Myxococcus Xanthus C-signaling

接触依赖的细胞-细胞信号在生物体从原核生物到人类的发育过程中是必不可少的，尽管其机制尚不清楚。本项目的目的是研究黄色粘球菌子实体发育所必需的C信号转导的分子基础。C-信号由细胞间的接触启动，并导致介导多细胞形态发生的基因表达的变化。CsgA是已知的唯一参与C-信号产生的蛋白质，它包含在短链酒精脱氢酶(SCAD)家族中发现的共同基序。丝氨酸似乎是这种酶的底物。CsgA或其酶产物可能在细胞间转移以启动C-信号转导，但信号成分的划分尚不清楚。CsgA的酶学将根据所使用的底物范围进行检查。每个反应的化学产物(S)将被检测、鉴定和提纯。将对产物的生物活性进行检测，以确定它们是否挽救了CsgA突变体的发育。最后，体外抑制CsgA酶活性的NAD+类似物将被用来确定CsgA酶活性是否对体内C信号转导至关重要。CsgA的亚细胞位置及其与其他蛋白质的相互作用将通过生化和遗传学方法确定，包括内外膜分离、显微镜和使用酵母双杂交系统来鉴定相互作用的蛋白质。最后，将开发一个模型来解释C信号。这个项目有望促进我们在几个重要领域的知识，包括原核发育和依赖接触的细胞相互作用。该项目还将对研究生和本科生进行实验室培训，其中一些将是少数民族和女性学生。结果将通过在会议上的陈述和在期刊上发表来传播。主要的好处将是提高一部分人口的科学素养，以及增进对原核生物生物学和多样性的理解。

Contact-dependent cell-cell signaling is essential for development of organisms from prokaryotes to humans though the mechanisms are poorly understood. The objective of this project is to examine the molecular basis of C-signaling, which is essential for fruiting body development of Myxococcus xanthus. C-signaling is initiated by contact between cells and leads to changes in gene expression that mediate multicellular morphogenesis. CsgA, the only known protein involved in C-signal production, contains consensus motifs found in the short chain alcohol dehydrogenase (SCAD) family. Serine appears to be a substrate for the enzyme. It is likely that either CsgA or its enzymatic product is transferred between cells to initiate C-signaling, but the compartmentalization of the signaling components remains unclear. The enzymology of CsgA will be examined with regard to the range of substrates utilized. The chemical product(s) of each reaction will be assayed, identified, and purified. The biological activity of the products will be examined to determine whether they rescue development of csgA mutants. Finally, NAD+ analogs that inhibit CsgA enzyme activity in vitro will be used to determine whether CsgA enzyme activity is essential for C-signaling in vivo. The subcellular location of CsgA and its interaction with other proteins will be determined by biochemical and genetic approaches including inner and outer membrane separation, microscopy, and the use of the yeast two hybrid system to identify interacting proteins. Finally, a model will be developed to explain C-signaling. This project is expected to advance our knowledge in several important areas including prokaryotic development and contact-dependent cell interactions. The project will also result in laboratory training of graduate and undergraduate students, some of whom will be minority and female students. The results will be disseminated through presentations at meetings and publication in journals. The major benefits will be an improvement in the scientific literacy of a segment of the population, and an improved understanding of prokaryotic biology and diversity.