Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Jul;65(7):2765–2771. doi: 10.1128/iai.65.7.2765-2771.1997

Cloning and characterization of a Prevotella melaninogenica hemolysin.

H E Allison 1, J D Hillman 1
PMCID: PMC175390  PMID: 9199448

Abstract

Hemolysins have been proven to be important virulence factors in many medically relevant pathogenic organisms. Their production has also been implicated in the etiology of periodontal disease. Hemolytic strain 361B of Prevotella melaninogenica, a putative etiologic agent of periodontal disease, was used in this study. The cloning, sequencing, and characterization of phyA, the structural gene for a P. melaninogenica hemolysin, is described. No extensive sequence homology could be identified between phyA and any reported sequence at either the nucleotide or amino acid level. As predicted from sequence analysis, this gene produces a 39-kDa protein which has hemolytic activity as measured by zymogram analysis. Unlike many Ca2+-dependent bacterial hemolysins, both the cloned and native PhyA proteins were enhanced by the presence of EDTA in a dose-dependent fashion with 40 mM EDTA allowing maximum activity. Ca2+ and Mg2+ were found to be inhibitory. The hemolytic activity also was found to have a dose-dependent endpoint. Through recovery of hemolytic activity from a spent reaction, this endpoint was shown to be the result of end product inhibition. This is the first report describing the cloning and sequencing of a gene from P. melaninogenica.

Full Text

The Full Text of this article is available as a PDF (781.1 KB).

Selected References

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

  1. Braun V., Focareta T. Pore-forming bacterial protein hemolysins (cytolysins). Crit Rev Microbiol. 1991;18(2):115–158. doi: 10.3109/10408419109113511. [DOI] [PubMed] [Google Scholar]
  2. Brendel V., Trifonov E. N. A computer algorithm for testing potential prokaryotic terminators. Nucleic Acids Res. 1984 May 25;12(10):4411–4427. doi: 10.1093/nar/12.10.4411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bulkacz J., Schuster G. S., Singh B., Scott D. F. Phospholipase A activity of extracellular products from Bacteroides melaninogenicus on epithelium tissue cultures. J Periodontal Res. 1985 Mar;20(2):146–153. doi: 10.1111/j.1600-0765.1985.tb00421.x. [DOI] [PubMed] [Google Scholar]
  4. Chu L., Bramanti T. E., Ebersole J. L., Holt S. C. Hemolytic activity in the periodontopathogen Porphyromonas gingivalis: kinetics of enzyme release and localization. Infect Immun. 1991 Jun;59(6):1932–1940. doi: 10.1128/iai.59.6.1932-1940.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fujiwara T., Nakagawa I., Morishima S., Takahashi I., Hamada S. Inconsistency between the fimbrilin gene and the antigenicity of lipopolysaccharides in selected strains of Porphyromonas gingivalis. FEMS Microbiol Lett. 1994 Dec 15;124(3):333–341. doi: 10.1111/j.1574-6968.1994.tb07305.x. [DOI] [PubMed] [Google Scholar]
  6. GIBBONS R. J., MACDONALD J. B. Hemin and vitamin K compounds as required factors for the cultivation of certain strains of Bacteroides melaninogenicus. J Bacteriol. 1960 Aug;80:164–170. doi: 10.1128/jb.80.2.164-170.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Green J., Sharrocks A. D., MacInnes J. I., Guest J. R. Purification of HlyX, a potential regulator of haemolysin synthesis, and properties of HlyX:FNR hybrids. Proc Biol Sci. 1992 Apr 22;248(1321):79–84. doi: 10.1098/rspb.1992.0045. [DOI] [PubMed] [Google Scholar]
  8. Grenier D., Mayrand D., McBride B. C. Further studies on the degradation of immunoglobulins by black-pigmented Bacteroides. Oral Microbiol Immunol. 1989 Mar;4(1):12–18. doi: 10.1111/j.1399-302x.1989.tb00400.x. [DOI] [PubMed] [Google Scholar]
  9. Gyka G., Gheţie V., Sjöquist J. Crosslinkage of antibodies to staphylococcal protein A matrices. J Immunol Methods. 1983 Feb 25;57(1-3):227–233. doi: 10.1016/0022-1759(83)90082-0. [DOI] [PubMed] [Google Scholar]
  10. Hillman J. D., Maiden M. F., Pfaller S. P., Martin L., Duncan M. J., Socransky S. S. Characterization of hemolytic bacteria in subgingival plaque. J Periodontal Res. 1993 May;28(3):173–179. doi: 10.1111/j.1600-0765.1993.tb01066.x. [DOI] [PubMed] [Google Scholar]
  11. Hofstad T. Virulence determinants in nonsporeforming anaerobic bacteria. Scand J Infect Dis Suppl. 1989;62:15–24. [PubMed] [Google Scholar]
  12. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  13. Joe A., Yamamoto A., McBride B. C. Characterization of recombinant and native forms of a cell surface antigen of Porphyromonas (Bacteroides) gingivalis. Infect Immun. 1993 Aug;61(8):3294–3303. doi: 10.1128/iai.61.8.3294-3303.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jun K. C., Barua P. K., Zambon J. J., Neiders M. E. Proteolytic activity in black-pigmented bacteroides species. J Endod. 1989 Oct;15(10):463–467. doi: 10.1016/s0099-2399(89)80025-1. [DOI] [PubMed] [Google Scholar]
  15. Kado C. I., Liu S. T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981 Mar;145(3):1365–1373. doi: 10.1128/jb.145.3.1365-1373.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Karunakaran T., Holt S. C. Cloning of two distinct hemolysin genes from Porphyromonas (Bacteroides) gingivalis in Escherichia coli. Microb Pathog. 1993 Jul;15(1):37–49. doi: 10.1006/mpat.1993.1055. [DOI] [PubMed] [Google Scholar]
  17. Kilian M. Degradation of immunoglobulins A2, A2, and G by suspected principal periodontal pathogens. Infect Immun. 1981 Dec;34(3):757–765. doi: 10.1128/iai.34.3.757-765.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Marsh P. D., McDermid A. S., McKee A. S., Baskerville A. The effect of growth rate and haemin on the virulence and proteolytic activity of Porphyromonas gingivalis W50. Microbiology. 1994 Apr;140(Pt 4):861–865. doi: 10.1099/00221287-140-4-861. [DOI] [PubMed] [Google Scholar]
  20. Mitchell C. G., Smith R., Lehner T. Recognition of carbohydrate and protein epitopes by monoclonal antibodies to a cell wall antigen from Streptococcus mutans. Infect Immun. 1987 Mar;55(3):810–815. doi: 10.1128/iai.55.3.810-815.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Moncla B. J., Braham P., Hillier S. L. Sialidase (neuraminidase) activity among gram-negative anaerobic and capnophilic bacteria. J Clin Microbiol. 1990 Mar;28(3):422–425. doi: 10.1128/jcm.28.3.422-425.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  23. Socransky S. S., Haffajee A. D. Microbial mechanisms in the pathogenesis of destructive periodontal diseases: a critical assessment. J Periodontal Res. 1991 May;26(3 Pt 2):195–212. doi: 10.1111/j.1600-0765.1991.tb01646.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES