JOURNAL OF BACTERIOLOGYVol. 88, No. 4, p. 858-864 October, 1964Copyright © 1964 American Society for Microbiology

Printed in U.S.A.

FUMARATE REDUCTION AND ITS ROLE IN THE DIVERSIONOF GLUCOSE FERMENTATION BY STREPTOCOCCUS FAECALIS'

R. H. DEIBEL2 AND MARILYN J. KVETKAS3

Division of Bacteriology, American Meat Institute Foundation, The University of Chicago,Chicago, Illinois

Received for publication 10 April 1964

ABSTRACT

DEIBEL, R. H. (American Meat InstituteFoundation, Chicago, Ill.), AND M. J. KVETKAS.Fumarate reduction and its role in the diversionof glucose fermentation by Streptococcus faecalis.J. Bacteriol. 88:858-864. 1964.-Fumarate divertsthe normal fermentation of glucose by Strepto-coccus faecalis FB82, as shown by the productionof increased amounts of CO2 , formate, acetate,and acetoin, and decreased formation of lactateand ethanol. Experiments with D-glucose-l-C'4,in which low levels of labeled CO2 were recovered,indicated that C-1 cleavage of the glucose moleculewas not involved. The presence of fumarateafforded consistently larger cell crops in growthstudies with glucose and other energy sources.On a molar growth-yield basis, anaerobicallygrown, glucose-fumarate cultures were equivalentto aerobically grown, glucose cultures. The reduc-tion of fumarate by cell suspensions indicated thatglucose, gluconate, and, to a lesser extent, glyceroland mannitol could serve as hydrogen donors.Several common metabolic inhibitors had noeffect upon the fumarate reductase system in cellsuspensions, although some sensitivity to acidicpH was noted. Significant levels of succinateoxidation activity were not detected. Fumaratereductase activity was demonstrated in all fiveS. faecalis strains tested. Distribution of thisability in S. faecium strains was variable, rangingfrom activity comparable with that of S. faecalisto total inactivity. The observations support theconclusion that fumarate functions as an alternatehydrogen acceptor, thus allowing pyruvate toparticipate in the energy-yielding phosphoro-clastic and dismutation pathways.

In a previous communication (Deibel, 1964b),1 Journal paper no. 277, American Meat In-

stitute Foundation.2 Present address: Division of Bacteriology,

Cornell University, Ithaca, N.Y.3 Present address: Biological and Medical Re-

search Division, Argonne National Laboratory,Argonne, Ill.

data were presented which suggested the abilityof fumarate to divert the normal homofermenta-tive dissimilation of glucose by Streptococcusfaecalis. In the presence of fumarate, the fermen-tation of glucose yielded significantly increasedquantities of C02, formate, acetate, and acetoin.To account for this radical alteration of endproducts, it was assumed that fumarate substi-tuted for pyruvate in the regeneration of oxidizednicotinamide adenine dinucleotide, and thusaverted the reduction of pyruvate to lactate. Thesubsequent catabolism of pyruvate via the dis-mutation and phosphoroclastic pathways wouldthen account for the observed alteration of endproducts.

It is the purpose of this communication tosubstantiate and to extend the above hypothesisand observations, as well as to integrate thesefindings with those of previous investigators.

MATERIALS AND METHODS

Strains. One strain of S. faecalis (FB82) wasused extensively in this study. It possessed themajority of physiological characteristics associ-ated with this species (Deibel, Lake, and Niven,1963). The source and characteristics of this andother enterococcal strains used were describedpreviously (Deibel et al., 1963).

Media. The strains were maintained by dailytransfer in APT medium (Difco). Most of theexperiments were performed with a complexmedium of the following composition: Tryptone(Difco), 10 g; yeast extract (Difco), 5 g; NaCl,5 g; K2HPO4, 5 g; distilled water, 1 liter; pH 7.2to 7.4. The energy source was variable, but unlessotherwise indicated it was added at the 1.0%level.

In some experiments a semisynthetic, casein-hydrolysate medium was employed (Deibel andNiven, 1964). Methionine was added to themedium, as Brodovsky, Utley, and Pearson(1958) showed that commercial preparations of

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FUMAI{ATE REDUCTION BY S. FAECALIS

casein hvdrolvsates may be deficient in thisamino acid.

Chemiticals. 1)-Glucose-i C14, sodium fumarate,and sodium pyruvate weIe obtained from Calbio-chem.

Analytical methods. In fermentation-balanceand in radioisotopic studies, the clarificationprocedure described by Neish (1952) was used.Volatile neutral products were determined bydistilling a 50-ml samnple of the clarified broth(pH 7.6 to 7.8). Approximately 200 ml of distil-late were collected over a period of 2.5 to 3.5 hr.The reaction flask was then acidified, and thedistillation was continued for the volatile acids.This sequence was employed to prevent some

acetoin from distilling with the volatile-acidfraction and interfering in the subsequent deter-mination of formate. Formate was estimated bythe imacrogravimetric procedure (Anonymous,1940). Acetate was calculated by difference fromthe total volatile acids. Previously, it was ob-served that formate and acetate were the onlyvolatile acids produced in the fermentation ofpyruvate by S. faecalis (Deibel and Niven, 1964).Fumarate was measured spectrophotometri-

cally at a wavelength of 240 m,u in appropriatelydiluted samples of the clarified fermentationbroth (Racker, 1950). Suceinate was estimatedmanometrically after continuous ether extractionof the sample for 48 hr (Uimbreit, Burris, andStauffer, 1957). Ethanol (volatile neutral distil-late) was determined enzymatically by the pro-

cedure of Bonnicksen and Lundgren (1957). Theenzyme preparation used in this method was

obtained from Sigma Chemical Co., St. Louis,Mo. Lactate was also estimated enzymatically,by the method of Scholz et al. (1959). The lacticdehydrogenase prep)aration was purchased fromCalbiochem. The excellent agreement betweenthis method and the chemical method of Barkerand Summerson (1941) promlpted continued use

of the less laborious enzymatic procedure. Acetoinwas estimated by the procedure described byNeish (1952).

Carbon dioxide was precipitated in a series of0.1 N barium hydroxide traps after acidificationof the culture medium and flushing with helium.The plrecipitate was collected by centrifugation,washed with cold distilled C02-free water, anddried to constant weight at 110 C.

Radioisotopic proceduires. The C'402 producedfrom the fermentation of D-glUcose-_-C'4 was

trapped in a series of tubes containing 0.1 N

sodium- hydroxide; 1-mIl portions of these samplesand of the fermentation media, suitably dilutedin 0.1 N sodium hydroxide, were dispensed on

stainless-steel planchets. Radioactivity was meas-

ured in a gas-flow counter.Preparation of cell suspension. Cultures were

grown in the conmplex medium, supplementedwith 1.0%7 sodium fumarate and 1.0% glycerol(or other energy source). The cells were harvestedby centrifugation, resuspended in phosphatebuffer (pH 7.0), and rapidly frozen in an ethyleneglycol bath at -20 C. Prior to use, the frozencells were rapidly thawed and resuspended inappropriate buffer. The cell concentration was

adjusted by dilution and comparison to a stand-ard curve relating optical density to dry weightof the cells.

Mliscellaneomis methods. Growth was estimatedby optical-density determinations in a Bausch &Lomb Spectronic-20 colorimeter at a wavelengthof 600 m,u. Methods of incubating under aerobicand anaerobic conditions were described previ-ously (Deibel, 1964a). All cultures were incubatedat 37 C.

RESULTS

Fate of C1 in glucose-1i C14. This experimlent was

performed to ascertain the possible diversion ofthe glucose fermentation by fumarate by a path-way other than the hexose diphosphate scheme.The fate of the various carbon atoms in alternatepathways of glucose metabolism has been re-

viewed (Gunsalus, Horecker, and Wood, 1955).Characteristically, the C, atom of glucose is foundin the methyl group of lactate (and presumablypyruvate) in the hexose diphosphate scheme, andin the C02 if metabolism occurs via the hexosemonol)hosphate scheme. Even if the fermentationis diver ted by fumarate as anticipated, the decar-boxylation of pyruvate would not be expected to

yield labeled CO2 from glucose-i-C'4.S. faecalis FB82 was grown in 50 ml of the

complex medium containing 1.0% glucose and2.0 m11c of glucose-I-C14 (specific activity = 7.4mc /mmole). A duplicate culture was preparedand sup)plemented with 1.0% sodium fumarate.Another set of cultures was prepared in an identi-cal manner but with the omission of the radio-active glucose. These cultures served as controlsfor subsequent analyses to determine the extentof diversion of the fermentation effected by

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DEIBEL AND KVETKAS

TABLE 1. Glucose fermentation balances with Strep-tococcus faecalis FB82, illustrating the

effect of fumarate

Substrate Product Amt*

mmoles

Glucoset 100Lactate 177.9Acetoin 0.7Ethyl alcohol 5.8Acetate 1.3Formate 2.9C02 6.8

Glucoset 100Fumarate 93.8

Succinate 98.7Lactate 126.8Acetoin 9.2Ethyl alcohol 0.09Acetate 45.9Formate 7.3C02 52.7

* All values corrected for endogenous metabo-lism.

t Carbon: recovery = 93.4%; O/R balance =1.27; Cl/C2 = 1.14.

$ Carbon recovery = 94.9%; O/R balance =1.02; Cl/C2 = 0.93.

TABLE 2. Growth response of Streptococcus faecalisFB82 to increasing levels of glucose in the

presence and absence of fumarate

Medium*Glucose in medium Basal Basal + fumarateBasal%(1.0%)

0 42t 480.1 63 850.2 73 1000.3 85 1220.4 105 1300.5 113 140

* The complex medium was employed (seeMaterials and Methods).

t Optical density X 100.

fumarate. The cultures were incubated anaero-

bically for 24 hr at 37 C, and the C02 producedwas trapped and counted.

In the presence of fumarate, only 0.52% of theinitial radioactivity was recovered in the C02, and79.9% of the activity remained in the clarifiedmedium. In the absence of fumarate, correspond-

ing values of 0.21% and 77.5% were obtained.Presumably, the remainder of the activity wasassociated with the cells which were removedin the clarification procedure.The results indicate that a C0 cleavage of the

glucose molecule is not effected in the presence offumarate. Thus, the most probable pathway ofglucose catabolism in the presence of fumarateis via the hexose diphosphate scheme.

Fermentation balances. Significant increases inthe quantities of acetate, formate, acetoin, andC02, and decreases in the quantities of lactateand ethanol, were observed in cultures with addedfumarate (Table 1). The ratio of glucose utilizedto fumarate reduced approached unity, indicatingthat approximately 50% of the total theoreticalC3-cleavage products of glucose were divertedfrom the normal pathway which ultimatelyresults in the reduction of pyruvate to lactate.The approximately quantitative recovery of suc-cinate tends to negate any fumarase activity, andthe alteration of end products effected by fuma-rate cannot be associated with this enzyme. Intoto, the results indicate that fumarate divertsthe fermentation by acting as an alternate hydro-gen acceptor, thus allowing the subsequent ca-tabolism of pyruvate via the phosphoroclasticand dismutation pathways.

Growth studies. The inclusion of fumarate inthe growth medium resulted in small but repro-ducible increases in the cell crop. A series ofincreasing glucose concentrations was preparedin the complex medium. A duplicate set was pre-pared in the same medium supplemented with fu-marate. The increased growth response of the fu-marate-containing cultures (Table 2) is in keepingwith the diversion hypothesis, as additional en-ergy-yielding reactions can be associated with py-ruvate catabolism. The studies were extended toinclude the gluconate, ribose, and pyruvate fer-mentations, since these compounds also serve asenergy sources for S. faecalis (Deibel et al., 1963)and involve catabolic pathways which differ fromthose of glucose. The results obtained with glu-conate and ribose paralleled those obtained withglucose, in that the fumarate-containing mediumafforded a larger cell crop as compared with themedium without fumarate. The formation ofslime, with pyruvate as the energy source, pre-cluded optical density determinations. The pro-duction and character-ization of this viscousmaterial has been discussed previously (Deibel,1964b).

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The increased cell crop effected by culturingthe organism with fumarate was measured interms of molar growth yields. Glucose-containingcultures were grown anaerobically with andwithout fumarate. In addition, a duplicate setof cultures was incubated aerobically on a

reciprocating shaking apparatus. To affordgreater stability of the cells in the subsequentrepeated washings, hydroxylysine (0.1 mg/ml)was added to the growth medium. Smith et al.(1962) demonstrated superior stability of entero-coccal cells when grown in the presence of thisamino acid.

Previously, Seeley and VanDemark (1951)demonstrated a 35% increase in the cell crop ofaerobically (as compared with anaerobically)grown cultures of S. faecalis. Thus, if this increasein cell crop can be equated with an alternativehydrogen acceptor (oxygen), the molar growthyield of aerobically grown, glucose cultures shouldapproximate that of anaerobically grown, glu-cose-fumarate cultures. The results of the experi-ment (Table 3) indicate a more efficient anaerobicutilization of glucose when the organism is cul-tured with fumarate. In addition, the results sug-

gest that Seeley and VanDemark's (1951)observation with aerobic cultures, and theanaerobic diversion effected with fumarate,reflect the same basic mechanism, which differsonly in final hydrogen acceptor (oxygen or fuma-rate). Although the molar growth yield obtainedwith the anaerobically grown, glucose-containingculture of S. faecalis was somewhat higher thanthat reported by Bauchop and Elsden (1960), therelationships described in this study are notaffected.Fumarate reduction with various hydrogen

donors. Subsequent experiments were performedto gain additional information on the enterococcalfumarate reductase system. In preliminarystudies, cell crops were grown with glycerol as theenergy source. Cell suspensions were prepared,and the ability of these preparations to reducefumarate with glvcerol as the hydrogen donorwas tested.The test system consisted of 25 Amoles of

sodium fumarate (1 ml), 50 ,umoles of glycerol(1 ml), and 1 ml of appropriately diluted cells.(A total volume of 4 ml was attained by additionof buffer.) All substrates and cell suspensions were

prepared with phosphate buffer (pH 7.0). Prepa-rations of cells alone, cells plus fumarate, andfumarate alone were included routinely for con-

TABLE 3. Comparison of growth yields of Stirepto-coccus faecalis FB82 under various

conditions of culture

Additions to basal medium*

Conditions Glucose Glucose + fumarateof (.%

incubation

Glucose Dry wt Molar Glucose Dry wt Molarutilized of cellst growth utilized of cellst growthyieldt yieldtmrnoles mg Z noles rng

Aerobic 1.70 74.4 43.8 1.69 81.6 48.3Anaero- 1.75 48.8 27.9 1 .75 82.4 47.1

bic

* Complex basal medium. All values correctedfor endogenous metabolism. Initial glucose con-centration = 1.75 mmoles/150 ml of medium.

t Cell crops were washed five times with dis-tilled water.

t Defined as mg (dry weight) of cells per mmoleof glucose utilized (Bauchop and Elsden, 1960).

trol purposes. The reaction mixtures were cen-trifuged (10 to 12,000 X g) to remove the cells,and residual fumarate in the supernatant fluidwas estimated spectrophotometrically. Generally,90 to 100% of the fumarate was reduced within1 hr at pH 7.0 with approximately 10 mg of cellsper reaction vessel. In this and subsequent cell-suspension experiments involving fumarate re-duction, the reaction vessels were incubatedroutinely under helium at 37 C.The activity of other hydrogen donors was

compared with that of glycerol. Cell suspensionswere prepared from cultures grown with mannitol,glycerol, glucose, or gluconate as the energysource. The concentration of cells employed wasadjusted so that less than maximal activity(4.7 mg of cells per reaction vessel) was obtained.All suspensions were adjusted to the same opticaldensity, and the respective substrates (50 ,umoles)for each cell suspension were employed. As inprevious experiments, a total volume of 4.0 mlwas attained by the addition of phosphate buffer(pH 7.0). Glucose and gluconate were found tobe the most efficient hydrogen donors, whereasglycerol and mannitol were approximately one-half as active (Table 4).

Initial results with the glucose system indicatedcomplete failure to reduce fumarate. In this ex-periment, the culture medium contained 1.0%glucose, and the final pH value of the mediumdropped to 4.2. As will be amplified below, the

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TABLE 4. Comparison of fumarate reductaseactivity with various hydrogen donorsa

Fumarate in cellHydrogen donorb suspension Fumarate

(50 Mmoles) reducedInitial Residualc

Asmoles jAmoles

Glycerol 27.5 22.9 16.7Sodium gluco- 27.5 19.0 31.0nate

Mlannitol 27.5 23.9 13.1Glucosed 27.5 27.5 0Glucosee 26.1 17.6 32.5

All cell suspensions (Streptococcus faecalisFB82) were adjusted to give 4.7 mg (dry weight)of cells per reaction vessel. Residual fumaratewas determined after 1 hr at 37 C.

b The cell crops were grown on the same energysource as the hydrogen donor.

c All values corrected for endogenous reduction.d Excessive acid production resulted in loss of

reductase activity.e Results obtained with cells grown under

pH-controlled conditions. See text.

low pH value probably destroyed the fumaratereductase activity. When the experiment wasrepeated, with 0.2%7, glucose in the growthmedium, an active system was obtained (Table 4).An attempt was made to determine the ability

of pyruvate to serve as a hydrogen donor forfumarate reduction. However, pyruvate (or per-haps its dimer) was observed to absorb signifi-cantly at 240 m,u, and thus further spectro-photometric studies with this substrate wereunsuccessful.The glycerol fumarate reductase system was

employed to determine the possible inhibitioneffected by suceinate. Varying the concentrationof suceinate from 10 to 500 ,umoles in the standardsystem (25 ,umoles of fumarate, 50 ,imoles ofglycerol, and 14.3 mg of cells in a total volume of4.0 ml; incubation for 1 hr at 37 C) was foundto have no effect upon the reduction of fumarate.

Various inhibitors (sodium sulfide, potassiumeyanide, sodium arsenite, sodium azide, Atabrine,and malonate) were tested with cells grown inglucose-fumarate and glycerol-fumarate to deter-mine their effect upon fumarate reduction. Inthe standard system described above, all inhibi-tors were added at a final concentration of 10-3 M,except for malonate (50 ,umoles). No significant

loss of fumarate reductase activity was demon-strated in either of the two cell suspensions withany of the inhibitors after incubation for 1 hrat 37 C.To ascertain the adaptive nature of the entero-

coccal fumarate reductase system, cells weregrown in the seemisvnthetic, casein hydrolysatemedium containing 0.15%o glucose; their activitywas compared with that of cells grown in thesame medium supplemented with 1.0%, sodiumfumarate. The glucose-fumarate-grown cell sus-pension was diluted to afford equivalent celldensity with the glucose-grown cell suspension,and each reaction vessel contained 20 mg (dryweight) of cells. Multiple tubes were preparedin the usual manner and, at periodic intervals,tubes were removed and placed in a boiling-waterbath to stop the reaction.

After an initial 5-min lag period, fumaratereduction was essentially linear with both cellsuspensions until 25 4moles of fumarate werereduced. The only significant difference observedwas that cells preadapted to fumarate accom-plished this reduction in 20 min whereas non-adapted cells required 30 min. It would appearthat more conielusive information regarding theadaptive nature of the fumarate reductase mustawait studies with cell-free extracts.

Effect of pH. In the standard system describedabove, glycerol-fumarate-grown cells (14.3 mgdry weight per reaction vessel), with glycerol(50 ,umoles) as the hydrogen donor, completereduction of 27.9 4moles of fumarate was ob-served at either pH 7 or 8 (phosphate buffer).

Diminished activity was observed at pH 6.0 inphosphate buffer (72.8% of the fumarate wasreduced). At pH 5.0 (acetate buffer), almostcomplete loss of activity was observed (only 3.7%of the fumarate was reduced). These results, inaddition to those obtained with a high glucoseconcentration, indicate that the fumarate re-ductase system is sensitive to acidic pH. Addi-tional studies with cell-free preparations areindicated.

Succinate oxidation. An experiment was per-formed to determine the ability of S. faecalisFB82 to oxidize succinate. The organism wasgrown aerobically in the complex medium con-taining 0.5% glucose supplemented with either1.0% sodium succinate or 1.0% sodium fumarate.The cell crops were harvested and tested for theirability to oxidize 50 Amoles of succinate under

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aerobic conditions in cell suspension. After a 4-hrincubation period, followed by centrifugation toremove the cells, fumarate was estimated spee-trophotometrically. Only 2.0 Amoles of fumarateaccumulated with the succinate-grown cells,indicating that only 4.0%7, of the suceinate hadbeen oxidized. No activity was observed withthe fumarate-grown cells. Consequently, it wouldappear that the organism has limited ability tooxidize succinate, in contrast with its ability toreduce fumarate.

Distribution of fumarate reductase among en-terococci. Ten representative strains of enterococeiwere cultured in the complex medium containing0.2%7, glucose and 0.5% sodium fumarate. Cellsuspensions were prepared and tested for fuma-rate reductase activity, with 50 ,imoles of glucoseas the hydrogen donor. In this experiment, 20 mgof cells per reaction vessel were used (total volumemiade to 4.0 ml with phosphate buffer at pH 7.0;incubation for 1 hr at 37 C). S. faecalis strainsR26, N83, K2A, 26C1, and 1OC1 reduced 95 to100% of the 25,umoles of fumarate added. Theresults with the S. faecium strains were variable.Strains F24 and K6A were devoid of activity,and strains Igau and R39 reduced 14.1 and 24.1%,respectively, of the added fumarate. Completereduction of the added fumarate was observedwith strain RIO.

Further studies with S. faecium (R10). Theobservation that S. faecium R10 possessed theability to metabolize fumarate prompted agrowth experiment identical with that presentedin Table 2. However, unlike the S. faecalis strains,no increase in the growth response of S. faeciumRIO was noted when the medium was supple-mented with fumarate. To some extent, thisresult was expected; this strain, like most otherS. faeciumi, strains, does not possess the ability toutilize pyruvate as an energy source (Deibel andNiven, 1964). Further study with S. faecium RIOto determine the fate of fuma-ate and the endproducts of the glycerol-fumarate fermentationare warranted.

DISCUSSION

The reduction of fumarate by enterococei wasfirst reported by Gunsalus (1947). He observedthat the anaerobic fermentation of glycerol wasenhanced significantly by fumarate; the quantita-tive recovery of suecinate supported the con-clusion that fumarate served as an alternate

hydrogen acceptor. Gunsalus also noted analteration of end products in the presence offumarate, with an increased accumulation ofoxidized compounds.

Kitahara, Fukui, and Misawa (1960) observedthe widespread occurrence of fumarase in variouslactobacilli and also detected fumarate disap-pearance in one of three enterocoecus strains. Ina previous study (Deibel, 1964b), no fumaraseactivity could be detected when S. faecalis FB82was cultured with labeled fumarate. In addition,the quantitative recovery of suceinate tends toreinforce these results. Thus, it is highly unlikelythat significant fumarase activity is associatedwith S. faecalis FB82.The results obtained in this study are in accord

with the hypothesis that fumarate diverts thefermentation by acting as an alternate hydrogenacceptor. This affords the further metabolismof the intermediate pyruvate through the phos-phoroclastic and dismutation pathways. T'here-fore, a more efficient utilization of substrate iseffected, as demonstrated by an increased cellcrop obtained in the presence of fumarate. 'lTheability of S. faecalis, but not S. faeciun, to utilizepyruvate as an energy source is the subject of aprevious communication (Deibel and Niven,1964).The parallelism between increased cell crop

and alternate hydrogen acceptor, as demon-strated in this study and that of Seeley andVanDemark (1951), merits comment. Previously,O'Kane (1950) observed the ability of lipoate(pyruvate oxidation factor) to affect radicallythe end products of glucose oxidation bY cellsuspensions of S. faecalis 10CL. W"hen lipoatewas added, acetate and CO2 were the chiefoxidized products. However, in the absence oflipoate, pyruvate either accumulated or it wasfurther metabolized to form acetoin. It wouldappear that fumarate as well as oxygen can divertthe normal glucose fermentation in such a mannerthat the subsequent metabolism of the lyruvateintermediate affords a larger cell crop, thus re-flecting a more efficient utilization of substrate.

Studies with cell suspensions indicate that theenterococcal fumarate reductase is somiiewhlatsensitive to acid. More definitive studies regard-ing pH lability and optimum, as well as possibleinhibition by various metabolic inhibitors, mustawait the employment of cell-free preparations.It is interesting that the more reduced substrates

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(i.e., glycerol and mannitol) do not effect a moreefficient reduction of fumarate than do glucose orgluconate. The data do not offer an explanationfor these results.The enterococcal enzyme catalyzing the reduc-

tion of fumarate is somewhat similar to that ofMicrococcus lactilyticus, as reported by Warringaet al. (1958). Enzyme systems from both speciesfavor the reduction of fumarate and evidencecomparatively weak oxidation of succinate. Untilcell-free studies are conducted with the enterococ-cal system, further comparison or contrast withthe enzyme obtained from other sources is notpossible.

ACKNOWLEDGMENT

This investigation was supported by PublicHealth Service grant AI-2499 from the NationalInstitute of Allergy and Infectious Diseases.

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BARKER, S. B., AND W. H. SUMMERSON. 1941. Thecolorimetric determination of lactic acid inbiological materials. J. Biol. Chem. 138:535-554.

BAUCHOP, T., AND S. R. ELSDEN. 1960. The growthof microorganisms in relation to their energysupply. J. Gen. Microbiol. 23:457-469.

BONNICKSEN, R., AND G. LUNDGREN. 1957. Com-parison of the ADH and the Widmark pro-cedures in forensic chemistry for determiningalcohol. Acta Pharmacol. Toxicol. 13:256-266.

BRODOVSKY, E. R., M. H. UTLEY, AND W. N.PEARSON. 1958. Methionine inadequacy ofcasein hydrolysate as a source of difficulty invitamin assays. Science 128:307-308.

DEIBEL, R. H. 1964a. Utilization of arginine as anenergy source for the growth of Streptococcusfaecalis. J. Bacteriol. 87:988-992.

DEIBEL, R. H. 1964b. The group D streptococci.Bacteriol. Rev. 28:330-366.

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1963. Physiology of the enterococci as relatedto their taxonomy. J. Bacteriol. 86:1275-1282.

DEIBEL, R. H., AND C. F. NIVEN, JR. 1964. Pyru-vate fermentation by [streptococcus] faecalis.J. Bacteriol. 88:4-10.

GUNSALUS, I. C. 1947. Products of anaerobic glyc-erol fermentation by streptococci [sic] faecalis.J. Bacteriol. 54:239-244.

GUNSALUS, I. C., B. L. HORECKER, AND W. A.WOOD. 1955. Pathways of carbohydratemetabolism in microorganisms. Bacteriol.Rev. 19:79-128.

KITAHARA, K., S. FUKUI, AND M. MISAWA. 1960.Preparation of L-malate from fumarate by anew process "enzymatic transcrystallization."J. Gen. Appl. Microbiol. (Japan) 6:108-116.

NEISH, A. C. 1952. Analytical methods for bac-terial fermentations. Report No. 46-8-3,2nd ed., Prairie Regional Laboratory, Saska-toon, Saskatchewan, Canada.

O'KANE, D. J. 1950. Influence of the pyruvateoxidation factor on the oxidative metabolismof glucose by Streptococcus faecalis. J. Bac-teriol. 60:449-458.

RACKER, E. 1950. Spectrophotometric measure-ments of the enzymatic formation of fumaricand cis-aconitic acids. Biochim. Biophys.Acta 4:211-214.

SCHOLZ, R., H. SCHMITZ, T. H. BUCHER, AND J.0. LAMPEN. 1959. Vber die Wirkung vonNystatin auf Backerhefe. Biochem. Z. 331:71-86.

SEELEY, H. W., AND P. J. VANDEMARK. 1951. Anadaptive peroxidation by Streptococcusfaecalis. J. Bacteriol. 61:27-35.

SMITH, W. G., M. NEWMAN, F. R. LEACH, AND L.M. HENDERSON. 1962. The effect of hydroxyly-sine on cell wall synthesis and cell stabilityin Streptococcus faecalis. J. Biol. Chem.237:1198-1202.

UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUF-FER. 1957. Manometric techniques. BurgessPublishing Co., Minneapolis.

WARRINGA, M. G. P. J., 0. H. SMITH, A. GUIDITTA,AND T. P. SINGER. 1958. Studies on succinatedehydrogenase. VIII. Isolation of a succinicdehydrogenase-fumaric reductase from anobligate anaerobe. J. Biol. Chem. 230.:97-109.

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