GENETICS & MOLECULAR BIOLOGY OF CHEMOTAXIS IN B SUBTILIS

遗传学

• 批准号: 3129944
• 负责人: George W. Ordal
• 金额: $ 17.12万
• 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 1991-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3129944
• 关键词: Bacillus subtilis; chemotaxis; chromosome deletion; cilium /flagellum motility; cyclic GMP; frameshift mutation; gel electrophoresis; gene complementation; gene deletion mutation; gene expression; genetic mapping; genetic markers; methylation; molecular cloning; mutant; operon; proteins; radioimmunoassay; receptor; scintillation counter; temperature sensitive mutant; transforming virus

In chemotaxis by Bacillus subtilis, the information that attractant has bound to chemoreceptors is transduced along a pathway, which includes loss of methyl groups from the methyl-accepting chemotaxis proteins (MCPs), and ultimately results in a transient increase of counterclockwise rotation by the flagella. It is very important to trace this pathway, to discover what proteins are involved and what their functions are. B. subtilis is perhaps the best organism for this work since we have, using purified methyltransferase and methylesterase, shown that attractants retard methylation and strongly enhance demethylation of MCPs in vitro. Only in B. subtilis have these enzymes been purified to homogeneity. We have also developed two complementation systems and have obtained evidence for over twenty new genes. Thus, the stage is now set to investigate proteins that modulate methylation reactions. Using this system, we will explore the mechanism of several novel events induced by attractant: transfer of methyl groups between MCPs, turnover of methyl groups on MCPs, and transfer to an intermediate acceptor before formation of methanol. The last step is blocked by repellents and is the first biochemical handle on their mechanism of action. Isolation and characterization of mutants, including those containing a transposon that may express Beta-galactosidase, and cloning the chemotaxis genes will greatly assist us. Once the genes are cloned, we will carry out a restriction analysis of chromosomal segments and correlate the genetic and physical maps. We will express the genes in E. coli minicells as radioactive proteins so that we can ascertain their size and hydrophobicity and search for protein/protein interactions, especially with the switch, which controls direction of flagellar rotation. By second-site reversion studies, we will also seek evidence for such interactions. Our understanding of the MCPs, which represent our major biochemical handle on chemotaxis, is hampered by lack of mutants and insufficient gene expression and, using the techniques of molecular biology, we exprect to remedy this. Our knowledge, not only of the MCPs, but of over twenty other chemotaxis proteins, will be enhanced by mapping and complementing the mutants and by varying levels of genes expression. Armed with a uniquely suitable in vitro system for evaluation the effects of chemotaxis proteins, we believe that a renewed emphasis on genetics and molecular biology will prove especially fruitful.

在枯草芽孢杆菌的趋化性中，吸引剂具有的信息 与化学感受器结合的途径被转导，其中包括损失 来自甲基趋化性蛋白（MCP）的甲基的甲基和 最终导致逆时针旋转的短暂增加 鞭毛。 追踪这一途径，发现什么非常重要 蛋白质涉及什么以及它们的功能是什么。 B.枯草厂也许是 自从我们拥有的这项工作的最佳生物，使用纯化 甲基转移酶和甲基酯酶，表明吸引剂延迟 甲基化并在体外强烈增强MCP的脱甲基化。 仅在 枯草芽孢杆菌的这些酶已被纯化为均匀性。 我们也有 开发了两个互补系统，并获得了过度的证据 二十个新基因。 因此，现在设定了阶段来研究蛋白质 调节甲基化反应。 使用此系统，我们将探索 吸引剂引起的几个新事件的机制：转移 MCP之间的甲基，MCP上甲基的周转和转移 在形成甲醇之前，要给中间受体。 最后一步是 被驱虫剂阻塞，是他们的第一个生化手柄 作用机理。 突变体的隔离和表征，包括 那些包含可能表达β-半乳糖苷酶的转座子的人，并且 克隆趋化基因将极大地帮助我们。 一旦基因为 克隆，我们将对染色体片段进行限制分析 并关联遗传和物理图。 我们将在 大肠杆菌小红霉作为放射性蛋白，以便我们可以确定它们 大小和疏水性以及寻找蛋白质/蛋白质相互作用， 特别是控制鞭毛方向的开关 旋转。 通过第二站点归还研究，我们还将寻求证据 这样的互动。 我们对代表我们的MCP的理解 缺乏突变体和 基因表达不足，并使用分子技术 生物学，我们征收以补救这一点。 我们的知识，不仅是MCP， 但是，通过映射将增强二十多种趋化蛋白 并补充突变体和不同水平的基因表达。 配备独特的体外系统以评估效果 关于趋化蛋白的，我们认为重点是遗传学和 分子生物学将被证明是特别富有成效的。