CHEMOTACTIC SENSORY TRANSDUCTION IN BACILLUS SUBTILIS

枯草芽孢杆菌的趋化感觉传导

• 批准号: 7072369
• 负责人: George W. Ordal
• 金额: $ 36.19万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-08-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7072369
• 关键词: Bacillus subtilis; SDS polyacrylamide gel electrophoresis; amidation /deamidation; asparagine; bacterial proteins; biological signal transduction; chemotaxis; enzyme activity; methylation; mutant; phosphorylation; proline; protein protein interaction; receptor binding; receptor expression

Our goal during the past thirty years of NIH funding has been to delineate the mechanism of chemotaxis in the Gram-positive bacterium Bacillus subtilis. During the past decade, the realization has emerged that the B. subtilis mechanism might be the ideal paradigm for understanding the mechanism in the broad sweep of Bacteria and Archaea since it is so similar to that in the Archaea. It is an ancient mechanism and close to the progenitor mechanism that existed just before the separation of the Bacteria and Archaea. Thus in studying this mechanism, we can see where the current mechanisms in Bacteria and Archaea came from and get insights into how they might currently work. The mechanism involves two proteins, found in the Archaea but not in Escherichia coli CheC and CheD. It also involves receptors that, like those in the Archaea, have two pairs of insertions of four turns of n-helix in two locations in the cytoplasmic region of the receptors, compared with what is found in E. coll. It also involves another protein CheV, found only in Bacteria but very widespread there, although not in E. coil. The B. subtilis mechanism has a large switch protein, FliY, whose C-terminal region is homologous to the small E. coli protein FliN but no one knew why the protein was so large. During the last funding period biochemical events associated with each of the novel proteins or with the receptor differences between B. subtilis and E. coli have been described so that the usefulness of the B. subtilis mechanism as a paradigm has become much enhanced. CheC and FliY are CheY-P phosphatases, CheD is a deamidase and stimulates CheC, whereas CheC inhibits CheD. CheV brings about adaptation by becoming phosphorylated. Methylation of receptors at nearby sites has opposite effects on the kinase. At this point, we wish to take our quest to understand this mechanism to a new level, namely, to refine our work on individual proteins and to understand how the various chemotaxis proteins interact to bring about excitation and adaptation--that is, chemotaxis. We have developed the genetic stocks and in vitro assays to do this. We propose to map out where each of the proteins interacts with the others and, using mutants, show the function of each interaction. We propose detailed characterization of receptor methylation to pin down the roles of each site. We propose experiments to clarify the roles of each of the enzymatic and binding functions of CheC, CheD, and CheV.

我们在过去三十年的NIH资金中的目标是描述革兰氏阳性细菌枯草菌中趋化性的机理。在过去的十年中，意识到枯草芽孢杆菌机制可能是了解细菌和古细菌广泛扫描机制的理想范式，因为它与古细菌非常相似。它是一种古老的机制，接近细菌和古细菌分离之前存在的祖细胞机制。因此，在研究这种机制时，我们可以看到细菌和古细菌中当前的机制来自何处，并了解它们目前​​的工作方式。该机制涉及两种蛋白质，在古细菌中发现 但不是在大肠杆菌chec和ched中。它还涉及受体，与古细菌一样，在受体的细胞质区域的两个位置，与E. coll中发现的那样，在两个位置的N螺旋中插入了四个旋转的N螺旋。它还涉及另一种蛋白质CHEV，仅在细菌中发现，但在那里非常广泛，尽管不在E.卷轴中。枯草芽孢杆菌机制具有大型开关蛋白，Fliy，其C末端区域与小的大肠杆菌蛋白Flin同源，但没人知道为什么该蛋白如此大。在最后的资金期间，已经描述了与枯草芽孢杆菌和大肠杆菌之间的每种新蛋白质或受体差异相关的生化事件，以便使枯草芽孢杆菌机制作为范式的有用性得到了大大增强。 Chec和Fliy是Chey-P磷酸酶，CHED是一种脱氨酶，刺激CHEC，而CHEC抑制了CHED。 CHEV通过磷酸化来适应适应。附近部位的受体甲​​基化对激酶的影响相反。在这一点上，我们希望采取我们的追求，以将这种机制理解到一个新的水平，即，完善我们在单个蛋白质上的工作，并了解各种趋化蛋白如何相互作用以引起激发和适应性（即趋化性）。我们已经开发了遗传库存和体外测定方法来做到这一点。我们建议绘制每种蛋白质与其他蛋白相互作用的位置，并使用突变体显示每种相互作用的功能。我们提出了受体甲基化的详细表征 固定每个站点的角色。我们提出了实验，以阐明CHEC，CHED和CHEV的酶促和结合功能的每个作用。