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

Abstract

In BruceUla abortus, a gene encoding a major cell envelope protein, omp2, is duplicated within a short segment of the large chromosomal DNA. Although both genes contain open reading frames, encoding proteins of high identity, expression from only one, omp2b, has been detected in laboratory-grown B. abortus. In the present study, we wished to determine whether omp2b encodes the previously studied BruceUa porin and to characterize the omp2a gene product. Experiments were performed with Escherichia coli transformants expressing either omp2a or omp2b. Our results indicated that both gene products localized to the outer membrane ofE. coli. Initial rates of transport of [14CJmaltose and growth rates in the presence of maltodextrins of defined size indicated an increased hydrophilic permeability of transformants expressing omp2a. These cells were also shown to grow on maltotetraose, a molecule with a molecular mass of 667 Da. Activity consistent with the formation of pores could not be demonstrated in transformants expressing omp2b. However, Omp2bformed oligomers resistant to heat denaturation up to 70°C in sodium dodecyl sulfate buffer, a property characteristic of bacterial porins. Overall, these results suggest that the omp2a gene product has pore-forming activity and that the omp2b gene encodes the previously characterized BruceUla porin. 3785 on July 31, 2020 by guest http://iai.asm.org/ Downloaded from

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

“…Omp2a on the other hand has no easily definable channel size with the transitions spanning a range from 650 pS down to 45 pS. This does not account for the increased sugar permeability observed previously [5]. Omp2a exhibits the properties of an unstable protein on SDS‐PAGE [5] and in this respect resembles the behaviour of eyelet deletions [16] in OmpC.…”

“…E. coli ECB611 (MC4100, Δ lamB ompF ::Tn5 ompC ::Tn10) was a kind gift from Spencer Benson [5] and was transformed with plasmids pAGF21 (expressing no porin), pAGF211 (expressing Omp2a) and pAGF201 (Omp2b) as previously described [5].…”

“…Porins were purified using Octyl‐POE detergent [12] with the modification that, following the final detergent extraction further column chromatography was not carried out. Expression levels can be revealed by immunoblots [5] and fractions were also assayed by pore formation.…”

“…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].…”

“…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]. Omp2a and Omp2b of B. abortus can be expressed in the outer membrane of E. coli and they exhibit properties of porins [5]. This paper describes results from Omp2 reconstitution into planar lipid bilayers and also a topology model of Omp2 and RIII created using methods known to produce accurate topologies for enteric and Rhodobacter porins whose structures are known [10].…”