S A N F A ~ O N , R., R. ROULLIARD et H. M. C. H E I C K . ~ ~ ~ ~ . The accumulation of succinate by the yeast Bretttrnomyco~ brrrxellet~sis. Can. J . Microbiol. 22: 2 13-220.

On a etudie le metabolisme de B~.ettcrtlotnyces brrr.rellet~sis afin de determiner le blocage metabolique responsable de I'accumulation de I'acetate observee dans des cultures de cette levure. L'acide succinique est le principal acide organique non-volatil dans les cultures qui se sont developpees sur du glucose. Ces cultures ont aussi un faible taux de succinique deshydrogenase (EC 1.3.99.1) et elles ne produisent pas de CO, a partirdes carbonesde I'ethanol. On en conclut que I'oxydation de I'tthanol est bloque au niveau de la succinique deshydrogenase. Si des cultures qui se sont developpees sur du glucose sont transferrees sur un milieu aI'Cthanol, ce blocage dans le metabolisme de I'ethanol est partiellement maitrise, le taux de succinique deshydrogenase augmente, la concentration du succinate intracellulaire diminue et le CO, est produit a partir du C-I de I'ethanol.

[Traduit par lejournal]

The accumulation of succinate by the yeast Brettanomyces bruxellensisl

ROGER S A N F A C O N , ~ ROGERROULLIARD, AND H. M. C . HEICK The Depcrrtt~ietlt of Biochemistry, Lor~crl U~~i~,ers i ty , Qlrebec City, Qr~e.

nnd Tl~e Depcrrtinetzt of Biochetnistiy, Utli1,ersity of Ottcrlva, Ot to~~cr , Cnncicln

Accepted October 9, 1975

S A N F A ~ O N , R., R. ROULLIARD, A N D H. M. C. HEICK. 1976. The accumulation of succinate by the yeast Brc,ttertro~njlces hrrr.rel1eti.si.r. Can. J . Microbiol. 22: 2 13-220.

The metabolism of Brettcrtlornyces brrrxellet~sis was investigated to determine the metabolic block responsible for the accumulation of acetate seen in culturesof this yeast. In glucose-grown cultures the major non-volatile intracellular organic acid was succinic acid. These cultures also had low levels of succinic dehydrogenase (succinate dehydrogenase, EC 1.3.99. I ) and did not produce CO, from the carbons of ethanol. It was concluded that a block in the oxidation of ethanol occurred at the level of succinic dehydrogenase. If glucose-grown cultures were transfer- red toethanol medium, the block in the metabolism of ethanol was partially overcome; the level of succinic dehydrogenase increased, the concentration of the intracellular succinate decreased, and CO, could be produced from C-l of ethanol.

Introduction The yeast, Brettanomyces, presents interesting

metabolic characteristics. Cultures of this yeast accumulate acidic products which lead to the death of the cells unless the culture medium is strongly buffered. Custers (7) has identified acetic acid as the major organic acid in the medium. This yeast also exhibits the phenomenon of stimulation of fermentation by oxygen known as the Custer's or negative Pasteur effect. It is the purpose of this communication to report evi- dence concerning the nature of the metabolic block which results in acetate accumulation.

Materials and Methods Yeast Strain

The species of yeast used these experiments was Brettatlomyces bruxellensis, American Type Culture Col- lection 10560. The strain was maintained by serial

'Received July 11, 1975. ZPresent address: Department of Obstetrics and Gyne-

cology, University of Western Ontario, London, Ontario.

transplants on agar medium of the foliowing composition: glucose 15 g, Difco yeast nitrogen base 6.7 g, calcium carbonate 5 g, agar 30 g, and water 1 litre.

Growt/z of Yeast Working cultures of the yeast were grown in sterile

medium containing Difco yeast nitrogen base, 6.7g/l, in 0.2 M potassium phosphate buffer, pH 6.6. The carbon source was either glucose, 15 g/l, or ethanol, 15 ml/l. The ethanol was added after sterilization.

Preliminary cultures were obtained by transferring loops of cells from a new (3- to 4-day-old) agar slant to 50 ml of glucose medium contained in a 500-ml Ehrlen- meyer flask. These cultures were incubated in a water- shaking bath and agitated continually for 72 h at a temperature of 27 "C. At this time the absorbance of the cultures was measured at 540 nm with a Gilford Model 301 spectrophotometer (Gilford Instruments, Oberlin, Ohio). The suspension of yeast was always diluted to obtain an absorbance between 0.5 and 1 unit. The ab- sorbance of these cultures varied from 1.5 to 3 O D units per millilitre. Working cultures were obtained by trans- ferring a voIume of the culture equivalent t o 20 O D units to 800 ml of medium contained in a 4-litre Ehrlenmeyer flask. These cultures were incubated at 27 OC with con- tinuous shaking in a water bath.

For experiments involving intact yeast cells the cultures

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214 CAN. J . MICRO9101 L. VOL. 22, 1976

were harvested by centrifugation after the desired growth period and washed twice with distilled water. The cells were then suspended in water a t a concentration of about 30 mg wet weight of yeast per millilitre (about 5 mg dry weight per millilitre).

Matzometric Techniques Gas exchange was determined by standard manometric

techniques with a differential respirometer (Gilson Medical Electronics). Each flask contained potassium phosphate buffer 0.125 mmol, substrate 62.5 mmol, yeast about 5 mg dry weight, in a final volume of 2.5 ml. Measurements were begun after the yeast had equilibrated in the substrate for 15 min.

Experiments were conducted in which the effect of different amounts of yeast per flask was determined. The Qo,: obtained with about 5 mg was the same as that obta~ned with 2-2.5 mg. Therefore, the larger amount of yeast was used in the experiments reported, because the larger respiratory changes allowed more precise deter- minations.

Measirret?iet~ts of Radioactive C 0 2 Preparation of the cells and the incubation medium

were the same as for the manometric experiments. The substrates were 0.025 M [I-' JC]ethanol or 0.025 M [2-"C]ethanol. Incubation of the flasks and the collection and determination of I4Co2 were done as described by Morsoli and BCgin-Heick (16).

Deterttlination of Organic Acids in Cells Cultures were harvested and washed as described above

and the cells were lyophylized. The organic acids were extracted and separated by elution from celite with increasing concentrations of 11-butanol in chloroform (8).

Electropllorcsis Electrophoresis of the yeast extract to observe the

alcohol dehydrogenase activity was done as described before (1 2).

Meanrrettlent of Et~iymatic Activity Extracts used for measuring enzymatic activity were

prepared in the following manner. After the growth period the cultures were harvested by centrifugation and washed twice with water. They were then taken up in 0.05 M potassium phosphate buffer, pH 7.4, at a ratio of about 5 ml of phosphate buffer per gram wet weight of yeast. The cells were then broken in a cold (2 "C) French pressure cell (American Instrument Company Inc.) by the use of a Carver tissue press (Fred S. Carver, Summit, New Jersey). A pressure of 20000 psi (1 psi = 703.07 kg/m2) was used. T o obtain sufficient breakage, the yeast had to be passed through the press twice. Micro- scopic examination showed that about 75% of the yeast

3Abbreviations: Qo2, microlitres of 0, consumed per hour per milligram dry weight; Qco,, microlitres of C 0 2 produced per hour per milligram dry weight; ethanol- cells, cells harvested from cultures grown 72 h in the glucose medium and then harvested and transferred to ethanol medium and allowed to incubate in this medium 22 h ; glucose-cells, cells harvested from cultures grown 72 h in the glucose medium; C/E ratio, ratio of the activity of alcohol dehydrogenase with cinnamyl alcohol a s substrate to the activity with ethanol a s substrate.

cells were broken. The homogenate was then centrifuged twice at 1500 x g for 10 min and the supernatant used for the measurement of enzymatic activity.

Succinic dehydrogenase (succinate:phenazine oxido- reductase. EC 1.3.99.1) was measured by the colorimetric method described by Skala et a/. (22). The sample was incubated for 15 min a t 37 "C in the following medium: potassium phosphate buffer, 50 mM, p H 7.4; phenazine methosulphate, 0 .025z; INT (2-p-iodophenyl-3-p-nitro- phenyl-5-phenyltetrazolium chloride), 0.1%; KCN, 1 mM; sucrose, 50 m M ; sodium succinate, 0.05 M. The reaction was stopped by the addition of 1 ml of 10% TCA and the formazan was extracted in 4 ml of ethyl acetate. Extinction was measured a t 490 nm. The extinction coefficient of formazan in the presence of phenazine methosulphate was taken as 20.1 x 106 cm2 mol-'.

Cytochrome oxidase (ferricytochrome c:oxygen oxido- reductase, EC 1.9.3.1) was measured by its ability to oxidize added reduced cytochrome c. Cytochrome c was reduced by dialyzing it in 0.1 M phosphate buffer, p H 7.0, in the presence of ascorbic acid for 24 h with three changes of buffer. The reduced solution of cytochrome c gave a ratio of extinction (absorbance) at 5501560 nm greater than 6. T o measure the cytochrome oxidase activity, 4.5 x mol of cytochrome c in 3 ml of potassium phosphate buffer were placed in each cuvette. Depending on the activity, a portion of the supernatant fraction corresponding to 20-200 pg protein was added. The reaction was allowed to proceed for 5-10min and then stopped by completely oxidizing the cytochrome c with a drop of saturated ferricyanide solution. The activity was then calculated according to the formula of Smith (23). The activity of the cytochrome oxidase was expressed as nanomoles of cytochrome c oxidized per minute per milligram of protein.

Acetyl-CoA synthetase (acetate:CoA ligase (AMP- forming), EC 6.2.1.1) was measured according t o the method of Keine and Jahnke (13). Aconitase (citrate- (isocitrate) hydro-lyase, EC 4.2.1.3) was measured ac- cording to the method of Racker (18). Citrate (si)- synthase (citrateoxaloacetate-lyase, EC 4.1.3.7) was mea- sured according to the method of Polakis and Bartley (17). Fumarase (L-malate hydro-lyase, EC 4.2.1.2) was mea- sured according to the method of Racker (18). Malate dehydrogenase (L-malate: NAD + oxydoreductase, EC 1 . I . 1.37) was measured according to the method of von Jagow and Klingenberg (24). Pyruvate kinase (ATP: pyruvate 2-0-phosphotransferase, E C 2.7.1.40) was mea- sured according to the method of Cartier et a/. (4). Isocitrate lyase (threo-D~-isocitrate glyoxylate-lyase, EC 4.1.3.1) was measured according to the method of Cooper and Beevers (5). The coefficient of extinction used was 2.2 x lo7 litres mol-I cm-' (2).

The methods used for the determination of alcohol dehydrogenase, fructose-1,6-diphosphatase, isocitrate de- hydrogenase, malate synthetase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase were those applied to the determination of the activity of these enzymes in Astasia lot~ga (1, 2).

Chemical Determit~atiot~s Protein was determined according to the method o f

Lowry et a/. (15). T o determine the glucose and ethanol concentrations in the culture medium, samples were with- drawn, centrifuged and the supernatant was diluted

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SANFACON ET AL.: SUCCINATE ACCUMULATION BY BRETTANOMYCES

TABLE 1. Glucose and ethanol metabolism during growth of culture of Brettano~nyces bruxeller~sir

Glucose Ethanol Age, Absorbance content of content of

Qo,

h at 540 nm medium, g/l medium, g/l Glucose Ethanol Endogenous

24 5 13 None detected 26 22 7 48 14 11 0.1 27 22 4 72 22 8.8 0 . 6 27 23 7 96 28 None detected 0 .4 23 19 10

NOTE: The cultures were grown for the indicated time interval. The absorbance at 540 nni n a s measured. Afler the cells were removed by centr~fugation, the glucose remaining in the medium and the ethanol produced were measured as described in Materials and Methods. For the manometric determinations each flask contained 4-5 rng dry weight ofyeast suspended in 0.05 M potassium phosphate buffer, pH 6.7. The values indicated are the mean ofthreecuItures grown independently. Variation from the mean was less than 5z.

appropriately. Glucose was determined enzymatically (Glucostat, Worthington). The ethanol content was determined spectrophotometrically by measuring the reduction of nicotinamide-adenine dinucleotide (NAD), using comnlercial alcohol dehydrogenase (3).

Source of Reager~ts Radioactive ethanol was obtained from the New

England Nuclear Corporation. Succinic acid, cytochrome c, phenazine methosulphate, p-iodonitrotetraiolium violet (INT) and bovine serum albumin were obtained from the Sigma Chemical Corporation, St. Louis, Missouri.

Results In Table 1 are shown some of the metabolic

characteristics of cultures of Brettat7omyces br~ixellensis grown on the glucose medium. The

growth of the culture stopped. The cell yield was about 15% of that of a comparable 72-h culture with glucose as the carbon source. Because of this poor growth on ethanol we investigated the effect of ethanol by first growing cultures on glucose medium, harvesting the cells under sterile con- ditions after 72 h of growth and reincubating them in an equal volume of ethanol medium for 22 h. All cultures were examined with a micro- scope for bacterial contamination. For all the experiments reported in this paper at least one determination was made in which penicillin and streptomycin were present during the period of incubation in ethanol. No significant differences

cultures remained in the logarithmic phase of were found if antibiotics were included. growth for the first 72 h. The generation time The effect of the transfer of the glucose-grown during this phase was calculated at 10 h. After cultures to theethanol medium on the respiratory 72 h, the rate ol" grqwth of the cultures began to characteristics is shown in Table 2. While there decrease. Sixty percent qf the original glucose was no change in the endogenous respiration, was still present at 72 h, b~ ; ? this glucose was the transfer to ethanol increased the glucose completely metabolized over the next 24 h. The ethanol content of the medium incrased rapidly between 48 h and 72 h but declined during the subsequent 24 h. The oxygen consumption of the cells changed very little during the first 72 h. Both glucose and ethanol stimulated the oxygen consumption, but glucose had a somewhat greater effect than ethanol. Between 72 and 96 h, the endogenous oxygen consumption increased slightly and the stimulation in the presence of the substrates declined somewhat. For the subsequent experiments, 72-h cultures were used since these cultures provided a good yield of cells, which respired actively.

Brettanonvces brrrxellet~sis grew poorly in media containing ethanol as the only carbon source. The generation time was about 26 h. Less than three generations occurred before the

stimulated oxygen consumption but decreased the corresponding CO, production. The RQ of 1.9 observed for the glucose-cells is consistent with the production of ethanol as reported in Table 1. After the transfer to ethanol the RQ for glucose declined to about one. This is consistent with the complete oxidation of glucose. Ethanol stimulated the oxygen consumption of the glu- cose-cells fourfold but the Q,, for ethanol was always slightly less than the Q,, for glucose, and the CO, production was only slightly stimulated by ethanol. After incubation in the ethanol medium, the Q,, for ethanol was equal to the Qo, for glucose and greater than the oxygen consumption by the glucose-cells observed in the presence of either substrate.

In order to determine the origin of the CO, produced in the presence of ethanol, experiments

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CAN. J . MICROBIOL. VOL. 22. 1976

TABLE 2. Respiration of Brettanotnyces bruxellensis grown on glucose medium and after transfer to ethanol medium

Growth conditions

Glucose (72 h) -+ Glucose (72 h) Ethanol (22 h)

Substrate Qo, Qco, RQ Qo, Qco, RQ

None 6 6 1 . O 6 6 1.0 Glucose 27 52 1.9 34 3 8 1.1 Ethanol 23 9 0 .4 33 19 0.6

NOTE: The yeasts were cultivated for 72 h, harvested and washed as described under Materials and Methods. One portion was used for the manometric experiments and another portion \\,as transferred to a volume of ethanol med i i~m equal to the volume o f glucose medium from which the transplanted cells were derived. The conditions for incubation are the same as those in Table I . T h e values indicated here a re tlie mean of three cultures grown indepen- dently. Variation from the mean was less than 5%.

were conducted using [l-'4C]ethanol and [2-14C]- ethanol. No radioactive CO, was recovered when the glucose-cells were incubated with either substrate. When the ethanol-cells were incubated with [2-14C]ethanol, again no radioactive CO, was recovered. However, with [I-'4C]ethanol as substrate, the production of CO, as measured by the recovery of 14C0, was 460 nmol h-' mg-' dry weight of cells. This corresponded to a Qco, of 10. Since the QCO2 for ethanol, determined manometrically, was 19 (Table 2) about half the CO, produced was derived from the first carbon of ethanol.

The respiratory characteristics of the two types of cells s~~ggested a block in the oxidation of ethanol which was at least partially overcome by the incubation in ethanol.

To help determine the step at which the metabolic block occurred, we investigated the accumulation of the intracellular non-volatile organic acids by separating the acids on a celite column (8). The major peak was eluted from the column with 300-400 ml of eluent. This is the region of the column at which succinic acid is eluted. To confirm the presence of succinic acid we recovered the product by evaporation of the solvent under vacuum and submitted it to infra- red spectroscopy. The spectrum obtained was identical with that of a spectrum obtained from an authentic sample of succinic acid (19). The yield of succinate was 14 mmol/gm dry weight of yeast. The dry weight of the yeast is about 20x of the wet weight. Assuming that 1.67 gm wet weight of yeast contains I ml of intracellular water (lo), the concentration of the succinate in the intracellular water would be about 4.5 mM.

After incubation of the cells in ethanol medium the concentration of succinate decreased by 50%. Some unidentified acidic products were eluted in the first 100 ml of eluent, but there were no peaks eluted corresponding to lactate or to citric acid cycle intermediates other than succinate.

The accumulation of succinate suggested a block in the metabolism of succinic acid. We therefore measured the level of succinate de- hydrogenase. We also measured the level of cytochrome oxidase which like succinate dehy- drogenase is found on the mitochondria1 mem- brane. The results are shown in Table 3. Initial attempts to measure succinic dehydrogenase activity were frustrated by the development of color in tubes without substrate. Dialysis of the sample prevented this high blank value in the measurement of succinic dehydrogenase but also resulted in the loss of 33% of the cytochrome oxidase activity. After dialysis a low level of succinate dehydrogenase was found. After trans- fer to ethanol the level of succinate dehydro- genase increased 2.6-fold and that of cytochrome oxidase by 20%.

The net accumulation of a citric acid cycle intermediate during growth on glucose requires that the cells have a method for producing four- carbon acids from three-carbon glycolytic inter- mediates. We therefore measured the level of pyruvate carboxylase and found the activity to be 64 nmol pyruvate transformed per minute per milligram of protein (Table 4).

Growth on ethanol requires a system of pro- ducing hexoses for structural components from a two-carbon source. In yeast the system involves the glyoxylate cycle and the reversal of the

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SANFACON ET AL.: SUCCINATE ACCUMULATION BY BRETTANOMYCES

TABLE 3. Activity of succinic dehydrogenase and cytochrome oxidase

Growth conditions

Glucose Glucose (72 h) (72 h) -.

Ethanol (22 h) Enzyme activity Before dialysis After dialysis After dialysis

Succinic dehydrogenase High blank 1 . O 2.6 Cytochrome oxidase 3 60 240 290

NOTE: The sample consisted of the 1500 x g supernatant of cells broken with the French pressure cell. Succinic dehydrogenase and cytochrome oxidase activities were determined as described in Materials and Methods. The specific activity of succinic dehydrogenase is expressed in nanomoles of formazan formed per minute per milligram of protein. The activity of cytochrome oxidase is expressed in nanomoles of cytochrome c oxidized per minute per milligram of protein. Samples were dialyzed at 4'C for 24 h against three changes of 1 litre of 0.05 M potassium phosphate buffer at p H 6.45.

TABLE 4. Effect of ethanol on the key enzymes of gluconeogenesis

Ratio of Specific activities specific activities,

(glucose (72 h) -+

Glucose (72 h) -+ ethanol (22 h))/ Enzyme Glucose (72 h) Ethanol (22 h) glucose (72 h)

Isocitrate lyase 2.3 12.3 5 .3 Malate synthetase 11 48 4 . 4 Pyruvate carboxylase 64 92 1 .4 Phosphoenol pyruvate carboxylase 27 28 1 .O Fructose-1,6-diphosphatase 30 50 1 .7

NOTE: The specific activities are expressed in nanomoles substrate transformed or product produced per minute per milligram of protein. The enzyme activity was measured in the 1500 x g supernatant. All values are the mean obtained for at least three different cultures. Variations in activity between cultures was less than 10%.

TABLE 5. The effect of ethanol on the activity of citric acid cycle enzymes

Ratio of Specific activities specific activities,

(glucose (72 h) -+

Glucose (72 h) + ethanol (22 h))/ Enzyme Glucose (72 h) Ethanol (22 h) glucose (72 h)

Citrate synthetase 1600 13 000 8 .1 Aconitase 18 125 7 .0 NAD-dependent isocitrate dehydrogenase 18 22 1.2 NADP-dependent isocitrate dehydrogenase 13 7 0 .5 Fumarase 345 630 1 .8 Malate dehydrogenase 58 980 17

NOTE: Specific activity is expressed in nanornoles substrate transformed or product formed per minute per milligram of protein. The sample consisted of the 1500 x g supernatant. Each value is the mean of determinations on at least three differ- ent cultures. The variation from the mean was less than IOW. The activity of citrate synthase was determined in a sample dialyzed as indicated for succinic dehydrogenase in Table 3. Dialysis doubled the value.

glycolytic sequence. The effect of ethanol on some of the key enzymes of gluconeogenesis is shown in Table 4. Ethanol had little effect on the level of activity of phosphenol pyruvate carboxy- kinase and fructose- l,6-diphosphatase. However, the level of the two enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, in- creased four- to fivefold. Both before and after

the transfer to ethanol, the enzyme of this group with the lowest activity was isocitrate lyase.

The effect of ethanol on the citric acid cycle enzymes is shown in Table 5. Citrate synthase had the highest level of activity. The level of this enzyme was increased by dialysis. The lowest levels of activity were found for isocitrate de- hydrogenase and aconitase. Transfer to ethanol

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CAN. J . MICROBIOL. VOL. 22, 1976

TABLE 6. The effect of ethanol on the activity of enzymes of glucose and ethanol metabolism

Ratio of Specific activities specific activities,

(glucose (72 h) + Glucose (72 h) -+ ethanol (22 h))/

Enzymes Glucose (72 h) Ethanol (22 h) glucose (72 h)

Pyruvate kinase 15 25 1 .7 Ethanol dehydrogenase 120 185 1.5 Cinnamyl alcohol dehydrogenase 50 90 1 .8 Acetyl-CoA synthetase 6 12 2 .0

NOTE: Specific activity is expressed in nanomoles substrate transformed or product produced per minute per milligram of protein. The sample consisted orthe I500 x g supernatant. The values are the mean ordeterminations on at least three different cultures. The variation rrom the mean was less than 10%.

produced large percentage increases in citrate synthase, aconitase, and malate dehydrogenase. The effect of the ethanol was much less marked on the NAD-dependent isocitrate dehydrogenase and fumarase. The low level of NADP-de~endent isocitrate dehydrogenase was decreased by the transfer to ethanol.

The effect of ethanol on the activitv of a number of other enzymes is shown in Table 6. Alcohol dehydrogenase activity was increased about 50%. There was little change in the ratio of activity with cinnamyl alcohol as substrate as compared with ethanol (C/E ratio). Electro- phoresis on polyacrylamide gel revealed two distinct bands: No change in this pattern, nor in the relative intensity of the two bands, was ob- served due to transfer to ethanol. The percentage increases in the levels of pyruvate kinase and acetyl-CoA synthetase were similar to the in- crease observed for alcohol dehydrogenase.

Discussion Custers (7) had already identified CO,, ethanol,

and acetate as metabolic products of Brettano- rnvces and he had also -determined that the acetate was produced via ethanol and not directly from pyruvate. The results presented above indicate that there is also an accumulation of succinate within the cells. Although succinic acid has been shown to be a major intracellular acid in a number of yeasts (6, 8, 9, 14), B. brirxellet7sis is unique in that succinic acid and acetic acid are the only organic acids involved in the inter- mediary metabolism of glucose that accumulate to any appreciable degree. No radioactive CO, could be detected when glucose-grown cells were incubated with labelled ethanol. There thus appears to be a block in the tricarboxylic acid cycle limiting the complete oxidation of ethanol

by this yeast. Of the enzymatic steps following succinate, the enzyme with the lowest level of activity was succinate dehydrogenase. This low level of succinic dehydrogenase may, therefore, be responsible for the accumulation of succinate.

The problem of accumulation of acetate is more complex and may be due to several factors. The available evidence indicates that the path- ways leading from acetate to succinate can only proceed at a limited rate. The activities of acetyl- CoA synthetase, aconitase, and isocitrate de- hydrogenase are low, thus limiting succinate production via the tricarboxylic acid cycle. Like- wise the low activity of isocitrate lyase would tend to limit succinate production via the gly- oxylate cycle. Thus a combination of very low levels of succinic dehydrogenase plus low levels of enzymes involved in the pathways between acetate and succinate could be responsible for the pattern of accumulation of organic acids. Other factors besides the level of enzyme activity may govern the flow of metabolites between acetate and succinate. The oxidation of ethanol to acetate results in the production of two molecules of reduced pyridine nucleotide. The further metabolism of acetate may then depend on the availability of oxidized pyridine nucleotide for the oxidation steps. Such a mechanism has been postulated as being responsible for the accumulation of acetate in Astasia exposed to high oxygen concentrations (16).

The concentration of succinate decreased in the ethanol-cells and radioactive CO, derived from ethanol could be detected. This occurred in spite of a large decrease in the activity of some of the enzymes leading to succinate. The increase in the enzymes leading to succinate does no: necessarily lead to a new increase in succinate concentration for, while citrate synthase activity

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SANFACON E T AL.: SUCCINATE AC

increased eightfold, it was already the highest activity observed in the glucose-grown cells. On the other hand the activity of acetyl-CoA syn- thetase, which catalyzes the first step in the utilization of acetate, increased less than that of succinate dehydrogenase. It should be remem- bered also that acetyl-CoA is at a crossroad of several metabolic pathways and other reactions besides those leading to succinate production may compete for this metabolite. The decrease in succinate was due probably to the increase in succinic dehydrogenase (2.6-fold) leading to the increased metabolism of this acid. The derivation of the radioactive CO, almost exclusively from C-1 of ethanol is consistent with the metabolism of ethanol through the glyoxylate cycle. The levels of both the key enzymes of the glyoxylate cycle increased four- to fivefold on exposure to ethanol, and in these cells isocitrate lyase had a higher specific activity than isocitrate dehydro- genase. These changes in enzyme levels may favor the metabolism of ethanol via the glyoxylate cvcle.

Because cycloheximide did not inhibit growth or protein synthesis in B. bruxellensis at a dose which affects S. cerevisiae, it was impossible to determine whether observed increases in enzyme activity represented the synthesis of new mole- cules or the activation of existing molecules. This notwithstanding, in addition to the stimulation of succinic dehydrogenase activity, exposure to ethanol medium (though it did not promote growth) resulted in the increase in activity of many of the enzymes associated with the inter- mediary metabolism of ethanol. The exposure to ethanol promoted the increase of both of the glyoxylate cycle enzymes by the same order of magnitude. The percentage change of the tri- carboxylic acid cycle enzymes was extremely variable. This may be an indication that in this yeast the activities of the former enzymes are controlled in a synchronous manner while those of the latter are not.

The regulation of alcohol dehydrogenase ac- tivity and the isozymes of this enzyme have been studied in a number of yeasts ( I 1, 12, 24, 20, 21, 25). In these yeasts exposure to ethanol medium resulted in the appearance of an isozyme of alcohol dehydrogenase which was virtually ab- sent in cultures where glucose served as the car- bon source. Brertanomyces, however, appears to have a single enzyme which moves as two electrophoretic bands, as noted for ADH-I of

Saccharomyces cerevisiae (12). This enzyme must function both in the synthesis and catabolism of ethanol. The 50% increase in alcohol dehydro- genase activity after exposure to ethanol was also much less than has been reported for other yeasts including Saccharomyces fragilis which like B. bruxellensis produces ethanol but grows poorly on ethanol as the carbon source (20).

Acknowledgments The financial support of the Medical Research

Council of Canada is gratefully acknowledged.

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2. BEGIN-HEICK, N. 1973. The localization of enzymes of intermediary metabolism in Asrrrsitr and Err,gIe,r~o. Biochem. J . 134: 607-616.

3. BONNISCHEN. R. 1965. Ethanol determination with alcohol dehydrogenase and DPN. 111 Methods in en- zymatic analysis. Etli/c,el hy H. U . Bergmeyer. Academic Press, Inc.. New York. N.Y.

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