Carpenter Beth M, West Abby L, Gancz Hanan, Servetas Stephanie L, Pich Oscar Q, Gilbreath Jeremy J, Hallinger Daniel R, Forsyth Mark H, Merrell D Scott, Michel Sarah L J
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences Bethesda, MD, USA.
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland Baltimore, Maryland, USA.
Front Microbiol. 2015 Jun 12;6:558. doi: 10.3389/fmicb.2015.00558. eCollection 2015.
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.
幽门螺杆菌镍响应调控蛋白（HpNikR）是一种依赖镍的转录因子，可直接调控这种重要胃部病原体中的多个基因。受HpNikR调控的一个关键基因是ureA，它编码脲酶。先前对HpNikR与ureA启动子（PureA）进行的体外DNA结合研究确定了一个高亲和力蛋白质/DNA结合所需的识别位点。为了确定该识别位点在体内的重要性，并鉴定ureA转录所需的关键DNA序列决定因素，在此，我们将这些体外研究结果转化为直接在幽门螺杆菌内进行的分析。使用一系列改变了PureA DNA靶点的绿色荧光蛋白（GFP）报告基因构建体，并结合缺乏关键调控蛋白的幽门螺杆菌突变菌株，我们证实了先前鉴定的HpNikR识别序列对依赖HpNikR的ureA转录的重要性。此外，我们鉴定出另一个因子，即HpArsRS双组分系统，它是ureA最大转录所必需的。虽然已知HpArsRS可响应酸休克调控ureA，但此前认为它独立于HpNikR发挥作用，且在中性pH条件下无作用。然而，我们对野生型、ΔnikR和ΔarsS单突变体以及ΔarsS/nikR双突变体菌株背景中ureA表达的定量PCR分析表明，缺失arsS时ureA的基础水平表达降低。此外，我们确定，在镍、低pH以及镍和低pH联合胁迫下，HpNikR和HpArsRS都是ureA最大表达所必需的。使用荧光各向异性对HpArsR-P与PureA DNA靶点进行的体外研究证实了蛋白质/DNA的直接结合相互作用。这些数据共同支持了一个模型，即HpArsRS和HpNikR在各种环境条件下协同相互作用以调控ureA转录。这是首次证明在中性pH条件下HpArsRS和HpNikR之间存在直接“串扰”。
