Identification and Characterization of Novel Helicobacter pylori apo-Fur-Regulated Target Genes
Identification and Characterization of Novel Helicobacter pylori apo-Fur-Regulated Target Genes
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DOI：
10.1128/jb.01026-13
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发表时间：
2013-12-01
影响因子：
3.2
通讯作者：
Merrell, D. Scott
中科院分区：
文献类型：
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作者：
Carpenter, Beth M.;Gilbreath, Jeremy J.;Merrell, D. Scott
在幽门螺杆菌中，铁摄取调节因子（Fur）已经进化出其他细菌中没有的额外调节功能;它可以抑制和激活铁结合和载脂蛋白形式的不同基因组。由于对脱辅基皮毛抑制的过程知之甚少，而且以前只鉴定了两个脱辅基皮毛抑制基因（pfr和sodB），因此我们试图扩大对这种类型调控的理解。利用已发表的基因组研究，我们选择了三个潜在的新的apo-Fur调节基因靶点：serB，hydA和细胞色素c（553）基因。转录分析证实，在铁的情况下，这些基因的毛皮依赖性抑制，以及在毛皮的情况下的去抑制。结合研究表明，apo-Fur直接与可疑的hydA和细胞色素c（553）启动子相互作用，但不与serB相互作用，serB随后被证明与pfr共转录; apo-Fur依赖性调节发生在pfr启动子。apo调控的启动子区域的比对显示了一个保守的，6 bp的共有序列（AAATGA）。DNA酶I足迹法显示，该序列位于pfr和hydA启动子的保护区内。此外，pfr启动子中的序列突变废除了Fur结合和DNA酶保护。同样，荧光各向异性研究和突变的共有序列的结合研究表明，该序列是重要的apo-Fur结合pfr启动子。这些研究一起扩展了已知的H. pylori的基因序列，并表征了首次报道的apo-Fur盒序列。
In Helicobacter pylori, the ferric uptake regulator (Fur) has evolved additional regulatory functions not seen in other bacteria; it can repress and activate different groups of genes in both its iron-bound and apo forms. Because little is understood about the process of apo-Fur repression and because only two apo-Fur-repressed genes (pfr and sodB) have previously been identified, we sought to expand our understanding of this type of regulation. Utilizing published genomic studies, we selected three potential new apo-Fur-regulated gene targets: serB, hydA, and the cytochrome c(553) gene. Transcriptional analyses confirmed Fur-dependent repression of these genes in the absence of iron, as well as derepression in the absence of Fur. Binding studies showed that apo-Fur directly interacted with the suspected hydA and cytochrome c(553) promoters but not that of serB, which was subsequently shown to be cotranscribed with pfr; apo-Fur-dependent regulation occurred at the pfr promoter. Alignments of apo-regulated promoter regions revealed a conserved, 6-bp consensus sequence (AAATGA). DNase I footprinting showed that this sequence lies within the protected regions of the pfr and hydA promoters. Moreover, mutation of the sequence in the pfr promoter abrogated Fur binding and DNase protection. Likewise, fluorescence anisotropy studies and binding studies with mutated consensus sequences showed that the sequence was important for apo-Fur binding to the pfr promoter. Together these studies expand the known apo-Fur regulon in H. pylori and characterize the first reported apo-Fur box sequence.