BACTERIAL CELL MEMBRANE--GROWTH AND CELL DIVISION

细菌细胞膜——生长和细胞分裂

• 批准号: 3284659
• 负责人: MOSELIO SCHAECHTER
• 金额: $ 29.73万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3284659
• 关键词: DNA binding protein; DNA footprinting; DNA methylation; DNA replication; DNA replication origin; Escherichia coli; Salmonella typhimurium; bacterial DNA; bacterial genetics; bacterial proteins; binding proteins; cell cycle; cell membrane; chromosome movement; electron microscopy; gel electrophoresis; genetic promoter element; membrane proteins; membrane structure; microorganism growth; mutant; protein biosynthesis; protein structure; radiotracer; receptor; synchronous cell division

Our long term objective is to understand how chromosomal segregation takes place in bacteria, i.e., what is the equivalent of mitosis in prokaryotes? We believe that such knowledge will be useful for a general understanding of cell function and, in particular, to help define new targets for antibacterial chemotherapy. This proposal is based on our observation that the replication origin of the Escherichia coli chromosome binds to the membrane for a defined period of time during the cell cycle. We have postulated that this time represent a biological clock during which the incipient progeny chromosomes become relegated to the two cell halves. During this period, origin DNA is hemimethylated, that is, it is newly replicated, but has not yet been modified by the major E. coli methylase, Dam. We have found a membrane protein we call Hob that binds uniquely to hemimethylated origin DNA. We have also delineated the gene for this protein, hob, to within a lambdal phage of the Kohara collection. The E. coli DNA inserted into this phage includes a gene, pcsA, involved in chromosome segregation. Cold sensitive mutants in pcsA cannot partition their chromosome and are defective in cell division. We wish to determine whether the Hob protein acts to anchor the replicative origin to the membrane (thus functioning as part of a bacterial kinetochore-equivalent). Because this is a novel topic, we must first carry out considerable biochemical and genetic groundwork. To this end, we will ask the following questions: What is the sequence of Hob? Are the hob and pcsA genes the same? Is hob essential? What are cytological features of hob mutants? We will then study the sequence in oriC that is recognized by Hob, the specificity of the interaction, and the state of aggregation of Hob in solution. Because the Hob protein is unlikely to function in isolation, we will determine what other proteins it interacts with by looking for extragenic suppressors of conditional hob mutations. We will determine the intracellular localization of the Hob protein and possible "suppressor" proteins. Our model makes a strong prediction about the time in the cell cycle when the Hob protein binds to the DNA. We will test this notion by in vivo footprinting techniques using cells synchronized in their DNA replication. This study will also reveal the DNA "occupancy time" of other proteins during the initiation and early replication of the E. coli chromosome. Ultimately, the function of the Hob protein must be studied by physiological means. We recognize that such studies are difficult and must rely on a firm biochemical and genetic knowledge of the system. Should we make sufficient progress during the period of this grant, we will study the activity and regulation of Hob during the cell cycle, in cultures growing at different rates, and in the presence of different amounts of oriC in the cell.

我们的长期目标是了解染色体分离是如何发生的 细菌中的位置，即原核生物中的有丝分裂相当于什么？ 我们相信这些知识对于一般理解是有用的 细胞功能，特别是帮助确定新的目标 抗菌化疗。 该提议基于我们的观察，即复制起源 大肠杆菌染色体在规定的时间内与膜结合 细胞周期中的时间。 我们假设这一次代表 一个生物钟，在此期间早期的后代染色体变成 被降级到两个半牢房。 在此期间，原始DNA是 半甲基化，即是新复制的，但尚未被复制 由主要的大肠杆菌甲基化酶 Dam 修饰。 我们发现了一层膜 我们称之为 Hob 的蛋白质，它独特地与半甲基化的原始 DNA 结合。 我们 还描绘了这种蛋白质的基因，hob，在 lambda 内 小原收藏的噬菌体。 插入该噬菌体的大肠杆菌 DNA 包括参与染色体分离的基因 pcsA。 冷敏感 pcsA 中的突变体无法分割其染色体并且在细胞中存在缺陷 分配。 我们希望确定 Hob 蛋白是否能够锚定复制 起源于膜（因此作为细菌的一部分 动粒等效）。 因为这是一个新颖的话题，我们必须首先 开展大量的生化和遗传学基础工作。 为此，我们 会问以下问题：Hob 的顺序是什么？ 是 hob和pcsA基因相同吗？ 灶台是必不可少的吗？ 什么是细胞学 滚刀突变体的特征？ 然后我们将研究 oriC 中的序列，即 Hob 认识到交互的特殊性以及状态 溶液中滚刀的聚集。 由于 Hob 蛋白不太可能单独发挥作用，因此我们将 通过寻找外源性来确定它与哪些其他蛋白质相互作用 条件hob突变的抑制因子。 我们将确定 Hob 蛋白的细胞内定位和可能的“抑制子” 蛋白质。 我们的模型对细胞周期中的时间做出了强有力的预测 Hob 蛋白与 DNA 结合。 我们将通过体内测试这个概念 使用DNA复制同步的细胞的足迹技术。 这项研究还将揭示其他蛋白质的DNA“占据时间” 在大肠杆菌染色体的起始和早期复制期间。 最终，必须通过以下方式研究 Hob 蛋白的功能： 生理手段。 我们认识到此类研究是困难的并且必须 依赖于牢固的生化和遗传系统知识。 我们应该 在本次资助期间取得足够的进展，我们将研究 Hob 在细胞周期、培养物中的活性和调节 以不同的速率，并且在存在不同量的oriC的情况下 细胞。