Vol. 43, No. 2 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1982, p. 311-318 0099-2240/82/020311-08$02.00/0 Effects of Polyenes, Detergents, and Other Potential Membrane Perturbants on an Osmotolerant Yeast, Saccharomyces rouxii WILFRED N. ARNOLD* AND BRENDA P. JOHNSON Department of Biochemistry, University of Kansas Medical Center, Kansas City, Kansas 66103 Received 27 July 1981/Accepted 18 October 1981 The osmotolerance of Saccharomyces rouxii 48-28 was confirmed with both NaCl- and KCl-fortified growth media, with more tolerance being exhibited for the potassium salt. Washed and buffered cells from unfortified medium were challenged with a variety of compounds (and also with physical treatments) that potentially would elicit membrane perturbations. The efficacy of these brief treatments was judged primarily by monitoring subsequent viability. Change in the degree of expression of P-fructofuranosidase (EC 3.2.1.26), which is cryptic in young cells of S. rouxii, was a second criterion. There was a linear correlation between cell death and enzyme expression for treatments with polyenes, deter- gents, some organic solvents which did not denature the enzyme, and various freeze-thaw regimens in graded amounts of glycerol. The species is relatively insensitive to polyene antimycotics, the order of decreasing effect being filipin, nystatin, and amphotericin B. S. rouxii was found to be less sensitive to osmotic shock than is Saccharomyces cerevisiae, but in neither species is P-fructofurano- sidase released to the medium. The sensitivity of S. rouxii to ionic detergents, but not to nonionic detergents, was rationalized as being due to cell wall discrimina- tion against larger micelles for the nonionic examples. This was confirmed by showing that protoplasts were sensitive to both classes. In cultures older than 5 days the normal agreement between colony-forming units and methylene blue exclusion (another test of viability) no longer held. Delayed fermentation of sucrose by S. rouxii, which is a diagnostic feature of the species, is explained by death of some cells, expression of their P-fructofuranosidase, and utilization of the monosaccharides by the surviving cells. Saccharomyces rouxii is one of a small num- ber of yeast species that can withstand high osmolality in the growth medium (19). This distinguishing feature of the osmotolerant spe- cies over other yeasts has long been of interest, but the physiological basis remains to be eluci- dated. A reasonable working hypothesis sup- poses special properties for the cell envelope (and in particular for the plasma membrane) of S. rouxii. Accordingly, we have undertaken a systematic study of the effects on S. rouxii cells of various classes of compounds and of physical treatments that might denature or perturb mem- branes. Another unusual feature of S. rouxii is the physically cryptic nature of its P-fructofuranosi- dase (2, 20), an enzyme generally expressed in yeast species by dint of its location in the periplasmic space (1). Circumstantial evidence lends some support to the location of cryptic ,B- fructofuranosidase in the periplasmic bodies within young cells of S. rouxii (4, 5). These vesicular structures are derived from the plasma membrane but remain attached through fine pedicels, thus thwarting separation from proto- plasts after cell wall dissolution (4). A pretreat- ment that could engender independent lysis of periplasmic bodies without concomitant loss of plasma membrane integrity would have experi- mental utility in the above application, and the possibility of finding a suitable perturbant was an additional impetus for the present investiga- tions. The experimental format was to subject standardized cell suspensions to brief treatments with potential membrane perturbants and then to monitor cell viability and ,-fructofuranosi- dase expression. MATERIALS AND METHODS Yeast. S. rouxii (University of California, Davis, Department of Food Science, no. 48-28) was main- tained on agar slants and handled as previously de- scribed (4). The yeast was routinely grown on yeast extract (0.3%, wt/vol), neopeptone (0.5%), and glu- cose (1%) (YNG medium) at 28 to 30°C. The experi- mental material was from a 72-h culture, except where 311 on July 17, 2020 by guest http://aem.asm.org/ Downloaded from
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Vol. 43, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1982, p. 311-3180099-2240/82/020311-08$02.00/0
Effects of Polyenes, Detergents, and Other PotentialMembrane Perturbants on an Osmotolerant Yeast,
Saccharomyces rouxiiWILFRED N. ARNOLD* AND BRENDA P. JOHNSON
Department ofBiochemistry, University of Kansas Medical Center, Kansas City, Kansas 66103
Received 27 July 1981/Accepted 18 October 1981
The osmotolerance of Saccharomyces rouxii 48-28 was confirmed with bothNaCl- and KCl-fortified growth media, with more tolerance being exhibited forthe potassium salt. Washed and buffered cells from unfortified medium werechallenged with a variety of compounds (and also with physical treatments) thatpotentially would elicit membrane perturbations. The efficacy of these brieftreatments was judged primarily by monitoring subsequent viability. Change inthe degree of expression of P-fructofuranosidase (EC 3.2.1.26), which is cryptic inyoung cells of S. rouxii, was a second criterion. There was a linear correlationbetween cell death and enzyme expression for treatments with polyenes, deter-gents, some organic solvents which did not denature the enzyme, and variousfreeze-thaw regimens in graded amounts of glycerol. The species is relativelyinsensitive to polyene antimycotics, the order of decreasing effect being filipin,nystatin, and amphotericin B. S. rouxii was found to be less sensitive to osmoticshock than is Saccharomyces cerevisiae, but in neither species is P-fructofurano-sidase released to the medium. The sensitivity of S. rouxii to ionic detergents, butnot to nonionic detergents, was rationalized as being due to cell wall discrimina-tion against larger micelles for the nonionic examples. This was confirmed byshowing that protoplasts were sensitive to both classes. In cultures older than 5days the normal agreement between colony-forming units and methylene blueexclusion (another test of viability) no longer held. Delayed fermentation ofsucrose by S. rouxii, which is a diagnostic feature of the species, is explained bydeath of some cells, expression of their P-fructofuranosidase, and utilization ofthe monosaccharides by the surviving cells.
Saccharomyces rouxii is one of a small num-ber of yeast species that can withstand highosmolality in the growth medium (19). Thisdistinguishing feature of the osmotolerant spe-cies over other yeasts has long been of interest,but the physiological basis remains to be eluci-dated. A reasonable working hypothesis sup-poses special properties for the cell envelope(and in particular for the plasma membrane) ofS. rouxii. Accordingly, we have undertaken asystematic study of the effects on S. rouxii cellsof various classes of compounds and of physicaltreatments that might denature or perturb mem-branes.Another unusual feature of S. rouxii is the
physically cryptic nature of its P-fructofuranosi-dase (2, 20), an enzyme generally expressed inyeast species by dint of its location in theperiplasmic space (1). Circumstantial evidencelends some support to the location of cryptic ,B-fructofuranosidase in the periplasmic bodieswithin young cells of S. rouxii (4, 5). Thesevesicular structures are derived from the plasma
membrane but remain attached through finepedicels, thus thwarting separation from proto-plasts after cell wall dissolution (4). A pretreat-ment that could engender independent lysis ofperiplasmic bodies without concomitant loss ofplasma membrane integrity would have experi-mental utility in the above application, and thepossibility of finding a suitable perturbant wasan additional impetus for the present investiga-tions. The experimental format was to subjectstandardized cell suspensions to brief treatmentswith potential membrane perturbants and thento monitor cell viability and ,-fructofuranosi-dase expression.
MATERIALS AND METHODS
Yeast. S. rouxii (University of California, Davis,Department of Food Science, no. 48-28) was main-tained on agar slants and handled as previously de-scribed (4). The yeast was routinely grown on yeastextract (0.3%, wt/vol), neopeptone (0.5%), and glu-cose (1%) (YNG medium) at 28 to 30°C. The experi-mental material was from a 72-h culture, except where
other ages are indicated in the text.- Harvesting andwashing of the cells was by sequential centrifugationand resuspension under aseptic conditions. The bufferemployed was 0.1 M sodium acetate-hydrochloric acid(pH 5.5). The final suspension was made to 20% (wetwt/vol) in the same buffer and is called "standard cellsuspension." Viability of cells was estimated some-times by the dye exclusion method (15) in conjunctionwith a hemacytometer. However, for the great major-ity of experiments, the number of live cells wasestimated by spreading diluted samples on YNG agarplates and counting colonies after 5 days at roomtemperature (23 to 25°C). Snail enzymes (Sigma Chem-ical Co., St. Louis, Mo.) were used for cell walldissolution in the preparation of protoplasts; debriswas removed by density gradient centrifugation aspreviously described (4).
Polyene antimycotics. Samples of nystatin (4,020 U/mg) and of amphotericin B (92.7%) were donated by E.R. Squibb and Sons, Princeton, N.J. A sample offilipin (crystalline complex, 66% pure) was donated bythe Upjohn Co., Kalamazoo, Mich. All polyene stocksolutions (50 mg/ml) were prepared in dimethyl sulfox-ide on the day of the experiment. Experiments were
performed under subdued lighting.Detergents. The following compounds (listed by
class, generic name, and trade name where appropri-ate) were used in this study. The nonionic detergentswere the following: sorbitan esters (Span 80), polyox-yethylene sorbitan esters (Tween 20 and 80), polyox-yethylene alcohols (Brij 35, 56, 58, and 98), all fromICI United States, Inc., Wilmington, Del.; polyox-yethylene-p-t-octyl phenol (Triton X-100) from Sigma;and octyl-,3-D-glucopyranoside from Calbiochem, LaJolla, Calif. The ionic detergents were the following:sodium dodecyl sulfate from Sigma; amine salt of alkylaryl sulfonate (Atlas G-3300) from ICI United States;sulfobetaines (Zwittergents 3-08, 3-10, 3-12, 3-14, and3-16) from Calbiochem; and cetyltrimethylammoniumbromide and cetylpyridinium bromide from Sigma.The bile salts, sodium deoxycholate, sodium taurocho-late, and the saponin digitonin were from Sigma.
Enzyme assay. ,B-Fructofuranosidase assays wereconducted at 30°C with sucrose as substrate. Specificactivities are expressed as International Units (IU) pergram (dry weight) of yeast (2). A 15-min pretreatmentof a given yeast sample with 0.1 volume of ethylace-tate (2) was taken as revealing the total enzymeconcentration; degrees of expression in companiontreatments are given as percentages of the total activi-ty. Heat inactivation of 13-fructofuranosidase withinstandard cell suspensions of S. rouxii was conductedaccording to previous methodology (1).
RESULTS
Osmotic pressure. S. rouxii will tolerate 50 to60% glucose (24) or 50% sucrose (20) in thegrowth medium. We have explored the effects ofa graded series of salt supplements on thegrowth of strain 48-28 and on the status of the I-
fructofuranosidase at term. These results aresummarized in Table 1. The inoculum for thesetwo trials was from YNG broth cultures, and thelaboratory strain was untrained in the sense thatit was carried on unfortified agar slants. Saltconcentration obviously affected yield (Table 1).KCl was better tolerated than NaCl, the latterhaving a more pronounced effect on lag periodas well as doubling time. A supplement of 2.5 MNaCl was not tolerated by this strain. At 2.5 MKCl, the growth pattern was biphasic, withreasonable growth rate being achieved only after140 h of apparent adaption (Table 1). There wasno discernible growth in the presence of 3.0 MKCl. The osmolality in the growth medium hadlittle direct effect on the degree of expression ofP-fructofuranosidase; in the case of 1.5 M NaCl,where expression was 62%, the fraction of deadcells by the dye exclusion criterion was about60%.
TABLE 1. Effects of osmolality on growth and ,B-fructofuranosidase status
Addition to Lago(h)bting(h) Term (h) Cellular 0-FructofuranosidaseAdditiontogh Doubling Term (h) yield (mg activity (IU/g [dry wt])
Another experiment was designed to test theeffect of osmotic shock. For this purpose, wechose cells from a 7-day culture with partialexpression. Part of a standard cell suspensionwas equilibrated with 2.0 M KCl (by sedimenta-tion and resuspension) and finally suspended tothe original volume in 2.0 M KCI. Samples (5 ml)of this material were placed in each of two 50-mlvolumetric flasks. A 5-ml sample of cells inoriginal buffer was placed in a third 50-ml flaskto serve as a control. All three samples werekept at room temperature for 90 min, at whichtime they were each diluted to 50 ml (finalvolume), and suitable volumes were taken for ,B-fructofuranosidase assay. One of the high-saltsamples was rapidly diluted with water, an at-tempt at osmotic shock, and resulted in 35%expression and 44% dead cells as judged by dyeexclusion. Slow dilution resulted in 38% expres-sion and 41% dead cells; the control sampledisplayed 24% expression and apparently 36%dead cells. Although the viability of the cells wasslightly lowered by the high osmotic pressureexcursion, the species was insensitive to thisosmotic shock.
Polyenes. We found that growth of log-phasecultures of S. rouxii was halted by addition of 25,ug of filipin, nystatin, or amphotericin B per ml.This was equivalent to 23 ,ug/mg (dry weight) ofyeast. Polyenes administered at 1/10 this con-centration did not inhibit growth. Stationary-phase cells were even more resistant (see be-low).Bulk treatments of washed cells from 2- or 3-
day cultures were made by adding 5 mg ofpolyene in 0.1 ml of dimethyl sulfoxide to 1.0 mlof 20% yeast suspension, i.e., about 125 ,ug/mg(dry weight) of yeast. Controls received water ordimethyl sulfoxide alone. Standard treatmentswere at room temperature for 20 min, at whichtime the cells were washed in sterile buffer and
TABLE 2. Effects of polyenes on the expression of,-fructofuranosidase in 3-day cells
a Polyenes (5 mg) were added in 0.1 ml of dimethylsulfoxide to 1.0 ml of 20% cell suspension, mixed, leftat room temperature for 20 min, and then washed withbuffer.
b Repetition of treatment in footnote a.c Standard ethyl acetate treatment.dAddition of 0.1 ml of dimethyl sulfoxide alone.
then assayed for ,3-fructofuranosidase expres-sion and, in some trials, for percent viability.The order of decreasing effect was filipin, nysta-tin, and amphotericin B. Another consistentfinding was no inactivation of P-fructofuranosi-dase by polyene pretreatments; i.e., in everycase posttreatment with ethyl acetate revealedthe full complement of cryptic activity.
In the experiment summarized in Table 2, theinitial treatments were done in triplicate so thattwo of the sets could be given a second exposureto polyenes or control solutions. Finally, one setreceived a third sequential treatment with ethylacetate. The results (Table 2) indicate that re-fractory cells remain resistant to further expo-sure, an increment of only 8% being effected byfilipin. Residual cryptic 1-fructofuranosidasewas not inactivated.
In another trial with 2-day cells the degree ofexpression of P-fructofuranosidase and the frac-tion of dead cells resulting from exposure topolyene were in close agreement (Table 3).Other trials indicated that susceptibility to nys-tatin was greatest in 1-day cells (late log phase),least in 2-day cells, and (somewhat surprisingly)intermediate in 3- or 4-day cells. A very similarset of relative enzyme activities for treated cellswas also obtained when raffinose was substitut-ed for sucrose in the assay mixture.
Detergents. A large number of detergents weretested against standard yeast suspensions overconcentration ranges that embraced the criticalmicellar concentration (CMC) in each case. Ex-posure time was 15 min at room temperature,after which the cells were washed in sterilebuffer and monitored for viability and ,-fructo-furanosidase expression. The results are sum-marized in Fig. 1. There was a strong inversecorrelation between viability and enzymeexpression as judged by the closeness of fit forthe data points and the line joining the referencepoint for ethyl acetate-treated cells (0% viabilityobserved, and expression set at 100%) to that foruntreated, live cells (100% viability, 0o expres-sion). The latter situation is approximated by
TABLE 3. Effects of polyenes on enzymeexpression and viability of 2-day cells
a Concentrations as indicated in Table 2.b Retive activity of P-fructofuranosidase.I Fraction of stained cells in the methylene blue test.d Mean ± standard error for 10 determinations.
FIG. 1. Effects of detergents on viability and en-zyme expression. Detergents are identified as follows:A, cetyltrimethylammonium bromide; B, cetylpyridin-ium bromide; C, sodium dodecyl sulfate; D, Zwitter-gent 3-16; E, digitonin; F, sodium deoxycholate; G,Zwittergent 3-10; H, octyl-p-glucoside; I, Triton X-100; J, sodium taurocholate. Concentrations were asindicated. The reference line is theoretical for a perfectinverse correlation and can be compared with a line ofbest fit for the 22 data points which has slope -0.996,intercept 102.5, and correlation coefficient -0.984.
cells from a 3-day culture (over 97% live cells bydye exclusion or plating and less than 3%expression).
Detergent treatments that were relatively inef-fective in eliciting expression were not detrimen-tal to the cryptic enzyme. For example, whendeoxycholate- or taurocholate-treated cells (16and 3% expression, respectively) were subse-quently treated with ethyl acetate, then 98% ofthe enzymatic activity was realized in both cas-es. It should be noted that detergent-treatedsuspensions were diluted with 4 volumes ofsterile buffer, and the bulk of the detergents wasremoved in the supernatants after centrifuga-tion. Pellets were resuspended to 5 ml in sterilebuffer. Samples for plating were diluted immedi-ately 100,000-fold, whereas samples for enzymeassay were not further diluted, may have re-tained traces of detergent, and thus extended theexposure through the processing and assay peri-ods. This may explain the datum for 0.25%digitonin (Fig. 1) which indicates a somewhathigher degree of expression (68%) than celldeath (42%).As discussed in further detail later, the cell
wall may provide an effective barrier to largemolecules, including the micelles of certain de-tergents. Accordingly, protoplasts were pre-pared by established methods (4) and $ubse-quently treated with selected detergents. Forexample, protoplasts in 2.0 M KCl were subject-
ed to a range of concentrations of Triton X-100for 15 min at room temperature. Analyses for P-fructofuranosidase activity were then performedin osmotically fortified medium and also afterguaranteed lysis of protoplasts by dilution inbuffer. Results indicated that all of the previous-ly cryptic activity was expressed by detergenttreatments at a concentration of 0.05% andabove, but 0.005% was ineffective (the criticalmicellar concentration for Triton X-100 is ap-proximately 0.015%). Effective treatments wereaccompanied by lysis of protoplasts; ineffectivetreatments did not cause lysis and did not inacti-vate the cryptic enzyme whose activity couldlater be revealed by lysis (dilution).As expected, protoplasts were susceptible to
ionic detergents at concentrations in the vicinityof the CMC. On the other hand, all of thepolyoxyethylene sorbitan esters and polyox-yethylene alcohols tested were no more effectivein causing lysis of protoplasts than they were inkilling intact cells, at least at the concentrationsemployed (0.05 to 0.25%).
Organic solvents. Compounds were chosen onthe basis of possible perturbation of membraneswithout concomitant enzyme denaturation.These were tested against standard cell suspen-sions at room temperature for 15 min. Afterremoval of test compounds by washing withsterile buffer and preparation of standard dilu-tions, the cells were tested as above. Samplesfrom treatments that did not elicit expressionwere subsequently given standard ethyl acetateexposure to test for remaining cryptic activityor, instead, apparent denaturation by the firsttreatment. The results of this survey can besummarized by grouping compounds accordingto the degree of expression compared with thatfor ethyl acetate, which is set at 100%. Statedconcentrations in parentheses are volume/vol-ume; otherwise, the cells were subjected tobuffer that had been saturated with the givencompound.
(i) Expression of 100 to 84%. The 100 to 84%expression group included ethyl acetate (9.1%),cellosolve acetate (9.1%), n-butanol, chloro-form, 2-phenoxy ethanol, and methyl cellosolve.All cells were killed by each of these treatments.Enzyme inactivation was low, the maximumsuffered being 7% in the case of n-butanol.
(ii) Expression of 61 to 34%. The 61 to 34%expression group included cyclohexanone, car-bon tetrachloride, and benzene. The viability oftreated cell populations generally was inverselyrelated to the degree of expression, althoughenzyme inactivation was significant (8 to 23%).
(iii) Expression of 10 to 2%. The 10 to 2%expression group included toluene, dimethylsulfoxide (9.1%), diethylene glycol (9.1%),methyl ethyl ketone (9.1%), p-dioxane (9.1%),
and acetone (9.1%). The viability of treated cellpopulations was 80 to 90%. Enzyme inactivationwas insignificant at these concentrations andthis exposure time.The resistance of S. rouxii cells to toluene and
to acetone was notable. We observed that expo-sure to 20% acetone for 15 min at room tempera-ture was attended by only 5% expression of 1B-fructofuranosidase activity, but 82% survival ofcells. At 40% acetone, there was 59% expressionand 41% inactivation, whereas no cells survivedthe treatment. When a fresh pellet of washedcells was resuspended in buffer saturated withtoluene (i.e., slightly less than saturated in thesuspension), held at room temperature for 15min, and then washed free of toluene, the sur-vival rate was 88%. On the other hand, cells didnot survive mixture with 0.1 volume toluene andoccasional vigorous shaking (emulsion) for 15min at room temperature.
Freeze-thaw treatments. The disruptive effectsof freezing and thawing on cells in suspensionwas explored with four different regimens. Rap-id freezing was achieved by submerging tubes(and swirling the contents) into an ethanol-dryice bath (-78°C). Alternatively, samples wereplaced in a -22°C box (requiring at least 15 minfor freezing) and left there for 2 h. Thawingcycles were either relatively fast (in a stirrredwater bath at 30°C) or relatively slow (in air at23°C). Further variations were provided by con-
Viability N
FIG. 2. Effects of freeze-thaw pretreatments of S.rouxii cells on the degree of expression of p-fructofur-anosidase and the subsequent viability of the cells asdetermined by plating. The four sets of data refer tobuffered cell suspensions fortified with glycerol as
indicated. The symbols refer to different freeze/thawregimens: 0, -78°C/23°C; *, -78°C/30°C; l, -22°C/30°C; *, -22°C/23°C. Untreated cells were deemed tobe 100% viable and exhibited 0.5% expression, where-as ethyl acetate-treated cells exhibited 0o viabilityand were deemed to exhibit 100%o expression; all othervalues are expressed as percentages of these limits.
ducting the treatments on cell suspensions in6.25, 12.5, 25, or 50% glycerol (as a potentialcryoprotectant).
Results are summarized in Fig. 2. The behav-ior of cells in 25% glycerol is not shown, but wasnot significantly different from that depicted for12.5% glycerol. There was no indication ofrelease of 1-fructofuranosidase to the mediumunder any of the regimens. In all cases, theremaining cryptic activity was revealed by thestandard ethyl acetate treatment, which indicat-ed no significant inactivation of the enzyme bythe primary treatments.The degree of expression of 1-fructofuranosi-
dase activity was increased by storing cells inthe frozen state at -22°C. For example, thedegree of expression was about 26% after 0.5 hand increased to 72% after 2 h. The inclusion of0.05% Triton X-100 in buffered cell suspensionsincreased the degrees of expression from 72 to88% or from 65 to 81% after cells had beenfrozen at -22°C for 2 h and then thawed at 30°Cor more slowly at 23°C, respectively.Heat treatments. Cell suspensions from 1-, 2-,
3-, and 10-day cultures were treated at 65°C. Theresults on expressed and total ,B-fructofuranosi-dase activities are summarized in Fig. 3. Thekinetics for total enzyme activity were similarfor cells up to 3 days old, but heat inactivationsusceptibility was increased in cells derivedfrom a 10-day culture. It is of interest to note thepartial expression of ,B-fructofuranosidase as aresult of heating; this phenomenon was morepronounced in 10-day cells (Fig. 3). Each ofthese trials was repeated on at least one (and insome cases two) additional independent cul-tures, and yielded similar results. Viability esti-mates indicated very low survival rates for cellssubjected to even 1-min treatments.
Untreated cells from the 10-day culture had11% expression of 1-fructofuranosidase and ex-hibited about 20% dead cells by dye uptakecriterion, but only 5% of the cells were capableof developing colonies on an agar plate. Thedisparity between dye exclusion and platingestimates, at least for older cells, prompted amore critical comparison and a reexamination ofaging in S. rouxii cells (see below).
Aging. For trials on membrane perturbation,we expressed estimates of viability as percent-ages by comparing numbers of colonies formedby treated cells with scores for similar amountsof control cells. For a critical comparison of dyeexclusion versus colony-forming ability in oldersamples, we were concerned about clumping ofcells. For 3-day cultures, the clumping problemwas eliminated by subjecting a 1% (wet wt/vol)suspension of washed cells to four separatebursts of sonication, each lasting 5 s and inter-spaced by similar intervals. The fraction of cells
FIG. 3. Effects of heating at 65°C on P-fructofuran-osidase activity of cells. Cells were derived from 1-day(A); 2-day (B); 3-day (C); or 10-day (D) cultures.Expressed (0) and total (O) enzyme activities aregiven as percentages of the value for the unheated,ethyl acetate-treated sample in each set.
that stained with methylene blue was always 3%or less, and the number of colonies formed wasclose to the expected value for complete viabili-ty.For 5-day and older cultures, the adopted
sonication pretreatment decreased the numberof observed aggregates, but some doublets, trip-lets, and larger groups of cell remained. Suchaggregates were scored in the hemacytometer assingle units in connection with estimating maxi-mum colony numbers obtainable with defineddilutions applied to agar plates. Representativeresults for cells from 5- to 13-day cultures areassembled in Table 4. One 5-day culture yieldedcomparable values for the two criteria of viabili-ty, but another showed a much lower value byplating in the face of only 5% of the cells takingup the dye in the standard methylene blue test.Lower values by plating than staining were theusual situation for 6- to 13-day cells. This is notdue to compromise by the sonciation procedure,because one particular 10-day culture revealed85% dye exclusion for untreated cells versus82% for sonicated samples compared with 1.7%versus 1.2%, respectively, in ability to grow intocolonies.Very old cultures (52 to 56 days) possessed
very few cells capable of subsequent growth.The typical number of colonies produced wasequivalent to 0.5% of the estimated (hemacy-tometer) total number of separate cells or aggre-gates in the sample applied to the plate. Evalua-
TABLE 4. Viability of S. rouxii cells
Age of culturea Viability (%)b by(days) Dye exclusionc Platingd
a Cells were grown in YNG medium at 28 to 30°C.b Mean ± standard error; all values based on cell
samples that had been lightly sonicated.c Methylene blue test; boiled controls gave 100%
stained cells for all samples except the 13-day culture,where 70% were intensely blue and 30% were onlypale blue.
d Based on numbers of colonies developed on agarplates and companion estimates in the hemacytometerof individual cells or aggregates as described in thetext.
tion of such samples by the dye exclusionmethod was not satisfactory because controlsamples that were preheated in a 100°C waterbath for 3 min were stained only light blue atbest, in contrast to the intense coloration ob-served with similarly treated cells from culturesup to 10 days old.
DISCUSSIONPresent results confirm and extend the dem-
onstration of osmotolerance in S. rouxii 48-28.The term osmophilicity has been used in some ofthe earlier literature but should be reserved forobligate examples as occasionally observed innature (17) or as generated in mutants (14). Theimmediately adaptive capacity of the presentstrain is remarkable, but not unique (9). Thegreater tolerance towards KCl compared withNaCl is also in agreement with Rodriguez-Na-varro (21). The high degree of crypticity for -fructofuranosidase in young cultures is not sig-nificantly influenced by the osmolality of themedium; those cases of increased expression(Table 1) were associated with older culturesand a proportional increase in the percentage ofdead cells. Apart from cases of extreme inhibi-tion, it can be concluded that growth rate doesnot have much influence on the concentration ofP-fructofuranosidase synthesized. Our experi-ence with S. rouxii also indicated that this spe-cies is not sensitive to osmotic shock. On theother hand, Saccharomyces cerevisiae cells ex-hibit decreased viability after shocking (6), butneither species liberates P-fructofuranosidase tothe medium.The relative sensitivities of different yeast
because experimentally determined minimuminhibitory concentrations vary so widely amongdifferent laboratories (11), and most investiga-tors have reported concentrations on a unitvolume of medium basis rather than the mean-ingful basis of unit weight of treated yeast. Thislaboratory reported that 0.24 ,ug/mg (dry weight)was fungistatic for the yeast-like organism His-toplasma capsulatum (7). By comparison, it issafe to infer from the present work that S. rouxiicells are inhibited by concentrations of polyeneat least 1 and possibly 2 orders of magnitudegreater than those required for S. cerevisiae andfor pathogenic yeasts (11). Furthermore, theorder of effectiveness against Candida albicans(10) is said to be amphotericin B > nystatin >filipin, which is the reverse order to that foundherein for S. rouxii.We found that even after two exposures to
filipin only 75% of the 0-fructofuranosidase ac-tivity of S. rouxii cells was expressed. Smallincrements evoked by repeated treatments indi-cated refractory cells in the overall populationrather than a need to saturate polyene absorp-tion sites (7). The similarities noted for degreesof expression when raffinose was used as alter-nate substrate to sucrose do not distinguish apossible difference in the size of the channels,which are supposedly evoked by the intercala-tion of different polyenes into the plasma mem-brane (18).The refractoriness of the S. rouxii cell toward
polyenes might be attributed to the lipid compo-sition of its plasma membrane. Although analy-ses of that plasma membrane are not available, itis worth noting that the sterol and sterol esterfractions of whole cells are not outstandinglydifferent from those found for other Saccharo-myces species (13). According to Gale et al. (10)the sensitivity of C. albicans to amphotericin Bcan be varied more than 300-fold by manipulat-ing the growth time and medium. These differ-ences were minimized after cell wall removal(10), and perhaps species differences also mayreside in the cell wall. Furthermore, Norman etal. (18) suggest that polyenes in aqueous solutionexist as micelles. With molecular weights rang-ing from 655 (filipin) to 926 (nystatin), only smalldegrees of aggregation might lead to exclusionby some yeast cell walls.The disruptive effect of detergents on biologi-
cal membranes depends in large part on theability of these compounds to dissolve lipid-soluble components into the hydrophobic interi-or of micelles (3). This sort of solubilizationoccurs only above the CMC of the mixture.Tukmachev et al. (23) studied two homologousseries of ionic detergents in connection with S.cerevisiae cells and concluded that most quanti-tative differences can be rationalized on the
basis of the size of micelles and their interactionwith cell wall porosity. Accordingly, we wouldpredict that any detergent with a reasonablehydrophile-lipophile balance number (12) wouldbe disruptive to yeast membranes provided thata concentration at or above the CMC is em-ployed and micellar size is not so large that thecell wall prevents access to the plasma mem-brane. Indeed, this seems to be the case with thepresent series of detergents and S. rouxii.The nonionic detergent Triton X-100 forms
spherical micelles of radius 4.8 nm (12). This isfive times the hydrodynamic radius for thresholdexclusion of polyethylene glycols by S. cerevi-siae cells (22) and may explain the observedresistance of S. rouxii cells to concentrations ofTriton X-100 above the CMC. On the otherhand, we found lysis of protoplasts in concentra-tions above the CMC (but not for 0.005%, whichis about one-third the CMC).The ionic detergents form micelles at 1 to 10
mM concentrations, but their micellar radius isusually about 1.5 nm (12). We found that CMCsof anionic and cationic detergents rapidly killedintact cells of S. rouxii. Lower concentrationselicited intermediate levels of viability and P-fructofuranosidase expression, supposedly re-flecting variable resistance within the yeast cellpopulation.The bile salts fall into a special category; they
form dimers to octomers in which these planar-polar molecules lie back to back. They do notpossess well-defined head groups and thus donot associate into roughly spherical micelles asis the case with other detergents (8). Free bilesalts form aggregates about 2 nm long and about0.7 nm in cross section. In the present case, thesodium salts of deoxycholate and taurocholatewere relatively undisruptive toward intact cellsof S. rouxii; digitonin had an intermediate effectin comparison with ionic detergents. All threecompounds caused disruption of protoplastswhen employed at concentrations above theirCMC.The disruptive effects of organic solvents on
biological membranes are well known, and sev-eral of the compounds tested here have beenused to induce autolysis in yeast. None wasfound to be more useful than ethyl acetate for ,B-fructofuranosidase expression. The apparentresistance of S. rouxii cells to 20% acetone for 15min may bear further comparisons with otheryeast species.The partial expression of P-fructofuranosidase
in S. rouxii after brief treatments at 65°C impliesthat the enzyme is less sensitive to denaturationthan is the membrane that confers crypticity.The physical stress of freezing and thawing inthe presence of 12.5% glycerol seemed to bemore deleterious toward some other vital func-
tion than membrane integrity as indicated by alower viability than was commensurate withenzyme expression. A recommendation (16) forincreased permeabilization of S. cerevisiae cellsby freezing in the presence of Triton X-100 wasnot as effective with S. rouxii. Treatments withpolyenes, detergents, most of the freeze-thawregimens, and those solvents which did notinactivate ,-fructofuranosidase all showedstrong inverse correlations between viability andenzyme expression. These results add to thebody of information (2, 4) on the physicallycryptic nature of the enzyme and point to amembrane barrier. However, none of thesetreatments can be deemed sufficiently discrimi-nating to give any support to independent lysisof periplasmic bodies, a suggested site for cryp-tic enzyme in young cells.The previous claim (2) that aged cultures had
higher degrees of expression than the apparentmortality must be reevaluated. Values for viabil-ity were based on dye exclusion which we nowfind to be in good agreement with colony countsonly for young cultures. We have no evidence athand to indicate that the cryptic P-fructofurano-sidase is ever expressed in live cells. Rather,aged populations can exploit sucrose in themedium (i.e., the classical delayed fermentation)by dint of the nonsynchronous death of somecells, the concomitant expression of their P-fructofuranosidase activity, and the utilizationof the monosaccharides by the remaining livecells.
ACKNOWLEDGMENTS
We acknowledge the technical assistance of John S. Lacyand Brenda J. Evans.
This work was supported by Public Health Service Grant Al13177 from the National Institutes of Allergy and InfectiousDiseases.
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