Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1992 Sep;60(9):3673–3681. doi: 10.1128/iai.60.9.3673-3681.1992

Characteristics and cariogenicity of a fructanase-defective Streptococcus mutants strain.

D L Wexler 1, J E Penders 1, W H Bowen 1, R A Burne 1
PMCID: PMC257376  PMID: 1500176

Abstract

Polymers of D-fructose produced by a variety of oral bacteria are believed to function as extracellular carbohydrate reserves. Degradation of these polysaccharides in plaque following exhaustion of dietary carbohydrates is thought to contribute to the extent and duration of the acid challenge to the tooth surface and thus to the initiation and progression of dental caries. Streptococcus mutans produces a fructanase, the product of the fruA gene, which is capable of degrading beta(2,6)- and beta(2,1)-linked fructans that are commonly synthesized by dental plaque microorganisms. To evaluate the role of the FruA protein in exopolysaccharide metabolism and to assess the contribution of this enzyme to the pathogenic potential of S. mutans, a fructanase-deficient strain of S. mutans was constructed. Inactivation of a cloned fruA gene was accomplished in Escherichia coli by using a mini-Mu dE transposon, and then an isogenic mutant of S. mutans UA159 was constructed by allelic exchange. Successful inactivation of fruA was confirmed through the use of biochemical assays, Western blotting (immunoblotting) with anti-recombinant FruA antisera, and Southern hybridization. The data indicated that FruA was the only fructan hydrolase produced by S. mutans UA159. Inactivation of fruA had no significant effects on glucosyltransferase or fructosyltransferase activity. In the rat caries model using animals fed a high-sucrose diet and ad libitum, there were no significant differences in the number or severity of smooth surface, sulcal, or root caries elicited by the fruA mutant and the wild-type organism.

Full text

PDF
3673

Images in this article

3676Image
on p.3676
3676Image
on p.3676

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bowen W. H., Amsbaugh S. M., Monell-Torrens S., Brunelle J. Effects of varying intervals between meals on dental caries in rats. Caries Res. 1983;17(5):466–471. doi: 10.1159/000260703. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Burne R. A., Rubinfeld B., Bowen W. H., Yasbin R. E. Tight genetic linkage of a glucosyltransferase and dextranase of Streptococcus mutans GS-5. J Dent Res. 1986 Dec;65(12):1392–1401. doi: 10.1177/00220345860650120301. [DOI] [PubMed] [Google Scholar]
  4. Burne R. A., Schilling K., Bowen W. H., Yasbin R. E. Expression, purification, and characterization of an exo-beta-D-fructosidase of Streptococcus mutans. J Bacteriol. 1987 Oct;169(10):4507–4517. doi: 10.1128/jb.169.10.4507-4517.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Castilho B. A., Olfson P., Casadaban M. J. Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons. J Bacteriol. 1984 May;158(2):488–495. doi: 10.1128/jb.158.2.488-495.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chassy B. M. A gentle method for the lysis of oral streptococci. Biochem Biophys Res Commun. 1976 Jan 26;68(2):603–608. doi: 10.1016/0006-291x(76)91188-8. [DOI] [PubMed] [Google Scholar]
  7. Chassy B. M., Bielawski R. M., Beall J. R., Porter E. V., Krichevsky M. I., Donkersloot J. A. Extracellular invertase in Streptococcus mutans and Streptococcus salivarius. Life Sci. 1974 Sep 15;15(6):1173–1180. doi: 10.1016/s0024-3205(74)80013-5. [DOI] [PubMed] [Google Scholar]
  8. Ebisu S., Kato K., Kotani S., Misaki A. Structural differences in fructans elaborated by streptococcus mutans and Strep. salivarius. J Biochem. 1975 Nov;78(5):879–887. doi: 10.1093/oxfordjournals.jbchem.a130993. [DOI] [PubMed] [Google Scholar]
  9. Ehrlich J., Stivala S. S., Bahary W. S., Garg S. K., Long L. W., Newbrun E. Levans: I. Fractionation, solution viscosity, and chemical analysis of levan produced by Streptococcus salivarius. J Dent Res. 1975 Mar-Apr;54(2):290–297. [PubMed] [Google Scholar]
  10. Gauthier L., Mayrand D., Vadeboncoeur C. Isolation of a novel protein involved in the transport of fructose by an inducible phosphoenolpyruvate fructose phosphotransferase system in Streptococcus mutans. J Bacteriol. 1984 Nov;160(2):755–763. doi: 10.1128/jb.160.2.755-763.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Germaine G. R., Schachtele C. F., Chludzinski A. M. Rapid filter paper assay for the dextransucrase activity from Streptococcus mutans. J Dent Res. 1974 Nov-Dec;53(6):1355–1360. doi: 10.1177/00220345740530061101. [DOI] [PubMed] [Google Scholar]
  12. Gold W., Preston F. B., Lache M. C., Blechman H. Production of levan and dextran in plaque in vivo. J Dent Res. 1974 Mar-Apr;53(2):442–446. doi: 10.1177/00220345740530024401. [DOI] [PubMed] [Google Scholar]
  13. Hamada S., Slade H. D. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Rev. 1980 Jun;44(2):331–384. doi: 10.1128/mr.44.2.331-384.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. KEYES P. H. Dental caries in the Syrian bamster. VIII. The induction of rampant caries activity in albino and golden animals. J Dent Res. 1959 May-Jun;38(3):525–533. doi: 10.1177/00220345590380031401. [DOI] [PubMed] [Google Scholar]
  15. KEYES P. H. Dental caries in the molar teeth of rats. II. A method for diagnosing and scoring several types of lesions simultaneously. J Dent Res. 1958 Nov-Dec;37(6):1088–1099. doi: 10.1177/00220345580370060901. [DOI] [PubMed] [Google Scholar]
  16. Kuramitsu H. K. Utilization of a mini-mu transposon to construct defined mutants in Streptococcus mutans. Mol Microbiol. 1987 Sep;1(2):229–232. doi: 10.1111/j.1365-2958.1987.tb00516.x. [DOI] [PubMed] [Google Scholar]
  17. LUCHSINGER W. W., CORNESKY R. A. Reducing power by the dinitrosalicylic acid method. Anal Biochem. 1962 Oct;4:346–347. doi: 10.1016/0003-2697(62)90098-2. [DOI] [PubMed] [Google Scholar]
  18. Larrimore S., Murchison H., Shiota T., Michalek S. M., Curtiss R., 3rd In vitro and in vivo complementation of Streptococcus mutans mutants defective in adherence. Infect Immun. 1983 Nov;42(2):558–566. doi: 10.1128/iai.42.2.558-566.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Loesche W. J. Role of Streptococcus mutans in human dental decay. Microbiol Rev. 1986 Dec;50(4):353–380. doi: 10.1128/mr.50.4.353-380.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Manly R. S., Richardson D. T. Metabolism of levan by oral samples. J Dent Res. 1968 Nov-Dec;47(6):1080–1086. doi: 10.1177/00220345680470061301. [DOI] [PubMed] [Google Scholar]
  21. Martin I., Débarbouillé M., Ferrari E., Klier A., Rapoport G. Characterization of the levanase gene of Bacillus subtilis which shows homology to yeast invertase. Mol Gen Genet. 1987 Jun;208(1-2):177–184. doi: 10.1007/BF00330439. [DOI] [PubMed] [Google Scholar]
  22. Miller C. H., Somers P. J. Degradation of levan by Actinomyces viscosus. Infect Immun. 1978 Oct;22(1):266–274. doi: 10.1128/iai.22.1.266-274.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Murchison H. H., Barrett J. F., Cardineau G. A., Curtiss R., 3rd Transformation of Streptococcus mutans with chromosomal and shuttle plasmid (pYA629) DNAs. Infect Immun. 1986 Nov;54(2):273–282. doi: 10.1128/iai.54.2.273-282.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ohta H., Kato H., Okahashi N., Takahashi I., Hamada S., Koga T. Characterization of a cell-surface protein antigen of hydrophilic Streptococcus mutans strain GS-5. J Gen Microbiol. 1989 Apr;135(4):981–988. doi: 10.1099/00221287-135-4-981. [DOI] [PubMed] [Google Scholar]
  25. Olsson J., Bowen W. Effect of diet on the colonization of the mouth by Actinomyces viscosus (T-6) in Osborne-Mendel rats. Arch Oral Biol. 1982;27(12):1087–1090. doi: 10.1016/0003-9969(82)90016-4. [DOI] [PubMed] [Google Scholar]
  26. Perry D., Kuramitsu H. K. Genetic transformation of Streptococcus mutans. Infect Immun. 1981 Jun;32(3):1295–1297. doi: 10.1128/iai.32.3.1295-1297.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Perry D., Kuramitsu H. K. Linkage of sucrose-metabolizing genes in Streptococcus mutans. Infect Immun. 1990 Oct;58(10):3462–3464. doi: 10.1128/iai.58.10.3462-3464.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Robrish S. A., Reid W., Krichevsky M. I. Distribution of enzymes forming polysaccharide from sucrose and the composition of extracellular polysaccharide synthesized by Streptococcus mutans. Appl Microbiol. 1972 Aug;24(2):184–190. doi: 10.1128/am.24.2.184-190.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rølla G., Oppermann R. V., Bowen W. H., Ciardi J. E., Knox K. W. High amounts of lipoteichoic acid in sucrose-induced plaque in vivo. Caries Res. 1980;14(4):235–238. doi: 10.1159/000260459. [DOI] [PubMed] [Google Scholar]
  30. SHILO M., WOLMAN B. Activities of bacterial levans and of lipopolysaccharides in the processes of inflammation and infection. Br J Exp Pathol. 1958 Dec;39(6):652–660. [PMC free article] [PubMed] [Google Scholar]
  31. Schilling K. M., Bowen W. H. Glucans synthesized in situ in experimental salivary pellicle function as specific binding sites for Streptococcus mutans. Infect Immun. 1992 Jan;60(1):284–295. doi: 10.1128/iai.60.1.284-295.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schroeder V. A., Michalek S. M., Macrina F. L. Biochemical characterization and evaluation of virulence of a fructosyltransferase-deficient mutant of Streptococcus mutans V403. Infect Immun. 1989 Nov;57(11):3560–3569. doi: 10.1128/iai.57.11.3560-3569.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shiroza T., Kuramitsu H. K. Sequence analysis of the Streptococcus mutans fructosyltransferase gene and flanking regions. J Bacteriol. 1988 Feb;170(2):810–816. doi: 10.1128/jb.170.2.810-816.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  35. Takamori K., Mizuno F., Yamamoto A., Etoh Y., Takahashi M., Takahashi N. Preliminary studies of fructan-hydrolyzing bacteria from human dental plaque. Microbiol Immunol. 1985;29(4):359–363. doi: 10.1111/j.1348-0421.1985.tb00834.x. [DOI] [PubMed] [Google Scholar]
  36. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Walker G. J., Hare M. D., Morrey-Jones J. G. Activity of fructanase in batch cultures of oral streptococci. Carbohydr Res. 1983 Feb 16;113(1):101–112. doi: 10.1016/0008-6215(83)88222-6. [DOI] [PubMed] [Google Scholar]
  38. Warner T. N., Miller C. H. Cell-associated levan of Actinomyces viscosus. Infect Immun. 1978 Feb;19(2):711–719. doi: 10.1128/iai.19.2.711-719.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wood J. M. The amount, distribution and metabolism of soluble polysaccharides in human dental plaque. Arch Oral Biol. 1967 Jul;12(7):849–858. doi: 10.1016/0003-9969(67)90107-0. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES