The expression of haemin-binding proteins (HBPs) in the outer membrane is one of the strategies used by Gram-negative bacteria to obtain iron from the host. No HBP has been described in Brucella spp. We investigated whether Omp31, an outer membrane protein from Brucella with homology to HBPs from Bartonella quintana, is an HBP. Soluble recombinant Omp31 bound specifically to haemin-agarose, while an unrelated Brucella protein (SurA) did not. A similar experiment showed that native Omp31 found in the Brucella suis membrane fraction also binds to haemin-agarose. Recombinant Omp31 was electrophoresed by SDS-PAGE, transferred to nitrocellulose, and incubated with a haemin solution. Haemin bound to Omp31 and to albumin (positive control) but not to SurA. IPTG-induced recombinant Escherichia coli cells expressing Omp31 on their membrane bound significantly more haemin than uninduced cells or controls carrying a similar plasmid without the omp31 gene, showing that Omp31 also binds haemin in a bacterial membrane environment. Viable Brucella ovis cells bound haemin in solution, and this binding was markedly inhibited by preincubation of cells with antibodies to Omp31 and to an exposed prominent loop of the protein, thus showing that Omp31 functions as an HBP in brucellae. To test whether the expression of Omp31 is iron-regulated, B. suis was grown in trypticase-soy broth (TSB) and in iron-depleted TSB. The expression of Omp31, as assessed by Western blot, was significantly higher in bacteria grown under iron limitation. Overall, these results show that Omp31 from B. suis, B. melitensis and B. ovis is an HBP, whose expression seems to be induced by iron limitation.
