1887

Abstract

SUMMARY: The two protein exotoxins secreted by are composed of three distinct components: protective antigen (PA), lethal factor (LF), and (o)edema factor (EF). We have developed a genetic strategy that permits us selectively to inactivate each of the genes coding for PA, EF or LF. This strategy involved the deletion of a portion of the structural gene and the insertion of an antibiotic resistance cassette. With this technique, double mutant strains of producing only one toxin component have been constructed. Characterization of the mutant strains indicated that they produced the expected single toxin protein. Using a simple, two-step protocol, we have purified PA, LF and EF to homogeneity from culture supernatants. These three mutant strains are potentially powerful tools for studying the individual effect of each toxin component and .

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-139-10-2459
1993-10-01
2024-04-24
Loading full text...

Full text loading...

/deliver/fulltext/micro/139/10/mic-139-10-2459.html?itemId=/content/journal/micro/10.1099/00221287-139-10-2459&mimeType=html&fmt=ahah

References

  1. Avakyan A.A., Katz L.N., Pavlova L.B. 1965; Structure and composition of the Bacillus anthracis capsule.. Journal of Bacteriology 90:1082–1095
    [Google Scholar]
  2. Beall F.A., Taylor M.J., Thorne C.B. 1962; Rapid lethal effects of the third component found upon fractionating the toxin of Bacillus anthracis. . Journal of Bacteriology 83:1274–1280
    [Google Scholar]
  3. Bragg T.S., Robertson D.L. 1989; Nucleotide sequence and analysis of the lethal factor gene (lef) from Bacillus anthracis. . Gene 81:45–54
    [Google Scholar]
  4. Cataldi A., Labruyère E., Mock M. 1990; Construction and characterization of a protective antigen deficient Bacillus anthracis strain.. Molecular Microbiology 4:1111–1117
    [Google Scholar]
  5. Escuyer V., Duflot E., Sezer O., Danchin A., Mock M. 1988; Structural homology between virulence-associated bacterial adenyate cyclases.. Gene 71:293–298
    [Google Scholar]
  6. Fish D. C., Klein F., Lincoln R. E., Walker J. S., Dobbs J. P. 1968 a; Pathophysiological changes in the rat associated with anthrax toxin.. Journal of Infectious Diseases 118:114–124
    [Google Scholar]
  7. Fish D. C., Mahlandt B. G., Dobbs J. P., Lincoln R. E. 1968b; Purification and properties of in vitro produced anthrax toxin components.. Journal of Bacteriology 95:907–918
    [Google Scholar]
  8. Green B.D., Battisti L., Koehler T.M., Thorne L.B., Ivins B.E. 1985; Demonstration of a capsule plasmid in Bacillus anthracis. . Infection and Immunity 49:291–297
    [Google Scholar]
  9. Hambleton P., Carman J.A., Melling J. 1984; Anthrax: the disease in relation to the vaccines.. Vaccine 2:125–132
    [Google Scholar]
  10. Iacono-Connors L.C., Schmaljohn C.S., Dalrymple J.M. 1990; Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.. Infection and Immunity 58:366–372
    [Google Scholar]
  11. Ivins B.E., Welkos S. 1986; Cloning and expression of the Bacillus anthracis protective antigen gene in Bacillus subtilis. . Infection and Immunity 54:537–542
    [Google Scholar]
  12. Labruyére E., Mock M., Ladant D., Michelson S., Gilles A.M., Laoïde B., Barzu O. 1990; Characterization of ATP and calmodulin-binding properties of a truncated form of Bacillus anthracis adenylate cyclase.. Biochemistry 29:4922–4928
    [Google Scholar]
  13. Ladant D. 1988; Interaction of Bordetellapertussis adenylate cyclase with calmodulin: identification of two separated calmodulin-binding domains.. Journal of Biological Chemistry 263:2612–2618
    [Google Scholar]
  14. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4.. Nature, London 227:680–685
    [Google Scholar]
  15. Leppla S.H. 1982; Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations in eucaryotic cells.. Proceedings of the National Academy of Sciences of the United States of America 793162–3166
    [Google Scholar]
  16. Leppla S.H. 1984; Bacillus anthracis calmodulin dependent adenylate cyclase: chemical and enzymatic properties and interactions with eucaryotic cells.. Advances in Cyclic Nucleotide and Protein Phosphorylation Research 17:189–198
    [Google Scholar]
  17. Leppla S.H. 1988; Production and purification of anthrax toxin.. Methods in Enzymology 165:103–116
    [Google Scholar]
  18. Mikesell P., Ivins B.E., Ristroph J.D., Dreier T.P. 1983; Evidence for plasmid-mediated toxin production in Bacillus anthracis. . Infection and Immunity 39:371–376
    [Google Scholar]
  19. Miller J.H. 1972 Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  20. Mock M., Labruyère E., Glaser P., Danchin A., Ullmann A. 1988; Cloning and expression of the calmodulin-sensitive Bacillus anthracis adenylate cyclase in Escherichia coli. . Gene 64:277–284
    [Google Scholar]
  21. Pezard C., Berche P., Mock M. 1991; Contribution of individual toxin components to virulence of Bacillus anthracis. . Infection and Immunity 59:3472–3477
    [Google Scholar]
  22. Quinn C.P., Shone C.C., Turnbull P.C.B., Melling J. 1988; Purification of anthrax-toxin components by high-performance anion exchange, gel filtration and hydrophobic-interaction chromatography.. Journal of Biochemistry 252:753–758
    [Google Scholar]
  23. Ristroph J.D., Ivins B.E. 1983; Elaboration of a new, defined culture medium.. Infection and Immunity 39:483–486
    [Google Scholar]
  24. Sambrook J., Maniatis T., Fritsch E. 1989 Molecular Cloning. A Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  25. Smith H., Stoner H.B. 1967; Anthrax toxin complex.. Federation Proceedings 261554–1557
    [Google Scholar]
  26. Stanley J.L., Smith H. 1961; Purification of factor I and recognition of the third factor of anthrax toxin.. Journal of General Microbiology 26:49–66
    [Google Scholar]
  27. Sterne M. 1939; The immunization of laboratory animals against anthrax.. Ondersterpoort Journal of Veterinary Science and Animal Industry 13:313–317
    [Google Scholar]
  28. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.. Proceedings of the National Academy of Sciences of the United States of America 764350–4354
    [Google Scholar]
  29. Trieu-Cuot P., Carlier C., Martin P., Courvalin P. 1987; Plasmid transfer by conjugation from Escherichia coli to Gram+ bacteria.. FEMS Microbiology Letters 48:289–294
    [Google Scholar]
  30. Trieu-Cuot P., Carlier C., Poyart-Salmeron C., Courvalin P. 1990a; A pair of mobilizable shuttle vectors conferring resistance to spectinomycin for molecular cloning in Escherichia coli and in Gram-positive bacteria.. Nucleic Acids Research 18:4296
    [Google Scholar]
  31. Trieu-Cuot P., Poyart-Salmeron C., Carlier C., Courvalin P. 1990 b; Nucleotide sequence of the erythromycin resistance gene of the conjugative transposon Tn1545. . Nucleic Acids Research 18:3660
    [Google Scholar]
  32. Uchida I., Sekizaki T., Hashimoto R., Tirakado N. 1985; Association of the encapsulation of Bacillus anthracis with a 60 megadalton plasmid.. Journal of General Microbiology 131:363–367
    [Google Scholar]
  33. Vodkin M.H., Leppla S.H. 1983; Cloning of the protective antigen gene of Bacillus anthracis. . Cell 34:693–697
    [Google Scholar]
  34. Welkos S. L., Friedlander A. M. 1988 a; Pathogenesis and genetic control of resistance to the Sterne strain of Bacillus anthracis. . Microbial Pathogenesis 4:53–69
    [Google Scholar]
  35. Welkos S. L., Friedlander A. M. 1988 b; Comparative safety and efficacity against Bacillus anthracis of protective antigen and live-vaccines in mice.. Microbial Pathogenesis 5:127–139
    [Google Scholar]
  36. Welkos S.L., Lowe J.R., Eden-Mccutchan F., Vodkin M., Leppla S.H., Schmidt J.J. 1988; Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis. . Gene 69:287–300
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-139-10-2459
Loading
/content/journal/micro/10.1099/00221287-139-10-2459
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