Regulation of gene expression in Borrelia burgdorferi

描述(申请人提供)：伯氏疏螺旋体细菌是莱姆病的病原体。伯氏杆菌可以合成几种不同的外表面蛋白，这些蛋白参与了流行病循环中的致病或传播。外表面蛋白质合成的调控在分子水平上还不是很清楚。这项应用建议评估这样一种假设，即顺式作用因子(如DNA超卷曲)和反式作用因子(如DNA结合抑制蛋白)调节外表面蛋白基因的表达以响应环境信号。外表面蛋白OspA和OspC的产生是相互调节的，这可能是Burgdorferi如何适应森林媒介和哺乳动物宿主的不同环境或影响环境之间的传播。一种SPAB操纵子启动子特异的反式作用蛋白被假设为抑制SPAB的转录。将对调节蛋白进行纯化和鉴定。SPAB启动子区域的顺式作用序列被假设为介导转录调控。这些地点将被绘制成地图并确定其特征。建筑DNA结合蛋白HBb被认为有助于调节OspC的表达。Hbb的功能将通过突变Hbb基因和OspC启动子区域的Hbb结合位点来探讨。OspC启动子将被一个可诱导的启动子系统取代，以在不干扰DNA超螺旋的情况下控制细胞OspC的水平，从而可以研究OspC和OspAB转录的偶联。FAC杂交启动子的OspC基因表达被认为是由IPTG调节的，并影响OspAB操纵子的表达。DNA超螺旋调控OspC表达的机制被认为涉及Hbb和OspC启动子区域的特定序列基序。将构建突变的SPC启动子。这些突变体对温度和DNA超螺旋的转录反应将被检测，以确定负责调控的顺式作用元件。这些研究的长期目标是了解外表面蛋白基因调控对环境信号的响应机制。

DESCRIPTION (provided by applicant): The bacterium Borrelia burgdorferi is a causative agent of Lyme disease. B. burgdorferi can synthesize several different outer surface proteins that are involved in pathogenesis or transmission during the enzootic cycle. The regulation of outer surface protein synthesis is not well understood at the molecular level. This application proposes to evaluate the hypothesis that cis-acting factors, such as DNA supercoiling, and trans-acting factors, such as DNA-binding repressor proteins, regulate the expression of outer surface protein genes in response to environmental signals. Production of outer surface proteins OspA and OspC is reciprocally regulated, which may be how B. burgdorferi adapts to the different environments of the tick vector and mammalian host or effects transmission between the environments. An ospAB operon promoter-specific trans-acting protein is hypothesized to repress ospAB transcription. The regulatory protein will be purified and identified. Cis-acting sequences in the ospAB promoter region are hypothesized to mediate transcriptional regulation. These sites will be mapped and characterized. The architectural DNA-binding protein Hbb is hypothesized to facilitate regulation of ospC expression. The function of Hbb will be probed by mutagenesis of the hbb gene and the Hbb binding site in the ospC promoter region. The ospC promoter will be replaced with an inducible promoter system to control cellular OspC levels without perturbing DNA supercoiling so that the coupling of ospC and ospAB transcription can be studied. ospC gene expression from the flac hybrid promoter is hypothesized to be regulated by IPTG and to influence ospAB operon expression. The mechanism by which DNA supercoiling regulates ospC expression is hypothesized to involve Hbb and specific sequence motifs in the ospC promoter region. Mutant ospC promoters will be constructed. The transcriptional response of these mutants to temperature and DNA supercoiling will be assayed in order to define the cis-acting elements responsible for regulation. The long-term objective of these studies is to understand the mechanism of outer surface protein gene regulation in response to environmental signals.