Autoregulation of Helicobacter pylori Fur revealed by functional analysis of the iron-binding site

Abstract

The ferric uptake regulator protein Fur regulates iron-dependent gene expression in bacteria. In Helicobacter pylori it has been shown to regulate iron-activated and iron-repressed genes. In this study, we show that H. pylori Fur protein regulates transcription from its own σ80 promoter Pfur in response to iron. Footprinting analysis shows that Fur binds at three distinct operators at Pfur overlapping and proximal to the promoter elements. Site-directed mutagenesis of the proposed iron-binding site of the protein results in derepression of Pfur and the loss of iron regulation. In vivo oligomerization assays reveals that the C-terminus of Fur is necessary for multimerization of the protein and that the mutations do not affect this activity. Molecular and phenotypic analysis of the mutant proteins provides evidence that the iron-binding site controls the specific affinity of Fur for the operators at Pfur and hence its repressive ability. In summary, the data presented are consistent with a model in which Fur acts as a rheostat of transcription to autoregulate its own expression in response to iron, which in turn controls expression of iron-induced and iron-repressed genes, providing maintenance of homeostasis.