Analysis of NikR and ArsR DNA-binding properties on urease promoters in the Hardy Helicobacter pylori strain CAN290

Helicobacter pylori ureases are essential enzymes that hydrolyze urea and allow the bacterium to survive in highly acidic gastric conditions. The transcriptional regulators NikR and ArsR modulate promoter activity in response to environmental signals, including changes in metal availability and biochemical state of the regulatory protein. Although these mechanisms have already been characterized in canonical H. pylori strains, their behaviour in the Hardy strain CAN290 remains uninvestigated. The Hardy strain persists in Indigenous populations from Siberia and the Americas and displays distinct genomic features, including genes involved in iron-associated urease activity and metal homeostasis, potentially reflecting adaptation to carnivore-associated gastric environments. This study investigates the DNA-binding properties of NikR and ArsR transcription factors at urease promoters in the H. pylori strain CAN290, with particular focus on the protein-DNA interactions under different binding conditions. NikR and ArsR proteins were expressed and purified, while promoter regions were amplified from CAN290 genomic DNA. Binding assays were performed through electrophoretic mobility shift assays (EMSA) under variable experimental conditions. EMSA analysis revealed that NikR binding to urease promoters exhibits a nickel-dependent trend, displaying different binding behaviours between the two target regions. Conversely, ArsR yielded no observable binding to either urease promoter, likely due to limitations in the in vitro phosphorylation efficiency. However, ArsR successfully bound to its own promoter in both states, suggesting that its autoregulatory mechanism is potentially phosphorylation independent. Overall, this work contributes to the understanding of transcription factor-DNA interactions in H. pylori strain CAN290, although further studies on the transcriptional regulatory mechanisms are required.