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. 1992 Sep;174(17):5639–5646. doi: 10.1128/jb.174.17.5639-5646.1992

Mechanism of Streptococcus mutans glucosyltransferases: hybrid-enzyme analysis.

Y J Nakano 1, H K Kuramitsu 1
PMCID: PMC206510  PMID: 1387395

Abstract

Streptococcus mutans GS5 expresses three glucosyltransferases (GTFs): GTF-I and GTF-SI, which synthesize water-insoluble glucans in a primer-independent manner, and GTF-S, which is responsible for the formation of primer-dependent soluble glucan. The amino acid sequences of the GTF-I and GTF-S enzymes exhibit approximately 50% sequence identity. Various hybrid genes were constructed from the structural genes for the enzymes, and their products were analyzed. Three different approaches were used to construct the hybrid enzymes: (i) ligation of DNA fragments containing compatible endonuclease restriction sites of the two genes at homologous positions; (ii) in vivo recombination between the homologous regions of each gene; and (iii) random fusion of DNA fragments from each gene generated following exonuclease III digestion of tandemly arranged fragments corresponding to the two functional domains of each enzyme. Hybrid GTFs composed of the sucrose-binding domain of one enzyme (GTF-I or GTF-S) with the glucan-binding domain of the other synthesized insoluble glucan exclusively in the absence of primer dextran. Insoluble glucan synthesis by some, but not all, of the GTF-S:GTF-I chimeric enzymes was stimulated by primer dextran T10 addition. In addition, glucan binding by the former but not latter group of hybrid GTFs was demonstrated. These results suggest that the glucan-binding domain alone does not solely determine primer dependence or independence or the structure of the resulting glucan product, although this carboxyl-terminal domain containing direct repeating units does appear to play a significant role in primer dependence.

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Selected References

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

  1. Abo H., Matsumura T., Kodama T., Ohta H., Fukui K., Kato K., Kagawa H. Peptide sequences for sucrose splitting and glucan binding within Streptococcus sobrinus glucosyltransferase (water-insoluble glucan synthetase). J Bacteriol. 1991 Feb;173(3):989–996. doi: 10.1128/jb.173.3.989-996.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aoki H., Shiroza T., Hayakawa M., Sato S., Kuramitsu H. K. Cloning of a Streptococcus mutans glucosyltransferase gene coding for insoluble glucan synthesis. Infect Immun. 1986 Sep;53(3):587–594. doi: 10.1128/iai.53.3.587-594.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Deich R. A., Metcalf B. J., Finn C. W., Farley J. E., Green B. A. Cloning of genes encoding a 15,000-dalton peptidoglycan-associated outer membrane lipoprotein and an antigenically related 15,000-dalton protein from Haemophilus influenzae. J Bacteriol. 1988 Feb;170(2):489–498. doi: 10.1128/jb.170.2.489-498.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ferretti J. J., Gilpin M. L., Russell R. R. Nucleotide sequence of a glucosyltransferase gene from Streptococcus sobrinus MFe28. J Bacteriol. 1987 Sep;169(9):4271–4278. doi: 10.1128/jb.169.9.4271-4278.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Giffard P. M., Simpson C. L., Milward C. P., Jacques N. A. Molecular characterization of a cluster of at least two glucosyltransferase genes in Streptococcus salivarius ATCC 25975. J Gen Microbiol. 1991 Nov;137(11):2577–2593. doi: 10.1099/00221287-137-11-2577. [DOI] [PubMed] [Google Scholar]
  8. Gilmore K. S., Russell R. R., Ferretti J. J. Analysis of the Streptococcus downei gtfS gene, which specifies a glucosyltransferase that synthesizes soluble glucans. Infect Immun. 1990 Aug;58(8):2452–2458. doi: 10.1128/iai.58.8.2452-2458.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gilpin M. L., Russell R. R., Morrissey P. Cloning and expression of two Streptococcus mutans glucosyltransferases in Escherichia coli K-12. Infect Immun. 1985 Aug;49(2):414–416. doi: 10.1128/iai.49.2.414-416.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hanada N., Kuramitsu H. K. Isolation and characterization of the Streptococcus mutans gtfC gene, coding for synthesis of both soluble and insoluble glucans. Infect Immun. 1988 Aug;56(8):1999–2005. doi: 10.1128/iai.56.8.1999-2005.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hanada N., Kuramitsu H. K. Isolation and characterization of the Streptococcus mutans gtfD gene, coding for primer-dependent soluble glucan synthesis. Infect Immun. 1989 Jul;57(7):2079–2085. doi: 10.1128/iai.57.7.2079-2085.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Honda O., Kato C., Kuramitsu H. K. Nucleotide sequence of the Streptococcus mutans gtfD gene encoding the glucosyltransferase-S enzyme. J Gen Microbiol. 1990 Oct;136(10):2099–2105. doi: 10.1099/00221287-136-10-2099. [DOI] [PubMed] [Google Scholar]
  13. Jones D. H., Howard B. H. A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction. Biotechniques. 1991 Jan;10(1):62–66. [PubMed] [Google Scholar]
  14. Kato C., Kuramitsu H. K. Carboxyl-terminal deletion analysis of the Streptococcus mutans glucosyltransferase-I enzyme. FEMS Microbiol Lett. 1990 Nov;60(3):299–302. doi: 10.1016/0378-1097(90)90321-g. [DOI] [PubMed] [Google Scholar]
  15. Kuramitsu H. K. Characterization of extracellular glucosyltransferase activity of Steptococcus mutans. Infect Immun. 1975 Oct;12(4):738–749. doi: 10.1128/iai.12.4.738-749.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuramitsu H., Ingersoll L. Immunological relationships between glucosyltransferases from Streptococcus mutans serotypes. Infect Immun. 1976 Sep;14(3):636–644. doi: 10.1128/iai.14.3.636-644.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Nogami T., Mizuno T., Mizushima S. Construction of a series of ompF-ompC chimeric genes by in vivo homologous recombination in Escherichia coli and characterization of the translational products. J Bacteriol. 1985 Nov;164(2):797–801. doi: 10.1128/jb.164.2.797-801.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schneider E., Walter C. A chimeric nucleotide-binding protein, encoded by a hisP-malK hybrid gene, is functional in maltose transport in Salmonella typhimurium. Mol Microbiol. 1991 Jun;5(6):1375–1383. doi: 10.1111/j.1365-2958.1991.tb00784.x. [DOI] [PubMed] [Google Scholar]
  22. Shiroza T., Ueda S., Kuramitsu H. K. Sequence analysis of the gtfB gene from Streptococcus mutans. J Bacteriol. 1987 Sep;169(9):4263–4270. doi: 10.1128/jb.169.9.4263-4270.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sung W. L., Zahab D. M. Site-specific recombination directed by single-stranded crossover linkers: specific deletion of the amino-terminal region of the beta-galactosidase gene in pUC plasmids. DNA. 1987 Aug;6(4):373–379. doi: 10.1089/dna.1987.6.373. [DOI] [PubMed] [Google Scholar]
  25. Tommassen J., van der Ley P., van Zeijl M., Agterberg M. Localization of functional domains in E. coli K-12 outer membrane porins. EMBO J. 1985 Jun;4(6):1583–1587. doi: 10.1002/j.1460-2075.1985.tb03820.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Ueda S., Shiroza T., Kuramitsu H. K. Sequence analysis of the gtfC gene from Streptococcus mutans GS-5. Gene. 1988 Sep 15;69(1):101–109. doi: 10.1016/0378-1119(88)90382-4. [DOI] [PubMed] [Google Scholar]
  28. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  29. Weber H., Weissmann C. Formation of genes coding for hybrid proteins by recombination between related, cloned genes in E. coli. Nucleic Acids Res. 1983 Aug 25;11(16):5661–5669. doi: 10.1093/nar/11.16.5661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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