We have isolated a recombinant clone harboring the chromosomal aroC gene, encoding chorismate synthase, from Vibrio anguillarum 775 by complementation of the Escherichia coli aroC mutant AB2849 which was transfected with a cosmid gene bank of the plasmidless V. anguillarum H775-3. The nucleotide sequence was determined, and an open reading frame that corresponds to a protein of 372 amino acids was found. The calculated mass of 40,417 Da was correlated with the size of the V. anguillarum aroC product detected in vitro. The homology of the V. anguillarum aroC gene to the aroC genes of E. coli and Salmonella typhi is 68% at the nucleotide level and 78% at the protein level. The expression of the aroC transcript is not regulated by iron, as determined by Northern (RNA) blot hybridization analysis. After insertion of an antibiotic resistance gene cassette within the cloned aroC gene, an aroC mutant of V. anguillarum was generated by allelic exchange. This mutant is deficient in the production of 2,3-dihydroxybenzoic acid (2,3-DHBA). Our bioassay and complementation experiments with this mutant demonstrate that the chromosome-mediated 2,3-DHBA is a precursor of the pJM1 plasmid-mediated siderophore anguibactin.