercepts with the curves theLD10 values (dose of NaCl to yield only 10% viability). Resultsfrom one typical experiment.

536 .

? 1997 by John Wiley & Sons, Ltd. . 13: 529–539 (1997)

In addition, one of the strains, CBS 2087, wasextreme in its response to salt. It had a phenomeno-logical threshold in the same range as the others;however, its response at higher concentrations ofsalt was less dramatic, resulting in an almost1000-fold difference in viability between this strainand strain Y41 at 1·6 -NaCl. This huge differencein salt stress tolerance between these strains wasconsistent with the results in a recent report ofyeast hypersensitivity at one single salt concen-tration, where the viability was found to be highlystrain dependent (Lewis et al., 1995). CBS 2087was also unusual because it would not grow at1·8 -NaCl. It thus displayed an unusually lowlevel of osmotic hypersensitivity while being ratherosmosensitive as far as its salt growth limit wasconcerned. This strain represents a rare variant ofthe yeast S. cerevisiae, and its cellular propertiesmight prove invaluable in the identification ofmolecular stress mechanisms to salt.

Osmotic shock resistance was not restricted tonon-proliferating cells

Non-growing stationary-phase cells are lessaffected by almost any kind of stress treatmentcompared to actively proliferating cells (Werner-Washburne et al., 1993). This was also true for asudden osmotic shock, and all strains tested exhib-ited improved tolerance after a starvation period.It has also been proposed that the mechanisms forstress resistance are not compatible with rapidgrowth since heat-shock resistance increasesgradually as cells grow more and more slowly; theresistance of starved stationary cultures might bethe extreme end of a continuum (Elliot andFutcher, 1993). However, in the present studyosmotic shock resistance was constitutive undercertain growth conditions, since the osmotichypersensitivity of exponentially growing cellscould be almost abolished either by the addition ofsalt to the growth media, or by growth on alterna-tive carbon sources. Apparently, the osmotichypersensitivity phenotype is not related to any ofthe strictly growth-linked functions in the cell, likeDNA replication or biosynthesis. Rather, it seemsas if some other strain-specific feature(s) sets therelative tolerance phenotype of a strain and thatgrowth conditions merely shift the response-scale,since strain-dependent differences in hypersensitiv-ity during basal growth, reflected in the respectiveLD10 values, was still apparent for starved cul-tures. The growth rate hypothesis has also been

partly falsified as growth on different nitrogensources results in various growth rates; leucineinstigates slow growth while still making cellshighly heat sensitive (Elliot and Futcher, 1993).Furthermore the growth rate of the salt-tolerantyeast D. hansenii does not influence the high saltplating efficiency (Larsson et al., 1990).

Accumulated glycerol is not the only cellulardeterminant of tolerance

Washing of salt-grown cells revealed an approxi-mately 50% contribution from the osmolyteglycerol to the saline growth-instigated resistanceto high NaCl plating. The cellular cause of theadditional 50% increase in survival is not known,but it could be hypothesized that expressionchanges linked to glucose derepression accountfor the additional tolerance since growth on non-fermentable carbon sources, at least for strainX2180–1A, drastically improved cellular resist-ance. Protein expression changes of strain Y41during steady-state growth in salt have beenstudied by two-dimensional polyacrylamide gelelectrophoresis, which revealed 13 proteins with atleast a two-fold change in expression (Norbeckand Blomberg, 1996). In addition to these saltresponders, two of the glycolytic enzymes down-stream of the triosephosphates, Tdh3p (glyceralde-hyde 3-phosphate dehydrogenase) and Eno2p(enolase), decrease significantly in amount and intheir corresponding specific activities. Both theseenzymes are repressed during growth on a non-fermentable carbon source (Bataille et al., 1988),thus conveying the impression of an overallslightly derepressed phenotype during salinegrowth. However, glucose repression, as studiedby invertase measurements, is only marginallyaffected by salt addition (Albertyn et al., 1994a).Furthermore, fully derepressed ethanol-grown Y41cells only marginally improved tolerance, whichmakes a slight salt-imposed derepression unlikelyas the sole cause of the glycerol-independentenhanced resistance of salt-grown cultures.It has been reported that Gpd1p overproduction

only has a major impact on the formationof glycerol under more derepressed conditions(Albertyn et al., 1994b). A vital role for decreasedexpression of enzymes in the lower part of glycolysisin the salt-instigated increase in glycerol productionhas been proposed (Norbeck and Blomberg, 1996).This would be in line with a resistance mechanisminvolving rapid production and accumulation of

537 .

? 1997 by John Wiley & Sons, Ltd. . 13: 529–539 (1997)

glycerol; only cells with a high capacity would re-cover from the dehydration-implemented cellulardamage. The increased amount and activity ofglycerol-3-phosphate dehydrogenase in salt-growncells favours such a resistance mechanism (Andreet al., 1991; Norbeck and Blomberg, 1996) and therecent identification of a salt-induced glycerol-3-phosphatase (Gpp2p) (Norbeck et al., 1996) furthersupports this assumption.Two other putative determinants of salt shock

tolerance have been suggested, factors with noclear connection to the glycerol story: (i) catalaseA, Ctt1p, an enzyme involved in the cellular pro-tection against hydroxyl radicals (Schuller et al.,1994), and (ii) trehalose, accumulation of whichpreserves cell membrane integrity in vitro upontotal dehydration (Crowe et al., 1984). Trehalosehas been proposed to have a protective effect oncells subjected to osmotic shock (MacKenzie et al.,1988) and dehydration stress (Hottiger et al.,1987). However, its role in salt shock tolerance hasbeen experimentally challenged, since resistantstationary-phase cultures of C. utilis retain toler-ance even after depletion of intracellular trehalose(Pardo et al., 1991).Little is known at present about the cellular

targets that initially are damaged by salt. Highconcentrations of NaCl may be mutagenic (Parkerand von Borstel, 1987). However, rad mutantsaffected in DNA repair do not display alteredosmotic hypersensitivity (A. Blomberg, unpub-lished data). Neither could any correlation be seenbetween ploidy and stress resistance; haploids(X2180–1A, X2180–1B and S288C) and diploids(SKQ2n) displayed similar hypersensitivity pro-files. These results make salt-instigated DNAdamage less likely as the initial event in theosmotic hypersensitivity phenomenon.It has also been proposed that cell shrinkage per

se would initiate death, since there is a correlationafter dehydration between cell volume and vi-ability (Morris et al., 1986). Plasmolysis is a plaus-ible cellular mechanism that could affect cellviability and also would explain the threshold forosmotic hypersensitivity. This phenomenon is dif-ficult to visualize by microscopic examinationin yeast cells (Morris et al., 1986). However, theconcentration at which incipient plasmolysisoccurs was extracted from the Boyle-van’t Hoffplot and the non-linear osmotic behaviour of cells(Meikle et al., 1988) and reported to be 0·39 -NaCl (2·48 MPa). Thus, the threshold values forthe different strains (0·75–1·1 -NaCl) did not

correlate well with the reported salt concentrationat plasmolysis, and if this response is the initialdeath target an additional increase in stress mag-nitude apparently has to be applied for the osmotichypersensitivity phenomenon to be manifested.

ACKNOWLEDGEMENTS

I thank Agneta Danielsson for technical help,Christer Larsson for guidance in performing therespiratory measurements, and Lennart Adlerand Joakim Norbeck for critical reading of themanuscript. This work was supported by grants01758–304 and 01758–305 from the NFR.

REFERENCES

Albertyn, J., Hohmann, S. and Prior, B. A. (1994a).Characterization of the osmotic-stress response inSaccharomyces cerevisiae: osmotic stress and glucoserepression regulate glycerol-3-phosphate dehydro-genase independently. Curr. Gen. 25, 12–18.

Albertyn, J., Hohmann, S., Thevelein, J. M. and Prior,B. A. (1994b). GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth un-der osmotic stress in Saccharomyces cerevisiae, and itsexpression is regulated by the high osmolarity glycerolresponse pathway. Mol. Cell. Biol. 14, 4135–4144.

Anand, J. C. and Brown, A. D. (1968). Growth ratepatterns of the so-called osmophilic and non-osmophilic yeasts in solutions of polyethylene glycol.J. Gen. Microbiol. 52, 205–212.

Andlid, T., Vazquez-Juarez, R. and Gustafsson, L.(1995). Yeast colonizing the intestine of rainbow trout(Salmo gairdneri) and turbot (Scophtalmus maximus).Microbial Ecol. 30, 12.

Andre, L., Hemming, A. and Adler, L. (1991). Osmo-regulation in Saccharomyces cerevisiae. Studies on theosmotic induction of glycerol production and glycerol3-phosphate dehydrogenase (NAD+). FEBS Lett.286, 13–17.

Bataille, N., Thoraval, D. and Boucherie, H. (1988).Two-dimensional gel analysis of yeast proteins: Ap-plication to the study of changes in the levels of majorpolypeptides of Saccharomyces cerevisiae dependingon the fermentable or nonfermentable nature ofcarbon source. Electrophoresis 9, 774–780.

Blomberg, A. and Adler, L. (1989). Roles of glyceroland glycerol-3-phosphate dehydrogenase (NAD+) inacquired osmotolerance of Saccharomyces cerevisiae.J. Bacteriol. 171, 1087–1092.

Blomberg, A. and Adler, L. (1992). Physiology of osmo-tolerance in fungi. Adv. Microbial Phys. 33, 145–212.

Blomberg, A., Larsson, C. and Gustafsson, L. (1988).Microcalorimetric monitoring of growth of Saccharo-myces cerevisiae: Osmotolerance in relation to physi-ological state. J. Bacteriol. 170, 4562–4568.

538 .

? 1997 by John Wiley & Sons, Ltd. . 13: 529–539 (1997)

Brown, A. D. (1978). Compatible solute and extremewater stress in eukaryotic micro-organisms. Adv.Microbial Phys. 17, 181–242.

Brown, T. A. and Trumpower, B. L. (1995). Strain-dependent variation in carbon source regulation ofnucleus-encoded mitochondrial proteins of Saccharo-myces cerevisiae. J. Bacteriol. 177, 1380–1382.

Crowe, J. H., Crowe, L. M. and Chapman, D. (1984).Preservation of membranes in anhydrobiotic organ-isms: the role of trehalose. Science 223, 701–703.

Edgley, M. and Brown, A. D. (1983). Yeast waterrelations: Physiological changes induced by solutestress in Saccharomyces cerevisiae and Saccharomycesrouxii. J. Gen. Microbiol. 129, 3453–3463.

Elliot, B. and Futcher, B. (1993). Stress resistance ofyeast cells is largely independent of cell cycle phase.Yeast 9, 33–42.

Fraenkel, D. G. (1982). Carbohydrate metabolism. InStrathern, J. N., Jones, E. W. and Broach, J. R. (Eds),The Molecular Biology of the Yeast Saccharomyces;Metabolism and Gene Expression. Cold Spring HarborLaboratory, Cold Spring Harbor, New York, pp.1–37.

Haller, T., Ortner, M. and Gnaiger, E. (1994). Arespirometer for investigating oxidative cell metab-olism: towards optimization of respiratory studies.Anal. Biochem. 218, 338–342.

Hottiger, T., Boller, T. and Wiemken, A. (1987). Rapidchanges of heat and desiccation tolerance correlatedwith changes of trehalose content in Saccharomycescerevisiae cells subjected to temperature shifts. FEBSLett. 220, 113–115.

Kielland-Brandt, M. C. (1994). Industrial Saccharo-myces yeasts. In Johnston, J. R. (Ed.), MolecularGenetics of Yeast; A Practical Approach. IRL Press,Oxford, pp. 247–259.

Larsson, C. and Gustafsson, L. (1993). The role ofphysiological state in osmotolerance of the salt-tolerant yeast Debaryomyces hansenii. Can. J. Micro-biol. 39, 603–609.

Larsson, C., Morales, C., Gustafsson, L. and Adler, L.(1990). Osmoregulation of the salt-tolerant yeastDebaryomyces hansenii grown in a chemostat at dif-ferent salinities. J. Bacteriol. 172, 1769–1774.

Lewis, J. G., Learmonth, R. P. and Watson, K. (1995).Induction of heat, freezing and salt tolerance by heatand salt shock in Saccharomyces cerevisiae. Micro-biology 141, 687–694.

MacKenzie, K. F., Blomberg, A. and Brown, A. D.(1986). Water stress plating hypersensitivity of yeasts.J. Gen. Microbiol. 132, 2053–2056.

MacKenzie, K. F., Singh, K. K. and Brown, A. D.(1988). Water stress plating hypersensitivity ofyeast: Protective role of trehalose in Saccharomycescerevisiae. J. Gen. Microbiol. 134, 1661–1666.

Meikle, A. J., Reed, R. H. and Gadd, G. M. (1988).Osmotic adjustment and the accumulation of organic

solutes in whole cells and protoplasts of Saccharo-myces cerevisiae. J. Gen. Microbiol. 134,

Morris, G. J., Winters, L., Coulson, G. E. and Clarke,K. J. (1986). Effect of osmotic stress on the ultra-structure and viability of the yeast Saccharomycescerevisiae. J. Gen. Microbiol. 132, 2023–2034.

Mortimer, R. K. and Johnston, J. R. (1986). Geneologyof principal strains of the yeast genetic stock centre.Genetics 113, 35–43.

Norbeck, J. and Blomberg, A. (1996). Protein expressionduring exponential growth in 0·7 -NaCl medium ofSaccharomyces cerevisiae. FEMSMicrobiol. Lett. 137,1–8.

Norbeck, J., Pahlmann, A.-K., Akhtar, N., Blomberg,A. and Adler, L. (1996). Purification and charac-terization of two isoenzymes of DL-glycerol3-phosphatase from Saccharomyces cerevisiae. Identi-fication of the corresponding GPP1 and GPP2 genesand evidence for osmotic regulation of Gpp2p expres-sion by the osmosensing mitogen-activated proteinkinase signal transduction pathway. J. Biol. Chem.271, 13875–13843.

Norkrans, B. (1966). Studies on marine occurring yeasts:growth related to pH, NaCl concentration and tem-perature. Arch. Mikrobiol. 54, 374–392.

Pardo, C., Lapena, M. A. and Gacto, M. (1991). On therole of trehalose in yeast cells subjected to hyper-osmotic shock. Microbiologia sem 7, 42–48.

Parker, K. R. and von Borstel, R. C. (1987). Base-substitution and frameshift mutagenesis by sodiumchloride and potassium chloride in Saccharomycescerevisiae. Mutation Res. 189, 11–14.

Schuller, C., Brewster, J. L., Alexander, M. R., Gustin,M. C. and Ruis, H. (1994). The HOG pathwaycontrols osmotic regulation of transcription via thestress response element (STRE) of the Saccharomycescerevisiae CTT1 gene. EMBO J. 13, 4382–4389.

Sherman, F. (1991). Getting started with yeast. InGuthrie, C. and Fink, G. R. (Eds), Methods inEnzymology (Guide to Yeast Genetics and MolecularBiology). Academic Press, London, pp. 1–25.

Slonimski, P. P., Perrodin, G. and Croft, J. H. (1968).Ethidium bromide induced mutation of the yeastmitochondria: complete transformation of cells intorespiratory deficient non-chromosomal ‘petites’. Bio-chem. Biophys. Res. Comm. 30, 232–239.

Werner-Washburne, M., Braun, E., Johnston, G. C. andSinger, R. A. (1993). Stationary phase in the yeastSaccharomyces cerevisiae. Microbiol. Rev. 57, 383–401.

Wieland, J., Nitsche, A. M., Strayle, J., Steiner, H. andRudolph, H. K. (1995). The PMR2 gene clusterencodes functionally distinct isoforms of a putativeNa+ pump in the yeast plasma membrane. EMBO J.14, 3870–3882.

O} lz, R., Larsson, K., Adler, L. and Gustafsson, L.(1993). Energy flux and osmoregulation of Saccharo-myces cerevisiae grown in chemostat under NaClstress. J. Bacteriol. 175, 2205–2213.

539 .

? 1997 by John Wiley & Sons, Ltd. . 13: 529–539 (1997)