A SINGLE AMINO-ACID CHANGE IN ANGR, A PROTEIN ENCODED BY PJM1-LIKE VIRULENCE PLASMIDS, RESULTS IN HYPERPRODUCTION OF ANGUIBACTIN

The siderophore anguibactin is produced in vivo in a diffusible form a nd is an important factor in the virulence of Vibrio anguillarum. The natural isolate V. anguillarum 531A is a hyperproducer of anguibactin when compared with the prototype strain V. anguillarum 775. The angR g ene was found to be responsible for this difference in levels of angui bactin produced. Nucleotide sequence analysis showed that the angR531A differed in a single nucleotide from the angR775 present in the proto type plasmid pJM1. This nucleotide substitution resulted in a change i n amino acid 267 from His in strain 775 to Asn in strain 531A. This am ino acid is located in a region between one of the two helix-turn-heli x domains and the neighboring leucine zipper. Mutations to replace His with either Leu or Gln, generated by site-directed mutagenesis, in am ino acid 267 resulted in strains for which the MIC of the iron chelato r ethylenediamine di(o-hydroxyphenyl) acetic acid were lower than for the proptotype 775 but higher than for iron uptake-deficient strains. In addition to its transcriptional activating function, AngR also comp lemented a mutation in the Escherichia coli entE gene, which encodes t he enterobactin biosynthetic enzyme 2,3-dihydroxybenzoate-AMP ligase. Therefore, AngR may also function in V. anguillarum as an EntE-like en zyme for the biosynthesis of anguibactin.