STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE SFID REGION OF ESCHERICHIA COLI

大肠杆菌SFID区的结构和功能分析

• 批准号: 6240542
• 负责人: ANN A MCPARTLAND
• 金额: $ 9.55万
• 依托单位: CALIFORNIA STATE UNIVERSITY HAYWARD
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 1998-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240542
• 关键词: Escherichia coli; bacterial genetics; cell cycle; gene mutation; genetic mapping; microorganism growth; molecular cloning; nucleic acid sequence; polymerase chain reaction; temperature sensitive mutant

The overall aim of this project is to probe the mechanisms regulating cell division in Escherichia coli. The proposed work focuses on the cellular role of the sfiD gene. Mutations at this locus prevent the action of two division inhibitors, Su1A and SfiC, whose synthesis is stimulated as part of the recA-dependent response to DNA damage. Su1A prevents "FtsZ ring formation," i.e. aggregation of the FtsZ cell division initiator protein on the cell envelope just prior to division. An sfiD mutation allows FtsZ ring formation in the presence of Su1A. The sfiD gene is located between kilobase pair 3,102 and 3,110 on the E. coli physical map, or roughly 63.7 minutes on the genetic map. A second phenotype associated with the sfiD mutations is extremely poor growth at temperatures below 29 degree. This "cold sensitivity" may be attributable to partial cell lysis at low temperature. In complementation studies, the mutant sfiD alleles are recessive to sfid plus. A number of the proposed experiments are directed toward structural analysis of the sfiD region. These include subcloning of complete and partial sfiD gene segments into phagemid vectors and DNA sequence determination. The nucleotide positions of mutant sfiD25 and sfiD27 alleles will be established using a combination of PCR, gene cloning and sequencing. In other experiments the effect of mutations in sfiD on the integrity of the cell wall will be examined, either directly or by response to antibiotic inhibitors of peptidoglycan synthesis. As a means of identifying genes whose products may interact with sfiD, the map positions of second site suppressor mutations that reverse the cold sensitivity phenotype will be determined. Finally, additional experiments designed to explore the physiological role of SfiD will include monitoring the effect of SfiD overproduction on division frequency and Su1A stability measurements in sfiD plus versus sfiD mutant strains.

本项目的总体目标是探讨细胞的调控机制， 大肠杆菌的分裂。 拟议的工作重点放在细胞 sfiD基因的作用 该位点的突变阻止了两个 分裂抑制剂，Su1A和SfiC，其合成被刺激作为部分 recA依赖的DNA损伤反应。 Su1A防止“FtsZ环” 形成”，即FtsZ细胞分裂起始蛋白的聚集 在细胞分裂前的细胞膜上 sfiD突变使得FtsZ 在Su1A存在下的环形成。 sfiD基因位于 在E.大肠杆菌物理图谱，或 在基因图谱上大约是63.7分钟 第二种表型与 在低于29 ℃的温度下， ℃下 这种“冷敏感性”可能归因于部分细胞溶解 在低温下。 在互补研究中，突变sfiD等位基因 是隐性的 一些拟议的实验是 针对sfiD区域的结构分析。 这些包括 将完整和部分sfiD基因片段亚克隆到 噬粒 载体和DNA序列测定。 的核苷酸位置， 将使用组合建立突变sfiD25和sfiD27等位基因 PCR、基因克隆和测序。在其他实验中， 将检查sfiD突变对细胞壁完整性的影响， 直接或通过对肽聚糖的抗生素抑制剂的反应 合成. 作为一种鉴定基因的手段， 使用sfiD，第二位点抑制突变的图谱位置， 将确定冷敏感性表型。 最后， 旨在探索SfiD生理作用的额外实验 将包括监测SfiD生产过剩对部门的影响 sfiD plus与sfiD突变体的频率和Su1A稳定性测量 菌株