1887

Abstract

strain P1059 is a highly virulent bacterium which causes fowl cholera in turkeys and chickens. A genomic library of P1059 DNA was constructed using pUC19, expressed in DH5α, and screened with chicken antisera generated against P1059. Twelve out of the 4100 clones screened were immunoreactive. Plasmids isolated from these twelve clones were transformed into CSR603 for maxicell analysis. Five proteins, with molecular masses of 34, 37, 43, 46 and 55 kDa, were expressed. Further work focused on the 43 kDa protein because it was expressed at levels detectable by SDS-PAGE and immunoblot analysis. The nucleotide sequence of the 1.8 kbp insert containing the gene encoding this protein was determined. The sequence contained three open reading frames (ORFs). The first ORF (ORF1) did not appear to code for any known protein. The second ORF (ORF2) encoded a protein of 403 amino acids (43662 Da). The deduced amino acid sequence showed 77% identity (84% similarity) with the tryptophan synthase β subunit (TrpB) of and The eight conserved regions of TrpB are observed in the enzyme, including the conserved lysine (Lys-88) and consensus sequence (GGGSNA) implicated in pyridoxal phosphate binding. The expression and identity of the TrpB were confirmed by complementation studies using W3110 The third ORF (ORF3) consisted of the first 77 nucleotides of the gene encoding the β-subunit of tryptophan synthase and overlapped the 3'-end of by 14 nucleotides. The deduced amino acid sequence of the 77 nucleotides of the TrpA had 68% identity (92% similarity) with the analogous region of TrpA from

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1996-01-01
2024-03-29
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References

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. 1990; Basic local alignment search tool. J Mol Biol 215:403–410
    [Google Scholar]
  2. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 1989 Short Protocols in Molecular Biology New York: Wiley-Interscience;
    [Google Scholar]
  3. Bardowski J., Ehrlich S. D., Chopin A. 1992; Tryptophan biosynthesis genes in Lactococcus lactis subsp. lactis. J Bacteriol 174:6563–6570
    [Google Scholar]
  4. Bollag D. M., Edelstein S. J. 1991 Protein Methods New York: Wiley-Liss;
    [Google Scholar]
  5. Crawford I. P. 1989; Evolution of a biosynthetic pathway: the tryptophan paradigm. Annu Rev Microbiol 43:567–600
    [Google Scholar]
  6. Creighton T. E., Yanofsky C. 1966; Association of the α and β2subunits of the tryptophan synthetase of Escherichia coli. J Biol Chem 241:980–990
    [Google Scholar]
  7. Dower W. F., Miller F., Ragsdale C. W. 1988; High efficiency transformation of Escherichia coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145
    [Google Scholar]
  8. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
    [Google Scholar]
  9. Hansen L. M., Hirsh D. C. 1989; Serum resistance is correlated with encapsulation of avian strains of Pasteurella multocida. Vet Microbiol 21:177–184
    [Google Scholar]
  10. Homchampa P., Strugnell R. A., Adler B. 1992; Molecular analysis of the aroA gene of Pasteurella multocida and vaccine potential of a constructed aroA mutant. Mol Microbiol 6:3585–3593
    [Google Scholar]
  11. Hu S. P., Felice L. J., Sivanandan V., Maheswaran S. K. 1986; Siderophore production by Pasteurella multocida. Infect Immun 54:804–810
    [Google Scholar]
  12. Hyde C.C., Ahmed S. A., Padlan E. A., Miles E. W., Davies D. R. 1988; Three dimensional structure of the tryptophan synthetase α2/β2 multienzyme complex from Salmonella typhimurium. J Biol Chem 263:17857–17871
    [Google Scholar]
  13. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  14. Lax A. J., Chanter N., Pullinger G. D., Higgins T., Staddon J. M., Rozengurt E. 1990; Sequence analysis of the potent mitogenic toxin of Pasteurella multocida. FEBS Lett 277:59–64
    [Google Scholar]
  15. Livrelli V., Peduzzi J., Joly B. 1991; Sequence and molecular characterization of the R.OB-1 beta-lactamase gene from Pasteurella haemolytica. Antimicrob Agents Chemother 35:242–251
    [Google Scholar]
  16. Lu Y. S., Aguila H. N., Lai W. C., Pakes S. P. 1991a; Antibodies to outer membrane proteins but not to lipopolysaccharide inhibit pulmonary proliferation of Pasteurella multocida in mice. Infect Immun 59:1470–1475
    [Google Scholar]
  17. Lu Y. S., Lai W.C., Pakes S. P., Nie L. C. 1991b; A monoclonal antibody against a Pasteurella multocida outer membrane protein protects rabbits and mice against pasteurellosis. Infect Immun 59:172–180
    [Google Scholar]
  18. Manoha F., Chevalier G., Wrdblewski H., Delamarche C. 1994; Cloning and expression of two Pasteurella multocida genes in Escherichia coli. Biochimie 76:9–14
    [Google Scholar]
  19. Miles E. W., Kawasaki H., Ahmed S. A., Morita H., Morita H., Nagata S. 1989; The p subunit of tryptophan synthase. J Biol Chem 264:6280–6287
    [Google Scholar]
  20. Mock M., Crasnier M., Duflot E., Dumay V., Danchin A. 1991; Structural and functional relationships between Pasteurella multocida and enterobacterial adenylate cyclases. J Bacteriol 173:6265–6269
    [Google Scholar]
  21. Murray M. G., Thompson W. F. 1980; Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325
    [Google Scholar]
  22. Nagata S., Hyde C. C., Miles E. W. 1989; The a subunit of tryptophan synthase. J Biol Chem 264:6288–6296
    [Google Scholar]
  23. Petersen S. K. 1990; The complete nucleotide sequence of the Pasteurella multocida toxin gene and evidence for a transcriptional repressor TxaR. Mol Microbiol 4:821–830
    [Google Scholar]
  24. Petersen S. K., Foged N. T. 1989; Cloning and expression of the Pasteurella multocida toxin gene, toxA, in Escherichia coli. Infect Immun 57:3907–3913
    [Google Scholar]
  25. Pohl S. 1981; Pasteurella multocida. Haemophilus, Pasteurella, and Actinobacillus245–254 Edited by Killian K., Fredericksen W., Biberstein E. L. London: Academic Press;
    [Google Scholar]
  26. Rhoades K. R., Rimler R. B. 1987; Effects of Pasteurella multocida endotoxins on turkey poults. Avian Dis 31:523–526
    [Google Scholar]
  27. Rhoades K. R., Rimler R. B. 1988; Toxigenicity and virulence of capsular serogroup D Pasteurella multocida strains isolated from turkeys. J Am Vet Med Assoc 192:1790
    [Google Scholar]
  28. Rimler R. B., Rhoades K. R. 1981; Lysates of turkey-grown Pasteurella multocida: protection against homologous and heterologous serotype challenge exposures. Am J Vet Res 42:2117–2121
    [Google Scholar]
  29. Rimler R. B., Rhoades K. R. 1989; Pasteurella multocida. Pasteurella and Pasteurellosis37–73 Edited by Adlam C., Rutter J. M. London: Academic Press;
    [Google Scholar]
  30. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  31. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
    [Google Scholar]
  32. Snipes K. P., Ghazikhanian G. Y., Hirsh D. C. 1987; Fate of Pasteurella multocida in the blood vascular system of turkeys following intravenous inoculation: comparison of an encapsulated, virulent strain with its avirulent, acapsular variant. Avian Dis 31:254–259
    [Google Scholar]
  33. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
    [Google Scholar]
  34. Truscott W. M., Hirsh D. C. 1988; Demonstration of an outer membrane protein with antiphagocytic activity from Pasteurella multocida of avian origin. Infect Immun 56:1538–1544
    [Google Scholar]
  35. Wijewardana T. G., Wilson C. F., Gilmour N. J., Poxton I. R. 1990; Production of mouse monoclonal antibodies to Pasteurella multocida type A and the immunological properties of a protective anti-lipopolysaccharide antibody. J Med Microbiol 33:217–222
    [Google Scholar]
  36. Wilson D. A., Crawford I. P. 1965; Purification and properties of the B component of Escherichia coli tryptophan synthetase. J Biol Chem 240:4801–4808
    [Google Scholar]
  37. Yanofsky C., Piatt T., Crawford I. P., Nichols B. P., Christie G. E., Horowitz H., VanCleemput M., Wu A. M. 1981; The complete nucleotide sequence of the tryptophan operon of Escherichia coli. Nucleic Acids Res 9:6647–6668
    [Google Scholar]
  38. Yutani K., Ogasawara K., Tsujita T., Kanemoto K., Matsumoto M., Tanaka S., Miyashita T., Matsushiro A., Sugino Y., Miles E. W. 1987; Tryptophan synthase a subunit glutamic acid 49 is essential for activity. J Biol Chem 262:13429–13433
    [Google Scholar]
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