Interaction of Bacillus subtilis Fur (ferric uptake repressor) with the dhb operator in vitro and in vivo

Bacillus subtilis contains three metalloregulatory proteins belonging to th e ferric uptake repressor (Fur) family: Fur, Zur, and PerR. We have overpro duced and purified Fur protein and analyzed its interaction with the operat or region controlling the expression of the dihydroxybenzoate siderophore b iosynthesis (dhb) operon. The purified protein binds with high affinity and selectivity to the dhb regulatory region. DNA binding does not require add ed iron, nor is binding reduced by dialysis of Fur against EDTA or treatmen t with Chelex. Fur selectively inhibits transcription from the dhb promoter by sigma(A) RNA polymerase, even if Fur is added after RNA polymerase holo enzyme. Since neither DNA binding nor inhibition of transcription requires the addition of ferrous ion in vitro, the mechanism by which iron regulates Fur function in vivo is not obvious. Mutagenesis of the fur gene reveals t hat in vivo repression of the dhb operon by iron requires His97, a residue thought to be involved in iron sensing in other Fur homologs, Moreover, we identify Bis96 as a second likely iron ligand, since a His96Ala mutant medi ates repression at 50 mu M but not at 5 mu M iron. Our data lead us to sugg est that Fur is able to bind DNA independently of bound iron and that the i n vivo role of iron is to counteract the effect of an inhibitory factor, pe rhaps another metal ion, that antagonizes this DNA-binding activity.