INFECTION AND IMMUNITY, Jan. 1985, p. 129-1340019-9567/85/010129-06$02.00/0Copyright C 1985, American Society for Microbiology

Effects of Oxygen on Pyruvate Formate-Lyase In Situ andSugar Metabolism of Streptococcus mutans and

Streptococcus sanguisTADASHI YAMADA,* SHOKO TAKAHASHI-ABBE, AND KAZUHIKO ABBE

Department of Oral Biochemistry, Tohoku University School of Dentistry, Sendai, 980 Japan

Received 11 July 1984/Accepted 27 September 1984

The strictly anaerobic metabolism of sugar in strains of Streptococcus mutans and Streptococcus sanguis wasstudied because deep layers of dental plaque are strictly anaerobic. Galactose-grown cells of these streptococcalstrains had higher pyruvate formate-lyase activity than did glucose-grown cells. Among these strains, twostrains of S. mutans had a significantly higher pyruvate formate-lyase activity than did the others. This enzymeis extremely sensitive to oxygen, and even in situ the enzyme was inactivated by exposure of the cells to air.Lactate was less than 50% of the total end product of the strictly anaerobic incubation of the galactose-growncells of S. mutans with excess glucose, and a significant amount of formate, acetate, and ethanol was producedthrough the catalysis of pyruvate formate-lyase. But the cells exclusively produced lactate when exposed to airfor 2 min before the anaerobic incubation. The metabolism of sorbitol by S. mutans was seriously impaired bythe exposure of the cells to oxygen, and the metabolic rate was reduced to less than 1/20 of that found understrictly anaerobic conditions because of the inactivation of pyruvate formate-lyase. S. sanguis produced asmaller amount of the volatile products from glucose than did S. mutans because of the low level of pyruvateformate-lyase. However, the pyruvate formate-lyase in situ in S. sanguis was less sensitive to oxygen than wasthat in S. mutans. Because of this low sensitivity, S. sanguis metabolized glucose more rapidly under aerobicconditions, whereas the rates of the aerobic and anaerobic metabolism of glucose by S. mutans were similar,which suggests that S. mutans rather than S. sanguis can sustain the rapid sugar metabolism in the deep layersof dental plaque.

The acids generated through the fermentation of sugar bythe microorganisms in dental plaque can decalcify the super-ficial layers of tooth enamel and can initiate dental caries.Since streptococci are major acidogenic agents in dentalplaque, the regulation of the acid production by streptococcihas attracted considerable attention in connection with theinitiation of dental caries.The redox potential in dental plaque falls during the

development of dental plaque on a clean enamel surface, andthe deep layers of dental plaque are highly anaerobic (15). Itis also quite clear that the acid produced in these layers isdirectly involved in the initiation of dental caries. Abbe et al.(1), have reported that under strictly anaerobic conditionsStreptococcus mutans produces a significant amount offormate, acetate, and ethanol as well as lactate, even in thepresence of excess glucose, and that the regulation ofpyruvate formate-lyase (formate acetyltransferase, EC2.3.1.54), as well as that of lactate dehydrogenase (EC1.1.1.27) (24, 25), plays a central role in the shift of thefermentation pattern of S. mutans. Pyruvate formate-lyaseis, however, extremely sensitive to oxygen, and this enzymein situ is inactivated and S. mutans produces exclusivelylactate when the intact cells are exposed to air for 2 min (1).Because Streptococcus sanguis is one of the predominant

inhabitants in the deep layers of dental plaque, the produc-tion of acid during the metabolism of sugars under anaerobicconditions is of considerable interest.

In the present study, the strictly anaerobic metabolism ofsugar by S. mutans and S. sanguis is studied, and the effect

* Corresponding author.

of oxygen on sugar metabolism by these streptococci isinvestigated.

MATERIALS AND METHODS

Microorganisms. The following strains were used: S. mu-tans strains NCIB 11723 (JC2 [5]), NCTC 10449, Ingbritt(16), BHT-2 (13), JH-145 (13), 6715 (8), HS6 (16), B13 (18),and C67-1 (19); S. sangguis NCTC 10904 and ATCC 10556;Streptococcus mitis NCTC 3165; and Streptococcus salivar-ius NCTC 8618. S. sanguis LPH1 and GSG2 were suppliedby J. Carlsson, University ofUmea, Umea, Sweden.

Atmospheric conditions. Unless otherwise noted, all of thefollowing procedures were carried out under anaerobic con-ditions in a glove box as described previously (1, 24).Growth conditions. Streptococci were grown at 35°C in an

anaerobic glove box (type NHC [N2, 80%; H2, 10%; CO2,10%]) in a medium containing, in 1 liter of 0.1 M potassiumphosphate buffer (pH 6.8): sugar (glucose, galactose, orsorbitol), 9.9 g; NH4HCO3, 2 g; sodium-L-glutamate, 1 g;dried extract of yeast (Daigo Eiyokagaku Ltd., Osaka,Japan), 2 g; L-cysteine-HCI, 0.1 g; MgSO4 .7H20, 0.2 g;NaCl, 0.01 g; MnSO4- 4H20, 0.01 g; and FeSO4 7H20,0.01 g (2). Cell growth was checked by measuring theturbidity of the culture at 660 nm, and cells were harvestedin the logarithmic growth phase when the turbidity reached0.8. These cells were washed twice with 0.04 M potassiumphosphate buffer (pH 6.8) in another type of an anaerobicglove box (type NH [N2, 90%; H2, 10%]). Cultures werechecked for purity by growing them on mitis salivarius agar

(Difco Laboratories, Detroit, Mich.) and blood agar plates.Sugar fermentation. Washed cells were suspended in 0.04

M potassium phosphate buffer (pH 6.8) containing 5 mM

129

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130 YAMADA, TAKAHASHI-ABBE, AND ABBE

MgCl2 and were incubated for 30 min at 35°C for starvation.The cells were washed and suspended again in the same

buffer. The reaction mixture (12 ml) contained 2 mmol ofpotassium phosphate buffer (pH 6.8); 60 pLmol of MgCl2; 360,umol of glucose, galactose, or sorbitol or 180 or 360 Fnmol ofsucrose; and cells (14.7 mg [dry weight]). Various volumesof air-saturated water were also added to the reactionmixture in some experiments. The air-saturated water was

placed in a tube stoppered with a tightly fitting screw cap

before being brought into the anaerobic glove box. Thesolubility of oxygen in the air-saturated water was calculatedfrom the experimental data by Hitchman (12). Reactionswere started by the addition of cell suspension and were run

at 35°C for 20 min.It was confirmed in advance that the pH in the reaction

mixtures was not changed during the 20-min incubationunder these experimental conditions. Not more than one-

third of the glucose added was consumed during the incuba-tion. The reaction was stopped by the addition of 1.2 ml of6.6 N perchloric acid. The mixture was then kept at 4°C for20 min and neutralized in air with potassium carbonate at0°C. The resultant precipitate was removed by centrifuga-tion, and the supernatant fluid was stored in air at -200Cuntil the end products were assayed.

All of these experiments were carried out two or more

times, and all of the results were reproducible.Analysis of end products. L-Lactate was estimated with

L-lactate dehydrogenase (rabbit muscle; Boehringer Mann-heim GmbH, Mannheim, Federal Republic of Germany)(10), and formate was estimated with formate dehydrogenase(Pseudomonas oxalaticus; EC 1.2.1.2; Sigma Chemical Co.,St. Louis, Mo.) as described by Quayle (21). Acetate andethanol were determined by a gas chromatography with a

hydrogen flame detector as described previously (24). Theseanalyses were carried out in air.

Preparation of cell-free extract and assays of enzymaticactivities. The frozen cell paste was thawed and suspended in40 mM potassium phosphate buffer (pH 6.8) and 20 mMdithiothreitol. The cells were disrupted by sonic oscillation(200 W, 2 A) for 10 min with ice-cold water circulatingaround the sonication cup, and the cell debris was removedby centrifugation at 17,500 x g for 30 min at 4°C. Thesupernatant fluid was designated cell-free extract. To carryout all of these sets of procedures under anaerobic condi-tions, the sonication cups and the centrifuge tubes were

tightly stoppered in an anaerobic glove box before theirremoval from the box, and after the sonication or thecentrifugation, the cups and the tubes were returned andunstoppered in the box. Before use, the tightness of the cupand tube stoppers was carefully checked by using methylvi-ologen solution as an indicator.The protein concentration in the cell-free extract was

estimated in air by the biuret method (17).The activities of lactate dehydrogenase and pyruvate

formate-lyase were estimated spectrophotometrically by re-

cording the change in absorption at 340 nm at 350C. Theassay system of lactate dehydrogenase contained 20 mMsodium pyruvate, 0.24 mM NADH, 0.5 mM fructose 1,6-bisphosphate, and cell-free extract in 50 mM morpholinepropanesulfonic acid-NaOH buffer (pH 6.8) (24). The assaymixture of pyruvate formate-lyase contained 20 mM sodiumpyruvate, 0.08 mM coenzyme A, 1 mM NAD, 6 mM sodiumDL-malate, 2 mM dithiothreitol, 1.1 U of citrate synthase (pigheart, EC 4.1.3.7) per ml, 22 U of malate dehydrogenase (pigheart, EC 1.1.1.37) per ml, and cell-free extract in 100 mMpotassium phosphate buffer (pH 7.6) (22). Each enzymatic

TABLE 1. Pyruvate formate-lyase and lactate dehydrogenaseactivities in the cell-free extract of oral streptococci grown on

glucose or galactose under strictly anaerobic conditions

Sp act (U/mg)b of the following enzyme(in cells grown on):

Strain (serotype)a Pyruvate Lactateformate-lyase dehydrogenase

Glucose Galactose Glucose Galactose

S. mutansNCIB 11723 (c) 2.9 10.7 23 24NCTC 10449 (c) 3.0 11.6 46 29Ingbritt (c) 2.4 2.2 37 22BHT-2 (b) 1.7 5.2 49 22JH-145 (b) 3.6 6.5 <0.04 <0.04B13 (d) 4.7 2.7 46 28C67-1 (g) NT 2.2 NT 196715 (g) 2.1 NG 9.7 NGHS6 (a) 3.7 4.9 51 23

S. sanguisNCTC 10904 1.3 5.1 63 31ATCC 10556 1.4 4.1 49 37LPH1 1.1 0.9 46 19GSG2 0.7 3.5 31 36

S. mitis NCTC 3165 1.6 2.1 25 31

S. salivarius 1.5 NG 55 NGNCTC 8618

a Serotypes of S. mutans are from references 3 and 20.b Data are the means of the results of two or more experiments. NT, Not

tested; NG, not grown.

activity was assayed with a double-beam spectrophotometer(Hitachi model 100-50, Hitachi Ltd., Tokyo, Japan) whichwas placed in the type NH anaerobic glove box. The electricsignals from the spectrophotometer were transferred to arecorder outside the anaerobic glove box. The anaerobicstorage of the cells at -20°C for a week had little effect onthe specific activities of these enzymes in the cell-freeextract.NADH oxidase activity was assayed in air by recording

the change in absorption at 340 nm as described by Carlssonet al. (7).

RESULTS

Pyruvate formate-lyase and lactate dehydrogenase of oralstreptococci grown on glucose or galactose under strictlyanaerobic conditions. All of the strains except S. mutansJH-145 had both pyruvate formate-lyase and lactate dehy-drogenase (Table 1). Strain JH-145 had no lactate dehydro-genase activity as reported by Johnson and Hillman (13).Most streptococcal strains had more pyruvate formate-lyaseand less lactate dehydrogenase when grown on galactosethan on glucose. The lactate dehydrogenases of all thesestreptococci were dependent on fructose 1,6-bisphosphatefor activity (data not shown), as were those of other strainsof streptococci (4, 23, 24). The pyruvate formate-lyaseactivity in the cell-free extract of the galactose-grown cellsof S. mutans NCIB 11723 and NCTC 10449 was much higherthan that of S. sanguis, S. mitis, and other strains of S.mutans.

Effect of a different concentration of oxygen on the fermen-tation of glucose, galactose, and sucrose by glucose- or galac-tose-grown cells. Both S. mutans and S. sanguis fermented

INFECT. IMMUN.

EFFECTS OF 02 ON SUGAR METABOLISM OF ORAL STREPTOCOCCI 131

End products(jpmol/m g cells) End products(ljmol/mg cells)4 3 2 1 0 0

S. mutans NCIB11723 Sugar fermented

Glucose

E^: Galactoset.S songuis NCTC10904

Glucose

Galactose

02 138 PM 02 0 PMLactate a Formate Acetate m Ethanol

FIG. 1. Amounts of fermentation end products per 20 min ofincubation of the galactose-grown cells of S. mutans and S. sanguiswith glucose or galactose under strictly anaerobic conditions or inthe presence of a half-saturated concentration (138 ,M) of 02 (1.7,umol of 02 and 14.7 mg [dry weight] of the cells were included in thereaction mixtures [12 ml] at the beginning of the reactions).

galactose mainly to formate, acetate, and ethanol (Fig. 1). Asignificant portion of the end products of the strictly an-

aerobic glucose metabolism of galactose-grown cells of S.mutans was composed of formate, acetate, and ethanol,even in the presence of excess glucose (Fig. 1). The molarratio of these products was 2:1:1, respectively, which sug-gests that these products were synthesized in the cellsthrough the catalysis of pyruvate formate-lyase.When sucrose was fermented by the galactose-grown cells

of S. mutans or S. sanguis under strictly anaerobic condi-tions, the ratio of lactate was lower than that from glucose

StrainsS. mutans

NCIB 11723

End products(jumol/mg cells)Sugar 0 1 2 3fermented

Glucose

SucroseSucrose(15 mM) I

S. songuisATCC 10556 Glucose -j..

Sucrose(15mM) I

* Lactate 0 Formate El Acetate E EthanolFIG. 2. Amounts of fermentation end products per 20 min of

incubation of the galactose-grown cells of S. mutans and S. sanguiswith glucose or sucrose under strictly anaerobic conditions. If notspecified, the concentration of glucose or sucrose included in thereaction mixture was 30 mM.

(Fig. 2). When 15 mM sucrose (equivalent to 30 mM glucosein weight per volume) was fermented, the ratio was evenlower.The galactose-grown cells of both S. mutans and S.

sanguis produced more formate, acetate, and ethanol fromglucose than did the glucose-grown cells (Table 2). Themolar ratio of these products, formate, acetate, and ethanol,was also 2:1:1. This difference between the products of thecells grown on galactose and on glucose could be ascribed tothe difference in the levels of pyruvate formate-lyase in thesecells (Table 1).

TABLE 2. Effect of oxygen concentration on the fermentation end products from glucose by the cells of S. mutans or S. sanguis grownon glucose or galactose

Carbon Oxygen Amtb (,umol/mg of cells [dry wt]Strains source for concna per 20 min of incubation) of: L-Lactate (ratio) per

growth4LM) ~~~~~~~~~~~~~~~~~~~~~totalproductsgrowth (>.M) L-Lactate Formate Acetate Ethanol

S. mutans NCIB 11723 Galactose 0 2.1 1.2 0.61 0.59 0.4746 2.8 1.0 0.57 0.44 0.5892 3.7 0.46 0.34 0.22 0.78138 3.9 0.20 0.18 0.12 0.89

Glucose 0 1.8 0.47 0.27 0.25 0.6546 2.2 0.23 0.15 0.13 0.8192 2.3 0.11 0.08 0.07 0.90138 2.4 0.08 ND ND 0.97

S. sanguis NCTC 10904 Galactose 0 2.6 0.50 0.29 0.29 0.6946 3.6 0.52 0.23 0.28 0.7892 4.4 0.49 0.33 0.30 0.80138 4.9 0.59 0.27 0.27 0.81

Glucose 0 0.8 0.06 ND ND 0.9346 2.1 0.05 ND ND 0.9892 3.5 0.1 ND ND 0.97138 4.3 0.1 ND ND 0.98

S. sanguis ATCC 10556 Galactose 0 3.3 0.49 0.23 0.30 0.7646 2.9 0.40 0.20 0.18 0.7992 2.9 0.34 0.22 0.22 0.79138 3.0 0.33 0.13 0.12 0.84

Glucose 0 1.5 0.05 0.02 0.03 0.9446 1.7 0.03 0.01 0.02 0.9792 1.7 0.01 ND ND 0.99138 1.8 0.02 0.01 0.01 0.98

0, 0.55, 1.10, and 1.7 p.mol of 0,, respectively, and 14.7 mg (dry weight) of cells were included in the reaction mixtures (12 ml) at the beginning of the reactions.b ND, Not detected.

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132 YAMADA, TAKAHASHI-ABBE, AND ABBE

End Products of End Products ofGlucose Atmospheric Sorbitol

((imol/mg cells) Conditons (pmol/mg cells)

~~~-~~~StrictlyAnaerobic

Anaerobic(Cells exposedito air)

Aerobic

Lactate E Formate ] Acetate Ethanol

FIG. 3. Amounts of fermentation end products per 20 min ofincubation of the sorbitol-grown cells of S. mutans NCTC 10449with sorbitol or glucose. The cells were kept under strictly an-aerobic conditions during the whole procedure in an experiment(strictly anaerobic). In another experiment (anaerobic), the cellsuspension (88 mg [dry weight] in 15 ml of phosphate buffer [pH6.8]) in a 100-mI Erlenmeyer flask was removed from the anaerobicglove box, shaken for 2 min in air, and returned to the anaerobicglove box through the entry box, where the air was removed with avacuum four times. The cell suspension was then incubated withsugar under strictly anaerobic conditions. Reactions were also runin air (aerobic). Other experimental conditions are described in thetext.

Effect of oxygen on the metabolism of sorbitol by S. mutans.Under strictly anaerobic conditions, S. mutans NCTC 10449fermented sorbitol to produce large amounts of formate andethanol in addition to small amounts of lactate and acetate(Fig. 3). The observation that the number of moles offormate produced was equal to that of the moles of ethanolplus acetate suggests that these products were also synthe-sized in the cells through the catalysis of pyruvate formate-lyase. The anaerobically grown cells fermented only a smallamount of sorbitol under aerobic conditions and produced asmall amount of lactate but none of the volatile products.The amount of lactate produced was similar to that producedduring the strictly anaerobic fermentation. Exposing thecells to air for only 2 min seriously impaired the anaerobicmetabolism of sorbitol, and only a trace amount of afermentation product (lactate) was detected. This exposureof the cells to air did not seriously impair glucose metabo-lism by S. mutans NCTC 10449, but it shifted end productsgreatly. Lactate was exclusively produced, presumably be-cause of the inactivation of the pyruvate formate-lyase in thecells.

Neither strain NCTC 10904 nor ATCC 10556 of S. sanguisgrew on sorbitol, and neither the galactose- nor the glucose-grown cells of these strains fermented sorbitol even understrictly anaerobic conditions.

Difference in the sensitivity of pyruvate formate-lyase in situin the cells of S. mutans and S. sanguis. When the glucose- orgalactose-grown cells of S. mutans NCIB 11723 were incu-bated with a higher concentration ofoxygen, smaller amountsof formate, acetate, and ethanol were produced from glucose(Table 2). Similarly, the production of these volatile prod-ucts by strain BHT-2 of S. mutans was reduced by exposingthe cells to air (Table 3), although this strain producedsmaller amounts of formate, acetate, and ethanol than didstrains NCIB 11723 and NCTC 10449 (Table 2; Fig. 3)presumably because of the low levels of pyruvate formate-lyase in this strain (Table 1). Metabolisms of sugars by S.sanguis NCTC 10904 and ATCC 10556 were, however, lesssensitive to oxygen (Table 2; Fig. 1), and the levels of

TABLE 3. Effect of oxygen on the fermentation end productsfrom glucose by cells of S. mutans grown on galactose

Oxygen Amta (,umol/mg of cells [dry wt] L-LactateStrain concn per 20 min of incubation) of: (ratio) per

(tLM) L-Lactate Formate Acetate Ethanol total products

BHT-2 0 3.6 0.72 0.43 0.41 0.70138b 4.2 0.53 0.25 0.20 0.81217C (in 5.7 ND ND ND 1.0

air)

JH-145 0 ND 1.9 0.91 1.0 0138b ND 1.7 0.74 0.79 0217C (in ND 0.12 0.14 0.19 0

air)a ND, Not detected.b 1.7 tLmol of 02 and 14.7 mg (dry weight) of the cells were included in the

reaction mixture (12 ml) at the beginning of the reactions.' 2.6 Lmol of 02 and 14.7 mg (dry weight) of the cells were included in the

reaction mixture (12 ml); the mixture was air saturated at 350C.

formate, acetate, and ethanol produced by these cells werenot changed markedly by the addition of oxygen in thereaction mixtures. S. sanguis LPH1 was resistant to oxygenas were the other strains of S. sanguis (data not shown). Tofind out whether the pyruvate formate-lyase in situ wasactually inactivated by exposing the cells to air, cell-freeextract was prepared after incubation of the cells withoxygen, and the pyruvate formate-lyase activity in thecell-free extract was assayed. S. mutans lost its pyruvateformate-lyase activity almost entirely after a 20-min incuba-tion of the cells in the reaction mixture half saturated with air(138 puM oxygen), irrespective of the presence of glucose(11% residual activity with glucose versus 9% residualactivity without glucose). But 56% of the original enzymeactivity still remained in the cells of S. sanguis after theincubation of the cells with oxygen (138 F±M) in the presence

100

4-

u 50

L4--a

c)0m

10time( min)

20

FIG. 4. Inactivation of pyruvate formate-lyase in the cell-freeextract of S. mutans and S. sanguis by a low concentration (14 puM)Of 02. The cell-free extract (0.7 mg of protein per ml) in 0.04 Mpotassium phosphate buffer (pH 6.8) was mixed with 1/20 volume ofair-saturated water at room temperature. A sample of the extractwas then taken for the assay of pyruvate formate-lyase activitybefore and after the addition of the air-saturated water. Symbols: 0,with the cell-free extract of S. mutans; 0, with the cell-free extractof S. sanguis.

@0

INFECT. IMMUN.

ol

EFFECTS OF 02 ON SUGAR METABOLISM OF ORAL STREPTOCOCCI 133

of glucose (versus 4% residual activity without glucose). Theenzyme activity in situ of both S. mutans and S. sanguis was

entirely lost by the incubation of the cells with oxygen in theabsence of glucose.The pyruvate formate-lyase in the cell-free extract of S.

mutans NCIB 11723 and S. sanguis NCTC 10904 was

studied for its sensitivity to oxygen. The time courses of theinactivation at a low concentration of oxygen were the same

for the enzyme from S. mutans and that from S. sanguis(Fig. 4). Therefore, the difference in oxygen-sensitivitybetween these enzymes in situ in the cells of S. mutans andS. sanguis did not seem to stem from the difference in thesensitivity of these enzymes themselves.

Since the rate of oxygen-uptake by streptococci is corre-lated with the level of the NADH oxidase activity (7), theNADH oxidase activity in the cell-free extract of the an-

aerobically grown cells of S. mutans NCIB 11723 and S.sanguis NCTC 10904 was estimated. There was no signifi-cant difference in the specific activity of NADH oxidase inthe cell-free extract of these cells (0.018 and 0.004 U/mg ofprotein, respectively). Thus, the difference in the sensitivityof the enzymes to oxygen was not ascribed to the differencein the capacity of the cells to consume oxygen.

Glucose fermentation of the lactate dehydrogenase-deficientstrain, S. mutans JH-145, and its parent strain, S. mutansBHT-2. As reported by Hillman (11), strain JH-145 of S.mutans lacked lactate dehydrogenase, but this strain hadpyruvate formate-lyase activity (Table 1). Under strictlyanaerobic conditions, the galactose-grown cells of S. mutansBHT-2 fermented glucose into formate, acetate, and ethanolin addition to a large amount of lactate. The galactose-growncells of strain JH-145 produced larger amounts of formate,acetate, and ethanol than did the cells of strain BHT-2, butno lactate was produced by strain JH-145 (Table 3). Themolar ratio of formate, acetate, and ethanol was 2:1:1,suggesting the involvement of pyruvate formate-lyase fortheir synthesis. Under aerobic conditions strain BHT-2, likethe other strains of S. mutans, fermented glucose to lactateexclusively, whereas strain JH-145 hardly metabolized glu-cose under aerobic conditions (Table 3). The pyruvateformate-lyase was considered to be the sole enzyme in-volved in the conversion of pyruvate into end products in themetabolism of sugar by strain JH-145.

DISCUSSION

The level of pyruvate formate-lyase largely affected thefermentation end products of the strictly anaerobic metabo-lism of sugar by oral streptococci in the presence of excess

sugar. Under strictly anaerobic conditions, strains of S.mutans, which had high levels of pyruvate formate-lyase,produced more formate, acetate, and ethanol than did strainsof S. sanguis or the other strains of S. mutans with lowerlevels of this enzyme (Tables 1 and 2; Fig. 1). The glucose-grown cells of streptococci, which had a lower level ofpyruvate formate-lyase than the galactose-grown cells (Ta-ble 1), produced less volatile products than did the galac-tose-grown cells (Table 2). Moreover, when pyruvate for-mate-lyase was inactivated by the exposure of the cells of S.mutans to air (see above), the end products were mostlylactate (Table 2; Fig. 1 and 3).The pyruvate formate-lyase of S. mutans was especially

sensitive to oxygen even in the intact cells. The 2-minexposure of the cells to air inactivated pyruvate formate-lyase, and sorbitol was hardly metabolized (Fig. 3). Pyruvateformate-lyase is essential for the anaerobic metabolism of

sorbitol because 1 mol more of NAD per mol or sorbitolmetabolized must be regenerated by the reduction of acetyl-coenzyme A into acetaldehyde and ethanol (1). Underaerobic conditions NAD could be regenerated by NADHoxidase, and so some lactate could be produced (Fig. 3). Themetabolism of mannitol is also greatly impaired by exposureof the cells of S. mutans to air (1). Thus, it seems importantto keep the cells under strictly anaerobic conditions duringthe whole experimental procedure when the acidogenicity ofsugar alcohols is studied.

Pyruvate formate-lyase in situ in the cells of S. sanguiswas less sensitive to oxygen than that in the cells of S.mutans, so that the amounts of formate, acetate, and ethanolproduced were the same whether the cells of S. sanguis wereexposed to air or not (Table 2; Fig. 1). This differentsensitivity between S. mutans and S. sanguis was not,however, ascribed to the difference in the sensitivity of thepyruvate formate-lyase itself, because the in vitro sensitivityof the pyruvate formate-lyase of S. sanguis to oxygen wassimilar to that of the pyruvate formate-lyase of S. mutans(Fig. 4). But, S. sanguis retained a significantly high activityof pyruvate formate-lyase during the glucose metabolismeven in the presence of oxygen, whereas S. mutans did not(see above). Cells of S. sanguis may possess some mecha-nism which prevents oxygen from inactivating pyruvateformate-lyase, but this mechanism has not been elucidatedin this study.Under strictly anaerobic conditions, the total amount of

acid produced by the lactate dehydrogenase-deficient mu-tant (JH-145) of S. mutans was similar to that produced byits parent strain, BHT-2, whereas very little acid was pro-duced by the mutant strain under aerobic conditions (Table3). Hillman (11) reports that lactate dehydrogenase-deficientmutants of S. mutans produce less titratable acid fromglucose, but it was suspected that the mutants produced lessacid because the reactions were run under aerobic condi-tions, and pyruvate formate-lyase had already been inacti-vated in his study.Because of the high level of pyruvate formate-lyase, some

strains of S. mutans rapidly metabolized glucose underanaerobic conditions as well as under aerobic conditions. Onthe other hand, S. sanguis cells could metabolize glucosemore efficiently under aerobic conditions than under an-aerobic conditions because of the low level of pyruvateformate-lyase and its low sensitivity to oxygen (Table 2;Fig. 1).

Since the deep layers of mature dental plaque are highlyanaerobic (9, 14, 15), these strains of S. mutans wereexpected to sustain the rapid production of acids in theanaerobic, deep layers of dental plaque, and S. sanguis wasexpected to produce acids more rapidly in the aerobic,superficial layers. This may be one of the reasons why S.sanguis is one of the first microorganisms which colonize onteeth (6). The rapid production of acid by S. mutans understrictly anaerobic conditions may confer a high cariogenicpotential on these strains of S. mutans, since the acidsproduced in the deep layers of dental plaque are directlyinvolved in the initiation and progression of dental caries.

ACKNOWLEDGMENTS

We thank Y. Mito for her valuable technical assistance.This study was supported in part by Grants-in-Aid for Scientific

Research (B) 57480340 and (A) 59440077 and Grant-in-Aid forDevelopmental Scientific Research 58870088 from the JapaneseMinistry of Education, Science, and Culture.

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134 YAMADA, TAKAHASHI-ABBE, AND ABBE

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