Synthesis of a broth levan by a cell-bound levansucrase from Streptococcus salivarius (SS2)

Archs

oral Bd.

Vol.

18, pp. 239-251,

1973.Pergamon Press. Printed inGreatBritain.

SYNTHESIS OF A BROTH LEVAN BY A CELL-BOUND LEVANSUCRASE FROM STREPTOCOCCUS SALIVARIUS W2) SUZANNE M. GARSZCZYNSKI and

J. R. EDWARDS

Department of Chemistry, Villanova University, Villanova, Pennsylvania 19085, U.S.A. Summary-The levansucrase from Streptococcus suliuurius (SS2) is a constitutive, cellbound enzyme. When SS2 is cultured in the absence of sucrose, neither puromycin nor chloramphenicol inhibit the synthesis of the levan upon the addition of sucrose to the media. The levansucrase is found associated with the cell-wall fraction of the cell following cellular disruption and differential centrifugation. The enzyme can be released from the cell-wall fraction and from intact whole cells by extraction with 8 M lithium chloride. The isolated levansucrase has a pH optimum of 5.6 and is independent of divalent cations for activity. INTRODUCTION THE correlation between certain streptococci which produce extracellular polysaccharides and the occurrence of dental caries has been established. The cariogenic streptococci may synthesize either levans or dextrans when cultured in the presence of sucrose. These polysaccharides are considered essential to plaque formation where they may act as a binder on the tooth surface (FITZGERALD and JORDAN, 1968; KEYES, 1968; GIBBONSand FITZGERALD,1969). Sucrose is the glycosyl donor for levan and dextran synthesis. Glucose and fructose support growth, but do not act as effective glycosyl donors for either levan or dextran synthesis (GIBBONSand BANGHART,1968; GIBBONSand NYGAARD,1968). CARLSSON(1970) has studied the synthesis of levan by a purified extracellular broth enzyme from Streptococcus mutans (JC2). Broth dextransucrases from several streptococci have also been studied (WOOD, 1967; CARLSSON,NEWBRUN and KRASSE, 1969). Cell-bound dextransucrases have been reported in washed-cell preparations which produce dextrans upon the introduction of sucrose to suspensions of streptococci (GUGGENHEIMand SCHROEDER, 1967; GIBBONSand FITZGERALD,1969) and of a lactobacillus (HAMMOND,1969). This paper describes several parameters involved in the biosynthesis of a levan by Streptococcus suliuurius (SS2): chemical composition of the levan, association of the levansucrase with the cells, constitutive nature of the levansucrase and solubilization of the levansucrase. MATERIALS

AND

METHODS

Bacterial strain Streptococcus salivarim (SS2) was obtained from Dr. R. J. Gibbons, Forsyth Dental Center, Boston, Mass. The culture was maintained on Eugonagar by bimonthly transfer. This strain has been shown to be cariogenic in rats (GIBBONSand BANGHART,1968). Broth cultures were routinely grown on trypticase soy broth (TSB) at 37°C.

239

240

SUZANNE

M.

GARSZCZYNSKIAND

J.R.EDWARDS

Chromatographic methods

Cultures of SS2 were grown in TSB supplemented with 5 per cent sucrose for 18-20 hr. The cells were removed by centrifugation and the levan precipitated by 2 volumes of ethanol. The levan was dissolved in water and reprecipitated twice. The levan was hydrolyzed with sulphuric acid (0.1 N, 100°C for 1 hr) and neutralized with powdered barium carbonate. The hydrolyzate was separated on Whatman No. 1 paper using as developer: n-propanol, ethyl acetate and water (6: I : 3, v/v) and the sugars were located using anilinediphenylamine-phosphoric acid spray (BAILEYand BOURNE,1960). This spray gives a differential colour assay for glucose and fructose. Thin-layer chromatograms were performed on silica gel G using as developer: 2-butanone, acetic acid and methanol (6: 2:2, v/v) and the components were located with S per cent sulphuric acid spray followed by heating. Concanavalin A preparations

Concanavalin

A was prepared

from jack bean meal according

to AGRAWALand GOLDSTEIN assay.

(I 967). Precipitin studies were performed using the standard Ouchterlony double-diffusion Dry weight determinations

Aliquots of cell suspensions 105°C.

in phosphate

buffer were dried to a constant weight in an oven at

Solutions

Unless otherwise specified, all per cent solutions are weight per volume. Typical cellpreparations

Cells used in buffer-sucrose suspensions were prepared in the following manner. The organisms were cultured in TSB containing 0.1 per cent glucose for 12-15 hr at 37”C, removed by centrifugation (5000 g, 5 min), and washed twice in phosphate-citrate buffer (0.05 M, pH 5.6). These cultures are normally in either the late logarithmic or early stationary phase of growth after 12-15 hr and are capable of actively synthesizing levan from sucrose. The cell suspension (5 ml, 0.85 mg/ml dry weight) was added to citrate-phosphate buffer (45 ml, 0.05 M, pH 5.6) containing sucrose (2.5 g) at 25°C. Aliquots (5 ml) were removed, chilled in ice-water, centrifuged (5000 g, 5 min), and the supematant dialysed exhaustively against distilled water. The dialysed sample was brought to known volume and the levan content analyzed by the phenol-sulphuric acid assay (Duaoors e’tal., 1956). The initial samples were removed about 1 min after the cells had been added to the sucrose-buffer system. This time lag and the activity of the cells in ice may account for the high initial value of levan production. No levan was synthesized using heat-killed cells (60°C 30 min) or by using an equivalent amount of glucose and fructose(l.25 geach).

The incorporation of [r4C]-leucine in the cellular material was used as a measure of protein synthesis and was determined as follows: Cell suspension (0.36 mg dry weight/ml) or growing cells (absorbance of approximately 0.4) were incubated at 37°C for periods up to 30 min with [r4C]-leucine (0.25 &i/ml). Aliquots (1 ml) were removed at various time intervals and added to filter membranes (Schleicher and Schuell, Bact-T-Flex, 25 mm, B-6) which had been soaked in phosphatecitrate buffer (0.05 M, pH 5.6) in which leucine (0.25 mg/ml) had been dissolved. The cells were washed with 15 ml of the leucine-phosphate-citrate buffer and then with 10 ml phosphate buffer. The membranes were dried at 65°C for 45 min and placed in 15 ml scintillation fluid (4 g PPO and 50 mg POPOP dissolved in 1.0 litre of toluene). The incorporation of [r4C]-leucine was determined in a Nuclear-Chicago scintillation counter. To this standard incubation mixture, either chloramphenicol, puromycin or fluoride was added to inhibit protein synthesis. Cellular disruption and fi-actionation

Cultures of Strep. salivarias (SS2) were grown for 1S hr in TSB with 0. I per cent glucose, but without sucrose at 37°C. The cells were removed by centrifugation, washed twice with 1 per cent saline and suspended in citrate-phosphate buffer (0.05 M, pH 5.6). The cellular suspension from 1 .O litre of culture was divided into two equal fractions of 30 ml. One fraction was disrupted by sonication for 15 mm at 0°C using a Branson Sonifer Model S125 (Branson Sonic Power, Danbury, Conn.). The

STREPTOCOCCAL CELL-BOUND LEVANSUCRASE

241

other cellular fraction was disrupted by glass beads in a Braun shaker Model MSK (Bronwill Scientific, Rochester, N.Y.). The cell suspension (15 ml) was shaken for 2 min with glass beads (15 ml, 0.012-0.015 mm) with liquid CO* cooling. After cellular disruption, the samples were subjected to differential centrifugation. The initial centrifugation sedimented undisrupted whole cells (3500 g for 10 min), the next centrifugation sedimented the cell-wall fraction (8000 g for 10 mitt); and the final centrifugation sedimented the cell membrane fraction (40,000 g for 30 min) (PARK and CHAT~ERJEE,1966). The subcellular fractions from the differential centrifugation, as well as the whole cells and the spent cell-free broth, were analysed for levan synthesis. Lithium chloride extraction Whole cells from 1 .O litre of culture of SS2 were washed twice with saline (1 per cent) and suspended in phosphate buffer (62 ml, 0.05 M, pH 5.6). The cells were subjected to extraction at various molarities of lithium chloride for 15 min at 4”C, 24°C and 37°C. Solid lithium chloride was added to the cell suspension and the suspension was stirred in an ice bath to absorb the heat of solution of the salt. The suspension was then stirred at the desired temperature. The cellular material was removed by centrifugation (3000 g for 5 min) and the soluble material was analysed for levansucrase activity after dialysis against 1 per cent glycine. A flocculent material formed on the surface during extraction with 8 M lithium chloride. Various salt solutions were tried in an attempt to dissolve the enzyme from the flocculent material. Sodium chloride (1 M) proved to be the best of the solubilization of the levansucrase. All cellular material, supematant and residue fractions, were analysed for protein by the Lowry method (LOWRY et al., 1951) and for levansucrase activity after dialysis against 1 per cent glycine. The release of total reducing sugar and total non-dialysable carbohydrate was measured. Measurement of enzyme activity A sodium chloride solution (1 M) of the solubilized levansucrase (9 ml) and sucrose (1 ml, 50 per cent) were incubated (37”C, 30 min). Sodium hydroxide (0.1 N, 0.5 ml) was added to an aliquot (1 ml) of the incubation mixture to terminate the reaction. The samples were analysed for carbohydrate after dialysis by the phenol-sulphuric acid method (DUBOIS et al., 1956) and for glucose by reducing power (SOMOGYI,1945) and by glucose oxidase (Sigma Chemical Bulletin No. 510). The enzymatic activity under varied conditions; i.e. various metal ions, buffers and hydrogen ion concentrations were studied with these general procedures. Materials Culture media were obtained from Baltimore Biological Laboratories, Baltimore, Maryland. Puromycin was obtained from Nutritional Biochemical Co., Cleveland, Ohio and chloroamphenicol from Sigma Chemical Co., St. Louis, Missouri. DL-Leucine-1-[I%] (2 &i/PM), 2,5-diphenyloxazole (PPO) and 1,4-bis-2-(5-phenyloxazolyl)-benzene (POPOP) were purchased from New England Nuclear, Boston, Mass. All other chemicals were reagent grade chemicals. Infrared spectra of the levan were obtained with a Perkin-Elmer spectrophotometer (model 225) as potassium bromide pellets. RESULTS

Analysis of levan

Levan synthesized by growing cultures and by cells suspended in sucrose-phosphate buffer was isolated. Paper and thin-layer chromatograms of the acid hydrolysate revealed fructose as the only hexose. The levans synthesized in phosphate buffer and during growth in TSB gave isoprecipitin bands in Ouchlerlony plates when diffused against concanavalin A. The reactivity of the levan with concanavalin A is indicative of a branched structure. Concanavalin A reaction with streptococcal dextrans has been reported by RBLLA (1971). The infrared spectra of the levan synthesized in buffer and in TSB were identical showing the characteristic absorption bands at 930, A.O.B. 18/2-e

242

SUZANNE

M. GARSZCZYNSKI AND J. R. EDWARDS

867 and 813 cm-‘. Periodate oxidation data (unpublished) is consistent with the fructose being linked 2,6. These results show that the levan produced in phosphate buffer is identical to that produced in TSB under growth conditions. Optimum concentration of sucrose for levan production Streptococcus saiivarius (SS2) was grown in TSB (24 hr at 37°C) containing 0.1 per cent glucose and various concentrations of sucrose. The cells were removed by centrifugation (5000 g, 5 min) and the broth levan was precipitated from the cell-free broth with 2 volumes of ethanol. The precipitate was dissolved in water, dialysed t

8.0:

6 O4.0z-o-

zo-

I-0: 0.8- d:0.6- $0.6: 0.4-io.4_ b 2 Z -I 02-:02-o

-6,2 -5.8

I

n

5-4 1 -

Z0.6-

-5 0

-004-4.2 - 0.02hr FIG. I. Typical growth of Strep. salivarius SS2 in broth supplemented with 5 per cent sucrose. Absorbance (450 nm) -O-O-; DNA &g/ml) --A----&; levan (mg/ml) -&--¤--;pH-+--+-.

exhaustively against distilled water, and assayed for carbohydrate by the phenolsulphuric acid assay. A sucrose concentration of 5-7 per cent yielded 11-12 mg of levan per mg dry weight of bacteria. This range of maximal yield is in agreement with dextran production (GIBBONS and BANGHART, 1967). Growth parameters

Various parameters were followed during growth to ascertain the correlation between cellular growth and levan production. An inoculum (1 per cent) of a 15-hr culture grown in TSB without sucrose was added to TSB containing 5 per cent sucrose and the culture was incubated at 37°C. As samples were removed, their absorbance was measured at 450 nm and the pH was determined potentiometrically. The dry weight of the twice washed cells was measured and the deoxyribonucleic acid (DNA) content was analysed by the diphenylamine-phosphoric acid assay (TANZER, Wool and KRICHJWSKY, 1969). The levan in the cell-free broth was precipitated by 2 volumes

STREITOCOCCAL

CELL-BOUND

243

LEVANSUCBASE

of ethanol, dialysed and assayed for carbohydrate (DUBOISet al., 1956). The amount of DNA, levan, and the turbidity increase at nearly the same rate during growth (Fig. 1). After a short lag period, the most rapid synthesis of levan occurs during the last half of the exponential growth phase. The production of levan ceases at about the same time that the cells stop growing. The pH of the culture was 4-4-4.2 after 24 hr of incubation. Sucrose additions during growth

Since there is a lag in the production of levan during growth, the question as to whether the levansucrase is a constitutive or induced enzyme was examined. Sucrose was added to aliquots (250 ml) of TSB cultures when the absorbance was 0 -2, 0 - 77 and l-1. The organisms were then allowed to continue to grow under normal incubation conditions. Aliquots (5 ml) from the various cultures were removed and

r .s% :

1

_--------------__-__-__-_________________*

5.0 4.0302.0 -c ‘, ;:; E

I 1

5 3

_

I

- 2.0 49-------_---______ .#

1 I.0 - 0.5

/

- 0.2

P

10.1

d I

0.05

2

4

6

6

10

12

14

16

16

20

22

24

hr FIG.

2. J_e.vansynthesis (mg/ml) after sucrose additions to aliquots of the culture at

absorbance

values of 0.2, --A----A; 0.77, -O-O-; absorbance of culture -0-

1.1, --m--m--; -0-.

analysed for the presence of levan in the cell-free broth. There was an immediate production of levan (Fig. 2) indicating that the levansucrase might be a constitutive enzyme. The quantity of levan synthesized in the first 2 samples was nearly equal to the quantity synthesized when sucrose was present throughout the experiment. Cell-bound levansucrase

The levansucrase was found to be associated with the cells and was not found in appreciable amounts in the broth. Cells from 5 ml of culture were suspended in phosphate-citrate buffer (5 ml, O-05 M, pH 5-6) and the amount of levan produced from sucrose (5 per cent) was compared to that of the cell-free broth. The cell suspensions produced 5 * 1 mg of levan while the broth produced O-14 mg of levan in the

244

SUZANNE M. GAR~ZCZYNSKI AND

J. R. EDWARDS

presence of sucrose. It is possible that the presence of the activity in the broth is from lysis of the cells. Eflect of pH and bufler

Cells suspended in a phosphatecitrate buffer covering the pH range from 4 a4 to 7 *4 showed little pH dependence for levan synthesis over this pH range (about 1 mg of levan per mg dry weight of cells). There was a 5-fold inhibition of levan synthesis when the cells are suspended in tris buffer (0.05 M, pH 7.8) compared to phosphate buffer (0.05 M, pH 7.4). This phenomenon has been reported by GIBBONSand NYGAARD(1968) in the synthesis of dextran by Strep. mutans, but is contrary to that found by CARLSSON (1970). Effect of puromycin and chloramphenicol

The synthesis of levan by washed cells and the effect of sucrose additions during growth are strongly suggestive of a constitutive enzyme, but does not preclude protein turnover and synthesis due to sucrose induction. To cell suspensions in phosphate buffer, either puromycin or chloramphenicol(20 pg/ml final concentration) was’added. Sucrose was then added and the quantity of levan synthesized determined (Table 1).

Time (min) 3 :: 30

Levan production/bacterial dry weight Puromycin Control (m&s) (mg/mg)

12 1.43 1.44

I.39 I .48 1.37 I.37

Chloramphenicol (melmg) 1.48 K2 1.48

There was no inhibition of levan synthesis by either antibiotic. The incorporation of [14C]-leucine into growing cells was followed to determine the permeability of the cells to puromycin and chloramphenicol. The incorporation of [14C]-leucine (Fig. 3) was completely inhibited by puromycin (20 pg/ml), chloramphenicol (20 pg/ml) and fluoride (20 pg/ml) indicating that the antibiotics could penetrate the cells. The cells which were cultured for 20 min in either puromycin, chloramphenicol or fluoride retained the ability to synthesize levan. These studies would substantiate that the levansucrase is a constitutive enzyme in SS2. Cell disruption and cell fractionation In contrast to the levansucrase of Strep. mutans which is released into the broth (CARLSSON,1970), the enzyme from SS2 is found associated with the cellular material. The cellular location of the enzyme was examined following cellular disruption. Sonication for time periods up to 15 min had little effect on the streptococcal cells and released little protein into the homogenate. There was little visible change in the

STRE~CDCCAL

245

CELL-BOUND LEVANSUCRASE

150 E 2 l_J 100 z c .f s 3

50

1

I1

111

10

I

20

30

II,

40

50

FIG. 3. Incorporation of [‘4C]-leucine into growing cultures. At IS min either puromycin, chloramphenicol or fluoride was added to the culture. l Control, U fluoride, 0 chloramphenicol, 0 puromycin.

cell morphology as examined by light microscopy. Since the bulk of the material was recovered in the unbroken cell fraction, this procedure was not pursued. The cells were readily disrupted by shaking with the glass beads in the Braun homogenizer. Approximately 95 per cent of the cells were disrupted with this technique and the homogenate was then subjected to differential centrifugation.

(loo

culture per cent)

I

centrifuge 3000 g/10 mill

4

4 Cell-free broth

Cells

(2.5 per cent)

(91.5 per cent)

I

(I) cellular disruption (2) CentrifUgC3000 g/10 tin

Unbroken cells (19’3 per cent)

Supernatant

I

centrifuge 10,000 g/10 tin

4 Cell

j. Supematant

walls

(69.6 per cent) centrifuge 40,000 g/30 min

j.

4

Cell membrane (5.8 per cent)

Suwmatant (2.8 per cent)

SUZANNE

246

M. GARSZCZYNSKI AND J. R. EDWARDS

The cellular fractions were separated by differential centrifugation (Table 2) and each fraction was analysed for enzyme activity by an increase in reducing power and by non-dialysable Ievan. The cell wal1 fraction contained the bulk of the levansucrase activity (69 -6 per cent) when compared to the other fractions. Tt was concluded that the enzyme was associated with the cell wall fraction. Lithium chloride extraction

Since high concentrations of lithium chloride have been successfully used in the solubilization of an autolysin from streptococcus cell walls (SHOCKMAN and MARTIN, 1968), extractions of the cell wall by lithium chloride were attempted in order to solubilize the levansucrase (Table 3). The bulk of the enzyme activity (93 per cent)

Concentration of LX1

Activity released mg levan formed (mg protein x min-I)

PeE$aaje

of

activity

could be released from the cell walls by 8 M lithium chloride. The levansucrase activity was present in the flocculent material that formed during the extraction and it could be solubilized with 1 M sodium chloride. Extractions of whole cells by lithium chloride were attempted so that cell walls would not need to be prepared prior to the extraction of the levansucrase. Various molarities of lithium chloride (2-8 M) and various temperatures (4-37°C) were used for extraction (Table 4). A pellet material and supernatant fraction were removed in

4°C Molarity of LiCl 2 5 8 supernatant flocculent* l

0.35 0.96 I.31 5.50

Temperature of extraction 24°C mg levan formed (mg protein x min-‘) ;:$j 1.17 3.60

37°C

0.12 0.36 0.87 2.50

Solubilized with 1 M sodium chloride.

all cases except the 8 M concentration where a flocculent fraction was also present. About 80 ger cent of the levansucrase activity that was originally present in the whole cells was released by the 8 M lithium chloride and was found in the flocculent material. The levansucrase was extracted from the flocculent material by 1 M sodium chloride (24 hr, 4°C).

STREPTOCOCCAL

CELL-BOUND

241

LEVANSUCRASE

Pretreatment of the whole cells by trypsin prior to extraction by lithium chloride was attempted to ease the release of the enzyme. Trypsin (1 mg/ml) was incubated at 4°C for 15 min with whole cells suspended in phosphate buffer (pH 5.6, 0.05 M). The cells were washed twice with 1 per cent saline and extracted with the various molarities of lithium chloride. About five times as much protein was released from the trypsinized cells, but the isolated material was completely inactivated. Characteristics of the solubilized levansucrase

The reaction conditions for maximal levansucrase activity and the nature of the enzyme was determined in the following series of experiments. The lithium chlorideextracted levansucrase showed great pH dependence with maximal activity at pH 5 *6

200

4.0

5.0

6.0

70

P”

FIG. 4. Dependence

of levansucrase activity on pH in phosphatexitrate

buffer.

(Fig. 4). This is in marked contrast to the lack of pH dependence of the cell-bound enzyme. The isolated levansucrase showed dependence on sucrose concentration with maximal activity at 5 per cent. Since various levansucrases have shown dependence on magnesium salts, a series of experiments were performed to study the effect of cations and anions. Different metal ions were added (1, 2 and 20 mM, final concentration) to the levansucrase along with buffered sucrose (pH 5 +6, 5 per cent). The presence of Mn2+ did not affect the activity of the enzyme (Table 5) while Ca2 +, Zn2 +, Mg2 +, Ni2 +, Co2 + and Cd2 + inhibited the activity to about the same extent. Ethylene diamine tetraacetic acid

SUZANNE

248

Control EDT-A= Mgz + Mn2+

g,$:

M.

GARSZCZYNSKI AND

100~0’ 11.4 54.6 100.0 49.2 52.0

100.0 10.5 18.0 100.0 18.0 17.0

100.0 73.5 31.0 98.0

100.0 24.0 28.0 21 .o 100.0 100~0 140.0 100.0

iii.0

c”l”-’

;gj:;

CN-

140.0 100.0

Mercaptoethanol

* Relative

enzyme

J. R. EDWARDS

i+~li

‘OK:

2.7 100.0 13.6 IO.0

ii.i

loo.0 20.0 12.0 17.0 I% 140.0 100.0

activity.

(EDTA) inhibited the enzyme, possibly through removal of a tightly bound metal ion. Cyanide stimulated the enzymatic activity while mercaptoethanol had no effect. Since neither Ag+ nor mercaptoethanol affected the levansucrase, it is likely that the enzyme does not have an essential sulphydryl group at the active site. DISCUSSION Streptococcus salivarius (332) produces a levan by a cell-associated levansucrase when cultured with sucrose. The levan may play a role in the formation of plaque and the adhesion of the plaque to the tooth surface (GUGGENHEIMand SCHROEDER, 1967). The levans have been reported to be degraded by mixed oral flora and used as an energy source (GIBBONS and BANGHART, 1967; DA COSTA and GIBBONS, 1968; HIGUCHIet al., 1970). We have found that cultures of SS2 do not degrade their levan

in the spent TSB-sucrose media for periods up to five days. Undoubtedly, there is a large amount of sucrose still available to support their metabolism in the absence of levan hydrolysis. The greater conversion of sucrose to levan (8-9 g/l by Strep. salivarius (SS2) compared to the conversion to dextran (0.8-l *Og/l) by Strep. mutans (GF71, GS5, HS6) may offset the hydrolysis of the levan by the oral flora and allow it to play an important role in plaque formation. The organism grows at about the same rate in the presence or absence of sucrose. Sucrose has been shown to be inhibitory in some strains of streptococci (GIBBONS, 1968; TANZERet al., 1969), but this has not been observed in this study or with several Strep. mutans strains in this laboratory. The rate of levan synthesis shows an initial lag during the first phase of cellular growth and then parallels cellular growth and declines. The levan synthesized in the cell suspensions is identical to the levan synthesized during growth since their chemical composition, infra-red spectra, and reaction with concanavalin A are identical. The levan has a molecular weight of about two million as measured by gel filtration on Sepharose 2B (unpublished data). This is similar to the general size reported by NEWBRUN and BAKER(1968) and CARLSSON (1970).

STRE-CAL

CELLBOUND LEVANSUCRA!3E

249

The levansucrase is a constitutive enzyme which can very rapidly convert sucrose into levan. This was demonstrated in growing cultures and in buffer suspensions of cells. Neither puromycin, chloramphenicol, nor fluoride inhibited the synthesis of levan by the cell suspensions supporting the constitutive nature of the enzyme. CARLSSON (1970) has isolated a broth levansucrase from Strep. mutuns which was produced in the absence of induction by sucrose. Several broth (extracellular) dextransucrases have been described in various strains of cariogenic streptococci (WOOD, 1967; CARLSSONet al., 1969). The cell-bound nature of the levansucrase of Strep. saliuarius differs from these and may be more related to the cell-bound dextransucrase in Strep. mutans (GIBBONSand NYGAARD,1968; GUGGENHEIM and SCHROEDER, 1967). The levansucrase of SS2 is localized in the cell wall fraction following cellular disruption by glass beads and differential centrifugation. Since the location of the enzyme is at the cell periphery, it could easily be responsible for the production of the extracellular levan. Trypsin treatment of the whole cells prior to lithium chloride extraction drastically decreased the activity of the enzyme. This suggests that the levansucrase is exposed to the solutes of the media and most likely resides outside the cytoplasmic membrane. Most of the dextransucrases studied (WOODand CRITCHLEY, 1966) and of levansucrases (CARLSSON,1970) have been isolated from the broth. GIBBONSand NYGAARD(1968) have shown that the dextrans synthesized at pH 5-7 by a cell-bound enzyme are less soluble than those produced by the broth enzyme. The levan produced by SS2 may have physical properties which may differ as markedly as those produced by Strep. mutans as one alters the pH. The ease of levansucrase release from whole cells by 8 M lithium chloride offers a possibility to compare the cell-bound enzyme and its levan with the broth enzyme and its products. The isolated levansucrase shows a marked pH dependence with maximal activity at 5.6 while that associated with the cells shows little pH dependence from pH 4.5 to 7.5. The broth dextransucrases from Strep. sunguis displayed a pH optima of S-37-O (CARLSSON et al., 1969) and from Strep. mutuns FAI of 6 (WOOD, 1967). GIBBONS and NYGAARD(1968) reported a cell-associated dextransucrase to have a pH optima of 6-8 and a broth levansucrase to have a pH optima of 5-8. The inhibition of the levansucrase by Mg2+ is markedly different from what is found with the Leuconostoc mesenteroides preparations. The lack of divalent ions caused a marked alteration of the dextran produced (NEELY, 1960). CARLSONet al. (1969) did not detect any apparent metal ion requirement for a purified dextransucrase. The loss of activity of the levansucrase by EDTA was also observed by CARLETON (1970) which may be caused by the removal of a tightly bound divalent ion which activated the enzyme. Divalent ions may play an important role in the synthesis of dextran during growth (BOWEN, 1971), but this effect is not evident with the isolated levansucrase. Work on the purification of the solubilized levansucrase is currently being pursued. Acknowledgements-This investigation was supported by funds provided by Villanova University and by a N.I.D.R. Grant DE03101-01 and by a National Science Foundation predoctoral fellowship (to SMG).

SUZANNEM. GARSZC~YNSKIANDJ. R. EDWARDS

250

Resume--La levansucrase du Streptococcus salivarius (SS2) est une enzyme constitutive, I& a la cellule. Quand le SS2 est cultive dans I’absence de la sucrose, ni la puromycine ni le chloramphtnicol n’inhibe la synthitse du levan, a moins qu’on ajoute la sucrose au milieu. La levansucrase est trouv&. assock% avec la fraction de la paroi de la cellule, suivant la scission cellulaire et la centrifugation differentielle. L’enzyme peut &tre rellthee de la fraction de la paroi cellulaire et de cellules entieres intactes par I’extraction avec du chlorure de lithium 8M. La levansucrase isolee a un pH optimum de 5,6 et est indtpendante de cations bivalents pour I’activite.

Zusanunenfassung-Die Levansaccharase des Streptococcus salivarius (SS2) ist ein grundlegendes, zellengebundenes Enzym. Wenn SS2 ohne Saccharose geztichtet wird, hemmen weder Puromycin noch Cloromycetin bei Zufiigen von Saccharose zu dem Mittel die Synthese von Levan. Es wird festgestellt, dass die Saccharase nach zellullirer Spaltung und Differentialzentrifungierung mit dem Zellenwandfragment verbunden ist. Das Enzym kann durch Extraktion mit 8 M Lithiumchlorid von dem Zellenwandfragment und von unversehrten ganzen Zellen freigegeben werden. Die abgesonderte Levansaccharase hat ein optimales pH von 5,6 und ist fur Aktivitlit von zweiwertigen Kationen unabhangig. REFERENCES AGRAWAL, B. B. L. and GOLDSTEIN,I. J. 1967. Isolation of concanavalin A by specific adsorption

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