1887

Abstract

, a Gram-negative periodontopathic bacterium, produces a leukotoxin belonging to the RTX family. The production of leukotoxin varies greatly among different strains of this species. In this paper the effects of growth rate and bicarbonate on the leukotoxin production by a toxin-production-variable strain (301-b) during growth in a chemostat were examined. When the bacterium was grown in anaerobic fructose-limited chemostat cultures (pH 7.0 and 37 °) at dilution rates ranging from 0.04 to 0.20 h in the absence and presence of 10 mM bicarbonate, it produced leukotoxin as a cluster of two polypeptides 113000 and 120000) and complexed with nucleic acids on the bacterial cell surface. The relationship between leukotoxin production and specific growth rate was analysed by plotting the specific rate of leukotoxin production in μg (mg dry wt) h] against . The plots were approximated to the linear relationships 2.7 − 0.058 and − 9.3 − 0.407 without and with bicarbonate, respectively. These relationships suggest that the apparent leukotoxin production is a result of both growth-rate-dependent production and growth-rate-independent decomposition. The cellular leukotoxin level was also followed after the change from chemostat to batch culture in the same fermenter. In batch culture leukotoxin production stopped immediately and the cellular toxin level rapidly decreased, suggesting toxin decomposition. From the slopes of the approximated linear relationships between and , a theoretical maximum leukotoxin yield ) was estimated as 2.7 and 9.3 μg (mg dry wt) in the absence and presence of 10 mM bicarbonate, respectively. The increased value in the cultures containing bicarbonate indicated that the addition stimulated the efficiency of leukotoxin synthesis up to about threefold. Further increases of bicarbonate concentration to between 20 and 40 mM had no effect on the total leukotoxin production, but the amount of extracellular leukotoxin increased with higher bicarbonate concentrations.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-142-4-963
1996-04-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/142/4/mic-142-4-963.html?itemId=/content/journal/micro/10.1099/00221287-142-4-963&mimeType=html&fmt=ahah

References

  1. Bartkus J.M., Leppla S.L. Transcriptional regulation of the protective antigen gene of Bacillus anthracis. Infect Immun 1989; 57:2295–2300
    [Google Scholar]
  2. Brogan J.M., Lally E.T., Poulsen K., Kilian M., Demuth D.R. Regulation of Actinobacillus actinomycetemcomitans leukotoxin expression: analysis of the promoter regions of leukotoxic and minimally leukotoxic strains. Infect Immun 1994; 62:501–508
    [Google Scholar]
  3. Caparon M.G., Geist R.T., Perez-Casal J., Scott J.R. Environmental regulation of virulence in group A streptococci: transcription of the gene encoding M protein is stimulated by carbon dioxide. J Bacteriol 1992; 174:5693–5701
    [Google Scholar]
  4. Di Rienzo J.M., Tsai C.-C., Shenker B.J., Taichman N.S., Lally E.T. Monoclonal antibodies to leukotoxin of Actinobacillus actinomycetemcomitans. Infect Immun 1985; 47:31–36
    [Google Scholar]
  5. Guthmiller J.M., Kolodrubetz D., Cagle M.P., Kraig E. Sequence of the IktB gene from Actinobacillus actinomycetemcomitans. Nucleic Acids Res 1990a; 18:5291
    [Google Scholar]
  6. Guthmiller J.M., Kraig E., Cagle M.P., Kolodrubetz D. Sequence of the IktD gene from Actinobacillus actinomycetemcomitans. Nucleic Acids R« 1990b; 18:5292
    [Google Scholar]
  7. Hunkapiller M.W., Hewick R.M., Dreyer W.J., Hood L.E. High -sensitivity sequencing with a gas-phase sequenator. Methods Entçymol 1983; 91:399–413
    [Google Scholar]
  8. Jenkins G.N. The Physiology and Biochemistry of the Mouth, 4th edn 1978 Oxford: Blackwell Scientific Publications;
    [Google Scholar]
  9. Koehler T.M., Dai Z., Kaufman-Yarbray M. Regulation of the Bacillus anthracis protective antigen gene: COa and a transacting element activate transcription from one of two promoters. J Bacteriol 1994; 176:586–595
    [Google Scholar]
  10. Kraig E., Dailey T., Kolodrubetz D. Nucleotide sequence of the leukotoxin gene from Actinobacillus actinomycetemcomitans: homology to the alpha-hemolysin/leukotoxin gene family. Infect Immun 1990; 58:920–929
    [Google Scholar]
  11. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685
    [Google Scholar]
  12. Lally E.T., Golub E.E., Kieba I.R., Taichman N.S., Rosenbloom J., Rosenbloom J.C., Gibson C.W., Demuth D.R. Analysis of the Actinobacillus actinomycetemcomitans leukotoxin gene. J Biol Chem 1989; 264:15451–15456
    [Google Scholar]
  13. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 1987; 262:10035–10038
    [Google Scholar]
  14. Nicaud J.-M., Mackman N., Gray L., Holland I.B. Regulation of haemolysin synthesis in E. coli determined by HLY genes of human origin. Mol & Gen Genet 1985; 199:111–116
    [Google Scholar]
  15. Ohta H., Kato K. Leukotoxic activity of Actinobacillus actinomycetemcomitans. In Periodontal Disease: Pathogens 6a Host Immune Responses 1991 Edited by Hamada S., Holt S.C., McGhee J.R. Tokyo: Quintesence Publishing; pp 143–154
    [Google Scholar]
  16. Ohta H., Kokeguchi S., Fukui K., Kato K. Leukotoxic activity in Actinobacillus (Haemophilus) actinomycetemcomitans isolated from periodontal disease patients. Microbiol Immunol 1987; 31:313–325
    [Google Scholar]
  17. Ohta H., Fukui K., Kato K. Effect of bicarbonate on the growth of Actinobacillus actinomycetemcomitans in anaerobic fructose-limited chemostat cultures. J Gen Microbiol 1989; 135:3485–3495
    [Google Scholar]
  18. Ohta H., Kato K., Kokeguchi S., Hara H., Fukui K., Murayama Y. Nuclease-sensitive binding of an Actinobacillus actinomycetemcomitans leukotoxin to the bacterial cell surface. Infect Immun 1991; 59:4599–4605
    [Google Scholar]
  19. Ohta H., Hara H., Fukui K., Kurihara H., Murayama Y., Kato K. Association of Actinobacillus actinomycetemcomitans leukotoxin with nucleic acids on the bacterial cell surface. Infect Immun 1993; 61:4878–4884
    [Google Scholar]
  20. Perez-Casal J., Caparon M.G., Scott J.R. Mry, a transacting positive regulator of the M protein gene of Streptococcus pyogenes with similarity to the receptor proteins of two-component regulatory systems. J Bacteriol 1991; 173:2617–2624
    [Google Scholar]
  21. Pirt S.J. Principles of Microbe and Cell Cultivation 1975 London: Blackwell Scientific Publications;
    [Google Scholar]
  22. Poulsen K., Theilade E., Lally E.T., Demuth D.R., Kilian M. Population structure of Actinobacillus actinomycetemcomitans: a framework for studies of disease-associated properties. Microbiology 1994; 140:2049–2060
    [Google Scholar]
  23. Sirard J.-C., Mock M., Fouet A. The three Bacillus anthracis toxin genes are coordinately regulated by bicarbonate and temperature. J Bacteriol 1994; 176:5188–5192
    [Google Scholar]
  24. Slots J. Salient biochemical characters of Actinobacillus actinomycetemcomitans. Arch Microbiol 1982; 131:60–67
    [Google Scholar]
  25. Slots J., Schonfeld S.E. Actinobacillus actinomycetemcomitans in localized juvenile periodontitis. In Periodontal Disease: Pathogens & Host Immune Responses 1991 Edited by Hamada S., Holt S.C., McGhee J.R. Tokyo: Quintesence Publishing; pp 53–64
    [Google Scholar]
  26. Spitznagel J. Jr, Kraig E., Kolodrubetz D. Regulation of leukotoxin in leukotoxic and nonleukotoxic strains of Actinobacillus actinomycetemcomitans. Infect Immun 1991; 59:1394–1401
    [Google Scholar]
  27. Strathdee C.A., Lo R.Y.C. Cloning, nucleotide sequence, and characterization of genes encoding the secretion function of the Pasteurella haemolytica leukotoxin determinant. J Bacteriol 1989; 171:916–928
    [Google Scholar]
  28. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76:4350–4354
    [Google Scholar]
  29. Tsai C.-C., McArthur W.P., Baehni P.G., Hammond B.F., Taichman N.S. Extraction and partial characterization of a leukotoxin from a plaque-derived gram-negative microorganism. Infect Immun 1979; 25:427–439
    [Google Scholar]
  30. Tsai G.-G., Shenker B.J., Di Rienzo J.M., Malamud D., Taichman N.S. Extraction and isolation of a leukotoxin from Actinobacillus actinomycetemcomitans with polymyxin B. Infect Immun 1984; 43:700–705
    [Google Scholar]
  31. Uchida I., Hornung J.M., Thorne C.B., Klimpel K.R., Leppla S.H. Cloning and characterization of a gene whose product is a /ra«-activator of anthrax toxin synthesis. J Bacteriol 1993; 175:5329–5338
    [Google Scholar]
  32. Zambon J. Actinobacillus actinomycetemcomitans in human periodontal disease. J Clin Periodontol 1985; 12:1–20
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-142-4-963
Loading
/content/journal/micro/10.1099/00221287-142-4-963
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error