Department of Microbiology and Immunology, Uniformed Services University of Health Sciences, Bethesda, MD, USA.
Mol Microbiol. 2013 Jul;89(2):304-23. doi: 10.1111/mmi.12278. Epub 2013 Jun 10.
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 wild-type (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 manganese-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.
幽门螺杆菌的铁摄取调节因子（Fur）是一种全局调节因子，对于在胃黏膜中定植和存活至关重要。H. pylori Fur 的独特之处在于它能够激活和抑制铁结合（Fe-Fur）和脱辅基（apo-Fur）形式的基因表达。在本研究中，我们结合随机和定点突变技术，确定了对 Fe-Fur 和 apo-Fur 功能都重要的氨基酸残基。我们鉴定了 25 个突变，这些突变影响了 Fe-Fur 的抑制作用，23 个突变影响了 apo-Fur 的抑制作用，这是通过转录分析 Fe-Fur 靶基因 amiE 和 apo-Fur 靶基因 pfr 来确定的。此外，其中 8 个突变还显著影响了细胞中 Fur 的水平。基于调控表型，我们选择了几个有代表性的突变进行进一步分析。在所选择的突变中，我们纯化了野生型（HpFurWT）和三种突变 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。通过圆二色性光谱学评估纯化蛋白的二级结构，原子吸收分光光度法评估铁结合，体外交联测定法评估锰取代和脱辅基条件下的寡聚化，荧光各向异性法评估 Fe-Fur 和 apo-Fur 靶序列的 DNA 结合。结果表明，N 端、S2 和 S3 区域在 Fur 结构-功能关系方面发挥着不同的作用。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，并提供了有关 H. pylori Fur 如何在铁结合和脱辅基形式的蛋白质中调节基因表达的机制见解。
