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Cloning and nucleotide sequence of the gene coding for the major 25-kilodalton outer membrane protein of Brucella abortus.

ABSTRACT: The cloning and sequencing of the Brucella abortus major 25-kDa outer membrane protein (OMP) is reported. The 25-kDa (group 3) OMP has been proposed, on the basis of amino acid composition, to be the counterpart of OmpA (D. R. Verstraete, M. T. Creasy, N. T. Caveney, C. L. Baldwin, M. W. Blab, and A. J. Winter, Infect. Immun. 35:979-989, 1982). However, the amino acid sequence predicted from the cloned B. abortus gene did not reveal significant homology with either OmpA sequences from different members of the family Enterobacteriaceae or other known protein sequences.

SUBMITTER: de Wergifosse P

PROVIDER: S-EPMC176829 | biostudies-other | 1995 Apr

REPOSITORIES: biostudies-other

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Cloning and nucleotide sequence of the gene coding for the major 25-kilodalton outer membrane protein of Brucella abortus.

de Wergifosse P P Lintermans P P Limet J N JN Cloeckaert A A

Journal of bacteriology 19950401 7

The cloning and sequencing of the Brucella abortus major 25-kDa outer membrane protein (OMP) is reported. The 25-kDa (group 3) OMP has been proposed, on the basis of amino acid composition, to be the counterpart of OmpA (D. R. Verstraete, M. T. Creasy, N. T. Caveney, C. L. Baldwin, M. W. Blab, and A. J. Winter, Infect. Immun. 35:979-989, 1982). However, the amino acid sequence predicted from the cloned B. abortus gene did not reveal significant homology with either OmpA sequences from different ...[more]

PMID: 7896724

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Project description:The gene coding for the major outer membrane protein (OMP) of 31 to 34 kDa, now designated Omp31, of Brucella melitensis 16M was cloned and sequenced. A B. melitensis 16M genomic library was constructed in lambda GEM-12 XhoI half-site arms, and recombinant phages expressing omp31 were identified by using the anti-Omp31 monoclonal antibody (MAb) A59/10F09/G10. Subcloning of insert DNA from a positive phage into pGEM-7Zf allowed the selection of a plasmid bearing a 4.4-kb EcoRI fragment that seemed to contain the entire omp31 gene under control of its own promoter. omp31 was localized within a region of the EcoRI insert of approximately 1.1 kb. Sequencing of this region revealed an open reading frame of 720 bp encoding a protein of 240 amino acids and a predicted molecular mass of 25,307 Da. Cleavage of the first 19 amino acids, showing typical features of signal peptides for protein export, leaves a mature protein of 221 amino acids with a predicted molecular mass of 23,412 Da. The predicted amino acid sequence of B. melitensis 16M Omp31 showed 35.2% identity with the RopB OMP of Rhizobium leguminosarum bv. viciae 248 and 34.3% identity with Omp25 of B. abortus 544. As in Brucella spp., Omp31 was located in the outer membrane of recombinant Escherichia coli, but its reported peptidoglycan association in Brucella cells was not detected in E. coli. The ability of Omp31 to form oligomers resistant to sodium dodecyl sulfate denaturation at low temperatures, a characteristic described for several bacterial porins, was observed in both B. melitensis and recombinant E. coli. The epitope recognized by the anti-Omp31 MAb A59/10F09/G10, for which a protective activity has been suggested, has been delimited to a region of 36 amino acids of Omp31 covering the most hydrophilic part of the protein. The availability of recombinant Omp31 and the identification of the antigenic determinant recognized by MAb A59/10F09/G10 will allow the evaluation of their potential protective activity and their potential for the development of subcellular vaccines against brucellosis.

Project description:The cloning of the gene(s) encoding a 36-kilodalton (kDa) cell envelope protein of Brucella abortus has been previously described (T. A. Ficht, S. W. Bearden, B. A. Sowa, and L. G. Adams, Infect, Immun. 56:2036-2046, 1988). In an attempt to define the nature of the previously described duplication at this locus we have sequenced 3,500 base pairs of genomic DNA encompassing this region. The duplication represented two similar open reading frames which shared more than 85% homology at the nucleotide level but differed primarily because of the absence of 108 nucleotides from one of the two gene copies. These two genes were read from opposite strands and potentially encoded proteins which are 96% homologous. The predicted gene products were identical over the first 100 amino acids, including 22-amino-acid-long signal sequences. The amino acid composition of the predicted proteins was similar to that obtained for the Brucella porin isolated by Verstreate et al. (D. R. Verstreate, M. T. Creasy, N. T. Caveney, C. L. Baldwin, M. W. Blab, and A. J. Winter, Infect. Immun. 35:979-989, 1982) and presumably represented two copies of the porin gene, tentatively identified as omp 2a (silent) and omp 2b (expressed). The homology between the two genes extended to and included Shine-Dalgarno sequences 7 base pairs upstream from the ATG start codons. Homology at the 3' ends extended only as far as the termination codon, but both genes had putative rho-independent transcription termination sites. Localization of the promoters proved more difficult, since the canonical procaryotic sequences could not be identified in the region upstream of either gene. Promoter activity was demonstrated by ligation to a promoterless lacZ gene in pMC1871. However, only one active promoter could be identified by using this system. A 36-kDa protein was synthesized in E. coli with the promoter in the native orientation and was identical in size to the protein produced in laboratory-grown B. abortus. When the promoter-containing fragment was inverted, a 33-kDa protein was expressed. These results were consistent with the predicted sizes based on the nucleotide sequences of the open reading frames in omp 2b and omp 2a. Whether this locus contains one active and one silent or cryptic porin gene, or two active Brucella porin genes expressed under different environmental conditions, is discussed.

Project description:The nucleotide sequences encoding the major 25-kDa outer membrane protein (OMP) (omp25 genes) of Brucella ovis 63/290, Brucella melitensis 16M, Brucella suis 1330, Brucella canis RM6/66, and Brucella neotomae 5K33 (all reference strains) were determined and compared with that of Brucella abortus 544 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The major difference found was between the omp25 gene of B. ovis and those of the other Brucella species; the B. ovis gene had a 36-bp deletion located at the 3' end of the gene. The corresponding regions of other Brucella species contain two 8-bp direct repeats and two 4-bp inverted repeats, which could have been involved in the genesis of the deletion. The mechanism responsible for the genesis of the deletion appears to be related to the "slipped mispairing" mechanism described in the literature. Expression of the 25-kDa outer membrane protein (Omp25) in Brucella spp. or expression from the cloned omp25 gene in Escherichia coli cells was studied with a panel of anti-Omp25 monoclonal antibodies (MAbs). As shown by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy, Omp25 was exported to the outer membrane in E. coli expressing either the truncated omp25 gene of B. ovis or the entire omp25 genes of the other Brucella species. Size and antigenic shifts due to the 36-bp deletion were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and by the differences in binding patterns in ELISA of the anti-Omp25 MAbs at the cell surface of E. coli cells harboring the appropriate gene and of cells of B. ovis and other Brucella species. In particular, MAbs directed against discontinuous epitopes of the entire Omp25 showed the absence of, or a significant reduction in, antibody reactivity with the B. ovis truncated Omp25. The results indicated that, as defined by the MAbs, exported Omp25 probably presents similar topologies in the outer membranes of E. coli and Brucella spp. and that the short deletion found in the omp25 gene of B. ovis has important consequences for the expression of surface B-cell epitopes which should be considered for the development of vaccines against B. ovis infection.

Dataset Information

Cloning and nucleotide sequence of the gene coding for the major 25-kilodalton outer membrane protein of Brucella abortus.

Publications

Cloning and nucleotide sequence of the gene coding for the major 25-kilodalton outer membrane protein of Brucella abortus.

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