Regulation of gene expression in Borrelia burgdorferi

描述（由申请人提供）：伯氏疏螺旋体是莱姆病的病原体。伯氏疏螺旋体可以合成几种不同的外表面蛋白，这些蛋白在地方性动物循环中参与发病或传播。外表面蛋白合成的调控在分子水平上还没有很好地理解。该应用程序旨在评估顺式作用因子（如DNA超螺旋）和反式作用因子（如DNA结合抑制蛋白）在响应环境信号时调节外表面蛋白基因表达的假设。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如何适应蜱媒介和哺乳动物宿主的不同环境或影响环境之间的传播。假设ospAB操纵子启动子特异性反式作用蛋白抑制ospAB转录。将对调节蛋白进行纯化和鉴定。假设ospAB启动子区域的顺式作用序列介导转录调控。这些地点将被绘制成地图并加以描述。据推测，结构dna结合蛋白Hbb可促进ospC表达的调控。Hbb的功能将通过Hbb基因的突变和Hbb在ospC启动子区域的结合位点来探测。ospC启动子将被一个诱导启动子系统取代，以控制细胞ospC水平而不干扰DNA超旋，以便研究ospC和ospAB转录的耦合。假设flac杂交启动子的ospC基因表达受IPTG调控并影响ospAB操纵子的表达。假设DNA超螺旋调节ospC表达的机制涉及Hbb和ospC启动子区域的特定序列基序。构建突变体ospC启动子。这些突变体对温度和DNA超卷曲的转录反应将被分析，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在响应环境信号时的调控机制。

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.