Abstract
The Fur protein is a global regulator of iron metabolism and other processes in many bacterial species. A key feature of the model of Fur function is the recognition of a DNA element within target promoters with similarity to a 19-bp AT-rich palindromic sequence called a Fur box. The irr gene from Bradyrhizobium japonicum is under the control of Fur. Here, we provide evidence that B. japonicum Fur (BjFur) binds to the irr gene promoter with high affinity despite the absence of DNA sequence similarity to the Fur box consensus. Both Escherichia coli Fur and BjFur bound a synthetic Fur box consensus DNA element in electrophoretic gel mobility shift assays, but only BjFur bound the irr promoter. BjFur maximally protected a 30-bp region in DNase I footprinting analysis that includes three imperfect direct repeat hexamers. BjFur formed a high mobility complex and a low mobility complex with DNA in electrophoretic gel mobility shift assays corresponding to occupancy by a single dimer and two dimers or a tetramer, respectively. A mutation in the downstream direct repeat DNA sequence allowed high mobility complex formation only. In vitro transcription from the wild type irr promoter or from a mutated promoter that allowed only dimer occupancy was repressed by Fur, indicating that the dimer can be a functional repressor unit. Our findings identify a novel DNA-binding element for Fur and suggest that the Fur box consensus may not completely represent the target sequences for bacterial Fur proteins as a whole. In addition, Fur binding to a target promoter is sufficient to repress transcription in vitro.
Highlights
Control of iron homeostasis is essential to most living organisms
B. japonicum Fur Binds to the irr Promoter—Previous work shows that the iron-dependent control of irr mRNA expression is aberrant in a fur mutant and that E. coli extracts that overexpress B. japonicum Fur (BjFur) bind to DNA corresponding to the irr gene upstream region [22]
This activity presumably allows BjFur to regulate the irr gene despite the absence of a Fur box consensus element in the irr promoter
Summary
Chemicals and Reagents—All chemicals were reagent grade and were purchased from Sigma, Fisher, VWR Scientific, West Chester, PA, or J. Purified His-Fur was eluted with phosphate elution buffer (250 mM imidazole, 300 mM NaCl, 10% glycerol, 50 mM NaPO4, pH 8). The His tag was cleaved by adding 3 units of thrombin (Novagen, Madison, WI) per 2 mg of His-Fur and incubating at room temperature for 2 h. EMSA reactions were analyzed on 5% nondenaturing polyacrylamide gels in electrophoresis buffer (20 mM bis-Tris borate, pH 7.5) that were prerun for 30 min at 200 V of constant voltage. DNA probes used to delimit sequence sufficient for Fur binding in the irr promoter were initially synthesized as overlapping fragments and compared by EMSA.
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