Identification and Characterization of Novel Helicobacter pylori apo-Fur-Regulated Target Genes

InHelicobacter pylori , the ferric uptake regulator (Fur) has evolved additional regulatory functions not seen in other bacteria; it can repress and activate different groups of genes in both its iron-bound andapo forms. Because little is understood about the process ofapo -Fur repression and because only twoapo -Fur-repressed genes (pfr andsodB ) have previously been identified, we sought to expand our understanding of this type of regulation. Utilizing published genomic studies, we selected three potential newapo -Fur-regulated gene targets:serB ,hydA , and the cytochromec 553 gene. Transcriptional analyses confirmed Fur-dependent repression of these genes in the absence of iron, as well as derepression in the absence of Fur. Binding studies showed thatapo -Fur directly interacted with the suspectedhydA and cytochromec 553 promoters but not that ofserB , which was subsequently shown to be cotranscribed withpfr ;apo -Fur-dependent regulation occurred at thepfr promoter. Alignments ofapo -regulated promoter regions revealed a conserved, 6-bp consensus sequence (AAATGA). DNase I footprinting showed that this sequence lies within the protected regions of thepfr andhydA promoters. Moreover, mutation of the sequence in thepfr promoter abrogated Fur binding and DNase protection. Likewise, fluorescence anisotropy studies and binding studies with mutated consensus sequences showed that the sequence was important forapo -Fur binding to thepfr promoter. Together these studies expand the knownapo -Fur regulon inH. pylori and characterize the first reportedapo -Fur box sequence.