Wen Yi, Feng Jing, Scott David R, Marcus Elizabeth A, Sachs George
Membrane Biology Laboratory, Department of Physiology, David Geffen School of Medicine at UCLA, VA Greater Los Angeles Healthcare System, Los Angeles, California 90073, USA.
J Bacteriol. 2006 Mar;188(5):1750-61. doi: 10.1128/JB.188.5.1750-1761.2006.
About 200 genes of the gastric pathogen Helicobacter pylori increase expression at medium pHs of 6.2, 5.5, and 4.5, an increase that is abolished or much reduced by the buffering action of urease. Genes up-regulated by a low pH include the two-component system HP0165-HP0166, suggesting a role in the regulation of some of the pH-sensitive genes. To identify targets of HP0165-HP0166, the promoter regions of genes up-regulated by a low pH were grouped based on sequence similarity. Probes for promoter sequences representing each group were subjected to electrophoretic mobility shift assays (EMSA) with recombinant HP0166-His(6) or a mutated response regulator, HP0166-D52N-His(6), that can specifically determine the role of phosphorylation of HP0166 in binding (including a control EMSA with in-vitro-phosphorylated HP0166-His(6)). Nineteen of 45 promoter-regulatory regions were found to interact with HP0166-His(6). Seven promoters for genes encoding alpha-carbonic anhydrase, omp11, fecD, lpp20, hypA, and two with unknown function (pHP1397-1396 and pHP0654-0675) were clustered in gene group A, which may respond to changes in the periplasmic pH at a constant cytoplasmic pH and showed phosphorylation-dependent binding in EMSA with HP0166-D52N-His(6). Twelve promoters were clustered in groups B and C whose up-regulation likely also depends on a reduction of the cytoplasmic pH at a medium pH of 5.5 or 4.5. Most of the target promoters in groups B and C showed phosphorylation-dependent binding with HP0166-D52N-His(6), but promoters for ompR (pHP0166-0162), pHP0682-0681, and pHP1288-1289 showed phosphorylation-independent binding. These findings, combined with DNase I footprinting, suggest that HP0165-0166 is an acid-responsive signaling system affecting the expression of pH-sensitive genes. Regulation of these genes responds either to a decrease in the periplasmic pH alone (HP0165 dependent) or also to a decrease in the cytoplasmic pH (HP0165 independent).
胃病原体幽门螺杆菌约200个基因在pH值为6.2、5.5和4.5的中等pH条件下表达增加，而这种增加会因脲酶的缓冲作用而消除或大幅降低。低pH上调的基因包括双组分系统HP0165 - HP0166，这表明其在某些pH敏感基因的调控中发挥作用。为了鉴定HP0165 - HP0166的靶标，根据序列相似性对低pH上调基因的启动子区域进行分组。用重组HP0166 - His(6)或突变的应答调节因子HP0166 - D52N - His(6)对代表每组的启动子序列探针进行电泳迁移率变动分析（EMSA），后者可特异性确定HP0166磷酸化在结合中的作用（包括用体外磷酸化的HP0166 - His(6)进行的对照EMSA）。发现45个启动子调控区域中的19个与HP0166 - His(6)相互作用。编码α - 碳酸酐酶、omp11、fecD、lpp20、hypA的基因以及两个功能未知基因（pHP1397 - 1396和pHP0654 - 0675）的七个启动子聚集在基因A组中，它们可能在细胞质pH恒定的情况下对周质pH变化作出反应，并在与HP0166 - D52N - His(6)的EMSA中表现出磷酸化依赖性结合。十二个启动子聚集在B组和C组中，它们的上调可能也取决于在pH值为5.5或4.5的中等pH条件下细胞质pH的降低。B组和C组中的大多数靶标启动子与HP0166 - D52N - His(6)表现出磷酸化依赖性结合，但ompR（pHP0166 - 0162）、pHP0682 - 0681和pHP1288 - 1289的启动子表现出磷酸化非依赖性结合。这些发现与DNA酶I足迹分析相结合，表明HP0165 - 0166是一个影响pH敏感基因表达的酸响应信号系统。这些基因的调控要么仅对周质pH的降低作出反应（依赖于HP0165），要么也对细胞质pH的降低作出反应（不依赖于HP0165）。
