Abstract
The Fur protein represses transcription of iron-responsive genes in bacteria. The discovery that Fur is a zinc metalloprotein and the use of surrogate metals for Fe(2+) for in vitro studies question whether Fur is a direct iron sensor. In the present study, we show that the affinity of Fur from Bradyrhizobium japonicum (BjFur) for its target DNA increases 30-fold in the presence of metal, with a K(d) value of about 2 nM. DNase I footprinting experiments showed that BjFur protected its binding site within the irr gene promoter in the presence of Fe(2+) but not in the absence of metal, showing that DNA binding is Fe(2+)-dependent. BjFur did not inhibit in vitro transcription from the irr promoter using purified components in the absence of metal, but BjFur repressed transcription in the presence of Fe(2+). Thus, BjFur is an iron-responsive transcriptional repressor in vitro. A regulatory Fe(2+)-binding site (site 1) and a structural Zn(2+)-binding site (site 2) inferred from the recent crystal structure of Fur from Pseudomonas aeruginosa are composed of amino acids highly conserved in many Fur proteins, including BjFur. BjFur mutants containing substitutions in site 1 (BjFurS1) or site 2 (BjFurS2) bound DNA with high affinity and repressed transcription in vitro in an Fe(2+)-dependent manner. Interestingly, only a single dimer of BjFurS2 occupied the irr promoter, whereas the wild type and BjFurS1 displayed one- or two-dimer occupancy. We suggest that the putative functions for metal-binding sites deduced from the structure of P. aeruginosa Fur cannot be extrapolated to bacterial Fur proteins as a whole.
Highlights
Control of iron homeostasis is essential to most living organisms
We suggest that the putative functions for metal-binding sites deduced from the structure of P. aeruginosa Fur cannot be extrapolated to bacterial Fur proteins as a whole
Previous work shows that the high mobility complex (HMC) and the low mobility complex (LMC) correspond to a Fur dimer and to two dimers or a tetramer, respectively [7]
Summary
Control of iron homeostasis is essential to most living organisms. Iron availability can be limited because it is predominantly in the ferric form in aerobic environments. Fur containing one Zn2ϩ atom is sufficient for DNA binding, and the affinity does not increase when the second Zn2ϩ site is occupied [8] These observations question whether Fur is a direct iron sensor. Several groups have shown that the structural Zn2ϩ atom of E. coli Fur is coordinated by Cys-92 and Cys-95 [9, 10] These cysteines are not conserved in many Fur proteins. Fur from Bradyrhizobium japonicum (BjFur) typifies bacterial Fur proteins in that it complements an E. coli Fur mutant, mediates iron-dependent control of gene expression, and binds to a so-called Fur box, a consensus Fur-binding DNA element [7, 15, 16]. The amino acids identified in metal binding in P. aeruginosa Fur are conserved in the
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