Biosynthetic and regulatory elements involved in the production of the siderophore vanchrobactin in Vibrio anguillarum

Abstract

Some Vibrio anguillarum strains produce a catechol-type siderophore named vanchrobactin, whose biosynthetic pathway has not been completely elucidated. In addition to the previously described genes vabA, vabC, vabB, vabE, vabF, vabS and vabH, in the present study we have identified the genes encoding a DAHP (3-deoxy-d-arabino-heptulosonate-7-phosphate) synthetase (vabG), a phosphopantheteinyl transferase (vabD), a LysR-family transcriptional regulator (vabR) and a putative siderophore receptor (fvtA). A deletion affecting vabG or vabD greatly reduced growth under iron-limiting conditions, whereas deletion of vabR did not have significant effects. Vanchrobactin production was abolished in the vabD mutant, whereas the vabG mutant retained a residual vanchrobactin production ability. Reverse transcriptase-mediated PCR indicated that this 11-gene cluster is organized into six iron-regulated transcriptional units. Transcriptional lacZ fusions demonstrated that the ferric uptake regulator (Fur) protein is the main iron-responsive regulator of these genes. Interestingly, the vabG gene was strongly iron-repressed, but Fur was not essential for this repression. In addition, the maximal expression from the vabG promoter was achieved only in the presence of an intact copy of vabR. Analysis of the beta-galactosidase activities of a fvtA : : lacZ fusion in a vabB mutant and in the presence of added vanchrobactin suggested that a ferric-vanchrobactin-dependent activator plays a positive regulatory role in transcription of the fvtA-vabD operon. This possibility is reinforced by the presence of a predicted AraC box upstream of fvtA. We propose that vanchrobactin biosynthesis is subjected to a complex regulatory circuitry aimed at adjusting vanchrobactin production for the maintenance of iron homeostasis in V. anguillarum.