Mutations to essential orphan response regulator HP1043 of Helicobacter pylori result in growth-stage regulatory defects.
Mutations to essential orphan response regulator HP1043 of Helicobacter pylori result in growth-stage regulatory defects.
复制标题
幽门螺杆菌的重要孤儿反应调节因子 HP1043 突变会导致生长阶段的调节缺陷。
DOI：
10.1128/iai.01193-12
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发表时间：
2013
影响因子：
3.1
通讯作者：
Hoffman,PaulS
中科院分区：
文献类型：
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作者：
Olekhnovich,IgorN;Vitko,Serhiy;Chertihin,Olga;Hontecillas,Raquel;Viladomiu,Monica;Bassaganya-Riera,Josep;Hoffman,PaulS
幽门螺杆菌造成胃粘膜的终身感染，胃粘膜被认为是对大多数微生物有害的小生境。虽然对胃酸和局部炎症的反应是了解的，但很少有人知道它们如何整合到细胞分裂和生长阶段基因表达的稳态控制中。在这里，我们调查的基本孤儿反应调节HP 1043，OmpR/PhoB转录调节子家族的成员，是唯一的Epsilonproteobacteria和缺乏磷酸化结构域。为了检验同二聚体中的构象变化可能导致基因表达缺陷的假设，我们寻找可能改变DNA结合效率的突变。两个引入突变（C215 S、C221 S）C末端到HP 1043（HP 1043 CC 11）的DNA结合结构域导致通过足迹法对其自身启动子的2倍更高的亲和力。对HP 1043晶体结构的建模研究表明，C215 S可能会影响螺旋-转角-螺旋结构域。用hp 1043 CC 11突变等位基因替换hp 1043等位基因导致蛋白水平降低2倍，尽管mRNA显著增加。这些突变并不影响小鼠模型的体外生长速率或定植效率。蛋白质组学分析（CC 11突变株与野生型）确定了许多表达差异，定量PCR进一步显示，11个的12个检测基因已失去了生长阶段的调控和6个基因包含HP 1043结合的启动子区域内的共识序列（毛皮，cagA，cag 23，flhA，翻转，andnapA）。我们的研究表明，影响DNA结合亲和力的突变可用于识别HP 1043调节子的新成员。
Helicobacter pylori establishes lifelong infections of the gastric mucosa, a niche considered hostile to most microbes. While responses to gastric acidity and local inflammation are understood, little is known as to how they are integrated into homeostatic control of cell division and growth-stage gene expression. Here we investigate the essential orphan response regulator HP1043, a member of the OmpR/PhoB subfamily of transcriptional regulators that is unique to the Epsilonproteobacteria and that lacks phosphorylation domains. To test the hypothesis that conformational changes in the homodimer might lead to defects in gene expression, we sought mutations that might alter DNA-binding efficiency. Two introduced mutations (C215S, C221S) C terminal to the DNA-binding domain of HP1043 (HP1043CC11) resulted in a 2-fold higher affinity for its own promoter by footprinting. Modeling studies with the crystal structure of HP1043 suggested that C215S might affect the helix-turn-helix domain. Genomic replacement of thehp1043allele with thehp1043CC11mutant allele resulted in a 2-fold decrease in protein levels, despite a dramatic increase in mRNA. The mutations did not affectin vitrogrowth rates or colonization efficiency in a mouse model. Proteomic profiling (CC11 mutant strain versus wild type) identified many expression differences, and quantitative PCR further revealed that 11 out of 12 examined genes had lost growth-stage regulation and that 6 of the genes contained HP1043 binding consensus sequences within the promoter regions (fur,cagA,cag23,flhA,flip, andnapA). Our studies show that mutations that affect DNA-binding affinity can be used to identify new members of the HP1043 regulon.