C Signal-Dependent Gene Expression in Myxococcus xanthus

黄色粘球菌中 C 信号依赖性基因表达

• 批准号: 0090478
• 负责人: Lee Kroos
• 金额: $ 36万
• 依托单位: Michigan State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2004-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0090478&HistoricalAwards=false
• 关键词: Signal; Dependent; Gene; Expression; Myxococcus

In nature, bacteria exist as members of complex communities. Cells interact in these communities, sending signals to each other and changing their behavior in response. These groups of bacteria play a vital role in recycling carbon, nitrogen, and other elements in the global ecosystem. Often, the mixed population of bacteria is on or in another living organism, and interacts with its host. Understanding how bacteria interact with each other and with their hosts is a fundamental challenge in biology. Myxococcus xanthus provides a very attractive experimental system to elucidate the molecular mechanisms of interactions between bacterial cells. Swarms of these bacteria glide over solid surfaces, secreting substances that kill and digest prey bacteria. When starved, M. xanthus cells move to aggregation centers and construct a nascent fruiting body. Within the fruiting body, rod-shaped cells differentiate into spherical, dormant spores. This spectacular developmental process is regulated by cell-cell interactions. M. xanthus is very amenable to genetic and biochemical analyses at the molecular level. In contrast, molecular analysis of cell-cell interactions in mixed populations of bacteria is extremely difficult. As methods to study complex communities improve, research to understand their mechanisms of signaling and response will be facilitated by the paradigms learned from model organisms like M. xanthus. Studies of C signaling during M. xanthus development are establishing a new paradigm for how bacterial cells can interact. C signaling involves CsgA, a protein produced in the developing cells that becomes associated with the cell surface. CsgA appears to have enzymatic activity, but the substrate is unknown. It has been proposed that either CsgA itself or the product of its enzymatic activity is exchanged upon end-to-end contact between cells. C signaling induces several responses in recipient cells. It appears to maintain the stringent response, preventing cells from resuming growth on amino acids that are produced early in development, so these amino acids are instead used for macromolecular synthesis during development. C signaling also regulates cell movements, allowing macroscopic patterns called ripples and aggregates to form, and it regulates developmental gene expression and sporulation. Intriguingly, the different responses to C signaling require different levels of CsgA, and the level of CsgA rises during development because C signaling stimulates CsgA production. Extensive end-to-end contacts between cells in the nascent fruiting body have been proposed to allow efficient C signaling. The high level of CsgA that results within the fruiting body may trigger sporulation. This research aims to determine the molecular mechanisms of developmental gene expression in response to C signaling. Preliminary studies have characterized the DNA regulatory regions of two loci that depend partially on C signaling for expression and one that depends absolutely on C signaling. All three regions have one or more sequences matching the consensus CAYYCCY (called the C box; Y means pyrimidine) near the transcriptional start site. The one that depends absolutely on C signaling also has an upstream repeat sequence that is needed for expression. Mutational analyses will be used to define the sequence features of the C box and the upstream repeat that are important for expression. To identify proteins directly involved in the regulation of C signal-dependent genes, several approaches will be used. Preliminary results showing that the FruA protein binds to promoter regions of C signal-dependent genes will be extended in collaboration with L. Sogaard-Andersen, using in vitro footprinting and electrophoretic mobility shift assays. The technique of in vivo cross-linking and immunoprecipitation will be adapted for use in bacteria to examine FruA binding to promoter regions. Yeast one-hybrid screens will be performed to identify proteins that bind specifically to DNA sequences shown by mutational analyses to be important for expression. Transcription of a C signal-dependent gene will be reconstituted in vitro to identify the form of RNA polymerase involved and its associated sigma factor. Insights gained from research in this area should find application in environmental cleanup, industrial and agricultural practices.

在自然界中，细菌作为复杂群落的成员而存在。细胞在这些群落中相互作用，相互发送信号并改变其行为作为响应。这些细菌群在全球生态系统中碳、氮和其他元素的循环利用中发挥着至关重要的作用。 通常，混合的细菌群存在于另一种活生物体之上或之内，并与其宿主相互作用。 了解细菌如何相互作用以及如何与宿主相互作用是生物学中的一个基本挑战。 黄色粘球菌提供了一个非常有吸引力的实验系统来阐明细菌细胞之间相互作用的分子机制。 这些细菌群在固体表面上滑动，分泌杀死和消化猎物细菌的物质。 当饥饿时，M. xanthus 细胞会移动到聚集中心并构建新生的子实体。在子实体内，杆状细胞分化成球形休眠孢子。 这一惊人的发育过程受到细胞间相互作用的调节。 M. xanthus 非常适合分子水平上的遗传和生化分析。 相比之下，对混合细菌群体中细胞与细胞相互作用的分子分析极其困难。 随着研究复杂群落的方法不断改进，从 M. xanthus 等模式生物中学到的范式将有助于了解其信号传导和响应机制的研究。 对 M. xanthus 发育过程中 C 信号传导的研究正在为细菌细胞如何相互作用建立一个新的范例。 C 信号传导涉及 CsgA，这是一种在发育细胞中产生的蛋白质，与细胞表面相关。 CsgA 似乎具有酶活性，但底物未知。有人提出，CsgA 本身或其酶活性产物在细胞之间端到端接触时发生交换。 C 信号传导在受体细胞中诱导多种反应。它似乎维持了严格的反应，防止细胞在发育早期产生的氨基酸上恢复生长，因此这些氨基酸在发育过程中被用于大分子合成。 C 信号传导还调节细胞运动，允许形成称为波纹和聚集体的宏观模式，并调节发育基因表达和孢子形成。 有趣的是，对 C 信号传导的不同反应需要不同水平的 CsgA，并且 CsgA 的水平在发育过程中会上升，因为 C 信号传导会刺激 CsgA 的产生。研究表明，新生子实体细胞之间广泛的端到端接触可以实现有效的 C 信号传导。子实体内产生的高水平 CsgA 可能会引发孢子形成。 本研究旨在确定发育基因表达响应 C 信号传导的分子机制。 初步研究已经确定了两个基因座的 DNA 调控区的特征，这两个基因座部分依赖于 C 信号传导进行表达，而一个基因座则完全依赖于 C 信号传导。 所有三个区域在转录起始位点附近都有一个或多个与共有 CAYYCCY（称为 C 盒；Y 表示嘧啶）匹配的序列。 完全依赖于 C 信号传导的序列还具有表达所需的上游重复序列。突变分析将用于定义对表达重要的 C 盒和上游重复的序列特征。 为了鉴定直接参与 C 信号依赖性基因调节的蛋白质，将使用多种方法。 初步结果表明，FruA 蛋白与 C 信号依赖性基因的启动子区域结合，将与 L. Sogaard-Andersen 合作，使用体外足迹和电泳迁移率变化分析进行扩展。体内交联和免疫沉淀技术将适用于细菌，以检查 FruA 与启动子区域的结合。 将进行酵母单杂交筛选，以鉴定与突变分析显示对表达很重要的 DNA 序列特异性结合的蛋白质。 C信号依赖性基因的转录将在体外重建，以鉴定所涉及的RNA聚合酶的形式及其相关的西格玛因子。 从该领域的研究中获得的见解应该可以应用于环境清理、工业和农业实践。