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. 1989 Dec;57(12):3907–3913. doi: 10.1128/iai.57.12.3907-3913.1989

Cloning and expression of the Pasteurella multocida toxin gene, toxA, in Escherichia coli.

S K Petersen 1, N T Foged 1
PMCID: PMC259925  PMID: 2680987

Abstract

A chromosomal DNA library of a toxigenic type D strain of Pasteurella multocida subsp. multocida was established in Escherichia coli. From this library two clones, SPE308 and SPE312, were identified by using a monoclonal antibody against the osteoclast-stimulating P. multocida toxin (PMT). Extracts of these clones showed cytopathic activity identical to that of extracts of toxigenic P. multocida. The recombinant plasmids, pSPE308 and pSPE312, directed the synthesis of a protein with an apparent molecular weight of 143,000 which could be specifically detected by anti-PMT antibody. The recombinant toxin, which was located in the cytoplasm of E. coli, was purified by affinity chromatography with immobilized monoclonal antibody and was shown to react in a manner identical to that of PMT in a quantitative structural test using a series of monoclonal antibodies as well as in all quantitative functional tests used, i.e., tests for dermonecrotic activity and mouse lethality and the embryonic bovine lung cell test for cytopathic activity. The gene encoding this toxic activity was named toxA and was found to be present in the chromosome of toxigenic strains only of P. multocida. A probe spanning the toxA gene therefore has potential in the diagnosis and surveillance of progressive atrophic rhinitis in pigs.

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Selected References

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

  1. Andrup L., Atlung T., Ogasawara N., Yoshikawa H., Hansen F. G. Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein. J Bacteriol. 1988 Mar;170(3):1333–1338. doi: 10.1128/jb.170.3.1333-1338.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chanter N., Rutter J. M., Mackenzie A. Partial purification of an osteolytic toxin from Pasteurella multocida. J Gen Microbiol. 1986 Apr;132(4):1089–1097. doi: 10.1099/00221287-132-4-1089. [DOI] [PubMed] [Google Scholar]
  4. Foged N. T., Nielsen J. P., Pedersen K. B. Differentiation of toxigenic from nontoxigenic isolates of Pasteurella multocida by enzyme-linked immunosorbent assay. J Clin Microbiol. 1988 Jul;26(7):1419–1420. doi: 10.1128/jcm.26.7.1419-1420.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Foged N. T. Quantitation and purification of the Pasteurella multocida toxin by using monoclonal antibodies. Infect Immun. 1988 Aug;56(8):1901–1906. doi: 10.1128/iai.56.8.1901-1906.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kimman T. G., Löwik C. W., van de Wee-Pals L. J., Thesingh C. W., Defize P., Kamp E. M., Bijvoet O. L. Stimulation of bone resorption by inflamed nasal mucosa, dermonecrotic toxin-containing conditioned medium from Pasteurella multocida, and purified dermonecrotic toxin from P. multocida. Infect Immun. 1987 Sep;55(9):2110–2116. doi: 10.1128/iai.55.9.2110-2116.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. MALAMY M. H., HORECKER B. L. RELEASE OF ALKALINE PHOSPHATASE FROM CELLS OF ESCHERICHIA COLI UPON LYSOZYME SPHEROPLAST FORMATION. Biochemistry. 1964 Dec;3:1889–1893. doi: 10.1021/bi00900a017. [DOI] [PubMed] [Google Scholar]
  10. Nakai T., Kume K. Purification of three fragments of the dermonecrotic toxin from Pasteurella multocida. Res Vet Sci. 1987 Mar;42(2):232–237. [PubMed] [Google Scholar]
  11. Nakai T., Sawata A., Kume K. Intracellular locations of dermonecrotic toxins in Pasteurella multocida and in Bordetella bronchiseptica. Am J Vet Res. 1985 Apr;46(4):870–874. [PubMed] [Google Scholar]
  12. Nakai T., Sawata A., Tsuji M., Kume K. Characterization of dermonecrotic toxin produced by serotype D strains of Pasteurella multocida. Am J Vet Res. 1984 Nov;45(11):2410–2413. [PubMed] [Google Scholar]
  13. Nakai T., Sawata A., Tsuji M., Samejima Y., Kume K. Purification of dermonecrotic toxin from a sonic extract of Pasteurella multocida SP-72 serotype D. Infect Immun. 1984 Nov;46(2):429–434. doi: 10.1128/iai.46.2.429-434.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
  15. Nilsson B., Uhlén M., Josephson S., Gatenbeck S., Philipson L. An improved positive selection plasmid vector constructed by oligonucleotide mediated mutagenesis. Nucleic Acids Res. 1983 Nov 25;11(22):8019–8030. doi: 10.1093/nar/11.22.8019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pedersen K. B., Elling F. The pathogenesis of atrophic rhinitis in pigs induced by toxigenic Pasteurella multocida. J Comp Pathol. 1984 Apr;94(2):203–214. doi: 10.1016/0021-9975(84)90041-0. [DOI] [PubMed] [Google Scholar]
  17. Rimler R. B., Brogden K. A. Pasteurella multocida isolated from rabbits and swine: serologic types and toxin production. Am J Vet Res. 1986 Apr;47(4):730–737. [PubMed] [Google Scholar]
  18. Rutter J. M., Luther P. D. Cell culture assay for toxigenic Pasteurella multocida from atrophic rhinitis of pigs. Vet Rec. 1984 Apr 21;114(16):393–396. doi: 10.1136/vr.114.16.393. [DOI] [PubMed] [Google Scholar]
  19. Rylatt D. B., Parish C. R. Protein determination on an automatic spectrophotometer. Anal Biochem. 1982 Mar 15;121(1):213–214. doi: 10.1016/0003-2697(82)90578-4. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Stanley K. K. Solubilization and immune-detection of beta-galactosidase hybrid proteins carrying foreign antigenic determinants. Nucleic Acids Res. 1983 Jun 25;11(12):4077–4092. doi: 10.1093/nar/11.12.4077. [DOI] [PMC free article] [PubMed] [Google Scholar]

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