Cloning, identification and expression of an entE homologue angE from Vibrio anguillarum serotype O1.

Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, is synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulence plasmid pJM1. angE, as one of the NRPS genes, is responsible for selecting and activating 2,3-dihydroxybenzoic acid (2,3-DHBA), an important precursor in anguibactin synthesis, into 2,3-DHBA-AMP by adenylylation in the presence of ATP. In this work, an entE homologue, angE, was identified on pEIB1 (a pJM1-like plasmid) from virulent V. anguillarum serotype O1 strain MVM425. A recombinant clone carrying the complete angE was able to complement an Escherichia coli entE mutant. The angE-encoded protein was overexpressed in E. coli and purified by a three-step procedure. Purified AngE was then used to establish an in vitro enzymatic reaction in which its enzymatic activity of 1-(5'-monophosphate adenyl) 2,3-dihydroxybenzoic acid ligase (2,3-DHBA-AMP ligase) was proved using HPLC to detect AMP formation in the reaction mixture. Moreover, evidence at the level of both transcription and translation confirmed that angE was actively expressed in vivo in V. anguillarum MVM425, and interestingly, unlike many other iron-uptake-system-related genes, its expression is not induced by a low iron concentration in the surrounding environment.