Under iron-limiting conditions, Bacillus subtilis (Bs) produces the siderophore 2,3-dihydroxybenzoate (DHB) to acquire extracellular iron. In Escherichia coli (Ec), DHB is a precursor of the siderophore enterobactin, which suggested that Bs may possess similar biosynthetic enzymes. The sequences of two overlapping Bs clones capable of complementing Ec enterobactin mutants [Grossman, T.H., Tuckman, M., Ellestad, S. and Osburne, M.S. (1993) Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: Relationship between B. subtilis sfpo and Escherichia coli entD genes. J. Bacteriol. 175, 6203-6211] were analyzed and five open reading frames were identified. These genes are located near 291 degrees on the Bs chromosome and have been termed dhbA, dhbC, dhbE, dhbB and dhbF, based on similarities to Ec ent homologs. Amino-acid identities between gene product homologs are: EntA and DhbA, 41%; EntC and DhbC, 35%; EntE and DhbE, 48%; EntB and DhbB, 54%; and EntF and DhbF, 29%. DhbC is also 35% identical to the Bs menaquinone-specific isochorismate synthase, MenF, illustrating an example of gene duplication. Operon disruption studies suggested that the dhb genes comprise an operon of at least four genes.