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

Iron is an essential and often growth-limiting nutrient for microorganisms. The rapid oxidation of ferrous to ferric iron, which is virtually insoluble at a nearly neutral pH, reduces the level of bioavailable iron far below the approximately 1 μM that most organisms require for optimal growth (4, 18, 28, 29). Consequently, many bacteria synthesize and excrete high-affinity iron chelators (siderophores) that can solubilize ferric iron and subsequently be imported by a corresponding ferri-siderophore uptake system (29). The expression of genes for siderophore biosynthesis and transport is repressed by added iron. In many bacteria, this repression is mediated by a metal-sensing DNA-binding protein, the ferric uptake repressor (Fur) protein (22, 23). Fur has been best characterized from Escherichia coli (designated FurEC), but homologs have been identified in numerous other bacteria (22, 23). In vivo, FurEC represses a large regulon of iron uptake functions when iron is present in excess (6). Most other metals are ineffective at eliciting repression in vivo, although Mn(II) leads to the repression of some, but not all, Fur-regulated genes (3, 5, 21, 32). In many gram-negative bacteria, selection for manganese-resistant (Mnr) mutants leads to mutations in fur, suggesting that the ability of Mn(II) to activate Fur for DNA binding may be a source of manganese toxicity, perhaps by inappropriately repressing iron uptake functions (21). FurEC is an ∼16-kDa dimeric protein with an amino-terminal DNA recognition domain and a carboxyl-terminal metal-binding domain (12, 37). In vitro studies have demonstrated that FurEC binds to a specific DNA target site, the fur box, and that this binding requires a divalent metal ion as a cofactor (3, 14). In the vast majority of studies, Mn(II) is used to activate Fur, since this ion, unlike Fe(II), is stable in the presence of oxygen. It is generally assumed that Mn(II)-Fur is functionally analogous to the Fe(II) form thought to mediate repression in vivo. However, the metal-binding sites of Fur are not yet well characterized. Fur binds two divalent ions per monomer, and binding is thought to involve a cluster of conserved histidine residues and two pairs of cysteines in the carboxyl-terminal metal-binding domain (12, 24). One site is apparently occupied by a tightly associated (structural) zinc ion, while the second site is thought to bind Fe(II) reversibly to regulate DNA binding (24). Bacillus subtilis contains three Fur homologs (8, 17) that regulate a peroxide stress response (PerR), zinc uptake (Zur), and iron uptake (Fur). Fur regulates the expression of several operons implicated in iron transport (8, 23), including the dihydroxybenzoate siderophore biosynthesis (dhb) operon. Transcription of the dhb operon is initiated from a single ςA-dependent promoter with an overlapping consensus fur box (33). Mutations in this fur box or in fur prevent the iron-mediated repression of dhb (8, 33). However, unlike its homologs from gram-negative organisms, B. subtilis Fur does not recognize Mn(II) as a corepressor in vivo, and selection for Mnr does not generate fur mutants (8, 9). We have overproduced and purified B. subtilis Fur and initiated a study of its interactions with both DNA and metal ions. Surprisingly, Fur is purified in an active zinc-containing form that does not require the addition of Fe(II) to bind DNA or to repress transcription. Nevertheless, genetic studies are consistent with a model in which iron does interact directly with Fur in vivo.