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. 1991 Nov;59(11):3863–3868. doi: 10.1128/iai.59.11.3863-3868.1991

Deletion of the BCSP31 gene of Brucella abortus by replacement.

S M Halling 1, P G Detilleux 1, F M Tatum 1, B A Judge 1, J E Mayfield 1
PMCID: PMC258969  PMID: 1937745

Abstract

The 31-kDa salt-extractable immunogenic protein, BCSP31, was deleted from several Brucella abortus strains by replacement with a marker gene encoding resistance to the antibiotics kanamycin and neomycin. The BCSP31 gene replacement plasmids, constructed with ColE1-derived vectors, were introduced by electroporation into B. abortus strain 19 (S19), into a rough variant of B. abortus S19, and into B. abortus S2308, and antibiotic-resistant transformants were isolated. B. abortus S19 is an attenuated strain used as a vaccine for prevention of bovine brucellosis in the United States, and B. abortus S2308 is a commonly used challenge strain. The antibiotic-resistant isolates were all obtained by recombination; none were spontaneous mutants. Loss of the gene encoding BCSP31 and presence of the marker gene were confirmed by Southern analysis. Vector sequences were either absent or linked to the genome, indicating that ColE1-derived plasmids are not maintained in B. abortus. Survival of B. abortus mutant strains in the macrophagelike cell line J774 and in HeLa cells was examined and shown to be indistinguishable from that of the parental strain.

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Selected References

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  1. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  2. Bricker B. J., Tabatabai L. B., Deyoe B. L., Mayfield J. E. Conservation of antigenicity in a 31-kDa Brucella protein. Vet Microbiol. 1988 Dec;18(3-4):313–325. doi: 10.1016/0378-1135(88)90096-x. [DOI] [PubMed] [Google Scholar]
  3. Bricker B. J., Tabatabai L. B., Judge B. A., Deyoe B. L., Mayfield J. E. Cloning, expression, and occurrence of the Brucella Cu-Zn superoxide dismutase. Infect Immun. 1990 Sep;58(9):2935–2939. doi: 10.1128/iai.58.9.2935-2939.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chomczynski P., Qasba P. K. Alkaline transfer of DNA to plastic membrane. Biochem Biophys Res Commun. 1984 Jul 18;122(1):340–344. doi: 10.1016/0006-291x(84)90480-7. [DOI] [PubMed] [Google Scholar]
  5. Detilleux P. G., Deyoe B. L., Cheville N. F. Penetration and intracellular growth of Brucella abortus in nonphagocytic cells in vitro. Infect Immun. 1990 Jul;58(7):2320–2328. doi: 10.1128/iai.58.7.2320-2328.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ficht T. A., Bearden S. W., Sowa B. A., Adams L. G. A 36-kilodalton Brucella abortus cell envelope protein is encoded by repeated sequences closely linked in the genomic DNA. Infect Immun. 1988 Aug;56(8):2036–2046. doi: 10.1128/iai.56.8.2036-2046.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fiedler S., Wirth R. Transformation of bacteria with plasmid DNA by electroporation. Anal Biochem. 1988 Apr;170(1):38–44. doi: 10.1016/0003-2697(88)90086-3. [DOI] [PubMed] [Google Scholar]
  8. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  9. Hoiseth S. K., Stocker B. A. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature. 1981 May 21;291(5812):238–239. doi: 10.1038/291238a0. [DOI] [PubMed] [Google Scholar]
  10. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  11. Marchioli C. C., Yancey R. J., Jr, Wardley R. C., Thomsen D. R., Post L. E. A vaccine strain of pseudorabies virus with deletions in the thymidine kinase and glycoprotein X genes. Am J Vet Res. 1987 Nov;48(11):1577–1583. [PubMed] [Google Scholar]
  12. Mayfield J. E., Bricker B. J., Godfrey H., Crosby R. M., Knight D. J., Halling S. M., Balinsky D., Tabatabai L. B. The cloning, expression, and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein. Gene. 1988;63(1):1–9. doi: 10.1016/0378-1119(88)90540-9. [DOI] [PubMed] [Google Scholar]
  13. Mekalanos J. J., Swartz D. J., Pearson G. D., Harford N., Groyne F., de Wilde M. Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature. 1983 Dec 8;306(5943):551–557. doi: 10.1038/306551a0. [DOI] [PubMed] [Google Scholar]
  14. Pugh G. W., Jr, Tabatabai L. B., Bricker B. J., Mayfield J. E., Phillips M., Zehr E. S., Belzer C. A. Immunogenicity of Brucella-extracted and recombinant protein vaccines in CD-1 and BALB/c mice. Am J Vet Res. 1990 Sep;51(9):1413–1420. [PubMed] [Google Scholar]
  15. Pugh G. W., Jr, Zehr E. S., Meador V. P., Phillips M., McDonald T. J., Deyoe B. L. Immunologic, histopathologic, and bacteriologic responses of five strains of mice to Brucella abortus strain 2308. Am J Vet Res. 1989 Mar;50(3):323–328. [PubMed] [Google Scholar]
  16. Smith L. D., Heffron F. Transposon Tn5 mutagenesis of Brucella abortus. Infect Immun. 1987 Nov;55(11):2774–2776. doi: 10.1128/iai.55.11.2774-2776.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Tabatabai L. B., Deyoe B. L. Specific enzyme-linked immunosorbent assay for detection of bovine antibody to Brucella abortus. J Clin Microbiol. 1984 Aug;20(2):209–213. doi: 10.1128/jcm.20.2.209-213.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Taketo A. DNA transfection of Escherichia coli by electroporation. Biochim Biophys Acta. 1988 Mar 31;949(3):318–324. doi: 10.1016/0167-4781(88)90158-3. [DOI] [PubMed] [Google Scholar]
  20. 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]

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