In Vitro Analysis of Protein-Operator Interactions of the NikR and Fur Metal-Responsive Regulators of Coregulated Genes inHelicobacter pylori

Abstract

Two important metal-responsive regulators, NikR and Fur, are involved in nickel and iron homeostasis and controlling gene expression in Helicobacter pylori. To date, they have been implicated in the regulation of sets of overlapping genes. We have attempted here dissection of the molecular mechanisms involved in transcriptional regulation of the NikR and Fur proteins, and we investigated protein-promoter interactions of the regulators with known target genes. We show that H. pylori NikR is a tetrameric protein and, through DNase I footprinting analysis, we have identified operators for NikR to which it binds with different affinities in a metal-responsive way. Mapping of the NikR binding site upstream of the urease promoter established a direct role for NikR as a positive regulator of transcription and, through scanning mutagenesis of this binding site, we have determined two subsites that are important for the binding of the protein to its target sequence. Furthermore, by alignment of the operators for NikR, we have shown that the H. pylori protein recognizes a sequence that is distinct from its well-studied orthologue in Escherichia coli. Moreover, we show that NikR and Fur can bind independently at distinct operators and also compete for overlapping operators in some coregulated gene promoters, adding another dimension to the previous suggested link between iron and nickel regulation. Finally, the importance of an interconnection between metal-responsive gene networks for homeostasis is discussed.

Characterization of Key Helicobacter pylori Regulators Identifies a Role for ArsRS in Biofilm Formation

“…The vast majority of these studies have focused on resistance to various stressful environments (10,18,27,60), identification of the regulon of genes controlled by each regulatory factor (16, 17, 19-21, 23, 61), or elucidation of the contribution of each regulator to colonization in an animal model (8)(9)(10)(11). Despite the evidence supporting the fact that the Fur, NikR, and ArsRS regulons show considerable overlap, very few studies (8,17,25,53) have attempted to analyze the combined effect of these regulatory factors in H. pylori biology. Herein, we constructed a series of isogenic regulatory knockout strains and evaluated H. pylori growth in vitro.…”

“…Examination of these transcriptome studies en masse demonstrates the vast and overlapping nature of the Fur, NikR, and ArsRS regulons. Furthermore, evidence suggests that these regulators affect expression of each other; nikR and arsR are part of the Fur regulon (23,24), whereas NikR appears to regulate fur (20,25). Given the overlapping nature of these regulatory cascades, it is likely not surprising that the role of each of these factors appears to be expanded compared to what is seen in other bacteria.…”

NikR–operator complex structure and the mechanism of repressor activation by metal ions

“…In H. pylori, which requires the nickel enzyme urease to survive and colonize the acidic gastric mucus of the human stomach, NikR plays a more complex regulatory role. Transcriptome analysis of a deletion mutant and in vitro promoter-binding assays have revealed that H. pylori NikR (HpNikR) regulates transcription of not only the nickel importer and urease enzyme, but also other regulatory networks, some through repression of the gene encoding the ferric uptake regulator (Fur) protein (5)(6)(7)(8).…”

Helicobacter pylori NikR Protein Exhibits Distinct Conformations When Bound to Different Promoters

“…in rich media, changes in gene expression are observed in response to ϳ100 M) (6, 9 -11). From these studies, it appears that the recognition sequences in each promoter are defined by a series of poorly conserved 6-bp imperfect inverted repeat half-sites separated by a 15-bp spacer (5,7,12). HpNikR binds to these promoters with a range of affinities (5, 12).…”