Effect of entF deletion on iron acquisition and erythritol metabolism by Brucella abortus 2308

Brucella abortus has been shown to produce two siderophores: 2,3-dihydroxybenzoic acid (2,3-DHBA) and brucebactin. Previous studies on Brucella have shown that 2,3-DHBA is associated with erythritol utilization and virulence in pregnant ruminants. The biosynthetic pathway and role of brucebactin are not known and the only gene shown to be involved so far is entF. Using cre-lox methodology, an entF mutant was created in wild-type B. abortus 2308. Compared with the wild-type strain, the delta entF strain showed significant growth inhibition in iron minimal media that became exacerbated in the presence of an iron chelator. For the first time, we have demonstrated the death of the delta entF strain under iron-limiting conditions in the presence of erythritol. Addition of FeCl3 restored the growth of the delta entF strain, suggesting a significant role in iron acquisition. Further, complementation of the delta entF strain using a plasmid containing an entF gene suggested the absence of any polar effects. In contrast, there was no significant difference in survival and growth between the delta entF and wild-type strains grown in the murine macrophage cell line J774A.1, suggesting that an alternate iron acquisition pathway is present in Brucella when grown intracellulary.