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Journal of Bacteriology, July 1999, p. 4299-4307, Vol. 181, No. 14
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Interaction of Bacillus subtilis Fur (Ferric Uptake Repressor) with the dhb Operator In Vitro and In Vivo

Nada Bsatdagger and John D. Helmann*

Section of Microbiology, Cornell University, Ithaca, New York 14853-8101

Received 12 March 1999/Accepted 29 April 1999

Bacillus subtilis contains three metalloregulatory proteins belonging to the ferric uptake repressor (Fur) family: Fur, Zur, and PerR. We have overproduced and purified Fur protein and analyzed its interaction with the operator region controlling the expression of the dihydroxybenzoate siderophore biosynthesis (dhb) operon. The purified protein binds with high affinity and selectivity to the dhb regulatory region. DNA binding does not require added iron, nor is binding reduced by dialysis of Fur against EDTA or treatment with Chelex. Fur selectively inhibits transcription from the dhb promoter by sigma A RNA polymerase, even if Fur is added after RNA polymerase holoenzyme. 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 that 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 His96 as a second likely iron ligand, since a His96Ala mutant mediates repression at 50 µM but not at 5 µM iron. Our data lead us to suggest that Fur is able to bind DNA independently of bound iron and that the in vivo role of iron is to counteract the effect of an inhibitory factor, perhaps another metal ion, that antagonizes this DNA-binding activity.

* Corresponding author. Mailing address: Section of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101. Phone: (607) 255-6570. Fax: (607) 255-3904. E-mail: jdh9{at}cornell.edu.

dagger Present address: Section of Genetics and Development, Cornell University, Ithaca, NY 14853.

Journal of Bacteriology, July 1999, p. 4299-4307, Vol. 181, No. 14
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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