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Dextran hydrolysis by a Fusobacterium strain isolated from human dental plaque
Arrhr
oroi Bid
Vol. 19. pp. 341 to 342. Pergamon Press 1974. Printed in Great Britain
DEXTRAN HYDROLYSIS STRAIN ISOLATED FROM
T.
DA
BY A FUSOBACTERlUM HUMAN DENTAL PLAQUE
COSTA,L. C. BIERand F. GAIDA
Department of Pathology, Microbiology Section, Federal University of Santa Maria, Santa Maria, R.G.S., Brazil
Summary-Mixed cultures ofdental plaque bacteria were found to catabolize dextran when grown anaerobically, but not aerobically. A strain of Fusobacterium jiisiforme was isolated from plaque cultures which possessed cell-bound cfextranase activity. The enzyme could be solubilized with desoxycholate, and it exhibited maximal activity at pH 6.8. Free glucose was the only end-product detected in enzyme-dextran reaction mixtures.
The test system adopted to determine dextranase The production of extracellular dextran from sucrose activity consisted of 4 ml of enzyme preparation, 2 ml by cariogenic bacteria (Snyder et al., 1955; Wood and Critchley, 1966; Gibbons et al., 1966) seems to play an of 0.067 M phosphate buffer (PH 6.8), and 2 ml of 1 per important role in the composition (Wood 1967) and cent dextran. Control mixtures containing (a) boiled enzyme and dextran, (b) dextran only and (c) dexdevelopment of dental plaques (Carlsson and Egelberg, tranase only were included. The mixtures were incu1965; Jordan and Keyes, 1966; Gibbons and Banghart, bated at 35”Cin a water bath, and samples were period1967). Dextran has been found to be relatively resistant to attack by salivary micro-organisms (Gibbons et al., ically removed over a 60 min period. These were deproteinized by the addition of ZnSO, and BaOH, 1966). However, Wood (1967) reported its partial cataand analyzed for reducing sugar (Somogyi, 1945). bolism when plaque was incubated without substrate, The Fusobucterium dextranase was found to be cellsuggesting that plaque bacteria may possess dextranase activity. This investigation was carried out to associated, and cell-free culture liquor exhibited no seek dextranase-producing bacteria in human dental activity. The enzyme could be obtained in a soluble form by treating sedimented cells from a 3 day culture plaque. Samples of dental plaques were obtained from nine with 5 per cent sodium desoxycholate. A typical cellassociated enzyme preparation liberated approxisubjects with active carious lesions. The samples were diluted and cultivated in the following medium: Promately 700 pg of reducing sugar/hr/ml of enzyme soluteose Peptone (Oxoid), 2 per cent; Phytone (Bait. Biol. tion from dextran. The liberation of reducing sugar was Labs.), 0.3 per cent; NaCl, 0.2 per cent; K,HPO,, 0.3 found to be linear with time for at least 1 hr, and to be per cent; KH,PO,, @l per cent; Yeast Extract, 0.1 per proportional to the concentration of enzyme under the cent. The medium was supplemented with 0.15 per cent conditions employed. Optimum enzyme activity was dextran (M. W. 5-40 x 106-Nutritional Biochemifound to occur at pH 6.8 as determined in phosphate cals Corp.) and adjusted to pH 7.2. Duplicate cultures buffer (Fig. 1). Solutions of the enzyme could be stored were incubated aerobically and anaerobically for 2-3 in the frozen state for several weeks without marked days. Dextran hydrolysis was determined by testing for loss of activity. The products formed during hydrolysis were anaresidual material precipitable with two volumes of ethanol in the cell-free culture supernatant liquor after lyzed by thin layer chromatography using Kieselguhr (Merck). Samples of enzyme-dextran reaction mixgrowth. Uninoculated dextran broth served as a contures were spotted on plates which were developed trol. It was found that the mixed anaerobic growth from with ethyl-acetate, acetic acid, water (3 :3 : 1). The chrolo-’ and 10V8 dilutions of two of the nine plaque sammatograms showed two well-defined spots when sprayed with aniline-diphenylamine. One was located ples studied hydrolyzed dextran. Five other anaerobic at the origin, and presumably was residual dextran. samples showed dextranase activity in lower plaque dilutions (10-3, 10m4).From a culture inoculated with The second spot migrated comparably to free glucose. a lo-’ dilution of plaque, a strain of Fusobacterium These data suggest that the Fusobacterium enzyme works similarly to the dextranase produced by intesfisiforme was isolated. This strain was used as a source tinal Bacteroides, which liberate free glucose from dexof dextranase in subsequent studies. Aerobic cultures tran (Hehre and Sery, 1952; Bayley and Clarke, 1959). of plaque failed to hydrolyze dextran. 341
342
T. da Costa, L. C. Bier and F. Gaida plaque bacteria can synthesize and also partly utilize dextran, it would appear that this polysaccharide could act as a carbohydrate storage compound.
.
I\
I’
Acknowledgements-This investigation was supported the National Research Council of Brazil.
l \
/
REFERENCES
‘\
.
6.0
6.4
.
6.6
7.2
by
76
4 6.0
PH
Fig. 1. Relative activity of Fusobacterium dextranase determined in phosphate buffer of varying pH.
The ability of dialyzed preparations of enzyme to hydrolyze in vitro grown plaques of Streptococcus mutuns strain GS5 (Gibbons et al., 1966) was also studied Plaques were formed by suspending stainless steel wire in sucrose broth cultures of this Streptococcus. When they were incubated with enzyme preparations for 60 min, approximately 280 ,ug of reducing sugar was released per hr/ml of enzyme solutions. Partial dispersion of the Strep. mutans plaque was also observed. The present investigation has shown that certain strains of Fusobacterium species present in dental plaque can catabolize dextrans. Since human dental
Bayley R. W. and Clarke T. J. 1959. A bacterial dextranase. Biochem. J. 72,49-54. Carlsson J. and Egelberg J. 1965. Effect of diet on early plaque formation in man. Odont. Reuy 16, 112-115. Gibbons R. J.. Berman K. S., Knoettner P. and Kapsimalis B. 1966. Dental caries and alveolar bone loss in gnotobiotic rats infected with capsule forming streptococci of human origin. Archs oral Biol. 11,549-560. Gibbons R. J. and Banghart S. 1967. Synthesis of dextran by cariogenic bacteria and its presence in human dental plaque. Archs ora Biol. 12. 1I-24. Hehre E. J. and Sery T. W. 1952. Dextran-splitting anaerobit bacteria from the human intestine. J. Bacterial. 63, 424426. Jordan H. V. and Keyes P. H. 1966. III citro methods for the study of plaque formation and carious lesions. Archs orul Biol. 11, 793-801. Snyder M. L., Hackedorn H. M.. Martin D. 0. and Johnston D. D. 1955. The synthesis of mutinous polysaccharide from sucrose by oral bacteria. J. dent. Res. 34, 368379. Somogyi M. 1945. Determination of blood sugar. J. biol. Chem. 160,69-73. Wood J. M. and Critchley P. 1966. The extracellular polysaccharide produced from sucrose by a cariogenic streptococcus. Archs oral Biol. 11, 1039-1042. Wood J. M. 1967. The amount, distribution and metabolism of soluble polysaccharide in human dental plaque. Archs oral Biol. 12, 849-858.
R&u&-De cultures mixtes de bactCries de plaque dentaire catabilisent le dextrane en anaCrobiose, mais non en aCrobiose. Une souche de Fusobacteriumfusiforme est isolCe de cultures de plaque: elle posdde une activite en dextranase, 1iCeaux cellules. L’enzyme a pu 2tre solubiliie B l’aide de dCsoxycholate et prCsente une activite maximale g pH 6.8. Du glucose libre est le seul produit terminal dttectC dans les m&nges de rCaction enzyme-dextrane. Zusammenfassung-Mischkulturen von Plaquebakterien bauten unter anaeroben Bedingungen, nicht jedoch unter aeroben, Dextran ab. Ein Stamm von Fuwbacterium fusiforme mit zellgebundener Dextranaseaktivitit wurde auf Plaque-Kulturen isoliert Das Enzym konnte mit Desoxycholat gel&t werden; es entwickelte maximale Aktivitit bei pH 6.8. Das einzige Endprodukt in Enzym-Dextran-Readtionsgemischen war freie Glukose.
oroi Bid
Vol. 19. pp. 341 to 342. Pergamon Press 1974. Printed in Great Britain
DEXTRAN HYDROLYSIS STRAIN ISOLATED FROM
T.
DA
BY A FUSOBACTERlUM HUMAN DENTAL PLAQUE
COSTA,L. C. BIERand F. GAIDA
Department of Pathology, Microbiology Section, Federal University of Santa Maria, Santa Maria, R.G.S., Brazil
Summary-Mixed cultures ofdental plaque bacteria were found to catabolize dextran when grown anaerobically, but not aerobically. A strain of Fusobacterium jiisiforme was isolated from plaque cultures which possessed cell-bound cfextranase activity. The enzyme could be solubilized with desoxycholate, and it exhibited maximal activity at pH 6.8. Free glucose was the only end-product detected in enzyme-dextran reaction mixtures.
The test system adopted to determine dextranase The production of extracellular dextran from sucrose activity consisted of 4 ml of enzyme preparation, 2 ml by cariogenic bacteria (Snyder et al., 1955; Wood and Critchley, 1966; Gibbons et al., 1966) seems to play an of 0.067 M phosphate buffer (PH 6.8), and 2 ml of 1 per important role in the composition (Wood 1967) and cent dextran. Control mixtures containing (a) boiled enzyme and dextran, (b) dextran only and (c) dexdevelopment of dental plaques (Carlsson and Egelberg, tranase only were included. The mixtures were incu1965; Jordan and Keyes, 1966; Gibbons and Banghart, bated at 35”Cin a water bath, and samples were period1967). Dextran has been found to be relatively resistant to attack by salivary micro-organisms (Gibbons et al., ically removed over a 60 min period. These were deproteinized by the addition of ZnSO, and BaOH, 1966). However, Wood (1967) reported its partial cataand analyzed for reducing sugar (Somogyi, 1945). bolism when plaque was incubated without substrate, The Fusobucterium dextranase was found to be cellsuggesting that plaque bacteria may possess dextranase activity. This investigation was carried out to associated, and cell-free culture liquor exhibited no seek dextranase-producing bacteria in human dental activity. The enzyme could be obtained in a soluble form by treating sedimented cells from a 3 day culture plaque. Samples of dental plaques were obtained from nine with 5 per cent sodium desoxycholate. A typical cellassociated enzyme preparation liberated approxisubjects with active carious lesions. The samples were diluted and cultivated in the following medium: Promately 700 pg of reducing sugar/hr/ml of enzyme soluteose Peptone (Oxoid), 2 per cent; Phytone (Bait. Biol. tion from dextran. The liberation of reducing sugar was Labs.), 0.3 per cent; NaCl, 0.2 per cent; K,HPO,, 0.3 found to be linear with time for at least 1 hr, and to be per cent; KH,PO,, @l per cent; Yeast Extract, 0.1 per proportional to the concentration of enzyme under the cent. The medium was supplemented with 0.15 per cent conditions employed. Optimum enzyme activity was dextran (M. W. 5-40 x 106-Nutritional Biochemifound to occur at pH 6.8 as determined in phosphate cals Corp.) and adjusted to pH 7.2. Duplicate cultures buffer (Fig. 1). Solutions of the enzyme could be stored were incubated aerobically and anaerobically for 2-3 in the frozen state for several weeks without marked days. Dextran hydrolysis was determined by testing for loss of activity. The products formed during hydrolysis were anaresidual material precipitable with two volumes of ethanol in the cell-free culture supernatant liquor after lyzed by thin layer chromatography using Kieselguhr (Merck). Samples of enzyme-dextran reaction mixgrowth. Uninoculated dextran broth served as a contures were spotted on plates which were developed trol. It was found that the mixed anaerobic growth from with ethyl-acetate, acetic acid, water (3 :3 : 1). The chrolo-’ and 10V8 dilutions of two of the nine plaque sammatograms showed two well-defined spots when sprayed with aniline-diphenylamine. One was located ples studied hydrolyzed dextran. Five other anaerobic at the origin, and presumably was residual dextran. samples showed dextranase activity in lower plaque dilutions (10-3, 10m4).From a culture inoculated with The second spot migrated comparably to free glucose. a lo-’ dilution of plaque, a strain of Fusobacterium These data suggest that the Fusobacterium enzyme works similarly to the dextranase produced by intesfisiforme was isolated. This strain was used as a source tinal Bacteroides, which liberate free glucose from dexof dextranase in subsequent studies. Aerobic cultures tran (Hehre and Sery, 1952; Bayley and Clarke, 1959). of plaque failed to hydrolyze dextran. 341
342
T. da Costa, L. C. Bier and F. Gaida plaque bacteria can synthesize and also partly utilize dextran, it would appear that this polysaccharide could act as a carbohydrate storage compound.
.
I\
I’
Acknowledgements-This investigation was supported the National Research Council of Brazil.
l \
/
REFERENCES
‘\
.
6.0
6.4
.
6.6
7.2
by
76
4 6.0
PH
Fig. 1. Relative activity of Fusobacterium dextranase determined in phosphate buffer of varying pH.
The ability of dialyzed preparations of enzyme to hydrolyze in vitro grown plaques of Streptococcus mutuns strain GS5 (Gibbons et al., 1966) was also studied Plaques were formed by suspending stainless steel wire in sucrose broth cultures of this Streptococcus. When they were incubated with enzyme preparations for 60 min, approximately 280 ,ug of reducing sugar was released per hr/ml of enzyme solutions. Partial dispersion of the Strep. mutans plaque was also observed. The present investigation has shown that certain strains of Fusobacterium species present in dental plaque can catabolize dextrans. Since human dental
Bayley R. W. and Clarke T. J. 1959. A bacterial dextranase. Biochem. J. 72,49-54. Carlsson J. and Egelberg J. 1965. Effect of diet on early plaque formation in man. Odont. Reuy 16, 112-115. Gibbons R. J.. Berman K. S., Knoettner P. and Kapsimalis B. 1966. Dental caries and alveolar bone loss in gnotobiotic rats infected with capsule forming streptococci of human origin. Archs oral Biol. 11,549-560. Gibbons R. J. and Banghart S. 1967. Synthesis of dextran by cariogenic bacteria and its presence in human dental plaque. Archs ora Biol. 12. 1I-24. Hehre E. J. and Sery T. W. 1952. Dextran-splitting anaerobit bacteria from the human intestine. J. Bacterial. 63, 424426. Jordan H. V. and Keyes P. H. 1966. III citro methods for the study of plaque formation and carious lesions. Archs orul Biol. 11, 793-801. Snyder M. L., Hackedorn H. M.. Martin D. 0. and Johnston D. D. 1955. The synthesis of mutinous polysaccharide from sucrose by oral bacteria. J. dent. Res. 34, 368379. Somogyi M. 1945. Determination of blood sugar. J. biol. Chem. 160,69-73. Wood J. M. and Critchley P. 1966. The extracellular polysaccharide produced from sucrose by a cariogenic streptococcus. Archs oral Biol. 11, 1039-1042. Wood J. M. 1967. The amount, distribution and metabolism of soluble polysaccharide in human dental plaque. Archs oral Biol. 12, 849-858.
R&u&-De cultures mixtes de bactCries de plaque dentaire catabilisent le dextrane en anaCrobiose, mais non en aCrobiose. Une souche de Fusobacteriumfusiforme est isolCe de cultures de plaque: elle posdde une activite en dextranase, 1iCeaux cellules. L’enzyme a pu 2tre solubiliie B l’aide de dCsoxycholate et prCsente une activite maximale g pH 6.8. Du glucose libre est le seul produit terminal dttectC dans les m&nges de rCaction enzyme-dextrane. Zusammenfassung-Mischkulturen von Plaquebakterien bauten unter anaeroben Bedingungen, nicht jedoch unter aeroben, Dextran ab. Ein Stamm von Fuwbacterium fusiforme mit zellgebundener Dextranaseaktivitit wurde auf Plaque-Kulturen isoliert Das Enzym konnte mit Desoxycholat gel&t werden; es entwickelte maximale Aktivitit bei pH 6.8. Das einzige Endprodukt in Enzym-Dextran-Readtionsgemischen war freie Glukose.