Abstract
Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (PureA) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the PureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, ΔnikR and ΔarsS single mutants as well as a ΔarsS/nikR double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the PureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct “cross-talk” between HpArsRS and HpNikR at neutral pH has been demonstrated.
Highlights
Helicobacter pylori is a microaerophilic, Gram negative pathogen that infects over half of the world’s human population (Marshall and Warren, 1984; Loughlin, 2003)
Prolonged H. pylori infection is associated with the development of gastritis, peptic ulcer disease, Mucosal-Associated Lymphoid Tissue (MALT) lymphoma and gastric adenocarcinoma (Marshall and Warren, 1984; Cover and Blaser, 1992; Sepulveda and Coelho, 2002; Loughlin, 2003; Kusters et al, 2006)
We show that the HpArsRS acid response regulatory system, which has previously been shown to regulate ureA expression in response to low pH, affects ureA transcription at neutral pH. This is the first time that HpArsR has been shown to regulate urease expression in conjunction with Helicobacter pylori NikR (HpNikR), and we propose that there is a cooperative interaction between these two regulators to control urease expression in H pylori
Summary
Helicobacter pylori is a microaerophilic, Gram negative pathogen that infects over half of the world’s human population (Marshall and Warren, 1984; Loughlin, 2003). In addition to the genes for which direct protein/DNA binding has been established, approximately 30 additional genes have been predicted to be regulated by HpNikR This is the first time that HpArsR has been shown to regulate urease expression in conjunction with HpNikR, and we propose that there is a cooperative interaction between these two regulators to control urease expression in H pylori
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