METAL REGULATION IN HOST COLONIZATION BY B BURGDORFERI

描述（改编自申请人的摘要）：与其他 细菌病原体必须克服宿主的铁限制， 建立一个成功的感染，伯氏疏螺旋体，病原体 莱姆病的病原体，能够绕过宿主体内的铁限制 通过最小化甚至消除对铁的需求。他们 通过消除包括重要的铁- 含有蛋白质和替代其他微量金属， 在B中发现的金属蛋白。burgdorferi。因此，他们 似乎不基于细胞内水平调节基因表达 在其他细菌病原体中也可以看到铁的存在。相反，B。burgdorferi 似乎是通过监测其他基因的水平来调节基因的表达。 金属，如锰或锌。去调查观察到的金属- 依赖于基因表达，PI已经鉴定并克隆了一个基因， 编码来自B的推定的金属依赖性阻遏蛋白（PerR）。 burgdorferi，并确定了目标序列，使用迁移率变化DNA- 结合分析该序列是一个启动子的上游91 bp。 推定的2基因操纵子编码谷氨酸转运蛋白（gltP）和 NADH过氧化物酶（npx），这表明PerR可能参与了 通过B调节氧化应激反应。burgdorferi。A PerR 从枯草芽孢杆菌中鉴定同源物介导细胞反应 氧化应激和金属饥饿的反应。到 了解这种调节蛋白在生存中的作用， B的响应。burgdorferi和确定它调节的其他基因， PI建议（1）表征PerR及其推定靶序列 使用迁移率改变DNA结合、引物延伸、DNA酶I 足迹法和甲基化/尿嘧啶干扰试验，（2）评估 PerR和Nox在B细胞氧化应激反应中的作用。burgdorferi 通过研究O2-，过氧化物和金属饥饿对 Nox的表达和（3）鉴定由PerR调节的另外的基因。

DESCRIPTION (Adapted from the Applicant's Abstract): Unlike other bacterial pathogens which must overcome host iron restriction to establish a successful infection, Borrelia burgdorferi, the causative agent of Lyme disease, is able to bypass iron limitation within a host by minimizing or perhaps even eliminating the need for iron. They accomplish this by eliminating pathways that include important iron- containing proteins and substituting other trace metals in metalloproteins that are found in B. burgdorferi. As a result, they do not appear to regulate gene expression based upon intracellular levels of iron as is seen in other bacterial pathogens. Instead, B. burgdorferi appears to regulate gene expression by monitoring levels of other metals, such as manganese or zinc. To investigate the observed metal- dependent gene expression, the PI has identified and cloned a gene encoding a putative metal-dependent repressor protein (PerR) from B. burgdorferi and identified a target sequence using a mobility shift DNA- binding assay. This sequence is 91 bp upstream of the start codon of a putative 2 gene operon encoding a glutamate transporter (gltP) and a NADH peroxidase (npx), suggesting that PerR may be involved in regulating an oxidative stress response by B. burgdorferi. A PerR homolog identified from Bacillus subtilis mediates cellular responses to oxidative stress and metal starvation in that bacterium. To understand the role this regulatory protein plays in the survival response of B. burgdorferi and to identify other genes it regulated, the PI proposed to (1) characterize PerR and its putative target sequence using mobility shift DNA-binding, primer extension, DNase I footprinting, and methylation/uracil interference assays, (2) assess the role of PerR and Nox in the oxidative stress response in B. burgdorferi by examining the effects of O2-, peroxide, and metal starvation on the expression of Nox and (3) identify additional genes regulated by PerR.