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. 1991 Nov;59(11):4238–4248. doi: 10.1128/iai.59.11.4238-4248.1991

Construction of minitransposons for constitutive and inducible expression of pertussis toxin in bvg-negative Bordetella bronchiseptica.

M J Walker 1, M Rohde 1, J Wehland 1, K N Timmis 1
PMCID: PMC259022  PMID: 1682257

Abstract

Appropriately detoxified pertussis toxin (PT) of Bordetella pertussis is considered to be an essential component of new-generation whooping cough vaccines, but the development of a procedure to obtain high levels of purified toxin has been and continues to be a major difficulty. To produce a system enabling the biological separation of PT from other virulence determinants of B. pertussis and the attainment of high yields of the toxin, minitransposons containing the PT operon were constructed and stably integrated into the chromosome of Bordetella virulence regulatory gene (bvg)-negative Bordetella bronchiseptica ATCC 10580. Since the minitransposons introduced into Bordetella spp. lack the cognate transposase function, they are unable to undergo further transposition events or mediate gene deletions and rearrangements that lead to strain instability. The TnPtacPT minitransposon contains the PT operon under the control of the tac promoter and directs IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible expression of PT in B. bronchiseptica ATCC 10580. The level of IPTG-induced PT expression was, however, lower than that found for the wild-type B. pertussis Tohama I strain. The TnfusPT minitransposon contains a promoterless PT operon which is only expressed after insertion of the transposon downstream of an appropriately oriented indigenous promoter. After "promoter probing" of B. bronchiseptica with the transposon, clones were screened for PT production by immunoblotting with specific monoclonal antibodies. One clone, designated B. bronchiseptica 10580:: TnfusPT1, expresses significantly higher levels of PT than does B. pertussis Tohama I. The recombinant toxin produced was biologically active in the Chinese hamster ovary cell-clustering assay. High-level expression of PT from a B. bronchiseptica host promoter should provide better yields of the toxin from bacteria not producing other bvg-regulated pathogenesis factors that may play a role in the undesired side effects of current pertussis vaccine preparations.

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Selected References

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  1. Aricò B., Rappuoli R. Bordetella parapertussis and Bordetella bronchiseptica contain transcriptionally silent pertussis toxin genes. J Bacteriol. 1987 Jun;169(6):2847–2853. doi: 10.1128/jb.169.6.2847-2853.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bartley T. D., Whiteley D. W., Mar V. L., Burns D. L., Burnette W. N. Pertussis holotoxoid formed in vitro with a genetically deactivated S1 subunit. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8353–8357. doi: 10.1073/pnas.86.21.8353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Brownlie R. M., Coote J. G., Parton R., Schultz J. E., Rogel A., Hanski E. Cloning of the adenylate cyclase genetic determinant of Bordetella pertussis and its expression in Escherichia coli and B. pertussis. Microb Pathog. 1988 May;4(5):335–344. doi: 10.1016/0882-4010(88)90061-7. [DOI] [PubMed] [Google Scholar]
  5. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  6. Burns D. L., Kenimer J. G., Manclark C. R. Role of the A subunit of pertussis toxin in alteration of Chinese hamster ovary cell morphology. Infect Immun. 1987 Jan;55(1):24–28. doi: 10.1128/iai.55.1.24-28.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Finan T. M., Kunkel B., De Vos G. F., Signer E. R. Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol. 1986 Jul;167(1):66–72. doi: 10.1128/jb.167.1.66-72.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Frank D. W., Parker C. D. Isolation and characterization of monoclonal antibodies to Bordetella pertussis. J Biol Stand. 1984 Oct;12(4):353–365. doi: 10.1016/s0092-1157(84)80060-8. [DOI] [PubMed] [Google Scholar]
  9. Gillenius P., Jätmaa E., Askelöf P., Granström M., Tiru M. The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells. J Biol Stand. 1985 Jan;13(1):61–66. doi: 10.1016/s0092-1157(85)80034-2. [DOI] [PubMed] [Google Scholar]
  10. Gross R., Rappuoli R. Positive regulation of pertussis toxin expression. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3913–3917. doi: 10.1073/pnas.85.11.3913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Herrero M., de Lorenzo V., Timmis K. N. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol. 1990 Nov;172(11):6557–6567. doi: 10.1128/jb.172.11.6557-6567.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hewlett E. L., Sauer K. T., Myers G. A., Cowell J. L., Guerrant R. L. Induction of a novel morphological response in Chinese hamster ovary cells by pertussis toxin. Infect Immun. 1983 Jun;40(3):1198–1203. doi: 10.1128/iai.40.3.1198-1203.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Keen N. T., Tamaki S., Kobayashi D., Trollinger D. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene. 1988 Oct 15;70(1):191–197. doi: 10.1016/0378-1119(88)90117-5. [DOI] [PubMed] [Google Scholar]
  14. Kimura A., Mountzouros K. T., Schad P. A., Cieplak W., Cowell J. L. Pertussis toxin analog with reduced enzymatic and biological activities is a protective immunogen. Infect Immun. 1990 Oct;58(10):3337–3347. doi: 10.1128/iai.58.10.3337-3347.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Knapp S., Mekalanos J. J. Two trans-acting regulatory genes (vir and mod) control antigenic modulation in Bordetella pertussis. J Bacteriol. 1988 Nov;170(11):5059–5066. doi: 10.1128/jb.170.11.5059-5066.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Lee C. K., Roberts A., Perrin S. Expression of pertussis toxin in Bordetella bronchiseptica and Bordetella parapertussis carrying recombinant plasmids. Infect Immun. 1989 May;57(5):1413–1418. doi: 10.1128/iai.57.5.1413-1418.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Locht C., Barstad P. A., Coligan J. E., Mayer L., Munoz J. J., Smith S. G., Keith J. M. Molecular cloning of pertussis toxin genes. Nucleic Acids Res. 1986 Apr 25;14(8):3251–3261. doi: 10.1093/nar/14.8.3251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Loosmore S. M., Zealey G. R., Boux H. A., Cockle S. A., Radika K., Fahim R. E., Zobrist G. J., Yacoob R. K., Chong P. C., Yao F. L. Engineering of genetically detoxified pertussis toxin analogs for development of a recombinant whooping cough vaccine. Infect Immun. 1990 Nov;58(11):3653–3662. doi: 10.1128/iai.58.11.3653-3662.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McGillivray D. M., Coote J. G., Parton R. Cloning of the virulence regulatory (vir) locus of Bordetella pertussis and its expression in B. bronchiseptica. FEMS Microbiol Lett. 1989 Dec;53(3):333–337. doi: 10.1016/0378-1097(89)90241-3. [DOI] [PubMed] [Google Scholar]
  21. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Monack D. M., Arico B., Rappuoli R., Falkow S. Phase variants of Bordetella bronchiseptica arise by spontaneous deletions in the vir locus. Mol Microbiol. 1989 Dec;3(12):1719–1728. doi: 10.1111/j.1365-2958.1989.tb00157.x. [DOI] [PubMed] [Google Scholar]
  23. Munoz J. J., Arai H., Cole R. L. Mouse-protecting and histamine-sensitizing activities of pertussigen and fimbrial hemagglutinin from Bordetella pertussis. Infect Immun. 1981 Apr;32(1):243–250. doi: 10.1128/iai.32.1.243-250.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nencioni L., Pizza M., Bugnoli M., De Magistris T., Di Tommaso A., Giovannoni F., Manetti R., Marsili I., Matteucci G., Nucci D. Characterization of genetically inactivated pertussis toxin mutants: candidates for a new vaccine against whooping cough. Infect Immun. 1990 May;58(5):1308–1315. doi: 10.1128/iai.58.5.1308-1315.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Robinson A., Irons L. I., Ashworth L. A. Pertussis vaccine: present status and future prospects. Vaccine. 1985 Mar;3(1):11–22. doi: 10.1016/0264-410x(85)90004-0. [DOI] [PubMed] [Google Scholar]
  27. Roth J., Bendayan M., Carlemalm E., Villiger W., Garavito M. Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. J Histochem Cytochem. 1981 May;29(5):663–671. doi: 10.1177/29.5.6166664. [DOI] [PubMed] [Google Scholar]
  28. Runeberg-Nyman K., Engström O., Löfdahl S., Ylöstalo S., Sarvas M. Expression and secretion of pertussis toxin subunit S1 in Bacillus subtilis. Microb Pathog. 1987 Dec;3(6):461–468. doi: 10.1016/0882-4010(87)90016-7. [DOI] [PubMed] [Google Scholar]
  29. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sato H., Sato Y., Ito A., Ohishi I. Effect of monoclonal antibody to pertussis toxin on toxin activity. Infect Immun. 1987 Apr;55(4):909–915. doi: 10.1128/iai.55.4.909-915.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sato Y., Arai H. Leucocytosis-promoting factor of Bordetella pertussis. I. Purification and characterization. Infect Immun. 1972 Dec;6(6):899–904. doi: 10.1128/iai.6.6.899-904.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stainer D. W., Scholte M. J. A simple chemically defined medium for the production of phase I Bordetella pertussis. J Gen Microbiol. 1970 Oct;63(2):211–220. doi: 10.1099/00221287-63-2-211. [DOI] [PubMed] [Google Scholar]
  33. Valentine R. C., Shapiro B. M., Stadtman E. R. Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli. Biochemistry. 1968 Jun;7(6):2143–2152. doi: 10.1021/bi00846a017. [DOI] [PubMed] [Google Scholar]
  34. Walker M. J., Birch R. G., Pemberton J. M. Cloning and characterization of an albicidin resistance gene from Klebsiella oxytoca. Mol Microbiol. 1988 Jul;2(4):443–454. doi: 10.1111/j.1365-2958.1988.tb00050.x. [DOI] [PubMed] [Google Scholar]
  35. Walker M. J., Guzmán C. A., Rohde M., Timmis K. N. Production of recombinant Bordetella pertussis serotype 2 fimbriae in Bordetella parapertussis and Bordetella bronchiseptica: utility of Escherichia coli gene expression signals. Infect Immun. 1991 May;59(5):1739–1746. doi: 10.1128/iai.59.5.1739-1746.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Walker M. J., Rohde M., Brownlie R. M., Timmis K. N. Engineering upstream transcriptional and translational signals of Bordetella pertussis serotype 2 fimbrial subunit protein for efficient expression in Escherichia coli: in vitro autoassembly of the expressed product into filamentous structures. Mol Microbiol. 1990 Jan;4(1):39–47. doi: 10.1111/j.1365-2958.1990.tb02013.x. [DOI] [PubMed] [Google Scholar]
  37. Walker M. J., Wehland J., Timmis K. N., Raupach B., Schmidt M. A. Characterization of murine monoclonal antibodies that recognize defined epitopes of pertussis toxin and neutralize its toxic effect on Chinese hamster ovary cells. Infect Immun. 1991 Nov;59(11):4249–4251. doi: 10.1128/iai.59.11.4249-4251.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Witvliet M. H., Burns D. L., Brennan M. J., Poolman J. T., Manclark C. R. Binding of pertussis toxin to eucaryotic cells and glycoproteins. Infect Immun. 1989 Nov;57(11):3324–3330. doi: 10.1128/iai.57.11.3324-3330.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  41. de Lorenzo V., Herrero M., Jakubzik U., Timmis K. N. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990 Nov;172(11):6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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