Regulation of gene expression in Borrelia burgdorferi

伯氏疏螺旋体基因表达的调控

• 批准号: 6679560
• 负责人: D. SCOTT SAMUELS
• 金额: $ 11.37万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-05 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6679560
• 关键词: Borrelia; DNA binding protein; DNA footprinting; Lyme disease; SDS polyacrylamide gel electrophoresis; binding sites; biological signal transduction; communicable disease transmission; gel mobility shift assay; gene environment interaction; gene expression; genetic regulatory element; ion exchange chromatography; lac operon; membrane proteins; molecular pathology; pathologic process; polymerase chain reaction; protein biosynthesis; regulatory gene; transcription factor

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.

描述（由申请人提供）：伯氏疏螺旋体细菌是莱姆病的病原体。伯氏疏螺旋体可以合成几种不同的外表面蛋白，这些蛋白参与地方性动物流行周期中的发病机制或传播。外表面蛋白质合成的调节在分子水平上尚不清楚。本申请旨在评估以下假设：顺式作用因子（例如 DNA 超螺旋）和反式作用因子（例如 DNA 结合阻遏蛋白）响应环境信号调节外表面蛋白基因的表达。外表面蛋白 OspA 和 OspC 的产生是相互调节的，这可能是伯氏疏螺旋体适应蜱媒介和哺乳动物宿主的不同环境或影响环境之间传播的方式。假设 ospAB 操纵子启动子特异性反式作用蛋白可抑制 ospAB 转录。调节蛋白将被纯化和鉴定。假设 ospAB 启动子区域中的顺式作用序列介导转录调节。这些地点将被绘制地图并进行表征。假设结构 DNA 结合蛋白 Hbb 可以促进 ospC 表达的调节。 Hbb 的功能将通过 hbb 基因和 ospC 启动子区域中的 Hbb 结合位点的诱变来探测。 ospC 启动子将被替换为诱导型启动子系统，以控制细胞 OspC 水平而不扰乱 DNA 超螺旋，以便研究 ospC 和 ospAB 转录的偶联。 推测 flac 杂合启动子的 ospC 基因表达受 IPTG 调节并影响 ospAB 操纵子表达。推测 DNA 超螺旋调节 ospC 表达的机制涉及 Hbb 和 ospC 启动子区域中的特定序列基序。将构建突变的ospC启动子。将分析这些突变体对温度和 DNA 超螺旋的转录反应，以确定负责调节的顺式作用元件。这些研究的长期目标是了解外表面蛋白基因调控响应环境信号的机制。