C Signal-Dependent Gene Expression in Myxococcus xanthus

在自然界中，细菌作为复杂群落的成员存在。细胞在这些群落中相互作用，相互发送信号并改变它们的行为作为回应。这些细菌群在全球生态系统中的碳、氮和其他元素的循环中发挥着至关重要的作用。 通常，细菌的混合种群在另一个活的生物体上或体内，并与其宿主相互作用。 了解细菌如何相互作用以及与宿主的相互作用是生物学中的一个基本挑战。 黄色粘球菌为阐明细菌细胞间相互作用的分子机制提供了一个非常有吸引力的实验体系。 成群的这些细菌在固体表面上滑行，分泌杀死和消化猎物细菌的物质。 饿了，M。xanthus细胞移动到聚集中心并构建新生子实体。在子实体内，杆状细胞分化成球形休眠孢子。 这个壮观的发育过程是由细胞间的相互作用调节的。 M. Xanthus非常适合在分子水平上进行遗传和生物化学分析。 相比之下，在混合细菌种群中进行细胞-细胞相互作用的分子分析是极其困难的。 随着研究复杂群落的方法的改进，从模式生物如M. xanthus C信号通路在M. Xanthus的发展正在为细菌细胞如何相互作用建立一个新的范例。 C信号涉及CsgA，一种在发育细胞中产生的蛋白质，与细胞表面结合。 CsgA似乎具有酶活性，但底物未知。已经提出，CsgA本身或其酶活性的产物在细胞之间的端对端接触时交换。C信号在受体细胞中诱导几种反应。它似乎维持了严格的反应，阻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用于大分子合成。 C信号还调节细胞运动，允许称为波纹和聚集体的宏观模式形成，并且它调节发育基因表达和孢子形成。 有趣的是，对C信号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上升，因为C信号刺激CsgA的产生。已经提出在新生子实体中细胞之间的广泛的端对端接触允许有效的C信号传导。在子实体内产生的高水平CsgA可触发孢子形成。 本研究旨在确定发育基因表达的分子机制，响应C信号。 初步的研究已经确定了两个基因座的DNA调控区的特征，这两个基因座的表达部分依赖于C信号传导，另一个基因座完全依赖于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CAYYCCY（称为C盒; Y表示嘧啶）一致的序列。 完全依赖于C信号传导的基因也有一个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盒和上游重复序列的序列特征。 为了鉴定直接参与调节C信号依赖基因的蛋白质，将使用几种方法。 初步结果表明，FruA蛋白结合到C信号依赖基因的启动子区将与L。Sogaard-Andersen，使用体外足迹法和电泳迁移率变化测定。体内交联和免疫沉淀的技术将适用于在细菌中检查FruA结合到启动子区域。 将进行酵母单杂交筛选以鉴定特异性结合DNA序列的蛋白质，所述DNA序列通过突变分析显示对表达是重要的。将在体外重建C信号依赖性基因的转录，以鉴定所涉及的RNA聚合酶的形式及其相关的σ因子。 从这一领域的研究中获得的见解应该在环境清理、工业和农业实践中得到应用。

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.