Regulation and Mechanism of M. xanthus Social Gliding

描述（由申请方提供）：IV型药丸（Tfp）是细菌毒力和生物膜形成的重要决定因素，生物膜形成是许多机会性和慢性细菌感染的原因。Tfp还介导细菌表面运动的一种形式，在黄色粘球菌中称为社会（S）滑动，在其他细菌物种中称为抽搐。Tfp在表面运动中的功能与它们在细菌致病性和生物膜形成中的功能相关。Tfp生物合成和功能所必需的基因突变导致细菌毒力、生物膜形成和Tfp介导的运动性同时缺陷。我们的长期目标是使用M。Xanthus S-运动作为研究Tfp介导的功能和信号传导的模型。S-能动性本身也值得进一步研究，因为它对M的发育过程很重要。苍耳多细胞子实体。除Tfp外，M. xanthus S-运动需要另一种细胞表面成分，称为细胞外原纤维。先前的研究表明，编码细菌趋化蛋白同源物的dif基因是纤维生物发生或产生的核心。最近的研究表明，Tfp生物合成和功能所需的转录因子基因也参与了M. xanthus我们建议采取以下行动。首先，我们将验证原纤维生物合成对TFP的要求。我们将进一步构建双突变体，以研究我们的假设所预测的在调控纤维生物发生中dif和dif基因之间的上位关系。其次，我们将使用酵母双杂交（Y2 H）系统和磷酸化研究，以检查如果Dif化学传感器样蛋白相互作用的物理和生物化学作为其对应物在细菌的趋化性，并确定任何新的相互作用，可能存在于Dif之间。第三，目前尚不清楚DifA，甲基接受化学受体蛋白（MCP）的同源物，定位于特定的亚细胞区域，以及DifA和Dif通路如何响应刺激。我们将通过免疫荧光显微镜研究DifA定位和正确的DifA定位对dif和p53基因的依赖性。我们还将构建和使用NarX-DifA融合来检查DifA修饰和Dif途径响应于硝酸盐刺激的信号传导特性。最后，将进行Y2 H和遗传筛选，以鉴定对原纤维调节和生物发生重要的新基因，特别是Dif蛋白的下游基因。

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.