APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1977, p. 115-119 Copyright © 1977 American Society for Microbiology
Effect of Tween 80
on
Vol. 34, No. 2 Printed in U.S.A.
Glucosyltransferase Production in
Streptococcus mutans YOSHINORI UMESAKI,* YASUO KAWAI, AND MASAHIKO MUTAI Yakult Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo, Japan
Received for publication 16 February 1977
Glucan production from sucrose by Streptococcus mutans OMZ 176 was stimulated approximately threefold in the presence of 0.1% Tween 80. When OMZ 176 was grown in a medium containing glucose, the glucosyltransferase level in the medium was also increased about fivefold in the presence of 0. 1% Tween 80. The glucosyltransferase level increased in proportion to the logarithm of the concentration of Tween 80 in the glucose medium. Tween 80 affected neither bacterial growth nor the activity of glucosyltransferase. The appearance of glucosyltransferase in the glucose medium was inhibited immediately by chloramphenicol and actinomycin D and, after a lag, by rifampin as well. It was observed that the fatty acid composition of the cells grown with Tween 80 was altered. These results suggest that Tween 80 stimulates glucosyltransferase synthesis either directly, or indirectly by promoting glucosyltransferase secretion.
The synthesis of water-insoluble glucan from sucrose by Streptococcus mutans has been considered to affect the development of dental plaque in the presence of sucrose (8, 9). Although many studies on the properties of the glucosyltransferase of S. mutans have been carried out (6, 7, 10), there is little information on glucosyltransferase formation and secretion. In the present study, it was found that the level of glucosyltransferase in the medium was markedly elevated by the addition of surfactants such as Tween 80 (polyethylene glycol sorbitan monooleate) and Emanon 4115 (polyethylene glycol monooleate). The enhancement of enzyme production by nonionic surfactants has been reported in many microorganisms (19, 23). Cellulase production in many fungi and pullulanase production inAerobacter aerogenes are increased 20-fold and 1.5-fold, respectively, by the addition of 0.1% Tween 80 (5). It is apparent that surfactants play a role in the production and/or secretion of microbial enzymes. Surfactants are contained in many foods, and their effects on glucosyltransferase activity and formation are important for dental health considerations. In this paper, we describe the enhancement of glucosyltransferase formation by S. mutans in the presence of Tween 80. MATERIALS AND METHODS Bacterial strain. S. mutans OMZ 176 was obtained from S. Kotani (Department of Microbiology, Dental School, Osaka University, Osaka, Japan). The serotype of this strain has been described by Perch et al. (18).
Chemicals. Tween 80 was purchased from Wako Pure Chemical Industries (Osaka, Japan); Emanon 4115 was from Kao-Atlas (Tokyo, Japan); clinical dextran was from E. Merck (Darmstadt, Germany); rifampin and actinomycin D were from Sigma Chemical Co. (St. Louis, Mo.); and chloramphenicol was from Sankyo Co. (Tokyo, Japan). Growth conditions. Cells were grown in Rogosa medium (4) containing 5% sucrose (sucrose medium) or 1% glucose (glucose medium) at 37°C. Preculture was carried out in glucose medium without Tween 80 for about 15 h. Growth was determined with a Klett-Summerson colorimeter equipped with a no. 66 filter. Assay of glucan. Glucan was isolated according to the method of Jeanes (13). The culture broth of the sucrose medium was centrifuged, and the precipitate was washed with 50 mM phosphate buffer, pH 6.0. KOH, 1 N, was added to solubilize the alkalisoluble polymer. After standing for 30 min at room temperature, the solution was centrifuged again to remove the cells and alkali-insoluble polymer. The supernatant thus obtained was neutralized with concentrated acetic acid and mixed with 2 volumes of ethanol. The glucan precipitate was isolated by centrifugation. The sugar content of suspensions containing alkali-soluble glucan was estimated by the phenol-sulfuric acid method (3). Assay of glucosyltransferase. The enzyme activity was determined by a modification of Hehre's assay method (6). The standard reaction mixture contained the following components: 0.1 M sucrose, 1 ml; 10 mg of clinical dextran per ml (average molecular weight, 17,700), 0.2 ml; and enzyme solution, 0.2 ml. Each component was dissolved in phosphate buffer (pH 6.0). The clinical dextran was added as a glucosyl acceptor. Enzyme solution was prepared by centrifuging culture broth at 10,000 x g for 5 min and dialyzing the supernatant against 115
116
UMESAKI, KAWAI, AND MUTAI
phosphate buffer. To estimate cell-bound glucosyltransferase, the cells were precipitated by centrifugation at 10,000 x g, washed with the phosphate buffer, and suspended in the same buffer. This suspension was used as the enzyme preparation. A sonified sample was prepared by disruption of the cells with a Kubota Insonator (model 200M, 9 kHz) at 200 W for 10 min. The reaction mixture was incubated at 37°C for 60 min, and the reaction was stopped by incubation in boiling water for 5 min. Reducing sugar released was estimated by the method of Somogyi (22) and was expressed in terms of glucose. One unit of enzyme activity was defined as that amount of glucosyltransferase which released 1.0 ,umol of reducing sugar per min from sucrose. In preliminary studies with media containing glucose and Tween 80, it had been found that the culture of strain OMZ 176 had no levansucrase and very low invertase activities. Therefore, the enzyme activity estimated in this study was attributed to glucosyltransferase exclusively. Tween 80 did not affect the enzyme activity at concentrations of up to 5 mg/ml. Analysis of fatty acid composition of the cells. Fatty acids of the cells were identified and determined by gas-liquid chromatography. Cells harvested at the late-logarithmic phase were washed three times with the phosphate buffer. Lipids were extracted from the cells according to the method of Bligh and Dyer (1) and were methylated with 5% HCl-methanol at 100°C for 60 min. The samples were injected into a Shimadzu GC-5A gas-liquid chromatograph equipped with a flame ionization detector. A glass column (2 mm by 2 m) packed with 10% polyethylene glycol succinate on Chromosorb W (Analytical Engineering Laboratories, Inc., Hamden, Conn.; 60 to 80 mesh) was used at 155°C, with a flow rate of N2 gas of 40 ml/min. Methylesters of known fatty acids prepared in the same way as the test samples were used as standards.
RESULTS Glucan production in the sucrose medium. The time course of glucan production by S. mutans OMZ 176 grown in a sucrose medium is shown in Fig. 1. Glucan production was greatly stimulated in the presence of Tween 80. The glucan was water insoluble and aggregated with the cells. It was separated from the cells and alkali-insoluble polymer with 1 N KOH. The turbidity of 1 N KOH-insoluble materials in the culture broth was the same in the absence and presence of Tween 80. This suggests that Tween 80 has no effect on the growth of OMZ 176 in the sucrose medium. Glucosyltransferase production in the glucose medium. The time course of glucosyltransferase production in the medium in the presence and absence of Tween 80 (1 mg/ml) is shown in Fig. 2. Although Tween 80 had no effect on bacterial growth, the level of glucosyltransferase in the medium was increased about
APPL. ENVIRON. MICROBIOL.
Time ( hr) FIG. 1. Glucan production in a sucrose medium. A preculture of S. mutans cells in glucose medium without Tween 80 was transferred with a 1 % inoculum to sucrose medium in the presence of (0) or in the absence of (0) 1 mg of Tween 80 per ml. Cultivation was carried out at 37°C.
i
200 -( VI
00
-
1; 50 t
32
4, 908
0
O0
(B)
.14
.04
2 I
.
.,,6,
(D
X
-
9/4 '
4 2 6 Time (hr) Time(hr) FIG. 2. Glucosyltransferase production in a glucose medium. A preculture of S. mutans in glucose medium without Tween 80 was transferred with a 5% inoculum to glucose medium with (a) or without (0) 1 mg of Tween 80 per ml. Other cultivation conditions were as same as in Fig. 1. (A) Bacterial growth; (B) glucosyltransferase production. 0
fivefold by the surfactant. Both the level of glucosyltransferase and the turbidity of the culture broth increased logarithmically between 2 and 4 h, after a lag phase of 2 h. The production of glucosyltransferase in the medium was closely associated with the growth of the bacteria. Effect of Tween 80 concentration on the enzyme level in the medium. The stimulation of the production of glucosyltransferase was dependent on the concentration ofTween 80 in the medium (Fig. 3). Under the conditions used, the enzyme activity that developed in the culture was more or less proportional to the logarithm of the concentration of added surfactant. Further, Tween 80 did not affect the activity of glucosyltransferase (data not shown). Accordingly, the increase in the activity in the medium reflects an increase in the enzyme level.
VOL. 34, 1977
GLUCOSYLTRANSFERASE PRODUCTION IN S. MUTANS
80, a decrease in n-myristate and an increase in octadecenoate were observed. With Emanon 4115, which enhanced glucosyltransferase production to the same extent as Tween 80, the increase in octadecenoate content in the cells was not as great as with Tween 80.
.-1
.I.. .04
1 y
(3
~
117
m-l5 .2 .5o0 too Concentration of Tween 80( mg/rni)
I
5DO
FIG. 3. Effect of the concentration of Tween 80 on glucosyltransferase production. The cultivation was carried out in glucose medium with various concentrations of Tween 80. The activity ofglucosyltransferase was assayed on cell-free culture fluid at late-log phase (at 4 h).
Cell-bound glucosyltransferase. When S. mutans cells are grown with glucose, almost all of the glucosyltransferase activity is released into the culture medium (6, 20). In the case of strain OMZ 176, the glucosyltransferase activity of washed cells was very low compared with that in the culture supernatant, regardless of the presence or absence of Tween 80 (Table 1). When preparations disrupted by sonication were tested, no glucosyltransferase activity was detected in the cell debris, although slight activity was found in the supernatant of the sonicated cells. The addition of Tween 80 did not affect the enzyme activity of the sonicated supernatant. For estimation of the intracellular level of the enzyme, the enzyme activity was determined from the difference between total reducing sugar and 1-glucose (21), because the cells have high invertase activity. Robrish et al. also observed no substantial difference between a broken-cell preparation and the unbroken cell mass (20). Sensitivity of glucosyltransferase production to antibiotics. The effect of antibiotics such as chloramphenicol, actinomycin D, and rifampin on glucosyltransferase production with Tween 80 and glucose was tested. Each antibiotic was added to the culture medium at the phase of maximum production of the enzyme. Chloramphenicol and actinomycin D stopped any further increase in glucosyltransferase in the medium immediately, whereas rifampin showed a slight lag period (Fig. 4). Fatty acid composition of the cells. The effect of surfactant on the fatty acid composition of the cells grown to the late-logarithmic phase was investigated (Table 2). The major fatty acids contained in the cells were n-myristate, n-palmitate, n-stearate, and octadecenoate. n-Palmitate accounted for about one-half of the total fatty acids. In the presence of Tween
DISCUSSION In this study, it was found that the addition of Tween 80 to glucose medium enhanced the level of glucosyltransferase in the culture broth of S. mutans (Fig. 2). The effects of surfaceactive compounds on glucosyltransferase (12) and in vitro plaque formation (24) ofS. mutans have been reported. Tween 80 (1%) was reported not to inhibit or activate glucosyltransferase activity, as was found in the present study. The observed effect ofTween 80 on glucosyltransferase level may have various causes. The possibility that some factors that stimulate TABLz 1. Distribution ofglucosyltransferase activity in cultures of S. mutans OMZ 176a Glucosyltransferase activity Tween 80 added (mg/mi)
0
(U/ml of culture) Culture me- Washed cells dium (A)
(B)
0.0201
0.0016
8.0
1 0.0705 0.0053 7.5 Cells from cultures at log phase were sedimented by centrifugation, washed with phosphate buffer, and suspended in the original volume of the buffer. The medium was dialyzed against the buffer. The activity was measured and expressed as described in Materials and Methods. a
200 1
~50.
(A)
'2
E
4
J.2 (B) J
.C~~~~~~~~~~~U 10 6 C2~~~~.06220~~~~~~~~~. 6' -02 4 06 Time (hr) Tirne(hr) FIG. 4. Effect of antibiotics on glucosyltransferase production in the medium. Antibiotics were added to the culture broth 3 h after cultivation with 1 mg of Tween 80 per ml. Symbols: A, actinomycin D (20 pg/ ml); A, chloramphenicol (100 pg/ml); 0, rifampin (50 pg/ml); 0, none. Arrow indicates the time of the addition of the antibiotics. (A) Bacterial growth; (B) glucosyltransferase production.
118
UMESAKI, KAWAI, AND MUTAI
APPL. ENVIRON. MICROBIOL.
TABLE 2. Fatty acid composition of S. mutans cells grown with Tween 80 or Emanon 4115a Fatty acid composition (%) of cells grown with:
Fatty acid iso-Myristate
Emanon 4115 (0.5 0.1 mg/ml 1.0 mg/ml mg/ml) Tr Tr Tr Tween 80 at:
None Tr
(14:0)b n-Myristate (14:0) iso-Palmitate (16:0) n-Palmitate (16:0) Palmitoleate (16:1) n-Stearate (18:0) Octadecenoate (18:1) Eicosenoate (20:1)
13.8
7.5
3.5
5.5
Tr
1.3
Tr
Tr
45.0
46.0
41.3
56.0
5.3
1.7
2.9
3.0
8.3
13.4
5.5
9.6
18.6
20.6
35.8
20.8
6.6
7.6
9.9
3.8
a Fatty acids were extracted from the cells grown with each surfactant and analyzed by using gas-liquid chromatograph. The figures are expressed as percentages, and Tr indicates that the value is below 1%. bNumber preceding colon indicates number of carbons; number after colon designates degree of unsaturation.
glucosyltransferase activity or convert the enzyme from a latent form to an active form are produced in the presence of Tween 80 was excluded by our finding that cell lysate and the culture supernatant with Tween 80 did not enhance the activity of the supernatant from a culture without Tween 80. Kuramitsu has demonstrated that most cellassociated dextransucrase from glucose-grown cells of S. mutans FA-1 is eluted with hypertonic salt solution (15). In the case of strain OMZ 176, the fraction of glucosyltransferase activity bound to the cells was very low compared with the total activity, including that in the medium (Table 1). When washed cells grown to the logarithmic phase without Tween 80 were incubated with Tween 80 (1 mg/ml) in phosphate buffer, no increase in the enzyme activity was observed in the buffer. Therefore, Tween 80 probably does not cause the solubilization of cell-bound glucosyltransferase into the medium. On the addition of actinomycin D and chloramphenicol, the increase in glucosyltransferase activity stopped immediately (Fig. 4). In Bacillus stearothermophilus (25) and Bacillus subtilis (14), the possibility that a-amylase precursor accumulates in the cell could be excluded by the immediate inhibition of enzyme formation by antibiotics such as chloramphenicol and actinomycin D. If Tween 80 acts to release enzyme accumulated within the cell or associated
with the cell, or if it converts the enzyme from an inactive form to an active form, the enzyme should continue to appear for some time after the cessation of enzyme synthesis. Thus, it appears that Tween 80 enhances the process of synthesis of glucosyltransferase, either directly or indirectly. The stimulation of enzyme production was not specific to Tween 80, because other nonionic surfactants such as Emanon 4115 and Tween 60 were also effective. The interaction of the surfactants with cells probably affects the structure and function of the cell membrane. It has been reported that permeabilities to various compounds are increased in Pseudomonas aeruginosa cells (2) and mycobacterial cells (17) grown in a medium containing Tween 80. In the case of S. mutans OMZ 176, the pattern of fatty acid composition of the cells was altered by Tween 80. In the presence of Tween 80, a decrease in the amount of n-palmitate and an increase of octadecenoate were observed, suggesting an increase in the fluidity of lipids. Although this result may not be relevant to the stimulatory effect of Tween 80 on the synthesis of glucosyltransferase, the membrane function of the cells might be altered as a result of the change in fatty acid composition such that synthesis of the enzyme is favored. Reese et al. suggested various mechanisms for the enhancement of enzyme yields by surfactants (5, 13). One explanation for the increased yield is that the leakiness of the cell membrane is increased. An increased rate of glucosyltransferase secretion also might result in increased synthesis. Another possibility is that the surfactant protects the enzyme from inactivation. However, in the case of the glucosyltransferase of S. mutans, Tween 80 did not affect its stability. The former mechanism may account for our observations. It is also possible that extracellular compounds that stimulate the process of synthesis of the enzyme become more permeable to the cell. Investigation of the mechanism by which Tween 80 accelerates glucosyltransferase formation is now in progress. ACKNOWLEDGMENTS We thank T. Enatsu, Department of Fermentation Technology, Osaka University, Osaka, Japan, and M. Akamatsu, National Institute of Health, Tokyo, Japan, for valuable suggestions and encouragement, and T. Yokokura of our laboratory for his advice during the preparation of the manuscript. LITERATURE CITED 1. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 2. Brown, M. R. W., and R. M. E. Richards. 1964. Effect
VOL. 34, 1977
3.
4.
5.
6.
GLUCOSYLTRANSFERASE PRODUCTION IN S. MUTANS
of polysorbate (Tween) 80 on the resistance of Pseudomonas aeruginosa to chemical inactivation. J. Pharmacol. 16(Suppl.):51-55. Dubois, M. K., A. Gilles, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350356. Efthymiou, C., and P. A. Hansen. 1962. An antigenic analysis of Lactobacillus acidophilus. J. Infect. Dis. 110:258-267. Faith, W. T., C. E. Neubeck, and E T. Reese. 1971. Production and applications of enzymes. Adv. Biochem. Eng. 1:79-111. Fukui, K., Y. Fukui, and T. Moriyama. 1974. Purification and properties of dextransucrase and invertase from Streptococcus mutans. J. Bacteriol. 118:796-
804. 7. Germaine, G. R., A. M. Chludzinski, and C. F. Schachtele. 1974. Streptococcus mutans dextransucrase: requirement for primer dextran. J. Bacteriol. 120:287294. 8. Gibbons, R. J., and S. B. Banghart. 1967. Synthesis of extracellular dextran by cariogenic bacteria and its presence in human dental plaque. Arch. Oral Biol. 12:11-24. 9. Gibbons, R. J., and R. J. Fitzgerald. 1969. Dextran induced agglutination of Streptococcus mutans and its potential role in the formation of microbial dental plaques. J. Bacteriol. 98:341-346. 10. Gugenheim, B., and E. Newbrun. 1969. Extracellular glucosyltransferase activity of an HS strain of Streptococcus mutans. Helv. Odontol. Acta 13:84-97. 11. Hehre, E. J. 1955. Polysaccharide synthesis from disaccharides. I. Dextransucrase. Methods Enzymol. 1:179-184. 12. Jablonski, W. M., and J. A. Hayashi. 1970. Inhibition of extracellular streptococcal enzymes. J. Dent. Res. 49:178. 13. Jeanes, A. 1965. Production of dextran from L. mesenteroides NRRL B-523. Methods Carbohydr. Chem. 5:124-127. 14. Kinoshita, S., H. Okada, and G. Terui. 1968. On the
15. 16. 17.
18.
19. 20.
21.
22. 23.
24. 25.
119
nature of a-amylase forming system in Bacillus subtilis. Stability of the mRNA for a-amylase. J. Ferment. Technol. 46:427-436. Kuramitsu, H. K. 1974. Characterization of cell-associated dextransucrase activity from glucose-grown cells of Streptococus mutans. Infect. Immun. 10:227-235. McCabe, M. M., and E. E. Smith. 1973. Origin of the cell-associated dextransucrase of Streptococcus mutans. Infect. Immun. 7:829-838. Paunesen, E., A. Ciolac-Negoescu, and G. Piscu. 1964. The effect of Tween 80 and penicillin on the physiochemical properties of the cell wall in mycobacteria. Acad. Repub. Pop. Rom. Stud. Cercet. Biochem. 7:184-190. Perch, B., E. Kjems, and T. Ravn. 1974. Biochemical and serological properties of Streptococcus mutans from various human and animal sources. Acta Pathol. Microbiol. Scand. Sect. B 82:357-370. Reese, E. T., and A. Maguire. 1969. Surfactants as stimulants of enzyme production by microorganisms. Appl. Microbiol. 17:242-245. Robrish, S. A., W. Reid, and M. I. Krichevsky. 1972. Distribution of enzymes forming polysaccharide from sucrose and the composition of extracellular polysaccharide synthesized by Streptococcus mutans. Appl. Microbiol. 24:184-190. Scales, W. R., L. W. Long, and J. R. Edwards. 1975. Purification of a glucosyltransferase complex from the culture broth of Streptococcus mutans FA-1. Carbohydr. Res. 42:325-338. Somogyi, M. 1945. A new reagent for the determination of sugars. J. Biol. Chem. 160:61-73. Takahashi, J., G. Abekawa, and K. Yamada. 1960. Effect of non-ionic surface active agents on mycelial form and amylase production of A. niger. Nippon Nogei Kagaku Kaishi 34:1043-1045. Turesky, S., I. Glickman, and R. Sandberg. 1972. In vitro chemical inhibition of plaque formation. J. Periodontol. 43:263-269. Welker, N. E., and L. L. Campbell. 1963. De novo synthesis of a-amylase by Bacillus stearothermophilus. J. Bacteriol. 86:1202-1210.
Effect of Tween 80
on
Vol. 34, No. 2 Printed in U.S.A.
Glucosyltransferase Production in
Streptococcus mutans YOSHINORI UMESAKI,* YASUO KAWAI, AND MASAHIKO MUTAI Yakult Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo, Japan
Received for publication 16 February 1977
Glucan production from sucrose by Streptococcus mutans OMZ 176 was stimulated approximately threefold in the presence of 0.1% Tween 80. When OMZ 176 was grown in a medium containing glucose, the glucosyltransferase level in the medium was also increased about fivefold in the presence of 0. 1% Tween 80. The glucosyltransferase level increased in proportion to the logarithm of the concentration of Tween 80 in the glucose medium. Tween 80 affected neither bacterial growth nor the activity of glucosyltransferase. The appearance of glucosyltransferase in the glucose medium was inhibited immediately by chloramphenicol and actinomycin D and, after a lag, by rifampin as well. It was observed that the fatty acid composition of the cells grown with Tween 80 was altered. These results suggest that Tween 80 stimulates glucosyltransferase synthesis either directly, or indirectly by promoting glucosyltransferase secretion.
The synthesis of water-insoluble glucan from sucrose by Streptococcus mutans has been considered to affect the development of dental plaque in the presence of sucrose (8, 9). Although many studies on the properties of the glucosyltransferase of S. mutans have been carried out (6, 7, 10), there is little information on glucosyltransferase formation and secretion. In the present study, it was found that the level of glucosyltransferase in the medium was markedly elevated by the addition of surfactants such as Tween 80 (polyethylene glycol sorbitan monooleate) and Emanon 4115 (polyethylene glycol monooleate). The enhancement of enzyme production by nonionic surfactants has been reported in many microorganisms (19, 23). Cellulase production in many fungi and pullulanase production inAerobacter aerogenes are increased 20-fold and 1.5-fold, respectively, by the addition of 0.1% Tween 80 (5). It is apparent that surfactants play a role in the production and/or secretion of microbial enzymes. Surfactants are contained in many foods, and their effects on glucosyltransferase activity and formation are important for dental health considerations. In this paper, we describe the enhancement of glucosyltransferase formation by S. mutans in the presence of Tween 80. MATERIALS AND METHODS Bacterial strain. S. mutans OMZ 176 was obtained from S. Kotani (Department of Microbiology, Dental School, Osaka University, Osaka, Japan). The serotype of this strain has been described by Perch et al. (18).
Chemicals. Tween 80 was purchased from Wako Pure Chemical Industries (Osaka, Japan); Emanon 4115 was from Kao-Atlas (Tokyo, Japan); clinical dextran was from E. Merck (Darmstadt, Germany); rifampin and actinomycin D were from Sigma Chemical Co. (St. Louis, Mo.); and chloramphenicol was from Sankyo Co. (Tokyo, Japan). Growth conditions. Cells were grown in Rogosa medium (4) containing 5% sucrose (sucrose medium) or 1% glucose (glucose medium) at 37°C. Preculture was carried out in glucose medium without Tween 80 for about 15 h. Growth was determined with a Klett-Summerson colorimeter equipped with a no. 66 filter. Assay of glucan. Glucan was isolated according to the method of Jeanes (13). The culture broth of the sucrose medium was centrifuged, and the precipitate was washed with 50 mM phosphate buffer, pH 6.0. KOH, 1 N, was added to solubilize the alkalisoluble polymer. After standing for 30 min at room temperature, the solution was centrifuged again to remove the cells and alkali-insoluble polymer. The supernatant thus obtained was neutralized with concentrated acetic acid and mixed with 2 volumes of ethanol. The glucan precipitate was isolated by centrifugation. The sugar content of suspensions containing alkali-soluble glucan was estimated by the phenol-sulfuric acid method (3). Assay of glucosyltransferase. The enzyme activity was determined by a modification of Hehre's assay method (6). The standard reaction mixture contained the following components: 0.1 M sucrose, 1 ml; 10 mg of clinical dextran per ml (average molecular weight, 17,700), 0.2 ml; and enzyme solution, 0.2 ml. Each component was dissolved in phosphate buffer (pH 6.0). The clinical dextran was added as a glucosyl acceptor. Enzyme solution was prepared by centrifuging culture broth at 10,000 x g for 5 min and dialyzing the supernatant against 115
116
UMESAKI, KAWAI, AND MUTAI
phosphate buffer. To estimate cell-bound glucosyltransferase, the cells were precipitated by centrifugation at 10,000 x g, washed with the phosphate buffer, and suspended in the same buffer. This suspension was used as the enzyme preparation. A sonified sample was prepared by disruption of the cells with a Kubota Insonator (model 200M, 9 kHz) at 200 W for 10 min. The reaction mixture was incubated at 37°C for 60 min, and the reaction was stopped by incubation in boiling water for 5 min. Reducing sugar released was estimated by the method of Somogyi (22) and was expressed in terms of glucose. One unit of enzyme activity was defined as that amount of glucosyltransferase which released 1.0 ,umol of reducing sugar per min from sucrose. In preliminary studies with media containing glucose and Tween 80, it had been found that the culture of strain OMZ 176 had no levansucrase and very low invertase activities. Therefore, the enzyme activity estimated in this study was attributed to glucosyltransferase exclusively. Tween 80 did not affect the enzyme activity at concentrations of up to 5 mg/ml. Analysis of fatty acid composition of the cells. Fatty acids of the cells were identified and determined by gas-liquid chromatography. Cells harvested at the late-logarithmic phase were washed three times with the phosphate buffer. Lipids were extracted from the cells according to the method of Bligh and Dyer (1) and were methylated with 5% HCl-methanol at 100°C for 60 min. The samples were injected into a Shimadzu GC-5A gas-liquid chromatograph equipped with a flame ionization detector. A glass column (2 mm by 2 m) packed with 10% polyethylene glycol succinate on Chromosorb W (Analytical Engineering Laboratories, Inc., Hamden, Conn.; 60 to 80 mesh) was used at 155°C, with a flow rate of N2 gas of 40 ml/min. Methylesters of known fatty acids prepared in the same way as the test samples were used as standards.
RESULTS Glucan production in the sucrose medium. The time course of glucan production by S. mutans OMZ 176 grown in a sucrose medium is shown in Fig. 1. Glucan production was greatly stimulated in the presence of Tween 80. The glucan was water insoluble and aggregated with the cells. It was separated from the cells and alkali-insoluble polymer with 1 N KOH. The turbidity of 1 N KOH-insoluble materials in the culture broth was the same in the absence and presence of Tween 80. This suggests that Tween 80 has no effect on the growth of OMZ 176 in the sucrose medium. Glucosyltransferase production in the glucose medium. The time course of glucosyltransferase production in the medium in the presence and absence of Tween 80 (1 mg/ml) is shown in Fig. 2. Although Tween 80 had no effect on bacterial growth, the level of glucosyltransferase in the medium was increased about
APPL. ENVIRON. MICROBIOL.
Time ( hr) FIG. 1. Glucan production in a sucrose medium. A preculture of S. mutans cells in glucose medium without Tween 80 was transferred with a 1 % inoculum to sucrose medium in the presence of (0) or in the absence of (0) 1 mg of Tween 80 per ml. Cultivation was carried out at 37°C.
i
200 -( VI
00
-
1; 50 t
32
4, 908
0
O0
(B)
.14
.04
2 I
.
.,,6,
(D
X
-
9/4 '
4 2 6 Time (hr) Time(hr) FIG. 2. Glucosyltransferase production in a glucose medium. A preculture of S. mutans in glucose medium without Tween 80 was transferred with a 5% inoculum to glucose medium with (a) or without (0) 1 mg of Tween 80 per ml. Other cultivation conditions were as same as in Fig. 1. (A) Bacterial growth; (B) glucosyltransferase production. 0
fivefold by the surfactant. Both the level of glucosyltransferase and the turbidity of the culture broth increased logarithmically between 2 and 4 h, after a lag phase of 2 h. The production of glucosyltransferase in the medium was closely associated with the growth of the bacteria. Effect of Tween 80 concentration on the enzyme level in the medium. The stimulation of the production of glucosyltransferase was dependent on the concentration ofTween 80 in the medium (Fig. 3). Under the conditions used, the enzyme activity that developed in the culture was more or less proportional to the logarithm of the concentration of added surfactant. Further, Tween 80 did not affect the activity of glucosyltransferase (data not shown). Accordingly, the increase in the activity in the medium reflects an increase in the enzyme level.
VOL. 34, 1977
GLUCOSYLTRANSFERASE PRODUCTION IN S. MUTANS
80, a decrease in n-myristate and an increase in octadecenoate were observed. With Emanon 4115, which enhanced glucosyltransferase production to the same extent as Tween 80, the increase in octadecenoate content in the cells was not as great as with Tween 80.
.-1
.I.. .04
1 y
(3
~
117
m-l5 .2 .5o0 too Concentration of Tween 80( mg/rni)
I
5DO
FIG. 3. Effect of the concentration of Tween 80 on glucosyltransferase production. The cultivation was carried out in glucose medium with various concentrations of Tween 80. The activity ofglucosyltransferase was assayed on cell-free culture fluid at late-log phase (at 4 h).
Cell-bound glucosyltransferase. When S. mutans cells are grown with glucose, almost all of the glucosyltransferase activity is released into the culture medium (6, 20). In the case of strain OMZ 176, the glucosyltransferase activity of washed cells was very low compared with that in the culture supernatant, regardless of the presence or absence of Tween 80 (Table 1). When preparations disrupted by sonication were tested, no glucosyltransferase activity was detected in the cell debris, although slight activity was found in the supernatant of the sonicated cells. The addition of Tween 80 did not affect the enzyme activity of the sonicated supernatant. For estimation of the intracellular level of the enzyme, the enzyme activity was determined from the difference between total reducing sugar and 1-glucose (21), because the cells have high invertase activity. Robrish et al. also observed no substantial difference between a broken-cell preparation and the unbroken cell mass (20). Sensitivity of glucosyltransferase production to antibiotics. The effect of antibiotics such as chloramphenicol, actinomycin D, and rifampin on glucosyltransferase production with Tween 80 and glucose was tested. Each antibiotic was added to the culture medium at the phase of maximum production of the enzyme. Chloramphenicol and actinomycin D stopped any further increase in glucosyltransferase in the medium immediately, whereas rifampin showed a slight lag period (Fig. 4). Fatty acid composition of the cells. The effect of surfactant on the fatty acid composition of the cells grown to the late-logarithmic phase was investigated (Table 2). The major fatty acids contained in the cells were n-myristate, n-palmitate, n-stearate, and octadecenoate. n-Palmitate accounted for about one-half of the total fatty acids. In the presence of Tween
DISCUSSION In this study, it was found that the addition of Tween 80 to glucose medium enhanced the level of glucosyltransferase in the culture broth of S. mutans (Fig. 2). The effects of surfaceactive compounds on glucosyltransferase (12) and in vitro plaque formation (24) ofS. mutans have been reported. Tween 80 (1%) was reported not to inhibit or activate glucosyltransferase activity, as was found in the present study. The observed effect ofTween 80 on glucosyltransferase level may have various causes. The possibility that some factors that stimulate TABLz 1. Distribution ofglucosyltransferase activity in cultures of S. mutans OMZ 176a Glucosyltransferase activity Tween 80 added (mg/mi)
0
(U/ml of culture) Culture me- Washed cells dium (A)
(B)
0.0201
0.0016
8.0
1 0.0705 0.0053 7.5 Cells from cultures at log phase were sedimented by centrifugation, washed with phosphate buffer, and suspended in the original volume of the buffer. The medium was dialyzed against the buffer. The activity was measured and expressed as described in Materials and Methods. a
200 1
~50.
(A)
'2
E
4
J.2 (B) J
.C~~~~~~~~~~~U 10 6 C2~~~~.06220~~~~~~~~~. 6' -02 4 06 Time (hr) Tirne(hr) FIG. 4. Effect of antibiotics on glucosyltransferase production in the medium. Antibiotics were added to the culture broth 3 h after cultivation with 1 mg of Tween 80 per ml. Symbols: A, actinomycin D (20 pg/ ml); A, chloramphenicol (100 pg/ml); 0, rifampin (50 pg/ml); 0, none. Arrow indicates the time of the addition of the antibiotics. (A) Bacterial growth; (B) glucosyltransferase production.
118
UMESAKI, KAWAI, AND MUTAI
APPL. ENVIRON. MICROBIOL.
TABLE 2. Fatty acid composition of S. mutans cells grown with Tween 80 or Emanon 4115a Fatty acid composition (%) of cells grown with:
Fatty acid iso-Myristate
Emanon 4115 (0.5 0.1 mg/ml 1.0 mg/ml mg/ml) Tr Tr Tr Tween 80 at:
None Tr
(14:0)b n-Myristate (14:0) iso-Palmitate (16:0) n-Palmitate (16:0) Palmitoleate (16:1) n-Stearate (18:0) Octadecenoate (18:1) Eicosenoate (20:1)
13.8
7.5
3.5
5.5
Tr
1.3
Tr
Tr
45.0
46.0
41.3
56.0
5.3
1.7
2.9
3.0
8.3
13.4
5.5
9.6
18.6
20.6
35.8
20.8
6.6
7.6
9.9
3.8
a Fatty acids were extracted from the cells grown with each surfactant and analyzed by using gas-liquid chromatograph. The figures are expressed as percentages, and Tr indicates that the value is below 1%. bNumber preceding colon indicates number of carbons; number after colon designates degree of unsaturation.
glucosyltransferase activity or convert the enzyme from a latent form to an active form are produced in the presence of Tween 80 was excluded by our finding that cell lysate and the culture supernatant with Tween 80 did not enhance the activity of the supernatant from a culture without Tween 80. Kuramitsu has demonstrated that most cellassociated dextransucrase from glucose-grown cells of S. mutans FA-1 is eluted with hypertonic salt solution (15). In the case of strain OMZ 176, the fraction of glucosyltransferase activity bound to the cells was very low compared with the total activity, including that in the medium (Table 1). When washed cells grown to the logarithmic phase without Tween 80 were incubated with Tween 80 (1 mg/ml) in phosphate buffer, no increase in the enzyme activity was observed in the buffer. Therefore, Tween 80 probably does not cause the solubilization of cell-bound glucosyltransferase into the medium. On the addition of actinomycin D and chloramphenicol, the increase in glucosyltransferase activity stopped immediately (Fig. 4). In Bacillus stearothermophilus (25) and Bacillus subtilis (14), the possibility that a-amylase precursor accumulates in the cell could be excluded by the immediate inhibition of enzyme formation by antibiotics such as chloramphenicol and actinomycin D. If Tween 80 acts to release enzyme accumulated within the cell or associated
with the cell, or if it converts the enzyme from an inactive form to an active form, the enzyme should continue to appear for some time after the cessation of enzyme synthesis. Thus, it appears that Tween 80 enhances the process of synthesis of glucosyltransferase, either directly or indirectly. The stimulation of enzyme production was not specific to Tween 80, because other nonionic surfactants such as Emanon 4115 and Tween 60 were also effective. The interaction of the surfactants with cells probably affects the structure and function of the cell membrane. It has been reported that permeabilities to various compounds are increased in Pseudomonas aeruginosa cells (2) and mycobacterial cells (17) grown in a medium containing Tween 80. In the case of S. mutans OMZ 176, the pattern of fatty acid composition of the cells was altered by Tween 80. In the presence of Tween 80, a decrease in the amount of n-palmitate and an increase of octadecenoate were observed, suggesting an increase in the fluidity of lipids. Although this result may not be relevant to the stimulatory effect of Tween 80 on the synthesis of glucosyltransferase, the membrane function of the cells might be altered as a result of the change in fatty acid composition such that synthesis of the enzyme is favored. Reese et al. suggested various mechanisms for the enhancement of enzyme yields by surfactants (5, 13). One explanation for the increased yield is that the leakiness of the cell membrane is increased. An increased rate of glucosyltransferase secretion also might result in increased synthesis. Another possibility is that the surfactant protects the enzyme from inactivation. However, in the case of the glucosyltransferase of S. mutans, Tween 80 did not affect its stability. The former mechanism may account for our observations. It is also possible that extracellular compounds that stimulate the process of synthesis of the enzyme become more permeable to the cell. Investigation of the mechanism by which Tween 80 accelerates glucosyltransferase formation is now in progress. ACKNOWLEDGMENTS We thank T. Enatsu, Department of Fermentation Technology, Osaka University, Osaka, Japan, and M. Akamatsu, National Institute of Health, Tokyo, Japan, for valuable suggestions and encouragement, and T. Yokokura of our laboratory for his advice during the preparation of the manuscript. LITERATURE CITED 1. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 2. Brown, M. R. W., and R. M. E. Richards. 1964. Effect
VOL. 34, 1977
3.
4.
5.
6.
GLUCOSYLTRANSFERASE PRODUCTION IN S. MUTANS
of polysorbate (Tween) 80 on the resistance of Pseudomonas aeruginosa to chemical inactivation. J. Pharmacol. 16(Suppl.):51-55. Dubois, M. K., A. Gilles, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350356. Efthymiou, C., and P. A. Hansen. 1962. An antigenic analysis of Lactobacillus acidophilus. J. Infect. Dis. 110:258-267. Faith, W. T., C. E. Neubeck, and E T. Reese. 1971. Production and applications of enzymes. Adv. Biochem. Eng. 1:79-111. Fukui, K., Y. Fukui, and T. Moriyama. 1974. Purification and properties of dextransucrase and invertase from Streptococcus mutans. J. Bacteriol. 118:796-
804. 7. Germaine, G. R., A. M. Chludzinski, and C. F. Schachtele. 1974. Streptococcus mutans dextransucrase: requirement for primer dextran. J. Bacteriol. 120:287294. 8. Gibbons, R. J., and S. B. Banghart. 1967. Synthesis of extracellular dextran by cariogenic bacteria and its presence in human dental plaque. Arch. Oral Biol. 12:11-24. 9. Gibbons, R. J., and R. J. Fitzgerald. 1969. Dextran induced agglutination of Streptococcus mutans and its potential role in the formation of microbial dental plaques. J. Bacteriol. 98:341-346. 10. Gugenheim, B., and E. Newbrun. 1969. Extracellular glucosyltransferase activity of an HS strain of Streptococcus mutans. Helv. Odontol. Acta 13:84-97. 11. Hehre, E. J. 1955. Polysaccharide synthesis from disaccharides. I. Dextransucrase. Methods Enzymol. 1:179-184. 12. Jablonski, W. M., and J. A. Hayashi. 1970. Inhibition of extracellular streptococcal enzymes. J. Dent. Res. 49:178. 13. Jeanes, A. 1965. Production of dextran from L. mesenteroides NRRL B-523. Methods Carbohydr. Chem. 5:124-127. 14. Kinoshita, S., H. Okada, and G. Terui. 1968. On the
15. 16. 17.
18.
19. 20.
21.
22. 23.
24. 25.
119
nature of a-amylase forming system in Bacillus subtilis. Stability of the mRNA for a-amylase. J. Ferment. Technol. 46:427-436. Kuramitsu, H. K. 1974. Characterization of cell-associated dextransucrase activity from glucose-grown cells of Streptococus mutans. Infect. Immun. 10:227-235. McCabe, M. M., and E. E. Smith. 1973. Origin of the cell-associated dextransucrase of Streptococcus mutans. Infect. Immun. 7:829-838. Paunesen, E., A. Ciolac-Negoescu, and G. Piscu. 1964. The effect of Tween 80 and penicillin on the physiochemical properties of the cell wall in mycobacteria. Acad. Repub. Pop. Rom. Stud. Cercet. Biochem. 7:184-190. Perch, B., E. Kjems, and T. Ravn. 1974. Biochemical and serological properties of Streptococcus mutans from various human and animal sources. Acta Pathol. Microbiol. Scand. Sect. B 82:357-370. Reese, E. T., and A. Maguire. 1969. Surfactants as stimulants of enzyme production by microorganisms. Appl. Microbiol. 17:242-245. Robrish, S. A., W. Reid, and M. I. Krichevsky. 1972. Distribution of enzymes forming polysaccharide from sucrose and the composition of extracellular polysaccharide synthesized by Streptococcus mutans. Appl. Microbiol. 24:184-190. Scales, W. R., L. W. Long, and J. R. Edwards. 1975. Purification of a glucosyltransferase complex from the culture broth of Streptococcus mutans FA-1. Carbohydr. Res. 42:325-338. Somogyi, M. 1945. A new reagent for the determination of sugars. J. Biol. Chem. 160:61-73. Takahashi, J., G. Abekawa, and K. Yamada. 1960. Effect of non-ionic surface active agents on mycelial form and amylase production of A. niger. Nippon Nogei Kagaku Kaishi 34:1043-1045. Turesky, S., I. Glickman, and R. Sandberg. 1972. In vitro chemical inhibition of plaque formation. J. Periodontol. 43:263-269. Welker, N. E., and L. L. Campbell. 1963. De novo synthesis of a-amylase by Bacillus stearothermophilus. J. Bacteriol. 86:1202-1210.
