Random and site-specific mutagenesis of the Helicobacter pylori ferric uptake regulator provides insight into Fur structure-function relationships.
Random and site-specific mutagenesis of the Helicobacter pylori ferric uptake regulator provides insight into Fur structure-function relationships.
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DOI：
10.1111/mmi.12278
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发表时间：
2013-07
影响因子：
3.6
通讯作者：
Merrell DS
中科院分区：
文献类型：
--
作者：
Gilbreath JJ;Pich OQ;Benoit SL;Besold AN;Cha JH;Maier RJ;Michel SL;Maynard EL;Merrell DS
幽门螺杆菌的铁摄取调节剂（Fur）是一种全局调节剂，对胃粘膜内的定植和存活至关重要。幽门螺杆菌在激活和抑制铁结合（Fe-Fur）和载脂蛋白（apo-Fur）形式的基因表达方面具有独特的能力。在目前的研究中，我们结合随机和位点特异性诱变来鉴定对Fe-Fur和apo-Fur功能都重要的氨基酸残基。通过对Fe-Fur靶基因amiE和载脂蛋白靶基因pfr的转录分析，我们确定了25个影响Fe-Fur抑制的突变和23个影响载脂蛋白fur抑制的突变。此外，其中8个突变也显著影响细胞中Fur的水平。基于调控表型，我们选择了几个有代表性的突变来进一步表征。在这些选择中，我们纯化了野生型（HpFurWT）和三个突变型Fur蛋白（HpFurE5A， HpFurA92T和HpFurH134Y），它们分别代表n端延伸，调节金属结合位点（S2）和结构金属结合位点（S3）的突变。用圆二色光谱法评估纯化蛋白的二级结构，用原子吸收分光光度法评估铁结合，用体外交联法评估铁取代和载子条件下的寡聚化，用荧光各向异性法评估DNA与Fe-Fur和载子- fur靶序列的结合。结果表明，n端、S2和S3区域在Fur的结构-功能关系中起着不同的作用。总的来说，这些研究提供了关于这些残基在Fur功能中的作用的新信息，并提供了幽门螺杆菌Fur如何调节铁结合和载脂蛋白形式的基因表达的机制见解。
The ferric uptake regulator (Fur) of Helicobacter pylori is a global regulator that is important for colonization and survival within the gastric mucosa. H. pylori Fur is unique in its ability to activate and repress gene expression in both the iron-bound (Fe-Fur) and apo forms (apo-Fur). In the current study we combined random and site-specific mutagenesis to identify amino acid residues important for both Fe-Fur and apo-Fur function. We identified 25 mutations that affected Fe-Fur repression and 23 mutations that affected apo-Fur repression, as determined by transcriptional analyses of the Fe-Fur target gene amiE, and the apo-Fur target gene, pfr. In addition, eight of these mutations also significantly affected levels of Fur in the cell. Based on regulatory phenotypes, we selected several representative mutations to characterize further. Of those selected, we purified the wildtype (HpFurWT) and three mutant Fur proteins (HpFurE5A, HpFurA92T, and HpFurH134Y), which represent mutations in the N-terminal extension, the regulatory metal binding site (S2) and the structural metal binding site (S3), respectively. Purified proteins were evaluated for secondary structure by circular dichroism spectroscopy, iron-binding by atomic absorption spectrophotometry, oligomerization in iron-substituted and apo conditions by in vitro cross-linking assays, and DNA binding to Fe-Fur and apo-Fur target sequences by fluorescence anisotropy. The results showed that the N-terminal, S2, and S3 regions play distinct roles in terms of Fur structure-function relationships. Overall, these studies provide novel information regarding the role of these residues in Fur function, and provide mechanistic insight into how H. pylori Fur regulates gene expression in both the iron-bound and apo forms of the protein.
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影响因子：
3.6
作者：
Pich OQ;Carpenter BM;Gilbreath JJ;Merrell DS
通讯作者：
Merrell DS
影响因子：
4.2
作者：
Grabowska AD;Wandel MP;Łasica AM;Nesteruk M;Roszczenko P;Wyszyńska A;Godlewska R;Jagusztyn-Krynicka EK
通讯作者：
Jagusztyn-Krynicka EK
影响因子：
3.6
作者：
Delany, I;Spohn, G;Scarlato, V
通讯作者：
Scarlato, V
影响因子：
3.5
作者：
Loprasert, S;Sallabhan, R;Mongkolsuk, S
通讯作者：
Mongkolsuk, S
影响因子：
3.1
作者：
Gancz, H;Censini, S;Merrell, DS
通讯作者：
Merrell, DS