Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Jul;175(14):4345–4353. doi: 10.1128/jb.175.14.4345-4353.1993

Molecular characterization of a negative regulator of Streptococcus sobrinus surface protein antigen gene.

I Takahashi 1, N Okahashi 1, S Hamada 1
PMCID: PMC204874  PMID: 8331066

Abstract

Mutans streptococci have been shown to give rise to variants in terms of expression of surface protein antigens by repeated subculturing of the organisms, which in turn induces changes in colonial morphologies. A 2,850-bp upstream region of the gene (pag) for a surface protein antigen, PAg, of Streptococcus sobrinus MT3791 was determined. Analysis of the nucleotide sequence revealed the existence of three open reading frames (ORFs) located upstream of the pag gene. ORF1 extended from an undetermined further upstream sequence to the termination codon TAG lying 1,943 bp upstream of the pag gene. ORF2, consisting of 609 bp lying 1,689 bp upstream of the pag gene, encoded a protein of 23,347 Da and a protein of 22,792 Da. The synthesis of these proteins (protein antigen regulators) was demonstrated by using the in vitro T7 RNA polymerase/promoter system. ORF3, extending from 314 bp upstream of the pag gene to 712 bp upstream of the pag gene, encoded a protein of 14,802 Da. Disruption of chromosomal ORF2 of parent strain MT3791 by allelic exchange resulted in isogenic mutants, termed PAREm-1 and PAREm-2, that synthesized larger amounts of cell-free and cell-associated PAg than did the parent strain. RNA dot blot analysis demonstrated that expression of PAg-specific mRNA transcripts by mutants PAREm-1 and PAREm-2 was about 32-fold higher than that by strain 3791. Mutants PAREm-1 and PAREm-2 were found to be more hydrophobic than strain MT3791. Resting cells of these mutants attached in larger numbers to saliva-coated hydroxyapatite than did those of the parent strain. These results suggest that protein antigen regulator regulates the expression of PAg gene in a negative fashion, affecting the colonization of tooth surfaces by the organism. Thus, ORF2 is concluded to be a negative regulator gene of PAg synthesis and was designated par.

Full text

PDF
4345

Images in this article

4350Image
on p.4350
4350Image
on p.4350
4350Image
on p.4350
4351Image
on p.4351
4351Image
on p.4351

Selected References

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

  1. Beattie D. T., Shahin R., Mekalanos J. J. A vir-repressed gene of Bordetella pertussis is required for virulence. Infect Immun. 1992 Feb;60(2):571–577. doi: 10.1128/iai.60.2.571-577.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Eifert R., Rosan B., Golub E. Optimization of an hydroxyapatite adhesion assay for Streptococcus sanguis. Infect Immun. 1984 May;44(2):287–291. doi: 10.1128/iai.44.2.287-291.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Forester H., Hunter N., Knox K. W. Characteristics of a high molecular weight extracellular protein of Streptococcus mutans. J Gen Microbiol. 1983 Sep;129(9):2779–2788. doi: 10.1099/00221287-129-9-2779. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  6. Hollingshead S. K., Fischetti V. A., Scott J. R. Size variation in group A streptococcal M protein is generated by homologous recombination between intragenic repeats. Mol Gen Genet. 1987 May;207(2-3):196–203. doi: 10.1007/BF00331578. [DOI] [PubMed] [Google Scholar]
  7. Holt R. G., Abiko Y., Saito S., Smorawinska M., Hansen J. B., Curtiss R., 3rd Streptococcus mutans genes that code for extracellular proteins in Escherichia coli K-12. Infect Immun. 1982 Oct;38(1):147–156. doi: 10.1128/iai.38.1.147-156.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Koga T., Asakawa H., Okahashi N., Takahashi I. Effect of subculturing on expression of a cell-surface protein antigen by Streptococcus mutans. J Gen Microbiol. 1989 Dec;135(12):3199–3207. doi: 10.1099/00221287-135-12-3199. [DOI] [PubMed] [Google Scholar]
  9. Koga T., Okahashi N., Takahashi I., Kanamoto T., Asakawa H., Iwaki M. Surface hydrophobicity, adherence, and aggregation of cell surface protein antigen mutants of Streptococcus mutans serotype c. Infect Immun. 1990 Feb;58(2):289–296. doi: 10.1128/iai.58.2.289-296.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. LaPolla R. J., Haron J. A., Kelly C. G., Taylor W. R., Bohart C., Hendricks M., Pyati J. P., Graff R. T., Ma J. K., Lehner T. Sequence and structural analysis of surface protein antigen I/II (SpaA) of Streptococcus sobrinus. Infect Immun. 1991 Aug;59(8):2677–2685. doi: 10.1128/iai.59.8.2677-2685.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Lindler L. E., Macrina F. L. Characterization of genetic transformation in Streptococcus mutans by using a novel high-efficiency plasmid marker rescue system. J Bacteriol. 1986 May;166(2):658–665. doi: 10.1128/jb.166.2.658-665.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Macrina F. L., Tobian J. A., Jones K. R., Evans R. P., Clewell D. B. A cloning vector able to replicate in Escherichia coli and Streptococcus sanguis. Gene. 1982 Oct;19(3):345–353. doi: 10.1016/0378-1119(82)90025-7. [DOI] [PubMed] [Google Scholar]
  14. Miller J. F., Mekalanos J. J., Falkow S. Coordinate regulation and sensory transduction in the control of bacterial virulence. Science. 1989 Feb 17;243(4893):916–922. doi: 10.1126/science.2537530. [DOI] [PubMed] [Google Scholar]
  15. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Murphy G. L., Connell T. D., Barritt D. S., Koomey M., Cannon J. G. Phase variation of gonococcal protein II: regulation of gene expression by slipped-strand mispairing of a repetitive DNA sequence. Cell. 1989 Feb 24;56(4):539–547. doi: 10.1016/0092-8674(89)90577-1. [DOI] [PubMed] [Google Scholar]
  17. Ogier J. A., Schöller M., Lepoivre Y., Gangloff S., M'Zoughi R., Klein J. P. A 40-kilodalton cell wall protein-coding sequence upstream of the sr gene of Streptococcus mutans OMZ175 (serotype f). Infect Immun. 1991 May;59(5):1620–1626. doi: 10.1128/iai.59.5.1620-1626.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Okahashi N., Koga T., Hamada S. Purification and immunochemical properties of a protein antigen from serotype g Streptococcus mutans. Microbiol Immunol. 1986;30(1):35–47. doi: 10.1111/j.1348-0421.1986.tb00919.x. [DOI] [PubMed] [Google Scholar]
  20. Okahashi N., Sasakawa C., Yoshikawa M., Hamada S., Koga T. Cloning of a surface protein antigen gene from serotype c Streptococcus mutans. Mol Microbiol. 1989 Feb;3(2):221–228. doi: 10.1111/j.1365-2958.1989.tb01811.x. [DOI] [PubMed] [Google Scholar]
  21. Perez-Casal J., Caparon M. G., Scott J. R. Mry, a trans-acting positive regulator of the M protein gene of Streptococcus pyogenes with similarity to the receptor proteins of two-component regulatory systems. J Bacteriol. 1991 Apr;173(8):2617–2624. doi: 10.1128/jb.173.8.2617-2624.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Russell M. W., Bergmeier L. A., Zanders E. D., Lehner T. Protein antigens of Streptococcus mutans: purification and properties of a double antigen and its protease-resistant component. Infect Immun. 1980 May;28(2):486–493. doi: 10.1128/iai.28.2.486-493.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Russell R. R. Wall-associated protein antigens of Streptococcus mutans. J Gen Microbiol. 1979 Sep;114(1):109–115. doi: 10.1099/00221287-114-1-109. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Stibitz S., Aaronson W., Monack D., Falkow S. Phase variation in Bordetella pertussis by frameshift mutation in a gene for a novel two-component system. Nature. 1989 Mar 16;338(6212):266–269. doi: 10.1038/338266a0. [DOI] [PubMed] [Google Scholar]
  27. Stout V., Gottesman S. RcsB and RcsC: a two-component regulator of capsule synthesis in Escherichia coli. J Bacteriol. 1990 Feb;172(2):659–669. doi: 10.1128/jb.172.2.659-669.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Takahashi I., Okahashi N., Sasakawa C., Yoshikawa M., Hamada S., Koga T. Homology between surface protein antigen genes of Streptococcus sobrinus and Streptococcus mutans. FEBS Lett. 1989 Jun 5;249(2):383–388. doi: 10.1016/0014-5793(89)80664-7. [DOI] [PubMed] [Google Scholar]
  30. Terleckyj B., Willett N. P., Shockman G. D. Growth of several cariogenic strains of oral streptococci in a chemically defined medium. Infect Immun. 1975 Apr;11(4):649–655. doi: 10.1128/iai.11.4.649-655.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tokuda M., Okahashi N., Takahashi I., Nakai M., Nagaoka S., Kawagoe M., Koga T. Complete nucleotide sequence of the gene for a surface protein antigen of Streptococcus sobrinus. Infect Immun. 1991 Sep;59(9):3309–3312. doi: 10.1128/iai.59.9.3309-3312.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES