In vitro and in vivo study of the binding activity of NikR to the promoter of the fecA3 gene of Helicobacter pylori.

In vitro and in vivo study of the binding activity of NikR to the promoter of the fecA3 gene of Helicobacter pylori. Simona Romagnoli, Francesca Agriesti, and Vincenzo Scarlato. Department of Biology, University of Bologna, Italy. NikR, a widespread nickel responsive transcriptional regulator, controls gene expression in response to nickel. In Helicobacter pylori, NikR is a global transcriptional regulator, that both activates and represses Ni-dependent target promoters to maintain proper intracellular nickel concentration in relation to environmental nickel availability. This double regulatory role is exerted by recognizing a proposed consensus sequence (TATWATT-N[11-13]-AATWATA) across the promoter region of interest. Herein, we report about the transcriptional regulation of fecA3, a putative nickel outer-membrane transporter. NikR turns off PfecA3 expression by binding to two adjacent operator sites, OpI and OpII, spanning nucleotides -44 to +32 of the promoter region. In vitro binding assays, showed that NikR binds cooperatively and with distinct affinity to the two operators and controls PfecA3 down-regulation in response to an excess of nickel, as well as its nickel-independent temporal expression throughout growth, as investigated by primer extension experiments. Sequence analysis of PfecA3 highlighted the presence of two moderately conserved consensus regions for NikR. However, hydroxyl radical footprinting experiments identified three sites of contact within each operator, namely two high-affinity quintuplets and a four-base low-affinity site within OpI and three four-base low affinity sites within OpII, revealing a so far unique array of binding sites for NikR. Mutagenesis of either one of the high-affinity sites strongly reduced the affinity of NikR for PfecA3 and also impaired the stability of the DNA-protein complex, resulting in a smeared slow-migrating complex as seen in gel shift assays, whereas a mild effect was observed when any of the low-affinity sites was mutated, suggesting that although the high-affinity sites are predominant, both operators contribute to the affinity and stability of the regulator-promoter complex.