DNA sequence and expression of the 36-kilodalton outer membrane protein gene of Brucella abortus

Abstract

The cloning of the gene(s) encoding a 36-kilodalton (kDa) cell envelope protein of BruceUa abortus has been previously described (T. A. Ficht, S. W. Bearden, B. A. Sowa, and L. G. Adams, Infect. Immun. 56:2036Immun. 56: -2046Immun. 56: , 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 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 MATERIALS AND METHODSBacterial strains and cultivation. B. abortus smooth strains 19 and 2308 were obtained from Billy Deyoe at the National 3281 on August 4, 2020 by guest http://iai.asm.org/ Downloaded from (XAR-5; Eastman Kodak Co.) for 24 to 48 h. RESULTS Restriction mapping. Genomic recombinants in X2001 containing Brucella DNA inserts of approximately 20 kbp were INFECT. IMMUN. on August 4, 2020 by guest http://iai.asm.org/ Downloaded from

The omp2 gene locus of Brucella abortus encodes two homologous outer membrane proteins with properties characteristic of bacterial porins

“…Cellular localization of Omp2a and Omp2b produced in E. coli. In a previous study, it was shown that the omp2b gene product was present in the CE of B. abortus but the omp2a gene product could not be detected (11). To determine the subcellular localization of both gene products, we used transformants of E. coli ECB611 expressing either omp2a or omp2b.…”

“…Bacterial strains included E. coli MC4100 (6) and ECB611 [MC4100(AlamB ompF::TnS ompC::TnlO)], which was the kind gift of Spencer Benson. ECB611 was transformed with plasmid pAGF201, pAGF211, or pAGF21 (11). ECB611(pAGF201) and ECB611(pAGF211) expressed the omp2b and omp2a genes of B. abortus, respectively.…”

“…cloned a gene encoding a major Brucella abortus cell envelope (CE) protein suggested to be the Brucella porin gene (10). Hybridization studies and DNA sequence analysis of the gene locus revealed the presence of duplicate genes, designated omp2a and omp2b, encoding proteins of 33 and 36 kDa (10)(11)(12). The 5' one-third of both genes is identical, and the 3' one-third exhibits 90% identity, while the middle sections differ mainly by the absence of a 108-bp segment in the omp2a sequence.…”

“…Only expression from the omp2b gene has been detected in B. abortus. In E. coli, expression of omp2a was obtained after inverting the KpnI restriction fragment which contains the omp2b promoter region and the 5' end of both genes (11). Because the omp2a gene product has not been identified in B. abortus, it is not known if omp2a is a silent or an active gene.…”

Brucella Omp2a and Omp2b porins: single channel measurements and topology prediction

“…are serious Gram‐negative pathogens of both domestic animals and humans. Their outer membrane proteins, Omp2 [5] together with RIII ( Rhizobium ) [6], form an amino acid sequence family which has many similar features [7], but no detectable sequence homology to the above families [2, 8]. Among Omp2 from Brucella species and biovars the most striking variation occurs in Omp2a of Brucella abortus (biovars 1, 2 and 4) where a deletion of 30 residues has occurred relative to the rest of the highly homologous Omp2 family [9].…”