Regulation and Mechanism of M. xanthus Social Gliding

描述(由申请人提供)：IV型药丸(TFP)是细菌毒力和生物膜形成的重要决定因素，是许多机会性和慢性细菌感染的原因。TFP还调节一种形式的细菌表面运动，称为社会(S)，在黄色粘球菌中滑动，在其他细菌物种中颤动。TFP在表面运动中的作用与其在细菌致病和生物被膜形成中的作用有关。TFP生物发生和功能所必需的基因突变会同时导致细菌毒力、生物膜形成和TFP介导的运动能力的缺陷。我们的长期目标是以黄花莲S运动为模型，研究TFP介导的功能和信号转导。S运动本身也值得进一步研究，因为它对黄花多细胞子实体的发育过程很重要。除了TFP外，黄花莲S的运动还需要另一种细胞表面成分，即胞外纤维。以前的研究表明，编码细菌趋化蛋白同源物的dif基因是生物发生或纤维产生的中心。最近的发现表明，TFP生物发生和功能所需的pil基因也参与了黄曲霉原纤维生物发生的调节。我们建议做以下工作。首先，我们将验证原纤维生物发生对TFP的要求。我们将进一步构建双突变体，以检验我们的假设所预测的dif和pil基因之间在纤维生物发生调控中的上位关系。其次，我们将使用酵母双杂交(Y2H)系统和磷酸化研究来检查Dif化学感觉样蛋白是否像它们在细菌趋化性中的对应物一样在物理和生物化学上相互作用，并确定Dif之间可能存在的任何新的相互作用。第三，目前尚不清楚接受甲基的化学受体蛋白(MCP)的同系物DIFA是否定位于特定的亚细胞区域，以及DIFA和Dif通路如何对刺激做出反应。我们将通过免疫荧光显微镜研究DIFA的定位以及DIFA正确定位对DIF和PIL基因的依赖。我们还将构建和使用NarX-DIFA融合来检测DIFA的修饰和Dif通路在硝酸盐刺激下的信号特性。最后，将进行Y2H和遗传筛选，以确定对纤维调节和生物发生重要的新基因，特别是Dif蛋白下游的新基因。

DESCRIPTION (provided by applicant): Type IV pill (Tfp) are important determinants of bacterial virulence and biofilm formation which is the cause of many opportunistic and chronic bacterial infections. Tfp also mediate a form of bacterial surface motility known as social (S) gliding in Myxococcus xanthus and twitching in other bacterial species. The function of Tfp in surface motility correlates with their function in bacterial pathogenicity and biofilm formation. Mutations in genes essential for Tfp biogenesis and function lead to simultaneous defects in bacterial virulence, biofilm formation and Tfp-mediated motility. Our long term goal is to use M. xanthus S-motility as a model to study Tfp-mediated functions and signaling. S-motility itself also warrants further studies on its own merit because it is important to the developmental process of M. xanthus multicellular fruiting bodies. Besides Tfp, M. xanthus S-motility requires another cell surface component known as extracellular fibrils. Previous studies showed that the dif genes, encoding homologues of bacterial chemotaxis proteins, are central to the biogenesis or production of fibrils. Recent findings suggest that the pil genes which are required for Tfp biogenesis and function are also involved in the regulation of fibril biogenesis in M. xanthus. We propose to do the following. First, we will verify the requirement of fibril biogenesis for Tfp. We will further construct double mutants to examine the epistatic relationships in the regulation of fibril biogenesis among dif and pil genes as predicted by our hypothesis. Second, we will use yeast two-hybrid (Y2H) system and phosphorylation studies to examine if the Dif chemosensory-like proteins interact with one another physically and biochemically as their counterparts in bacterial chemotaxis and to identify any novel interactions that may exist among Dif. Third, it is unknown whether DifA, a homologue of methyl-accepting chemoreceptor proteins (MCP), localizes to specific subcellular regions and how DifA and the Dif pathway respond to stimulation. We will study DifA localization and the dependence of correct DifA localization on dif and pil genes by immunofluorescence microscopy. We will additionally construct and use NarX-DifA fusions to examine DifA modification and the signaling properties of the Dif pathway in response to stimulation by nitrate. Finally, Y2H and genetic screens will be carried out to identify new genes important for fibril regulation and biogenesis, especially downstream of Dif proteins.