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. 1991 Jan;173(2):720–726. doi: 10.1128/jb.173.2.720-726.1991

Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation.

E M Barry 1, A A Weiss 1, I E Ehrmann 1, M C Gray 1, E L Hewlett 1, M S Goodwin 1
PMCID: PMC207064  PMID: 1987161

Abstract

In these studies, the Bordetella pertussis adenylate cyclase toxin-hemolysin homology to the Escherichia coli hemolysin is extended with the finding of cyaC, a homolog to the E. coli hlyC gene, which is required for the production of a functional hemolysin molecule in E. coli. Mutations produced in the chromosome of B. pertussis upstream from the structural gene for the adenylate cyclase toxin revealed a region which was necessary for toxin and hemolytic activities of the molecule. These mutants produced the 216-kDa adenylate cyclase toxin as determined by Western blot (immunoblot) analysis. The adenylate cyclase enzymatic activities of these mutants were equivalent to that of wild type, but toxin activities were less than 1% of that of wild type, and the mutants were nonhemolytic on blood agar plates and in in vitro assays. The upstream region restored hemolytic activity when returned in trans to the mutant strains. This genetic complementation defined a gene which acts in trans to activate the adenylate cyclase toxin posttranslationally. Sequence analysis of the upstream region defined an open reading frame with homology to the E. coli hlyC gene. In contrast to E. coli, this open reading frame is oriented oppositely from the adenylate cyclase toxin structural gene.

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Selected References

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  1. Au D. C., Masure H. R., Storm D. R. Site-directed mutagenesis of lysine 58 in a putative ATP-binding domain of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis abolishes catalytic activity. Biochemistry. 1989 Apr 4;28(7):2772–2776. doi: 10.1021/bi00433a005. [DOI] [PubMed] [Google Scholar]
  2. Boehm D. F., Welch R. A., Snyder I. S. Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes. Infect Immun. 1990 Jun;58(6):1959–1964. doi: 10.1128/iai.58.6.1959-1964.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Confer D. L., Eaton J. W. Phagocyte impotence caused by an invasive bacterial adenylate cyclase. Science. 1982 Sep 3;217(4563):948–950. doi: 10.1126/science.6287574. [DOI] [PubMed] [Google Scholar]
  4. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X. W., Finlay D. R., Guiney D., Helinski D. R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. doi: 10.1016/0147-619x(85)90068-x. [DOI] [PubMed] [Google Scholar]
  6. Felmlee T., Pellett S., Lee E. Y., Welch R. A. Escherichia coli hemolysin is released extracellularly without cleavage of a signal peptide. J Bacteriol. 1985 Jul;163(1):88–93. doi: 10.1128/jb.163.1.88-93.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Felmlee T., Pellett S., Welch R. A. Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J Bacteriol. 1985 Jul;163(1):94–105. doi: 10.1128/jb.163.1.94-105.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Glaser P., Elmaoglou-Lazaridou A., Krin E., Ladant D., Bârzu O., Danchin A. Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis. EMBO J. 1989 Mar;8(3):967–972. doi: 10.1002/j.1460-2075.1989.tb03459.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Glaser P., Ladant D., Sezer O., Pichot F., Ullmann A., Danchin A. The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coli. Mol Microbiol. 1988 Jan;2(1):19–30. [PubMed] [Google Scholar]
  11. Glaser P., Sakamoto H., Bellalou J., Ullmann A., Danchin A. Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis. EMBO J. 1988 Dec 1;7(12):3997–4004. doi: 10.1002/j.1460-2075.1988.tb03288.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gross R., Rappuoli R. Pertussis toxin promoter sequences involved in modulation. J Bacteriol. 1989 Jul;171(7):4026–4030. doi: 10.1128/jb.171.7.4026-4030.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hewlett E. L., Urban M. A., Manclark C. R., Wolff J. Extracytoplasmic adenylate cyclase of Bordetella pertussis. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1926–1930. doi: 10.1073/pnas.73.6.1926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Laoide B. M., Ullmann A. Virulence dependent and independent regulation of the Bordetella pertussis cya operon. EMBO J. 1990 Apr;9(4):999–1005. doi: 10.1002/j.1460-2075.1990.tb08202.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Masure H. R., Au D. C., Gross M. K., Donovan M. G., Storm D. R. Secretion of the Bordetella pertussis adenylate cyclase from Escherichia coli containing the hemolysin operon. Biochemistry. 1990 Jan 9;29(1):140–145. doi: 10.1021/bi00453a017. [DOI] [PubMed] [Google Scholar]
  17. Nicaud J. M., Mackman N., Gray L., Holland I. B. Characterisation of HlyC and mechanism of activation and secretion of haemolysin from E. coli 2001. FEBS Lett. 1985 Aug 5;187(2):339–344. doi: 10.1016/0014-5793(85)81272-2. [DOI] [PubMed] [Google Scholar]
  18. Nicosia A., Rappuoli R. Promoter of the pertussis toxin operon and production of pertussis toxin. J Bacteriol. 1987 Jun;169(6):2843–2846. doi: 10.1128/jb.169.6.2843-2846.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pearson R. D., Symes P., Conboy M., Weiss A. A., Hewlett E. L. Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin. J Immunol. 1987 Oct 15;139(8):2749–2754. [PubMed] [Google Scholar]
  20. Rogel A., Schultz J. E., Brownlie R. M., Coote J. G., Parton R., Hanski E. Bordetella pertussis adenylate cyclase: purification and characterization of the toxic form of the enzyme. EMBO J. 1989 Sep;8(9):2755–2760. doi: 10.1002/j.1460-2075.1989.tb08417.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stibitz S., Black W., Falkow S. The construction of a cloning vector designed for gene replacement in Bordetella pertussis. Gene. 1986;50(1-3):133–140. doi: 10.1016/0378-1119(86)90318-5. [DOI] [PubMed] [Google Scholar]
  22. Strathdee C. A., Lo R. Y. Cloning, nucleotide sequence, and characterization of genes encoding the secretion function of the Pasteurella haemolytica leukotoxin determinant. J Bacteriol. 1989 Feb;171(2):916–928. doi: 10.1128/jb.171.2.916-928.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wagner W., Vogel M., Goebel W. Transport of hemolysin across the outer membrane of Escherichia coli requires two functions. J Bacteriol. 1983 Apr;154(1):200–210. doi: 10.1128/jb.154.1.200-210.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wandersman C., Delepelaire P. TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4776–4780. doi: 10.1073/pnas.87.12.4776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Weiss A. A., Falkow S. Genetic analysis of phase change in Bordetella pertussis. Infect Immun. 1984 Jan;43(1):263–269. doi: 10.1128/iai.43.1.263-269.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weiss A. A., Goodwin M. S. Lethal infection by Bordetella pertussis mutants in the infant mouse model. Infect Immun. 1989 Dec;57(12):3757–3764. doi: 10.1128/iai.57.12.3757-3764.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Weiss A. A., Hewlett E. L., Myers G. A., Falkow S. Pertussis toxin and extracytoplasmic adenylate cyclase as virulence factors of Bordetella pertussis. J Infect Dis. 1984 Aug;150(2):219–222. doi: 10.1093/infdis/150.2.219. [DOI] [PubMed] [Google Scholar]
  29. Weiss A. A., Hewlett E. L., Myers G. A., Falkow S. Tn5-induced mutations affecting virulence factors of Bordetella pertussis. Infect Immun. 1983 Oct;42(1):33–41. doi: 10.1128/iai.42.1.33-41.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Welch R. A., Pellett S. Transcriptional organization of the Escherichia coli hemolysin genes. J Bacteriol. 1988 Apr;170(4):1622–1630. doi: 10.1128/jb.170.4.1622-1630.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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