Excellence in Research: Coupling of DNA Replication and Cell Division at Low Temperature in E. coli

卓越研究：大肠杆菌低温 DNA 复制与细胞分裂的耦合

• 批准号: 2101124
• 负责人: Pamela Jones
• 金额: $ 49.03万
• 依托单位: Winston-Salem State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2101124&HistoricalAwards=false
• 关键词: Excellence; Research; Coupling; DNA; Replication

Adverse environmental conditions such as extreme temperatures can have deleterious effects on cellular structures, processes and growth. Bacteria often encounter temperatures that are at or near their minimum temperature of growth. At temperatures just above the minimum temperature of growth, bacteria adapt and grow by inducing physiological changes that counteract the damaging effects of the cold stress. The long term goal of this project is to characterize the adaptive response to cold stress and identify factors that determine the minimum temperature of growth. In the bacterium Escherichia coli, an adaptive physiological change in response to low temperature is a decrease in cell size. Several proteins that function in DNA replication and cell division are specifically required for this adaptation. This research project aims to determine the connection between replication and cell division that facilitates growth and small cell size at cold temperatures. Broader impacts include the importance of understanding bacterial survival at cold temperatures because of our use of refrigeration in preserving and selling food. Additionally, the project will broaden participation by providing closely-mentored, hands-on research experience to train undergraduate students from groups that are underrepresented in STEM fields.The E. coli minimum temperature of growth is just above 8oC. The cells adapt to low temperature by increasing cell division and thereby reducing cell size. Compared to the large rods formed at 37oC, wild type E. coli are smaller rods at temperatures just above 8oC and filamentous rods at temperatures below the minimum temperature of growth. Late cell division proteins FtsN and DedD, which stimulate septal peptidoglycan synthesis and septation, are specifically required for growth and the formation of small rods at low temperature. A nucleoid-associated protein that enhances DNA replication is also required for growth and the production of the small rods. This project aims to test the hypothesis that replication and cell division are intricately linked, facilitating the small cell size and enabling growth at low temperatures. This hypothesis will be tested using two approaches. Flow cytometric and nucleoid staining analyses of the various cold-sensitive mutants will be performed to investigate the requirement for specific cell division and nucleoid-associated proteins for DNA replication at low temperature. Genetic screens will be done to identify suppressors of the cold-sensitive mutations, thereby elucidating the proteins required to couple DNA replication and cell division at low temperature. This research project should give novel insights into the differential requirements for the progression of the cell cycle as a function of temperature.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

极端温度等不利环境条件会对细胞结构、过程和生长产生有害影响。 细菌经常遇到等于或接近其生长最低温度的温度。 在略高于生长最低温度的温度下，细菌通过诱导生理变化来适应和生长，从而抵消冷应激的破坏性影响。 该项目的长期目标是表征对冷应激的适应性反应，并确定决定最低生长温度的因素。 在大肠杆菌中，响应低温的适应性生理变化是细胞尺寸减小。 这种适应特别需要几种在 DNA 复制和细胞分裂中起作用的蛋白质。 该研究项目旨在确定复制和细胞分裂之间的联系，从而促进低温下的生长和缩小细胞尺寸。 更广泛的影响包括了解细菌在低温下生存的重要性，因为我们使用冷藏来保存和销售食品。 此外，该项目还将通过提供密切指导的实践研究经验来扩大参与范围，以培训来自 STEM 领域代表性不足的群体的本科生。大肠杆菌的最低生长温度略高于 8oC。 细胞通过增加细胞分裂来适应低温，从而减小细胞尺寸。 与 37°C 时形成的大杆相比，野生型大肠杆菌在略高于 8°C 的温度下形成较小的杆，在低于最低生长温度的温度下形成丝状杆。 晚期细胞分裂蛋白 FtsN 和 DedD 可刺激间隔肽聚糖的合成和分隔，是低温下生长和小杆形成所特别需要的。 小棒的生长和产生还需要一种增强 DNA 复制的核样相关蛋白。该项目旨在检验复制和细胞分裂错综复杂地联系在一起的假设，从而促进细胞尺寸变小并能够在低温下生长。 该假设将使用两种方法进行检验。 对各种冷敏感突变体进行流式细胞术和类核染色分析，以研究低温下 DNA 复制对特定细胞分裂和类核相关蛋白的需求。 将进行遗传筛选来识别冷敏感突变的抑制因子，从而阐明在低温下耦合 DNA 复制和细胞分裂所需的蛋白质。 该研究项目应该对细胞周期进展作为温度函数的不同要求提供新颖的见解。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。