RpoS and gene expression in Borrelia burgdorferi

伯氏疏螺旋体的 RpoS 和基因表达

• 批准号: 6881367
• 负责人: RAMESH RAMAMOORTHY
• 金额: $ 20万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6881367
• 关键词: Borrelia; DNA binding protein; DNA directed RNA polymerase; Lyme disease; acid base balance; acidity /alkalinity; bacterial RNA; bacterial genetics; disease /disorder etiology; gel mobility shift assay; gene expression; genetic library; genetic regulation; genetic transcription; protein structure function; recombinant proteins; temperature; ticks; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): Borrelia burgdorferi, the etiologic agent of Lyme disease, cycles in nature between two evolutionarily diverse hosts, an invertebrate tick vector and a vertebrate host. The proliferation and maintenance of the spirochete in the two hosts is guided by several adaptive strategies involving the shuffling of proteins in response to changing environmental conditions. In this respect, molecular changes in the spirochete associated with tick feeding is of particular significance as they precede vertebrate infection. Tick feeding results in a dramatic burst in the spirochetal population, with accompanying changes in protein composition. These changes can be duplicated in vitro by shifting a spirochetal culture to a higher temperature and lower pH environment. One of the key proteins induced under these conditions is the transcription factor RpoS. Our long-term goal is to investigate the molecular events triggered in B. burgdorferi in response to changes in environmental temperature and pH. Our short-term objective is to exploit the in vitro model to develop a better understanding of this phenomenon. Specifically, the goals of this proposal are to (1) investigate the mechanism of regulation of RpoS, (2) identify chromosomal RpoS-regulated genes, (3) characterize the role of RpoS in the regulation of the chromosomal RpoS-regulated genes in vivo. The regulation of RpoS expression will be examined, as we and others have determined that its own expression is responsive to changes in temperature and pH. We have identified a putative DNA-binding protein by electromobility shift assay that specifically binds to the rpoS 5' sequence. Initially, a response regulator protein RRP-2 will be examined as a putative candidate, failing which the unknown protein will be screened for and identified. The role of this DNA-binding protein in the expression of RpoS will be defined in vitro. We will also identify new chromosomal RpoS-regulated proteins by an in vitro screening assay. This assay will involve in vitro transcription using recombinant B. burgdorferi RNA polymerases loaded with sigma D or sigma S. Finally, the regulation of expression of these newly identified genes by RpoS will be partially characterized in vivo. These studies will shed light on the mechanism guiding the flow of genetic information, and identify new effector proteins, in Lyme disease spirochetes poised to infect the vertebrate host.

描述（由申请人提供）：伯氏疏螺旋体（Borrelia burgdorferi）是莱姆病的病原体，在自然界中在两种进化上不同的宿主（无脊椎动物蜱媒介和脊椎动物宿主）之间循环。两种宿主中螺旋体的增殖和维持是由几种适应性策略指导的，这些策略涉及蛋白质的改组以响应不断变化的环境条件。在这方面，与蜱进食相关的螺旋体的分子变化具有特别重要的意义，因为它们发生在脊椎动物感染之前。蜱的摄食导致螺旋体数量急剧增加，并伴随着蛋白质组成的变化。通过将螺旋体培养物转移到较高温度和较低 pH 环境，可以在体外复制这些变化。在这些条件下诱导的关键蛋白质之一是转录因子 RpoS。我们的长期目标是研究伯氏疏螺旋体响应环境温度和 pH 值变化而触发的分子事件。我们的短期目标是利用体外模型更好地理解这种现象。具体来说，该提案的目标是（1）研究RpoS的调节机制，（2）鉴定染色体RpoS调节基因，（3）表征RpoS在体内调节染色体RpoS调节基因中的作用。我们将检查 RpoS 表达的调节，因为我们和其他人已经确定其自身的表达对温度和 pH 的变化有反应。我们通过电迁移分析鉴定了一种假定的 DNA 结合蛋白，该蛋白特异性结合 rpoS 5' 序列。最初，反应调节蛋白 RRP-2 将作为假定的候选蛋白进行检查，否则将筛选和鉴定未知蛋白。这种 DNA 结合蛋白在 RpoS 表达中的作用将在体外得到确定。我们还将通过体外筛选试验鉴定新的染色体 RpoS 调节蛋白。该测定将涉及使用装载有 sigma D 或 sigma S 的重组伯氏疏螺旋体 RNA 聚合酶进行体外转录。最后，将在体内部分表征 RpoS 对这些新鉴定的基因的表达调节。这些研究将揭示引导遗传信息流动的机制，并在准备感染脊椎动物宿主的莱姆病螺旋体中鉴定新的效应蛋白。