Isolation of the group 1 outer membrane protein gene of Brucella abortus and evidence for its location within a complex operon with similarity to the macromolecular biosynthesis II operon of Escherichia coli

Abstract

The role of outer membrane proteins (OMPs) in the pathogenesis of a number of gram-negative bacteria is widely reported in the literature, however, molecular studies of the facultative, intracellular pathogen Brucella abortus have provided limited information regarding the involvement of OMPs in the virulence of this infectious bacterium. In this investigation a gene encoding an 87kDa protein has been isolated from $lambda$2001 genomic DNA libraries derived from B. abortus strains S19 and S2308. The molecular mass of this protein corresponds with the previously described B. abortus group 1 OMPs. Inserts from the $lambda$2001 recombinants were subcloned into the plasmid vector pBR322 and expressed in E. coli. Western blot analysis of E. coli transformants shows the protein, designated Omp1, to be localized in the cell envelope. Transposon mutagenesis of the recombinant plasmids enabled the 5$spprime$ and 3$spprime$ ends of the omp1 gene to be more clearly defined and provided evidence that omp1 may be transcribed as part of a polycistronic messenger RNA. Results of the mutagenesis experiments show that expression of the omp1 gene product is interrupted by transposon insertions as much as two kilobases proximal to the omp1 coding region. DNA sequence analysis of the region upstream of omp1 revealed the presence of an 810 bp open reading frame (ORF) with a high degree of similarity to the cdsA gene of E. coli encoding CDP-diglyceride synthetase located in the minute 4 region of the E. coli chromosome. Results of complementation experiments designed to rescue the pH-sensitive phenotype of an E. coli cdsA mutant suggest that the upstream ORF is the B. abortus cognate cdsA gene. Finally, an examination of the genetic organization of the B. abortus omp1 locus revealed a number of similarities to the structure of the E. coli minute 4 region. Preliminary sequence analysis of regions downstream of the omp1 gene has provided additional evidence that omp1 is part of a complex operon whose genes are involved in B. abortus cell growth and/or division.