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J. Biol. Chem., Vol. 279, Issue 31, 32100-32105, July 30, 2004

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The Ferric Uptake Regulator (Fur) Protein from Bradyrhizobium japonicum Is an Iron-responsive Transcriptional Repressor in Vitro^*

Yali E. Friedman and Mark R. O'Brian

From the Department of Biochemistry and Witebsky Center for Microbial Pathogenesis and Immunology, State University of New York, Buffalo, New York 14214

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 Fe²⁺ 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 Fe²⁺ but not in the absence of metal, showing that DNA binding is Fe²⁺-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 Fe²⁺. Thus, BjFur is an iron-responsive transcriptional repressor in vitro. A regulatory Fe²⁺-binding site (site 1) and a structural Zn²⁺-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 Fe²⁺-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.

Received for publication, May 3, 2004 , and in revised form, May 13, 2004.

^* This work was supported by National Science Foundation Grant MCB-0089928, United States Department of Agriculture Grant 2003-35319-13269, and National Institutes of Health Grant GM-067966 (to M. R. O'B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, 140 Farber Hall, State University of New York, Buffalo, NY 14214. Tel.: 716-829-3200; Fax: 716-829-2725; E-mail: mrobrian{at}buffalo.edu.

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