Vol. 16, No. 2 Printed in U.S.A.

INFECTION AND IMMUNITY, May 1977, p. 637-648 Copyright © 1977 American Society for Microbiology

Streptococcus mutans Dextransucrase: Functioning of Primer Dextran and Endogenous Dextranase in Water-Soluble and Water-Insoluble Glucan Synthesis GREG R. GERMAINE,* SUSAN K. HARLANDER, WOON-LAM S. LEUNG, AND CHARLES F. SCHACHTELE Division of Oral Biology* and Microbiology Research Laboratories, School of Dentistry, and Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455

Received for publication 22 December 1976

The extracellular enzyme activities of Streptococcus mutans 6715 that synthesize glucans from sucrose were concentrated and partially purified by ammonium sulfate precipitation and gel permeation column chromatography. Polyacrylamide gel analysis demonstrated that all of the major proteins precipitated by ammonium sulfate were quantitatively recovered in the high-molecularweight, enzyme-containing aggregates found in the void volume of the gel column. Anion-exchange column chromatography was used to fractionate the aggregates into preparations, a and /3, which produced water-insoluble and water-soluble glucans, respectively. Polyacrylamide gel analysis showed that a and ,3 contained unique proteins and dextransucrase (EC 2.4.1.5) activities. Studies on the time course of glucan synthesis by a demonstrated that this enzyme preparation contained dextranase activity, which partially degraded nascent alcohol-insoluble glucan into alcohol-soluble products that were subsequently reincorporated into insoluble product. The ,3 enzyme preparation contained no detectable dextranase activity. Mixing experiments in the absence of primer dextran demonstrated that the dextranase activity present in a could modify glucan production by /. CsCl density gradient analysis of product glucans demonstrated that exogenous primer dextrans were used as acceptor molecules by both the a and /8 enzyme preparations, and that water-soluble glucans synthesized by /8 could be converted into water-insoluble glucans by a. It is proposed that the structural heterogeneity of the native glucans produced from sucrose by S. mutans is a result of the concerted action of glucan-forming dextransucrases and endohydrolytic dextranase activity.

Streptococcus mutans synthesizes extracellular glucans from sucrose. These polysaccharides may be divided into two groups, watersoluble glucans and water-insoluble glucans. Glucans in either solubility group contain glucopyranosyl moieties linked a-(1--6) and a(1->3) (2, 7, 19, 20, 22, 23, 29, 30). Since glucans with similar proportions of either linkage may exhibit dissimilar water solubility properties, the distribution of such linkages in a glucan molecule may be a solubility determinant (23). The a-(1-6) and a-(1-*3) linkages may both be present as main chain linkages and as branch points (2, 7, 19, 20, 23, 29). The glucan composition (linkage proportion and solubilities) is variable both within and among strains of S. mutans (22, 23). The basis of this heterogeneity has not been established. Furthermore, the mechanism(s) by which S. mutans produces glucans with the linkage and solubility parameters outlined above is unknown. The most fun-

damental question -how many enzymes are involved in total glucan synthesis? -remains unanswered. This is due, in part, to the presence in S. mutans culture supernatants of high-molecular-weight aggregates which contain all or most of the extracellular glucosyltransferase activity produced by this organism (8, 9, 11, 13, 17, 18, 21). Determination of the glucosyltransferase content of the aggregates [especially with respect to the activity that synthesizes a(1-33) linkages] must depend upon aggregate disruption. The aggregates synthesize both soluble and insoluble glucans (8, 9, 13, 18). In addition, S. mutans produces an extracellular endohydrolytic dextranase (EC 3.2.1.11) (5, 14, 31). A significant effect of the presence of exogenous dextranase (Spicaria violaceae) during glucan synthesis by S. mutans dextransucrase on the quantity of a-(1--+3) linkages in the product glucans has been reported (7). Thus, the endogenous S. mutans dextranase may con-

637

638

GERMAINE ET AL.

ceivably play a role in determining the final linkage composition and solubility properties of the sucrose-derived glucans. In this communication we describe an effective procedure for functionally separating the extracellular water-soluble and water-insoluble glucan-synthesizing activities from S. mutans 6715. In addition, unique interactions with primer dextran are described for each activity. Finally, evidence for the participation of endogenous dextranase in the synthesis of waterinsoluble glucan is presented. MATERIALS AND METHODS Bacterium and growth conditions. S. mutans strain 6715 was grown as previously described (10) except that 0.4 M fructose was substituted for glucose in the growth medium (11). Dextransucrase assay. Conversion of the glucosyl moiety of ['4C]sucrose to alcohol-insoluble glucan was measured as described previously (12). One unit of dextransucrase is that amount of enzyme causing the polymerization of 1 ,umol of sucrose-derived glucose per min at 37°C. Modifications of the assay for particular experiments are described below. Gel permeation chromatography. Bio-Gel A 1.5 M column chromatography of ammonium sulfateprecipitated enzyme preparations was performed as described previously (11) except that the column dimensions were 2.6 by 40 cm. Fractions (5.7 ml) were collected at a flow rate of 0.43 ml/min. The absorbance of the fractions at 540 nm was measured with a Spectronic 20 (Bausch and Lomb, Rochester, N.Y.). Enzyme activity was measured by adding 10 Al of each fraction to 50 gl of standard reaction mixture and, after 10 min of incubation at 37°C, removing a 25-1.l sample for processing (12). Fractions containing enzyme were pooled, adjusted to 60% (wt/vol) saturation with ammonium sulfate, and stored overnight at 4°C. Insoluble material was collected by centrifugation (24,000 x g, 20 min, 4°C), dissolved in 0.01 M sodium acetate buffer (pH 5.5, buffer SA), and dialyzed for 18 to 24 h against large volumes of the same buffer. The concentrated enzyme was stored at -20°C. Ion-exchange chromatography. Dialyzed concentrated enzyme from the Bio-Gel column was washed into an SA buffer-equilibrated, 1.8- by 18-cm diethylaminoethyl (DEAE)-cellulose (Whatman DE32, Reeve Angel, Clifton, N.J.) column. After washing with SA buffer (186 ml), elution was performed by application of a linear 0 to 0.5 M NaCl gradient. Fractions (2.8 ml) were collected and analyzed for absorbance at 280 nm in a Zeiss spectrophotometer (model PMQII, Carl Zeiss Inc., New York, N.Y.). Enzyme activity was determined as described for the Bio-Gel column except that only 5 ,ul of each fraction was added to 50 Al. of reaction mixture. The enzyme activity in pooled fractions was concentrated by ammonium sulfate precipitation as described above. Polyacrylamide gel electrophoresis. Slab polyacrylamide gel electrophoresis and the detection of Coomassie brilliant blue staining bands and dex-

INFECT. IMMUN. transucrase activity within the gel were performed as described previously (3, 10). Water-soluble and water-insoluble glucan production. Reaction conditions were identical to those used for measuring dextransucrase activity, except that the final volume of the reactants was always 250 ,u. Assays were terminated by the addition of 10 ,ul of a 250 mM silver nitrate solution (3) and placing of the reaction tubes on ice. Triplicate 10-,ul samples were immediately removed, and total alcohol-insoluble glucan was determined using the paper disk assay (12). Reaction tubes were then centrifuged at 2,500 rpm (IEC clinical centrifuge, Damon/IEC, Needham Heights, Mass.) for 5 min at 25°C. The supernatant was removed, and 10-,A samples were processed in triplicate as before to determine the amount of non-sedimentable or water-soluble glucan. Water-insoluble glucans were calculated as the difference between total and water-soluble glucan. Control experiments demonstrated that: (i) silver nitrate at 10 mM and cooling of the reaction tubes effectively stopped glucan synthesis (95% inhibition) for at least 1 h; (ii) 3H-labeled, water-soluble dextran (molecular weight of 70,000; New England Nuclear Corp., Boston, Mass.) remained greater than 99% soluble under our assay conditions; and (iii) washing by suspension and centrifugation of waterinsoluble glucan, followed by solubilization in 1 N NaOH and counting on paper disks, confirmed that our assay by difference accurately reflected the amount of this product synthesized under our assay conditions. CsCl density gradient centrifugation of dextransucrase product glucans. Reaction mixtures containing the appropriate enzyme preparation and primer (T2000, T10, or no primer) in a total volume of 125 ,ul were incubated in covered 5-ml cellulose nitrate centrifuge tubes at 37°C for the desired time. Next, 4.9 ml of a 56% (wt/wt) CsCl solution was added with thorough mixing. The solutions were centrifuged (SW 25.1 rotor, Sorvall OTD ultracentrifuge) at 15°C for 21 to 24 h at 48,000 rpm. The gradients were fractionated as before (11) at a rate of 0.4 ml/min. Portions of each fraction were pipetted onto paper disks and batch washed in methanol to remove unreacted ['4C]sucrose and [14C]fructose side product (12). Refractive index (n) measurements were made on every fifth fraction to determine the CsCl density gradient. Chemical assays. Protein and hexose were estimated as before (10).

RESULTS Analysis of high-molecular-weight dextransucrase-containing aggregates. We have previously demonstrated (11, 27) that gel-permeation column chromatography of ammonium sulfate-concentrated S. mutans 6715 culture supernatants yields all of the sucrose-dependent, glucan-synthesizing activity in a high-molecular-weight form, which elutes in or near the void volume of the columns. Typical results from a preparative Bio-Gel A 1. 5 M column run

S. MUTANS DEXTRANSUCRASE

VOL. 16, 1977

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were present in approximately equivalent amounts in the concentrated Bio-Gel void volume preparation (Fig. 2). The three proteins not detected in the Bio-Gel enzyme were present in low concentrations in the ammonium sulfate preparation. Thus, almost all of the proteins precipitated from cell-free S. mutans culture supernatants with 60% ammonium sulfate exist in high-molecular-weight aggregates.

Dextransucrase-containing aggregates

were

not created during ammonium sulfate precipitation. An S. mutans cell-free culture supernatant was passed through Bio-Gel A 1.5 M, and

C 10

20

30

40

50

Fraction FIG. 1. Bio-Gel A 1.5 M chromatography of ammonium sulfate-precipitated and dialyzed dextransucrase. VO refers to the column void volume, and the cross-hatched area indicates fractions (12 to 15) that

turned visibly turbid during enzyme assay. Symbols: (0) dextransucrase activity; (0) absorbance at 540 nm.

are presented in Fig. 1. Material present in the concentrated ammonium sulfate enzyme preparation that gave a slight brown color was monitored by measuring the absorbance at 540 nm. The bulk of this material was retained by the gel until most of the enzyme activity had been eluted and probably represents residual growth medium components not removed during salt precipitation and dialysis. During assay of sucrose-dependent glucan synthesis by Bio-Gel column fractions, it was consistently noted that samples from the fractions on the leading edge of the enzyme activity peak developed marked turbidity (cross-hatched portion of the peak, fractions 12 to 15). Although other column fractions (16 to 19) showed higher levels of polymerizing activity, turbidity was never observed during the assay of these fractions. The activity involved in the formation of turbidity always appeared to be of a greater molecular weight than the remainder of the glucan-forming activity. The enzyme-containing fractions (12 to 22) were pooled and concentrated by ammonium sulfate precipitation. Recovery of enzyme from Bio-Gel was consistently greater than 90% and the preparations had specific activities ranging from 2.5 to 3.0 dextransucrase units/ mg of protein. The proteins in the concentrated Bio-Gel enzyme were analyzed by polyacrylamide gel electrophoresis and compared to the proteins present in the original ammonium sulfate-precipitated enzyme preparation. All but 3 of the 20 protein bands found in the latter preparation

a b FIG. 2. Polyacrylamide gel separation of the proteins in the Bio-Gel (a) and ammonium sulfate (b) enzyme preparations. Samples run on the gel contained 14 pg of protein. The arrow on the left indicates the position of the bovine serum albumin standard run in an adjacent lane of the gel.

640

GERMAINE ET AL.

the elution of the enzyme was monitored by assaying each fraction for several hours under standard conditions. The bulk of the dextransucrase activity was found in the void volume of the column (data not shown). In addition, culture supernatants concentrated by Amicon filtration (PM 30 filter, Amicon Corp., Lexington, Mass.) were shown by passage through Bio-Gel to contain the aggregated enzyme complexes. Thus, the enzyme exists in the original culture supernatants as an aggregate with a molecular weight greater than 1.5 x 106. Fractionation of dextransucrase by ion-exchange chromatography. When the concentrated enzyme from Bio-Gel was subjected to DEAE ion-exchange chromatography, two peaks of enzyme activity were resolved by salt gradient elution (Fig. 3). The first activity peak (designated a) came off the column when the NaCl concentration reached 0.1 M and consisted of fractions that always turned turbid during assay with sucrose (cross-hatched peak, fractions 90 to 100). The second peak of enzyme activity (designated ,8) emerged when the NaCl concentration was 0.2 M and never showed turbidity during assay. Hereafter, enzymatic activity derived from the DEAE chromatography peaks will be referred to simply as a and /3. The 280-nm absorbance profile of the DEAE column revealed a peak of protein corresponding to the position of elution of the a enzyme, a major protein peak that was eluted after a, and several broad peaks of protein associated with the /3 enzyme. The fractions from each activity peak were pooled, concentrated by ammonium sulfate precipitation, and assayed for glucansynthesizing activity. The specific activities of the a and /3 preparations shown in Fig. 3 were

INFECT. IMMUN.

3.0 and 7.2 enzyme units/mg of protein, respectively, and are representative of such preparations. Polyacrylamide gel analysis of a and ,B. Figure 4 presents the protein and dextransucrase activity patterns obtained by electrophoresis of the a and / enzymes. There is one predominant Coomassie brilliant blue-stained area in the a preparation and four minor bands with higher mobility in the gel (Fig. 4, lane a). In situ analysis with radioactive sucrose of an adjacent area of the slab gel demonstrated that the glucan-synthesizing activity in a is associated with the amorphous band (Fig. 4, lane b). No additional glucan-producing activity in the a lane could be detected by prolonged autoradiography. The /8 enzyme preparation contained a mixture of at least 13 proteins (Fig. 4, lane c), with an enrichment for several of the bands present in the Bio-Gel preparation (Fig. 2). In marked contrast to a, /8 contains two distinct glucan-synthesizing activity bands (Fig. 4, lane d). These activities (/31 and /2) correspond to the two most heavily stained bands in the corresponding /8 protein pattern (lane c). Note that /31 and /32 are identical to the soluble glucan-producing major and minor forms of the S. mutans dextransucrase we previously described (3, 4, 10). Thus, the a and /8 enzyme preparations from DEAE can be clearly distinguished by the presence of both unique protein and glucan-producing activity bands. Based on the sensitivity of the in situ radioactivity assay, it would appear that cross-contamination between the a and /3 enzyme preparations is minimal. Analysis of glucan synthesis by a and f8. When assayed at equivalent protein concentrations in the presence of primer dextran (T10) a and /8 were shown to produce physically unique glucans from sucrose (Fig. 5). The a enzyme synthesized large quantities of glucan and the reaction tubes became turbid. The turbidity 6 -t03 06 t and glucan were sedimented by low-speed cenE 0 trifugation (Fig. 5a). Only a small quantity of soluble glucan was produced by a. In marked contrast, the /3 enzyme produced large amounts z of soluble glucan and only a small quantity of water-insoluble material (Fig. 5b). Prolonged incubation of /3 (24 h) never resulted in the formation of turbidity or accumulation of more than 5% water-insoluble polysaccharide. Thus, the unique glucan-synthesizing activities found Fraction in the a and /3 preparations (Fig. 4) produce FIG. 3. DEAE-cellulose chromatography of dex- polysaccharides readily distinguishable by transucrase from Bio-Gel. The cross-hatched area indicates fractions (90 to 100) that developed visible their solubility in water. During studies in which the concentrations of turbidity during enzyme assay. Symbols: (0) dexa and /3 and substrate were altered, several transucrase activity; (0) absorbance at 280 nm; (---) additional distinguishing features of these enNaCl gradient.

S. MUTANS DEXTRANSUCRASE

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irn-.

641

derived glucose was polymerized within 5 min (Fig. 7, open circles). However, throughout the remaining 4 h of incubation a partial disappearance and reappearance of radioactive glucan was noted. It appeared that the polysaccharide was being degraded to methanol-soluble fragments, which were subsequently converted back into methanol-insoluble glucan. Two such conversion cycles were observed over the 4-h incubation. In Fig. 8, glucan production by more dilute concentrations of a was monitored at frequent intervals. In every case the rapidly synthesized b

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FIG. 5. Time course of water-insoluble (0) and water-soluble (0) glucan production from sucrose by the a (a) and 8 (b) enzyme preparations in the presence of T10 primer. a and /8 were present at 6.5 and 6.3 Mg of protein/0.25 ml per tube, respectively.

FIG. 4. Polyacrylamide gel analysis of a and /3 enzyme fractions: (a) protein staining pattern of a; (b) dextransucrase activity gel of a; (c) protein staining pattern of /3; (d) dextransucrase activity of (3. n

zyme preparations were noted. At high enzyme concentrations /8 used greater than 90% of the substrate in less than 5 min when primer dextran was included in the reaction mixture (Fig. 6, open circles). The product glucan was completely stable over the 4-h observation period. In the absence of T10, the extent of the reaction reached about 70% of the primed level and the product glucan was again stable (Fig. 6, closed circles). As mentioned above, such reaction mixtures never developed a turbid appearance, even after 24 h of incubation. The rate and extent of glucan synthesis by a under identical conditions was very different from that by /8. In the absence of primer dextran, glucan synthesis by a was severely depressed (Fig. 7, closed circles), and almost 50% of the glucan synthesized in the first 90 min appeared to be degraded during subsequent incubation. In the presence of dextran, at least 80% of the sucrose-

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Hours FIG. 6. Glucan synthesis from sucrose by the /8 enzyme preparation. Twenty-five microliters (40 pg of protein) of the preparation was added to 75 1d of the standard reaction mixture (devoid of primer dextran) and 25 pi of either 0.05 M sodium acetate buffer, pH 5.5 (-), or 25 pi of dextran T10, 3.3 mgl ml (0). At the indicated times, 10-pl samples were removed to paper disks and processed as before (12) to determine total glucan.

642

GERMAINE ET AL.

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Hours FIG. 7. Glucan synthesis from sucrose by the a The details of the experiment of Fig. 6 except that 25 pl of the a preparation (30 Mg of protein) served as the enzyme source. Symbols: (0) with dextran; (0) without dexenzyme preparation. are identical to those

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tion of enzyme (Fig. 8, closed circles), it is clear that the degradative activity overshadows glucan formation, and after 60 min less than 10% of the polysaccharide present at 15 min was alcohol insoluble. Thus, in contrast to /, the a enzyme preparation contains an activity capable of severely affecting the alcohol solubility of the glucans it produces from sucrose. To determine whether the degradative activity in a could also affect glucan production by ,/, an enzyme-mixing experiment was performed under incubation conditions identical to those in Fig. 6 and 7, except that T10 primer dextran was not included. When both a and /8 were present, the glucan produced by /8 was partially degraded to an alcohol-soluble form (Fig. 9). In addition, and in contrast to the lack of turbidity development in the tubes containing a and /8 alone, the tube containing the mixture of enzymes developed extensive turbidity. The presence of the activity in a that is necessary for water-insoluble glucan production causes the product of the /3 enzyme to become water insoluble during incubation with sucrose. To determine whether a could cause the conversion of preformed water-soluble / glucan into a water-insoluble form, a was added to a reaction mixture that had been incubated with ,B until the maximum amount of glucan had accumulated (Fig. 10, closed circles). This glucan remained water soluble for at least 60 min (Fig. 10, open circles), and no turbidity developed during more prolonged incubation.

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Minutes FIG. 8. Metabolism of glucans produced from sucrose by various concentrations of a. The details of the experiment are the same as for Fig. 6, except that primer dextran was present in every case. The concentrations of a were 16.2 (0), 8.1 (0), and 3.2 (A) pg of protein per 25 p1 of enzyme added to the reaction mixture.

alcohol-insoluble glucan was extensively degraded during subsequent incubation. Although less than one-fifth of the sucrose available for polymerization had been used after 15 min of incubation with the highest concentra-

0

30

60

90

Minutes

FIG. 9. Glucan synthesis by the a and /8 preparations alone and in combination. Twenty-five microliters of either the a (0) or ,3 (0) preparation was combined with 75 pi of the standard reaction mixture (devoid of primer dextran) and 25 ,4 of sodium acetate buffer, pH 5.5. In addition, 25 p1 ofeach enzyme preparation was added to 75 ,l of the standard reaction mixture (A). At the indicated times, 10-,i samples were removed and the amount ofglucan present was determined.

VOL. 16, 1977

2,

S. MUTANS DEXTRANSUCRASE

0

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Minutes FIG. 10. Effect of addition of the a preparation on preformed glucan synthesized by the 8 preparation. Twenty-five microliters of the /3 preparation was combined with 300 pl of standard reaction mixture (no primer dextran) and incubated at 37°C. At the indicated times, 5-pi samples were removed and assayed for glucan (a). At 40 min, 75 p1 of the assay mixture was removed and combined with 25 pl of 0.05 M sodium acetate buffer, pH 5.5. After a 10-min incubation at 37°C to ensure completion ofglucan synthesis, 25 pi ofthe a preparation was added (denoted by arrow). Thereafter, 5-pl samples were removed at the indicated times (0, inset) and glucan was determined. The labels of the ordinate and abscissa of the inset are the same as on the main figure.

We reported earlier (11) that dextran T2000 formed a band in CsCl density gradients. In contrast, dextran T10 failed to form a band (Fig. 11) and was rather uniformly distributed throughout the gradient. The inability ofT10 to form a band is due to its low molecular weight (ca. 104). This fundamental difference in the behavior of dextrans that differed in molecular weight was used in the CsCl density gradient experiments that follow. Band formation by glycogen in CsCl density gradients has also been reported (1, 28) and is included in Fig. 11 for reference. CsCl gradient analysis of glucans synthesized by a and /8 under primed and unprimed conditions is shown in Fig. 12. When highmolecular-weight dextran (T2000) is present in the reaction mixture, the soluble product glucan synthesized by /8 forms a sharp band at n = 1.4030 (Fig. 12a, open circles). Glucan synthesized by /3 in the absence of any primer dextran also forms a sharp band, but in a position of lower buoyant density (n = 1.3990) (Fig. 12a, closed circles). Glucan synthesized by /8 in the presence of low-molecular-weight primer dextran T10 distributed in a very broad band (Fig. 12b, closed circles), with evidence of two glucan types as judged by (i) the broad peak in the high-density area (fractions 9 to 10) similar to the product from T2000-primed reactions and

643

(ii) the peak in the low-density area (fractions 14 to 15) similar to the glucan obtained from unprimed reactions. Glucans synthesized by a in the presence of T10 (Fig. 12b, open circles) or T2000 (data not shown) were identical and exhibited a buoyant density similar to that observed for unprimed /3-synthesized glucans. Although the products of primed a reactions and unprimed ,8 reactions exhibited a similar distribution in the CsCl gradients, the a product glucans always produced a turbid band (Fig. 12b, denoted by crosshatching). Turbid bands of product glucans were never seen with /8-directed synthesis. These results supp6rt our data (Fig. 4 and 5), which demonstrated the lack of cross-contamination between the glucan-synthesizing activities in our a and /8 enzyme preparations. The T2000- and T10-primed a reactions never gave any glucan products typical of primed /8 reactions. The CsCl distribution characteristics of glucan from T2000- and T10-primed synthesis by /3 always reflected the CsCl distribution characteristics of the primer dextran used. These observations clearly indicate that the added primer dextrans function as acceptors in /3 reactions. Since the CsCl band position of glucans synthesized by a seemed to bear no relationship to the band position of primer dextran that was added, it was possible that an endogenous primer might be present in a that dictated the ultimate solubility characteristics of the product. Therefore, /8-directed glucan synthesis was

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Fraction FIG. 11. CsCl density gradient centrifugation of dextran T10 (), dextran T2000 (0), and glycogen (x). One hundred microliters of stock polysaccharide solutions (5 mg/ml) was thoroughly mixed with 4.9 ml of 56% (wtlwt) CsCl and centrifuged as described in the text. Refractive index (n) measurements (0) are included for reference. Top of the gradient is to the right (fraction 27).

644

INFECT. IMMUN.

GERMAINE ET AL.

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Fraction FIG. 12. CsCl density gradient centrifugation of the glucans synthesized by the a and ,B preparations in both the presence and absence ofprimer dextrans. (a) Glucans synthesized by the /3 preparation in the presence (0) and absence (a) of primer dextran T2000. Twenty-five microliters of the /3 preparation was combined in cellulose nitrate centrifuge tubes with 75 pi of the standard reaction mixture (no primer) and 25 td of either 0.05 M sodium acetate buffer, pH 5.5 (-), or dextran T2000 (5 mg/ml in 0.05 M sodium acetate buffer, pH 5.5) (0). After incubation at 37°C for 3 h, CsCl was added as in Fig. 11, the tubes were centrifuged and fractionated, and the methanol-insoluble radioactivity was determined on 25-pi samples of the gradient fractions. Visible bands were not formed in either gradient. (b) Glucans synthesized by either a (0) or / (@) in the presence ofprimer dextran T10. Twenty-five microliters of the enzyme preparations was combined with 75 /u of the standard reaction mixture and 25 p4 of dextran T10 (5 mg/ml in 0.05 M sodium acetate buffer, pH 5.5). After incubation at 37°C for 3 (a) and 5 (/3) h, the reaction contents were processed as in (a). A sharp, turbid band was observed in the gradient from the a reaction mixture (denoted by cross-hatching). (c) Glucans synthesized by /3 in the presence of heat-inactivated a (a) and dextran T2000 (0). Twenty-five microliters of the /3 preparation was combined with 75 pi of standard reaction mixture (no primer) and 25 /4 of either dextran T2000 (5 mg/ml in 0.05 M sodium acetate buffer, pH 5.5) or heat-inactivated (100°C for 10 min) a. Enzyme reactions proceeded for 3 h at 37°C before centrifugation. Visible turbid bands were not present in either gradient.

examined in the presence of heat-inactivated a (Fig. 12c). A T2000-primed reaction was included for reference. It is evident that the product obtained from the reaction "primed" with heat-inactivated a was identical (solubility and position in the gradient) to that obtained from simply the unprimed /8 synthesis (cf. Fig. 12a and c). Thus, it appeared unlikely that an endogenous acceptor was present in a. From the data in Fig. 6, 7, and 9 it was not possible to determine whether dextran included in reaction mixtures with a actually functioned as a primer. It was possible that the dextran acted as an activator without serving as an acceptor. Since the T2000-primed glucan products of a and ,B are clearly distinguishable on the basis of buoyant density and solubility, we tested whether the primed glucan product of /8 could be incorporated into the insoluble glucan product of a. ['4C]glucan synthesized by /3 in the presence of dextran T2000 was purified by CsCl density gradient centrifugation and used as a primer source in a- and /3-directed glucan

synthesis. In this case, non-radioactive sucrose was used as substrate; thus, the only radioactivity present in the reaction mixtures was in the primer glucan. The CsCl density gradient profiles of the radioactive product glucans are shown in Fig. 13. The [14C]glucan used as primer was centrifuged alone (Fig. 13a) and exhibited the typical banding position seen earlier (Fig. 12) for T2000-primed / glucans. The product glucan from the /8-containing reaction mixture exhibited a slightly decreased buoyant density and a broader band width (Fig. 13b) than the original glucan. Turbidity was not seen in this gradient. In the reaction mixture containing a (Fig. 13c), turbidity was noted and was directly associated with the radioactive glucan in the gradient (turbidity denoted by cross-hatching). These data clearly indicate that dextran added to the a reaction mixtures is incorporated into insoluble product glucans. Finally, it should be noted that 94% of the radioactive primer glucan was recovered in the products of the /3-directed glucan synthesis,

645

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whereas only 60% was recovered in the a-directed reactions. Thus, the primer glucan was partly degraded by an activity present in the a preparation, as already evidenced by the data in Fig. 7 through 9. DISCUSSION Separation and characterization of a and 18 dextransucrase. The presence of the S. mutans dextransucrase activity in high-molecularweight aggregates has been observed by many investigators (8, 9, 11, 13, 17, 18, 21, 27). In general, it has been demonstrated that enzyme activities having the higher molecular weight produces water-insoluble products, whereas soluble glucan is associated with lower-molecular-weight enzyme (8, 9, 13, 17, 18). Our results in Fig. 1 support this concept. Although enzyme purification procedures involving gel filtration have been moderately successful in fractionating the dextransucrase into distinct activities, effective quantitative separation into fractions producing only water-soluble or water-insoluble glucan has not been obtained. By selecting the enzyme in the fractions at the leading edge of Bio-Gel columns, Mukasa and Slade (21) isolated an enzyme complex capable of synthesizing glucan that was greater than 95% water insoluble. By incorporating carbohydrases or low-molecular-weight dextrans into f-

S. mutans cultures during enzyme production, Kuramitsu (18) obtained dextransucrase from culture supernatants which could be fractionated by Bio-Gel into activities capable of synthesizing 56 and 97% water-insoluble glucan, respectively. We have demonstrated that dextransucrase activities capable of synthesizing either watersoluble or water-insoluble glucan may be obtained by ion-exchange column chromatography (Fig. 2). As a consequence of having grown the bacteria with fructose as the carbon source (11), the aggregates from the Bio-Gel column were partially disrupted by gradient salt development of DEAE. Two major species of dextransucrase were obtained. The a and ,3 forms of dextransucrase from DEAE produced almost exclusively either water-insoluble or water-soluble product (see Fig. 5). It is clear from polyacrylamide gel analyses that essentailly all of the proteins precipitated from the original cell-free culture supernatant by 60% ammonium sulfate were present in high-molecular-weight complexes (-1.5 x 106; Fig. 2). This finding suggests that most of the extracellular proteins produced by S. mutans either have strong affinities for each other or readily adhere to some extracellular entity or entities. At this time we have no clear explanation for the formation of the multicomponent

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10

20

Fraction FIG. 13. Incorporation of isolated (&synthesized, preformed soluble glucan into a-synthesized, insoluble glucan. Soluble radioactive glucan synthesized by the preparation in the presence ofprimer dextran T2000 was centrifuged in a 56% (wtlwt) CsCl density gradient; the glucan band was localized by radioassay of the gradient fractions. Fractions containing glucan were pooled and dialyzed overnight against 2 liters of 0.05 M sodium acetate buffer, pH 5.5. The sample was taken to dryness by evaporation with a gentle air stream at 35°C. The residue was taken up in 300 pi of 0.05 M sodium acetate buffer, pH 5.5. Next, 25 td of the preparation or the a preparation was added to 105 p1 of a reaction mixture containing non-radioactive sucrose (to give a final concentration of 20 mM) and the purified (3synthesized [14C]glucan (3 x 104 cpm). The reaction was incubated at 37°C for 2 h. A marked turbidity developed within 40 min of incubation in the reaction containing the a preparation. CsCl was added, and centrifugation and fractionation of the gradient proceeded as before. In this case, 100-pl samples of each fraction were assayed for radioactivity. A heavy, turbid band was observed in the gradient derived from the reaction mixture (c, denoted by cross-hatching). A turbid band was not seen in the gradient derived from the 8 reaction mixture (b). The purified [14C]glucan used as a primer dextran in this experiment was recentrifuged in CsCl as a control (a). a

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aggregates. Previous studies with a chemically defined culture medium (27) eliminated the possibility that the aggregates are solely due to the conversion to glucan of contaminating sucrose in the cultures. However, to obtain aggregates that can be effectively disrupted by salt, one must use conditions that either eliminate sucrose from the medium (18, 27) or prevent glucan formation (11, 18). The polyacrylamide gel protein patterns and enzyme activity patterns in Fig. 4 demonstrate that a and ,3 contain unique protein and enzyme activities. The amorphous band of enzyme activity in the a preparation is the predominant protein species, whereas /3 contains a greater number of proteins and two enzyme activity bands (,B, and 32). These bands correspond to the previously studied water-soluble glucan-producing activities (3, 4, 10). Although a trace of amorphously banding protein material can be detected in some /3 preparations (for example, see Fig. 4c) we have not been able to detect dextransucrase activity associated with this material (Fig. 4d). Similarly, we have never detected /8 enzyme activity bands in a preparations even under conditions where 10 to 20 times as much material was applied to the gel. Effect of primer dextran on glucan formation by a and fJ. In addition to having readily distinguishable products and distinct protein and dextransucrase compositions, the a and /3 enzyme preparations possess unique properties with respect to both primed and unprimed glucan synthesis. Examination and comparison of the CsCl density gradient behavior of primer dextrans and the glucans produced during primed synthesis by /3 indicates that the products are extended primers. Due to its size, lowmolecular-weight primer (T1O) fails to form a band in CsCl (Fig. 11). Similarly, the product glucans of T10-primed ,8 reactions failed to form sharp bands, but instead gave a broad distribution with evidence for two glucan products (Fig. 12b). When high-molecular-weight primer (T2000) that forms a band in CsCl is used with /3, the product glucans also form a band in CsCl (Fig. 12b). Thus, in the case of /3, product glucans of primed syntheses reflect the CsCl banding characteristics of the priming species. This situation is in marked contrast to primed a glucan synthesis. Irrespective of the primer used, a single CsCl distribution is seen (Fig. 12b). Thus, in the case of a, product glucans do not reflect the CsCl banding characteristics of the priming species. Glucan synthesis by both a and /3 is markedly stimulated by primer dextran. Due to the large concentration of /3 enzyme, the experi-

INFECT. IMMUN.

ment shown in Fig. 6 does not reflect the stimulation of /8-directed synthesis. In reactions where the enzyme is diluted (-40-fold) the inclusion of primer dextran results in almost a 20fold increase in the rate of glucan synthesis. In the case of a, the data in Fig. 7 clearly illustrate the enhancement of glucan synthesis by inclusion of primer dextran in the reaction mixture. Kuramitsu (18) recently reported that glucosyltransferase preparations from S. mutans GS-5, which synthesized both soluble and insoluble glucans from sucrose, were also stimulated by primer dextran. The degree of dextran stimulation of soluble glucan synthesis was of the same order we observed. However, the fold stimulation of insoluble glucan synthesis observed by Kuramitsu was two to three (18). Thus, it was suggested that an endogenous primer was present in the enzyme preparation that synthesized insoluble glucan. In comparison to Kuramitsu's preparation, our a enzyme was subjected to an additional purification step (ion-exchange chromatography) and was prepared in a medium free of dextrans. Since our a preparation is stimulated at least 10-fold by primer dextran (Fig. 7) and does not prime /8 (Fig. 13), we conclude an endogenous primer is most likely not present in our preparation of insoluble glucan-synthesizing activity. In the absence of primer dextran, net glucan accumulation by a is essentially absent (Fig. 7). Although initial glucan synthesis occurs at a very low rate (Fig. 7 and 9), further incubation results in product degradation. For example, after 5 h of incubation glucan bands in CsCl are not seen (data not shown). Glucan synthesis by ,3 does occur in the absence of primer dextrans (Fig. 6); however, as noted above, primer stimulation of glucan synthesis depends upon enzyme concentration. The CsCl behavior of unprimed 3-synthesized glucan is also unique. The glucan assumed a position in the gradient essentially equivalent to the insoluble glucan product of a (Fig. 12). As noted above, primed glucan synthesis by a (as judged by CsCl density gradient centrifugation) is unimodal. In addition to dextrans T10 and T2000, T2000-primed glucan synthesized by ,3 will also stimulate glucan synthesis by a. We observed that radioactivity present in the soluble glucan primer was incorporated into the insoluble glucan product of a (Fig. 13). This is in agreement with Kuramitsu (18), who also showed that soluble glucan could be converted to insoluble glucan by S. mutans glucosyltransferase. We also observed, however, that only 60% of the added radioactivity in the glucan primer was recovered in the a glucan product

VOL. 16, 1977

(Fig. 13c). This observation further suggests that the a enzyme contains glucanase activity. Endogenous dextranase and glucan formation by a and p. Guggenheim and Burckhardt have reported (14) that the dextranase activity produced by S. mutans OMZ 176 partially coeluted from DEAE with the first of two dextransucrase activity peaks. This observation correlates with our results (Fig. 6-10), which demonstrate that the a form of the S. mutans 6715 dextransucrase contains significant levels of glucan-hydrolyzing activity and that a contains undetectable quantitites of this activity. The main products of the endohydrolytic S. mutans dextranase are isomaltosaccharides containing four to six glucosyl residues per molecule (5). Since our assay of glucan synthesis detects methanol-insoluble glucans, and methanol insolubility of oligosaccharides commences at a degree of polymerization of 4 to 6, it is likely that the instability of the glucans produced by a (Fig. 7 and 8) results from degradation of nascent glucan by the endohydrolytic dextranase. The results (Fig. 9), which demonstrate that mixing of a with /3 causes the unprimed glucans synthesized by /8 to become unstable, support this proposal and emphasize the functional potential of dextranase in crude enzyme preparations. There would appear to be several possible mechanisms by which the glucan fragments produced by the dextranase in a can be reinserted into methanol-insoluble products (Fig. 7-9). Hehre and co-workers (15, 16, 33) have proposed that glycoside hydrolases can function by a glycosylation or glycosyl-hydrogen interchange reaction mechanism. With dextranases three possible expressions of this mechanism are hydrolysis, transglycosylation to acceptors other than water, and condensation of isomaltodextrins. Evidence has been presented that both exohydrolytic and endohydrolytic dextranases purified from various sources can act in any combination of these reactions depending upon the substrate, substrate concentration, and reaction conditions. Walker and Builder (34) have shown that the exohydrolytic dextranase from S. mitis can catalyze transfer reactions when incubated with concentrated isomaltosaccharides. Sawai and Niwa (26) demonstrated that a dextranase from Arthrobacter globiformis released successive isomaltose units from the nonreducing ends of dextrans and could cause transisomaltosylation reactions resulting in formation of isomaltotetraose and isomaltotriose (when degradation occurred in the presence of glucose). In addition, this enzyme can catalyze condensation reactions among isomaltodextrins. These authors (26)

S. MUTANS DEXTRANSUCRASE

647

emphasize the relative ease of isomaltose condensation by the dextranase, since the reaction is exergonic with a free energy change value of approximately -5,000 joules/mol (24, 32). An endodextranase from a pseudomonad (25) was shown to have marked synthetic activity at high substrate concentrations, causing condensation of low-molecular-weight isomaltodextrins in various combinations. Walker and Dewar (35) have shown that the Penicillium lilacinum endodextranase may cause condensation of isomaltose, followed by cleavage to glucose and isomaltotriose. Another possible mechanism for glucan modification during product synthesis could involve the release of oligosaccharides, which could then be reincorporated as a-(1--+3)-linked branches in a-(1-->6)-linked glucan chains. Evidence for this type of interaction has been presented by Ebert and Brosche (6) using dextransucrase from Leuconostoc mesenteroides. It is clear that further work with the S. mutans dextranase will be necessary to establish its metabolic capacity and ability to modify the glucans produced by S. mutans. The immediate significance of our results, which suggest the functioning of endogenous dextranase in S. mutans glucan production, is that studies on the S. mutans dextransucrase should either take into account the presence of this activity or demonstrate its absence in dextransucrase preparations. The presence of dextranase activity as part of the high-molecularweight, dextransucrase-containing aggregates and its presence in our water-insoluble glucansynthesizing a enzyme preparation indicate that this hydrolytic enzyme co-purifies with the dextransucrase via complexing with the extracellular material involved in aggregate formation. Although we have obtained from strain 6715 dextransucrase that is dextranase-free and capable of synthesizing water-soluble glucan (,8), we have as yet been unable to obtain water-insoluble glucan-forming activity (a) free of dextranase. Based on the evidence presented in this manuscript, and the observation that endodextranase production is characteristic of essentially every strain of S. mutans that has been tested (5, 31), it is not unlikely that this enzyme plays a significant role in glucan production and may partially explain the glucan heterogeneity observed in this microbial species. Thus, the relative quantities of synthetic (glucosyltransferases) and degradative (dextranases) activities, together with levels of substrates and/or alternate acceptors, may profoundly influence the solubility properties and quantity of glucan produced by S. mutans.

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ACKNOWLEDGMENTS This work was supported by Public Health Service grants DE 03654 and DE 04171 from the National Institute of Dental Research. C.F.S. was the recipient of Public Health Service career development award K4-DE-42,859 from the same institute. LITERATURE CITED 1. Brunk, C. F., and P. C. Hanawalt. 1966. Glycogen satellite bands in isopycnic CsCl gradients. Exp. Cell Res. 42:406-408. 2. Ceska, M., K. Granath, B. Normann, and B. Guggenheim. 1972. Structural and enzymatic studies on glucans synthesized with glycosyltransferases of some strains of oral streptococci. Acta Chem. Scand. 26:2223-2230. 3. Chludzinski, A. M., G. R. Germaine, and C. F. Schachtele. 1974. Purification and properties of dextransucrase from Streptococcus mutans. J. Bacteriol. 118:17. 4. Chludzinski, A. M., G. R. Germaine, and C. F. Schachtele. 1976. Streptococcus mutans dextransucrase: purification, properties, and requirement for primer dextran. J. Dent. Res. 55:C75-C86. 5. Dewar, M. D., and G. J. Walker. 1975. Metabolism of the polysaccharides of human dental plaque. Caries Res. 9:21-35. 6. Ebert, K. H., and M. Brosche. 1967. Origin of branches in native dextrans. Biopolymers 5:423-430. 7. Ebisu, S., A. Misaki, K. Kato, and S. Kotani. 1974. The structure of water-insoluble glucans of cariogenic Streptococcus mutans formed in the absence and presence of dextranase. Carbohydr. Res. 38:374-381. 8. Fukui, K., Y. Fukui, and T. Moriyama. 1974. Purification and properties of dextransucrase and invertase from Streptococcus mutans. J. Bacteriol. 118:796-804. 9. Fukui, K., Y. Fukui, and T. Moriyama. 1974. Some immunochemical properties of dextransucrase and invertase from Streptococcus mutans. Infect. Immun. 10:985-990. 10. Germaine, G. R., A. M. Chludzinski, and C. F. Schachtele. 1974. Streptococcus mutans dextransucrase: requirement for primer dextran. J. Bacteriol. 120:287294. 11. Germaine, G. R., and C. F. Schachtele. 1976. Streptococcus mutans dextransucrase: mode of interaction with high-molecular-weight dextran and role in cellular aggregation. Infect. Immun. 13:365-372. 12. Germaine, G. R., C. F. Schachtele, and A. M. Chludzinski. 1974. Rapid filter paper assay for the dextransucrase activity from Streptococcus mutans. J. Dent. Res. 53:1355-1360. 13. Gibbons, R. J. 1972. Presence of an invertase-like enzyme and a sucrose permeation system in strains of Streptococcus mutans. Caries Res. 6:122-131. 14. Guggenheim, B., and J. J. Burckhardt. 1974. Isolation and properties of a dextranase from Streptococcus mutans OMZ 176. Helv. Odontol. Acta 18:101-113. 15. Hehre, E. J., D. S. Genghof, and G. Okada. 1971. The a-amylases as glycosylases, with wider catalytic capacities than envisioned or explained by their representation as hydrolases. Arch. Biochem. Biophys. 142:382-393. 16. Hehre, E. J., G. Okada, and D. S. Genghof. 1969. Configurational specificity: unappreciated key to understanding enzymic reversions and de novo glycosidic bond synthesis. I. Reversal of hydrolysis by a-, ,3- and glucoamylases with donors of correct anomeric

INFECT. IMMUN. form. Arch. Biochem. Biophys. 135:75-89. 17. Kuramitsu, H. K. 1974. Characterization of cell-associated dextransucrase activity from glucose-grown cells of Streptococcus mutans. Infect. Immun. 10:227-235. 18. Kuramitsu, H. K. 1975. Characterization of extracellular glucosyltransferase activity of Streptococcus mutans. Infect. Immun. 12:738-749. 19. Lewicki, W. J., L. W. Long, and J. R. Edwards. 1971. Determination of the structure of a broth dextran produced by a cariogenic streptococcus. Carbohydr. Res. 17:175-182. 20. Long, L. W., and J. R. Edwards. 1972. Detailed structure of a dextran from a cariogenic bacterium. Carbohydr. Res. 24:216-217. 21. Mukasa, H., and H. D. Slade. 1974. Mechanism of the adherence of Streptococcus mutans to smooth surfaces. III. Purification and properties of the enzyme complex responsible for adherence. Infect. Immun. 10:1135-1145. 22. Newbrun, E. 1972. Extracellular polysaccharides synthesized by glucosyltransferases of oral streptococci. Caries Res. 6:132-147. 23. Nisizawa, T., S. Imai, H. Akada, M. Hinoide, and S. Araya. 1976. Extracellular glucans produced by oral streptococci. Arch. Oral Biol. 21:207-213. 24. Ono, S., K. Hiromi, and K. Takahashi. 1965. Calorimetric studies on hydrolysis of glucosides. I. Heats of hydrolysis of maltose and phenyl a-maltoside. J. Biochem. 57:799-807. 25. Richards, G. N., and M. Streamer. 1974. Studies on dextranases. IV. Mode of action of dextranase D, on oligosaccharides. Carbohydr. Res. 32:251-260. 26. Sawai, T., and Y. Niwa. 1975. Transisomaltosylation activity of a bacterial isomalto-dextranase. Agric. Biol. Chem. 39:1077-1083. 27. Schachtele, C. F., S. K. Harlander, and G. R. Germaine. 1976. Streptococcus mutans dextransucrase: availability of disaggregated enzyme after growth in a chemically defined medium. Infect. Immun. 13:1522-1524. 28. Segovia, Z. M. M., F. Sokol, I. L. Graves, and W. W. Ackermann. 1965. Some properties of nucleic acids extracted with phenol. Biochim. Biophys. Acta 95:329-340. 29. Sidebotham, R. L. 1974. Dextrans. Adv. Carbohydr. Chem. Biochem. 30:371-444. 30. Sidebotham, R. L., H. Weigel, and W. H. Bowen. 1971. Studies on dextrans and dextranases. Dextrans elaborated by cariogenic organisms. Carbohydr. Res. 19:151-159. 31. Staat, R. H., and C. F. Schachtele. 1974. Evaluation of dextranase production by the cariogenic bacterium Streptococcus mutans. Infect. Immun. 9:467-469. 32. Takahashi, K., Y. Yoshikawa, K. Hiromi, and S. Ono. 1965. Calorimetric studies on hydrolysis of glucosides. II. Heats of hydrolysis of a-1,6 glucosidic linkages. J. Biochem. 58:251-254. 33. Takeshita, M., and E. J. Hehre. 1975. The capacity of aamylases to catalyze the nonhydrolytic degradation of starch and glycogen with formation of novel glycosylation products. Arch. Biochem. Biophys. 169:627637. 34. Walker, G. J., and J. E. Builder. 1967. Metabolism of the reserve polysaccharide of Streptococcus mitis. Properties of a-1,6-glucosidase. Biochem. J. 105:937942. 35. Walker, G. J., and M. D. Dewar. 1975. The action pattern ofPenicillium lilacinum dextranase. Carbohydr. Res. 39:303-315.