Microbiol.Res. (1998) 153, 179- 184

© Gustav Fischer Verlag

Polyamine metabolism in Saccharomyces cerevisiaeexposed to ethanol

Dale Walters, Tracy Cowley

Departmentof Plant Biology, PlantScience Division,The ScottishAgricultural College, Auchincruive, NrAyr, KA65HW, UK

Accepted : April 6, 1998

Abstract

Growth of theyeast Saccharomycescerevisiae was unaffectedby upto 24hexposure to ethanolconcentrations ranging fromI% to 9%, butwas reduced following exposure to 12% etha­nol. Concentrations of the polyamines putrescine, cadaverineand spermidine were not affected by a 24 h exposure to 12%ethanol, although there was a significant increase in sperminelevel. These changes were accompanied by significant in­creases intheactivitiesofthe polyamine biosynthetic enzymesornithine decarboxylase (ODC) and S-adcnosylmethionincdecarboxylase (AdoMetDC) and in the flux of label fromornithine into the polyamines. Formation of the cadaverinederivatives aminopropylcadaverine and N,N-bis(3-aminopro­pyl)cadaverine was greatly increasedin yeast exposed to 12%ethanol for24 h, probably via theaction of ODC, AdoMetDCand the aminopropyltransferases. Exposure to 12% ethanolalso led to substantial reductions in the uptake of putrescineand spermidine and theaminoacid methionine.

Key words: ethanol - polyamines - cadaverine derivatives ­Saccharomyces cerevisiae

Introduction

Ethanol is producedas a result of microbial fermentationof carbohydrates and it acts as a chemical stress follow­ing accumulation in the growth medium. Yeasts havebeen traditionally employed for ethanol production andnot suprisingly therefore, most studies on ethanol stresshave focused on these fungi, especially Saccharomycesspecies (Misra, 1993).Fungi differ in their ability to growin the presence of ethanol and, in general, those fungiknown to produce ethanol i.e. yeasts, include species

Corresponding author: D.Walters

which are comparatively resistant to ethanol and cangrow at concentrations of up to 12% (v/v)( Misra, 1993).

Ethanol induces a number of changes in the physio­logy of fungal cells. For example, ethanol interacts withthe plasma membrane and perturbs the organisation ofmembrane lipids by lowering the transition temperatureand increasing membrane fluidity (Jain and Wu, 1977).Ethanol is also known to inhibit the uptake of variousnutrients e.g. the non-competitive inhibition of uptake ofglucose, amino acids and ammonium ions (Leao andVanUden, 1983 ; Thomas and Rose, 1979). With the ex­ception of glucose uptake in S. cerevisiae,which occursvia facilitated diffusion, ethanol-induced inhibition ofnutrient uptake may reflect its effects on the protonmotive force across the plasma membrane (Misra,1993).Various studies have also shown that ethanol caninduce the same set of proteins that are induced by heatshock in S. cerevisiae (Plesset et ai., 1982). Stress pro­teins are also known to be induced in the yeast Candidaalbicans and the filamentous fungus Neurosporacrassaexposed to ethanol (Misra, 1993).

The diamine putrescine, the triamine spermidine andthe tetraamine spermine are widely distributed in nature.Fungal polyamine biosynthesis commences with thedecarboxylation of ornithine in a reaction catalyzed byornithine decarboxylase (ODC) Walters, 1995). Spermi­dine and spermine are formed by the addition of anaminopropyl moiety to putrescine and spermidinerespectively, in reactions catalyzed by the aminopropyl­transferases, spermidinesynthase andspermine synthase.The aminopropyl moiety, decarboxylated S-adenosyl­methionine, is the result of the decarboxylation ofS-adenosylmethionine by S-adenosylmethionine decar­boxylase (AdoMet DC). Although the function of poly­amines is not known with any certainty (Davis et al.,1992), they are known to be essential for growth. Thus, a

Microbial. Res. 153 (1998) 2 179

null mutant of S. cerevisiae unable to form spermidineand spermine had an absolute requirement for theseamines for growth (Balasundaram et al., 1991). Severalstudies have indicated that polyamines might be involvedin protection of cells and cell components from oxidativedamage (Khan etal., 1992a, b; Minton et al., 1990) andBalasundaram et al. (1993) showed that spermidine andspermine were important in protecting S. cerevisiae fromoxygen toxicity. Polyamines are also known to interactwith membranes and exert an important role in majorbiological membrane functions (Schuber, 1989). Giventhe importance of polyamines in fungal growth, protec­tion of fungal cells from oxidative stress, and their effectson membranes, we decided to examine polyamine meta­bolism in S. cerevisiae exposed to ethanol.

Materials and methods

Fungal growth. S. cerevisiae was grown routinely inYPAD medium (1% yeast extract; 2% peptone; 2% dex­trose and 0.04% adenine sulphate) and to study the ef­fects of ethanol on polyamine metabolism, this mediumwas supplemented with 1-12% ethanol (v/v). Flaskswere stoppered to minimise the evaporation of ethanol.Cultures were incubated at 30 ° with rotary shaking andgrowth curves obtained by measuring the optical densityof the cultures at 600 nm in a Gallenkamp Visi-Specspectrophotometer after 2, 4, 6, 8 and 24 h.

Polyamine analysis. The method used was adapted fromSmith (1991). Briefly, yeast cells (from 20 ml ofYPADmedium) were harvested as described above, suspendedin 3 ml of 10% perchloric acid and sonicated for 10 cy­cles of 10 s on120s off (Soniprep 150, MSE). Test-tubeswere kept on ice during sonication. The extract wascentrifuged for 15 min at 5000 x g at 4°C and 100 III ofthe supernatant added to 200 III saturated Na2C03 and400 IIIdansyl chloride (10 mg/ml in acetone). This mix­ture was incubated in darkness at 60°C for 20 min, afterwhich 100 III proline (100 mg/ml) was added. After10 min incubation in the dark at room temperature,dansylated polyamines were extracted in 500 III toluene.Aliquots (25 ul) of the toluene phase were spottedonto activated LK6D silica gel plates (Whatman) anddeveloped in chloroform/triethylamine (12: 1, v/v).Polyamines were visualized under ultra-violet light andscraped off into 4 ml ethyl acetate. Fluorescence wasmeasured at an excitation wavelength of 365 nm and anemission wavelength of 506 nm.

Enzyme assays. ODC and AdoMetDC activities wereassayed as described by Foster and Walters (1990) usingcrude enzyme extracts prepared by suspending yeastcells (from 20 ml of YPAD medium) in 3 ml of theextraction buffer. The LDC assay was similar to the

180 Microbial. Res. 153 (1998) 2

ODC and AdoMethDC assays described in Foster andWalters (1990) except that the extraction medium con­tained 50 mM Tris-HCI, 0.5 mM EDTA and 5 mMdithiothreitol, pH 8.0, and the reaction medium contain­ed 10 mM Tris-HC1, 1 mM dithiothreitol, 0.1 mMEDTA, 0.1 mM pyridoxal phosphate,S mM lysine and0.125 llCi [U-14C]lysine.

Polyamine oxidase (PAD) was assayed using a me­thod adapted from Okuyama & Kubayashi (1961). Crudeenzyme extract was prepared from yeast cells as de­scribed above using an extraction medium containing100 mM potassium phosphate buffer and 2 mM dithio­threitol, pH 8.0. The extract was centrifuged at 20,000 x gfor 20 min at 4°C amd 0.5 ml of supernatant added to0.5 ml of 100 mM potassium phosphate buffer (pH 8.0)containing I mM spermidine, 0.15 llCi [14C]spermidine(Amersham International, UK) and 30 ug catalase. Sam­ples were incubated at 37°C for 30 min and the reactionthen stopped by the addition of 1 ml of 4 M NaOH.Reaction products were extracted in 2 ml toluene, mixedfor 20 s and a I ml aliquot of the (upper) toluene phasetransferred to 10 ml scintillant for radioactive countingas described above. Enzyme activity was expressed aspmol product mg protein" h!

In virto incorporation of labelled ornithine and lysineinto polyamines. ODC and LDC assays were performedas described in Foster and Walters (1990) and after thereactions were terminated, 100 III aliquots of the reac­tion medium were used for polyamine analysis as de­scribed above. This procedure allowed the flux of labelfrom ornithine into putrescine, spermidine and sper­mine, and from lysine into cadaverine, APC and 3APCto be determined.

Uptake ofpolyamines and methionine. Yeast cells from20 ml of YPAD medium were harvested as describedabove, resuspended in 5 ml of fresh YPAD medium andincubated at 30°C for 40 min in a reciprocal shaker.Then, 0.125 llCi of either [3H]methionine (NEN Re­search Products, UK), [14C]putrescine or [14C]spermi­dine (Amersham International, UK) was added and theflasks incubated for 40 min in a reciprocal shakerat 30°e. Following incubation, the yeast cells wereharvested as described above and washed twice with10 ml of distilled water by resuspending the pellet andcentrifuging. After the final wash, the pellet was de­posited in a glass scintillation vial containing 2 mlSoluene 100 (Packard), shaken on a rotamixer, incubat­ed for 3 h at 60°C and then left overnight at room tem­perature. Finally, 10 ml of Hionic-Fluor scintillationfluid (Packard) was added to each vial and radioactivitycounted on a 1900 TR liquid scintillation analyzer(Packard).

Statistical analysis. All values are the means of fourreplicates and experiments were repeated twice with

Table 3. Effect of 12% ethanol on ODC and AdoMetDCactivities in S. cerevisiae following a 24 h exposure

similar results. Data presented are from a representativeexperiment. Significance was determined using theStudent's t-test, Treatment Enzyme activity (pmal CO2 [mg protein]-l h- l)

ODC AdoMetDC

Significant differences are shown at P < 0.001**

Discussion

although there was a significant increase in the flux oflabel from lysine through to the higher cadaverine deri­vatives APC and 3APC (Table 4). There was also a verysubstantial (320%) increase in PAO activity in yeastgrown in 12% ethanol for 24 h (Table 5).

Although the effects of ethanol on growth of yeasts hasbeen studied extensively and it is known that someyeasts are comparatively resistant to ethanol, the kinet­ics of yeast growth in the presence of ethanol remainscontroversial (Misra, 1993). For example, some workers(Ghosh and Tyagi, 1979) have suggested a linear rela-

49 ± 1.9134 ± 8.7**

103 ± 10.3170 ± 0.5**

Uptake ofpolyamines and methionine

Exposure of S. cerevisiae to 12% ethanol for 24 h led tosubstantial reductions in the uptake of polyamines andmethionine (Table 6). Thus, putrescine uptake was re­duced by 92%, spermidine uptake by 70% and methio­nine uptake by 86%.

Control12% ethanol

Polyamine metabolism

Exposure of S. cerevisiae to 12% ethanol for 24 h pro­duced no significant changes in the concentrations ofputrescine, cadaverine and spermidine, although sper­mine concentration increased by 22% (Table 2). This in­crease in spermine was associated with increased poly­amine biosynthesis: a 65% increase in ODC activity anda 173% increase in AdoMetDC activity (Table 3), and anincreased flux of label from ornithine through to sper­mine (data not shown). Interestingly, activity of thecadaverine biosynthetic enzyme LDC was not signi­ficantly affected by exposure to 12% ethanol for 24 h,

Growth ofS. cerevisiae

Yeast was grown in ethanol concentrations ranging from1-12% and measurements made over a 24 h period.Yeast growth was unaffected by exposure to 1-9%ethanol for 24 h (Table 1: only data for 4% ethanolshown), whereas yeast growth was reduced by exposureto 12% ethanol over the entire experimental period(Table 1). Very little yeast growth occurred at ethanolconcentrations above 12% (data not shown). In view ofthese results, all experiments on polyamine metabolismand uptake were performed using yeast exposed to 12%ethanol for 24 h.

Results

Table1. Growth of S. cerevisiae exposed to 4% and 12% ethanol for 2,4,6,8, and 24 h

Treatment

Control4% ethanol12% ethanol

Optical Density at 600 nm

2h 4h 6h 8h 24h

0.46 ± 0.040 0.64 ± 0.073 0.97 ± 0.087 1.23 ± 0.090 1.36 ± 0.140.37 ± 0.043 0.52 ± 0.080 0.87 ± 0.074 1.03 ± 0.079 1.32 ± 0.110.26 ± 0.019* 0.36 ± 0.024* 0.46 ± 0.028* 0.59 ± 0.047* 0.67 ± 0.051*

Significant differences are shown at P < 0.001*

Table 2. Effect of 12% ethanol on polyamine concentrations in S. cerevisiae following a 24 h exposure

Treatment Polyamine concentration (nmol g' f.wt)

Putrescine Cadaverine Spermidine Spermine

Control12% ethanol

796 ± 98.4882 ± 32.9

439 ± 66.1510 ± 25.7

295 ± 10.3228 ± 11.5

630 ± 44.2805 ± 4.5**

Significant differences are shown at P < 0.001**

Microbiol. Res. 153 (1998) 2 ] 8]

Table 4. LDC activityand incorporation of labelledlysine into cadaverine derivatives in S. cerevisiae exposedto 12%ethanolfor 24 h

Treatment

Control12%ethanol

LDC activity(nmolCO2 [mg protein]:' h-i

1.2± 0.280.9 ± 0.11

Radioactivity in polyamine(dpm [mgprotein]")

APC

7.5 ± 8.9110 ±12.1*

3APC

110± 12.9190±1O.8**

Significantdifferences are shownat P < 0.01* and P < 0.001 **

Significantdifferences are shown at P < 0.001 **

Table 5. PAO activityinS. cerevisiae exposedto 12%ethanolfor 24 h

tionship between growth rate and extracellular ethanolconcentration, while others (Toda et al., 1987) have sug­gested that there is a critical concentration of ethanolabove which yeast cells are unable to grow e.g. 12.5%for S. cerevisiae. Our results agree with the latterauthors, since there was no effect on yeast growth at con­centrations of ethanol below 12%. Futher, althoughyeast growth was reduced by 64% following a 24 h ex­posure to 12% ethanol, there was very little yeast growthat concentrations above 12%.

Growth of yeast in 12% ethanol for 24 h had no effecton the intracellular concentrations of putrescine, cadav­erine and spermidine, but spermine concentration didincrease significantly, probably as a result of the sub­stantial increases in ODC and AdoMetDC activities.This increased polyamine biosynthesis far exceeded themodest increase in spermine. The three-fold increase inPAO activity was probably induced by the increase inspermine concentration, and may have been responsiblefor spermine levels not rising further. The role, if any, ofspermine in yeast exposed to 12% ethanol is not known.Nevertheless, it is well known that ethanol alters fattyacid and sterol composition of the plasma membrane in

Treatment

Control12%ethanol

PAO activity(pmolproduct [mgprotein]:' h')

32 ± 7.4135± 8.9**

S. cerevisiae and increases membrane fluidity (Misra,1993). It is also well known that polyamines, especiallyspermine, influence and possibly modulate membranefunctions (Schuber, 1989). For example, spermine hasbeen shown to stabilize protoplasts and spheroplastsagainst osmotic shocks (Mager, 1959; Tabor, 1962) andin Escherichia coli a dose-dependent lipid immobiliza­tion was observed with spermine (Souzu, 1986). It wasspeculated that this might have been the result of brid­ging between integral proteins and lipid binding sites.Polyamines are also known to modulate glycerolipidbiosynthesis required for membrane protection duringcell growth (Schuber, 1989). Whether the increasedspermine, and indeed the increased formation of higherderivatives of cadaverine (APC and 3APC) are associat­ed with altered membrane function in S. cerevisiae is notknown.

Increasing the ethanol concentration is also likely toincrease the stress imposed on the yeast by reducingwater availability (water activity) of the growth medium.For example, 5% or 10% ethanol would result in a wateractivity of 0.98 or 0.96 respectively. Decreasing the wateractivity of the medium has been shown to lead to theaccumulation of polyols in yeast e.g. glycerol in S. cere­visiae and arabitol in Zygosaccharomyces rouxii (Rose,1987). The increased spermine and cadaverine deriva­tives obtained in this work may be in response to adecrease in water activity in the ethanol amended media.

The formation of APC and 3APC was greatly increas­ed in yeast exposed to 12% ethanol for 24 h. Since LDCactivity did not change, while ODC and AdoMetDCactivities increased, it seems that biosynthesis of thesecompounds in S. cerevisiae occurred via the action of

Table 6. Effectsof exposure to 12% ethanol for 24 h on uptakeof putrescine, spermidine and methionine in S. cerevisiae.

Treatment

Control12%ethanol

Uptakedpm g-l f.wt)

Putrescine

6900 ± 451608 ± 52**

Spermidine

13500± 9814125 ± 312**

Methionine

7018 ± 3851050± 85**

Significantdifferences are shown at P < 0.001 **

182 Microbial. Res. 153 (1998) 2

ODC, AdoMetDC and the aminopropyltransferases. Thisagrees with recent work from this laboratory (Walters andCowley, 1996) amd is further evidence that the higherderivatives of cadaverine are not formed in this fungusfrom aspartic-f-semialdehyde as observed for somefungi (Zarb and Walters, 1994). Previous work has shownthat APC and 3APC formation is greatly increased invarious fungi exposed to, for example, elevated tempera­tures (Walters and Cowley, 1996; Zarb and Walters,1994). Whether APC and 3APC have any role to play inthe yeast response to ethanol remains to be discovered.

The effects of ethanol on fungal growth are due, inpart, to its effects on nutrient uptake (Misra, 1993). Thus,ethanol is known to be a powerful non-competitiveinhibitor of the uptake of, for example, glucose, maltose,amino acids and ammonium ions (Misra, 1993). Expos­ure to 12% ethanol for 24 h resulted in very substantialreductions in the uptake of putrescine and spermidineand of the amino acid methionine in S. cerevisiae. Putre­scine and spermidine are known to be actively taken upby a number of fungi (Davis and Ristow, 1988; Spathaset al., 1982; West and Walters, 1991) while methionine,like most amino acids, is taken up actively by many fungie.g. N. crassa (Whitaker, 1976). It is no suprise thereforethat ethanol reduced the uptake of the two amines andmethionine, since ethanol-induced reductions in uptakeare throught to be the result of an effect on the formationand maintenance of the proton motive force across theplasma membrane (Eddy, 1982).

S. cerevisiae is relatively tolerant of ethanol and in­deed, exposure to 12% ethanol for 24 h reduced growthby just 24%. It will be important to determine whetherthe observed changes in spermine level, and APC and3APC formation, are related to this tolerance of S. cere­visiae to ethanol, and also to compare these changeswith polyamine metabolism in a fungus showing muchgreater sensitivity to ethanol.

AcknowledgementsSAC receives funding from the Scottish Office Agriculture,Environment & Fisheries Department.

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