Analysis of Fur binding to operator sequences within the Neisseria gonorrhoeae fbpA promoter

The gene encoding Neisseria gonorrhoeae periplasmic binding protein FbpA contains two regions whose sequences exhibit homology with the Escherichia coli ferric uptake regulator protein (Fur) consensus binding sequence. In this study, DNase I footprinting experiments were employed to characterize the operator sequences within the fbpA promoter region to which E. coli Fur binds. A 160-bp fragment encompassing the promotor region and the putative iron boxes of the fbpA promoter was incubated with Fur, DNaseI was added, and the products of these reactions were sequenced to identify nucleotide peaks that were protected. At 50 nM Fur, a protected region that spanned 33 bp and extended 19 bp upstream and 8 bp downstream of the -35 region of the fbpA promoter was observed. At higher concentrations of Fur (75 and 100 nM), an extension of this protected region upstream of the -35 region was observed. Introduction of a plasmid carrying an fbpA-cat transcriptional fusion in E. coli H1717 (Fur+) resulted in an 88% induction of chloramphenicol acetyltransferase expression under conditions of iron restriction; however, chloramphenicol acetyltransferase expression was not responsive to iron in E. coli H1745 (Fur-), indicating that transcriptional regulation of fbpA in response to iron occurs via the negative regulator Fur. The extent of the fbpA operator sequence (42 bp), as defined by our footprinting analysis, would suggest the binding of two Fur repressor dimers.