Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1994 Sep;62(9):3633–3639. doi: 10.1128/iai.62.9.3633-3639.1994

Molecular cloning, nucleotide sequence, and occurrence of a 16.5-kilodalton outer membrane protein of Brucella abortus with similarity to pal lipoproteins.

A Tibor 1, V Weynants 1, P Denoel 1, B Lichtfouse 1, X De Bolle 1, E Saman 1, J N Limet 1, J J Letesson 1
PMCID: PMC303012  PMID: 8063379

Abstract

Recombinant lambda gt11 phages were selected by screening a genomic library of Brucella abortus DNA with monoclonal antibodies specific for a 16.5-kDa Brucella outer membrane protein (Omp16). The corresponding gene, named pal, was subcloned on a 0.7-kb AluI fragment. Immunoblotting confirmed the expression of a recombinant Omp16 in the transformants. DNA sequence analysis revealed an open reading frame of 168 codons. The deduced amino acid sequence agrees with an internal peptide sequence of native Omp16 and contains a potential lipoprotein signal peptide cleavage site, giving rise to a predicted mature protein of 144 amino acids. The predicted sequence of Omp16 also shows a remarkable degree of similarity to the sequences of three peptidoglycan-associated bacterial lipoproteins. In immunoblotting with a monoclonal antibody specific for Omp16, we demonstrated that Omp16 was expressed in the 34 Brucella strains tested, representing all six species and known biovars.

Full text

PDF
3633

Images in this article

3635Image
on p.3635

Selected References

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

  1. Bundle D. R., Gidney M. A., Perry M. B., Duncan J. R., Cherwonogrodzky J. W. Serological confirmation of Brucella abortus and Yersinia enterocolitica O:9 O-antigens by monoclonal antibodies. Infect Immun. 1984 Nov;46(2):389–393. doi: 10.1128/iai.46.2.389-393.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Caroff M., Bundle D. R., Perry M. B. Structure of the O-chain of the phenol-phase soluble cellular lipopolysaccharide of Yersinia enterocolitica serotype O:9. Eur J Biochem. 1984 Feb 15;139(1):195–200. doi: 10.1111/j.1432-1033.1984.tb07994.x. [DOI] [PubMed] [Google Scholar]
  3. Chen R., Henning U. Nucleotide sequence of the gene for the peptidoglycan-associated lipoprotein of Escherichia coli K12. Eur J Biochem. 1987 Feb 16;163(1):73–77. doi: 10.1111/j.1432-1033.1987.tb10738.x. [DOI] [PubMed] [Google Scholar]
  4. Chou P. Y., Fasman G. D. Conformational parameters for amino acids in helical, beta-sheet, and random coil regions calculated from proteins. Biochemistry. 1974 Jan 15;13(2):211–222. doi: 10.1021/bi00699a001. [DOI] [PubMed] [Google Scholar]
  5. Cloeckaert A., Jacques I., Bosseray N., Limet J. N., Bowden R., Dubray G., Plommet M. Protection conferred on mice by monoclonal antibodies directed against outer-membrane-protein antigens of Brucella. J Med Microbiol. 1991 Mar;34(3):175–180. doi: 10.1099/00222615-34-3-175. [DOI] [PubMed] [Google Scholar]
  6. Cloeckaert A., Jacques I., de Wergifosse P., Dubray G., Limet J. N. Protection against Brucella melitensis or Brucella abortus in mice with immunoglobulin G (IgG), IgA, and IgM monoclonal antibodies specific for a common epitope shared by the Brucella A and M smooth lipopolysaccharides. Infect Immun. 1992 Jan;60(1):312–315. doi: 10.1128/iai.60.1.312-315.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cloeckaert A., Kerkhofs P., Limet J. N. Antibody response to Brucella outer membrane proteins in bovine brucellosis: immunoblot analysis and competitive enzyme-linked immunosorbent assay using monoclonal antibodies. J Clin Microbiol. 1992 Dec;30(12):3168–3174. doi: 10.1128/jcm.30.12.3168-3174.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cloeckaert A., Zygmunt M. S., de Wergifosse P., Dubray G., Limet J. N. Demonstration of peptidoglycan-associated Brucella outer-membrane proteins by use of monoclonal antibodies. J Gen Microbiol. 1992 Jul;138(7):1543–1550. doi: 10.1099/00221287-138-7-1543. [DOI] [PubMed] [Google Scholar]
  9. Cloeckaert A., de Wergifosse P., Dubray G., Limet J. N. Identification of seven surface-exposed Brucella outer membrane proteins by use of monoclonal antibodies: immunogold labeling for electron microscopy and enzyme-linked immunosorbent assay. Infect Immun. 1990 Dec;58(12):3980–3987. doi: 10.1128/iai.58.12.3980-3987.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deich R. A., Metcalf B. J., Finn C. W., Farley J. E., Green B. A. Cloning of genes encoding a 15,000-dalton peptidoglycan-associated outer membrane lipoprotein and an antigenically related 15,000-dalton protein from Haemophilus influenzae. J Bacteriol. 1988 Feb;170(2):489–498. doi: 10.1128/jb.170.2.489-498.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Delforge D., Depiereux E., De Bolle X., Feytmans E., Remacle J. Similarities between alanine dehydrogenase and the N-terminal part of pyridine nucleotide transhydrogenase and their possible implication in the virulence mechanism of Mycobacterium tuberculosis. Biochem Biophys Res Commun. 1993 Feb 15;190(3):1073–1079. doi: 10.1006/bbrc.1993.1158. [DOI] [PubMed] [Google Scholar]
  12. Depiereux E., Feytmans E. MATCH-BOX: a fundamentally new algorithm for the simultaneous alignment of several protein sequences. Comput Appl Biosci. 1992 Oct;8(5):501–509. doi: 10.1093/bioinformatics/8.5.501. [DOI] [PubMed] [Google Scholar]
  13. Depiereux E., Feytmans E. Simultaneous and multivariate alignment of protein sequences: correspondence between physicochemical profiles and structurally conserved regions (SCR). Protein Eng. 1991 Aug;4(6):603–613. doi: 10.1093/protein/4.6.603. [DOI] [PubMed] [Google Scholar]
  14. Ficht T. A., Bearden S. W., Sowa B. A., Adams L. G. DNA sequence and expression of the 36-kilodalton outer membrane protein gene of Brucella abortus. Infect Immun. 1989 Nov;57(11):3281–3291. doi: 10.1128/iai.57.11.3281-3291.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fuchs P., Breitling F., Dübel S., Seehaus T., Little M. Targeting recombinant antibodies to the surface of Escherichia coli: fusion to a peptidoglycan associated lipoprotein. Biotechnology (N Y) 1991 Dec;9(12):1369–1372. doi: 10.1038/nbt1291-1369. [DOI] [PubMed] [Google Scholar]
  16. Green B. A., Metcalf B. J., Quinn-Dey T., Kirkley D. H., Quataert S. A., Deich R. A. A recombinant non-fatty acylated form of the Hi-PAL (P6) protein of Haemophilus influenzae elicits biologically active antibody against both nontypeable and type b H. influenzae. Infect Immun. 1990 Oct;58(10):3272–3278. doi: 10.1128/iai.58.10.3272-3278.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Green B. A., Quinn-Dey T., Zlotnick G. W. Biologic activities of antibody to a peptidoglycan-associated lipoprotein of Haemophilus influenzae against multiple clinical isolates of H. influenzae type b. Infect Immun. 1987 Dec;55(12):2878–2883. doi: 10.1128/iai.55.12.2878-2883.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gómez-Miguel M. J., Moriyón I. Demonstration of a peptidoglycan-linked lipoprotein and characterization of its trypsin fragment in the outer membrane of Brucella spp. Infect Immun. 1986 Sep;53(3):678–684. doi: 10.1128/iai.53.3.678-684.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gómez-Miguel M. J., Moriyón I., López J. Brucella outer membrane lipoprotein shares antigenic determinants with Escherichia coli Braun lipoprotein and is exposed on the cell surface. Infect Immun. 1987 Jan;55(1):258–262. doi: 10.1128/iai.55.1.258-262.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hayashi S., Wu H. C. Lipoproteins in bacteria. J Bioenerg Biomembr. 1990 Jun;22(3):451–471. doi: 10.1007/BF00763177. [DOI] [PubMed] [Google Scholar]
  21. Jacques I., Cloeckaert A., Limet J. N., Dubray G. Protection conferred on mice by combinations of monoclonal antibodies directed against outer-membrane proteins or smooth lipopolysaccharide of Brucella. J Med Microbiol. 1992 Aug;37(2):100–103. doi: 10.1099/00222615-37-2-100. [DOI] [PubMed] [Google Scholar]
  22. Jacques I., Olivier-Bernardin V., Dubray G. Induction of antibody and protective responses in mice by Brucella O-polysaccharide-BSA conjugate. Vaccine. 1991 Dec;9(12):896–900. doi: 10.1016/0264-410x(91)90010-4. [DOI] [PubMed] [Google Scholar]
  23. Klein P., Somorjai R. L., Lau P. C. Distinctive properties of signal sequences from bacterial lipoproteins. Protein Eng. 1988 Apr;2(1):15–20. doi: 10.1093/protein/2.1.15. [DOI] [PubMed] [Google Scholar]
  24. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Limet J. N., Bosseray N., Garin-Bastuji B., Dubray G., Plommet M. Humoral immunity in mice mediated by monoclonal antibodies against the A and M antigens of Brucella. J Med Microbiol. 1989 Sep;30(1):37–43. doi: 10.1099/00222615-30-1-37. [DOI] [PubMed] [Google Scholar]
  27. Limet J., Plommet A. M., Dubray G., Plommet M. Immunity conferred upon mice by anti-LPS monoclonal antibodies in murine brucellosis. Ann Inst Pasteur Immunol. 1987 May-Jun;138(3):417–424. doi: 10.1016/s0769-2625(87)80052-1. [DOI] [PubMed] [Google Scholar]
  28. Lin J., Adams L. G., Ficht T. A. Characterization of the heat shock response in Brucella abortus and isolation of the genes encoding the GroE heat shock proteins. Infect Immun. 1992 Jun;60(6):2425–2431. doi: 10.1128/iai.60.6.2425-2431.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ludwig B., Schmid A., Marre R., Hacker J. Cloning, genetic analysis, and nucleotide sequence of a determinant coding for a 19-kilodalton peptidoglycan-associated protein (Ppl) of Legionella pneumophila. Infect Immun. 1991 Aug;59(8):2515–2521. doi: 10.1128/iai.59.8.2515-2521.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Marck C. 'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 1988 Mar 11;16(5):1829–1836. doi: 10.1093/nar/16.5.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Meikle P. J., Perry M. B., Cherwonogrodzky J. W., Bundle D. R. Fine structure of A and M antigens from Brucella biovars. Infect Immun. 1989 Sep;57(9):2820–2828. doi: 10.1128/iai.57.9.2820-2828.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mizuno T. A novel peptidoglycan-associated lipoprotein (PAL) found in the outer membrane of Proteus mirabilis and other Gram-negative bacteria. J Biochem. 1981 Apr;89(4):1039–1049. [PubMed] [Google Scholar]
  34. Montaraz J. A., Winter A. J., Hunter D. M., Sowa B. A., Wu A. M., Adams L. G. Protection against Brucella abortus in mice with O-polysaccharide-specific monoclonal antibodies. Infect Immun. 1986 Mar;51(3):961–963. doi: 10.1128/iai.51.3.961-963.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Munson R. S., Jr, Granoff D. M. Purification and partial characterization of outer membrane proteins P5 and P6 from Haemophilus influenzae type b. Infect Immun. 1985 Sep;49(3):544–549. doi: 10.1128/iai.49.3.544-549.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nielsen K., Cherwonogrodzky J. W., Duncan J. R., Bundle D. R. Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Brucella abortus or vaccinated with strain 19. Am J Vet Res. 1989 Jan;50(1):5–9. [PubMed] [Google Scholar]
  37. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  39. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Vanfleteren J. R., Raymackers J. G., Van Bun S. M., Meheus L. A. Peptide mapping and microsequencing of proteins separated by SDS-PAGE after limited in situ acid hydrolysis. Biotechniques. 1992 Apr;12(4):550-2, 554, 556-7. [PubMed] [Google Scholar]
  42. Vinals C., Depiereux E., Feytmans E. Prediction of structurally conserved regions of D-specific hydroxy acid dehydrogenases by multiple alignment with formate dehydrogenase. Biochem Biophys Res Commun. 1993 Apr 15;192(1):182–188. doi: 10.1006/bbrc.1993.1398. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES