In vitro analysis of protein-operator interactions of the NikR and fur metal-responsive regulators of coregulated genes in Helicobacter pylori.
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.
Delany
Isabel
I
Molecular Immunology Unit, Chiron Vaccines, Siena, Italy.
Ieva
Raffaele
R
Soragni
Alice
A
Hilleringmann
Markus
M
Rappuoli
Rino
R
Scarlato
Vincenzo
V
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016480:Helicobacter pylori / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D021122:Protein Subunits; D012097:Repressor Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014510:Urease / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D021122:Protein Subunits; D012097:Repressor Proteins; D015534:Trans-Activators; C068477:ferric uptake regulating proteins, bacterial; D014510:Urease