1887

Abstract

The glucosyltransferases of are recognized as important virulence factors for this cariogenic bacterium. To study the expression of the gene of in biofilms, a promoter ()–green fluorescent protein (GFP) reporter system was developed. A shuttle vector harbouring a :: cassette was introduced into GS-5, and the expression of GFP by the transformed cells was confirmed by fluorescence microscopy. Furthermore, confocal laser scanning microscopy was carried out on biofilms attached to polystyrene plates; enhanced expression was observed in various microcolonies across these biofilms. To further test the hypothesis that expression is upregulated in biofilms, flow cytometry analysis was done on planktonic and biofilm cells; this analysis showed an approximately five-fold increase in expression in the biofilm cells relative to the planktonic cells. Real-time (TaqMan) PCR analysis confirmed that expression in the biofilm cells was enhanced relative to the planktonic cells. Previously, it has been suggested that the gene might be co-transcribed with . Therefore, RT-PCR analysis was performed on -expressing ; this analysis demonstrated that was co-transcribed with . These results indicated that GFP expression can be utilized to examine gene regulation in biofilm formation.

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2002-11-01
2019-10-20
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References

  1. Acebo, P., Nieto, C., Corrales, M. A., Espinosa, M. & Lopez, P. ( 2000; ). Quantitative detection of Streptococcus pneumoniae cells harbouring single or multiple copies of the gene encoding the green fluorescent protein. Microbiology 146, 1267-1273.
    [Google Scholar]
  2. Aoki, H., Shiroza, T., Hayakawa, M., Sato, S. & Kuramitsu, H. K. ( 1986; ). Cloning of a Streptococcus mutans glucosyltransferase gene coding for insoluble glucan synthesis. Infect Immun 53, 587-594.
    [Google Scholar]
  3. Brehm, J., Salmond, G. & Minton, N. ( 1987; ). Sequence of the adenine methylase gene of the Streptococcus faecalis plasmid pAMβ1. Nucleic Acids Res 15, 3177.[CrossRef]
    [Google Scholar]
  4. Burne, R. A., Chen, Y. Y. & Penders, J. E. ( 1997; ). Analysis of gene expression in Streptococcus mutans in biofilms in vitro. Adv Dent Res 11, 100-109.[CrossRef]
    [Google Scholar]
  5. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. ( 1994; ). Green fluorescent protein as a marker for gene expression. Science 263, 802-805.[CrossRef]
    [Google Scholar]
  6. Cormack, B. P., Valdivia, R. H. & Falkow, S. ( 1996; ). FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33-38.[CrossRef]
    [Google Scholar]
  7. Davey, M. E. & O’Toole, G. A. ( 2000; ). Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 64, 847-867.[CrossRef]
    [Google Scholar]
  8. Dunny, G. M., Lee, L. N. & LeBlanc, D. J. ( 1991; ). Improved electroporation and cloning vector system for gram-positive bacteria. Appl Environ Microbiol 57, 1194-1201.
    [Google Scholar]
  9. Goodman, S. D. & Gao, Q. ( 2000; ). Characterization of the gtfB and gtfC promoters from Streptococcus mutans GS-5. Plasmid 43, 85-98.[CrossRef]
    [Google Scholar]
  10. Hanada, N. & Kuramitsu, H. K. ( 1988; ). Isolation and characterization of the Streptococcus mutans gtfC gene, coding for synthesis of both soluble and insoluble glucans. Infect Immun 56, 1999-2005.
    [Google Scholar]
  11. Hanada, N. & Kuramitsu, H. K. ( 1989; ). Isolation and characterization of the Streptococcus mutans gtfD gene, coding for primer-dependent soluble glucan synthesis. Infect Immun 57, 2079-2085.
    [Google Scholar]
  12. Hansen, M. C., Palmer, R. J.Jr, Udsen, C., White, D. C. & Molin, S. ( 2001; ). Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration. Microbiology 147, 1383-1391.
    [Google Scholar]
  13. Hudson, M. C. & Curtiss, R., 3rd ( 1990; ). Regulation of expression of Streptococcus mutans genes important to virulence. Infect Immun 58, 464–470.
    [Google Scholar]
  14. Kutyavin, I. V., Afonina, I. A., Mills, A. & 11 other authors ( 2000; ). 3′-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res 28, 655–661.[CrossRef]
    [Google Scholar]
  15. Lawrence, J. R. & Neu, T. R. ( 1999; ). Confocal laser scanning microscopy for analysis of microbial biofilms. Methods Enzymol 310, 131-144.
    [Google Scholar]
  16. Loesche, W. J. ( 1986; ). Role of Streptococcus mutans in human dental decay. Microbiol Rev 50, 353-380.
    [Google Scholar]
  17. Perry, D., Wondrack, L. M. & Kuramitsu, H. K. ( 1983; ). Genetic transformation of putative cariogenic properties in Streptococcus mutans. Infect Immun 41, 722-727.
    [Google Scholar]
  18. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. & Cormier, M. J. ( 1992; ). Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229-233.[CrossRef]
    [Google Scholar]
  19. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  20. Sato, Y., Yamamoto, Y., Suzuki, R., Kizaki, H. & Kuramitsu, H. K. ( 1991; ). Construction of scrA::lacZ gene fusions to investigate regulation of the sucrose PTS of Streptococcus mutans. FEMS Microbiol Lett 63, 339-345.
    [Google Scholar]
  21. Shiroza, T. & Kuramitsu, H. K. ( 1993; ). Construction of a model secretion system for oral streptococci. Infect Immun 61, 3745-3755.
    [Google Scholar]
  22. Skillman, L. C., Sutherland, I. W., Jones, M. V. & Goulsbra, A. ( 1998; ). Green fluorescent protein as a novel species-specific marker in enteric dual-species biofilms. Microbiology 144, 2095-2101.[CrossRef]
    [Google Scholar]
  23. Smorawinska, M. & Kuramitsu, H. K. ( 1995; ). Primer extension analysis of Streptococcus mutans promoter structures. Oral Microbiol Immunol 10, 188-192.[CrossRef]
    [Google Scholar]
  24. Ueda, S., Shiroza, T. & Kuramitsu, H. K. ( 1988; ). Sequence analysis of the gtfC gene from Streptococcus mutans GS-5. Gene 69, 101-109.[CrossRef]
    [Google Scholar]
  25. Watnick, P. & Kolter, R. ( 2000; ). Biofilm, city of microbes. J Bacteriol 182, 2675-2679.[CrossRef]
    [Google Scholar]
  26. Yamashita, Y., Bowen, W. H., Burne, R. A. & Kuramitsu, H. K. ( 1993; ). Role of the Streptococcus mutans gtf genes in caries induction in the specific-pathogen-free rat model. Infect Immun 61, 3811-3817.
    [Google Scholar]
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