Characterization of the interaction between Fur and the iron transport promoter of the virulence plasmid in Vibrio anguillarum

The expression of iron transport genes fatDCBA in Vibrio anguillarum strain 775 is negatively regulated by two iron-responsive repressors, the Fur pro tein and the antisense RNA, RNA alpha. Here we report the identification of the promoter for the iron transport genes and studied the interaction betw een the V. anguillarum Fur protein and this promoter. The iron transport pr omoter was localized in a region approximately 300 base pairs upstream of f atD by both primer extension and S1 mapping analysis. High activity of the promoter was measured in response to iron depletion in the wild-type strain when a promoter-lacZ fusion was examined, whereas the promoter was constit utive in the Fur-deficient strain. Gel retardation and DNase I fool;print a nalysis showed that Fur binds specifically to two contiguous sites comprisi ng the promoter region and the region downstream of the transcription start site. The identified Fur binding sites showed a low degree of homology to each other as well as to the consensus sequence for the Escherichia coli Fu r protein. DNase I footprints pattern suggested a sequential interaction of Fur with these two sites that renders a protection in the template strand and a hyper sensitivity to the nuclease in the nontemplate strand. The peri odicity of the hypersensitive sites suggested that the promoter DNA undergo es a structural change upon binding to Fur, which might play a role in the repression of gene expression.