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