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

极端温度等不利环境条件可能会对细胞结构、过程和生长产生有害影响。细菌经常遇到处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，这些变化抵消了冷应激的破坏性影响。该项目的长期目标是表征对冷胁迫的适应性反应，并确定决定生长最低温度的因素。在大肠杆菌中，对低温作出反应的一种适应性生理变化是细胞大小减小。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。这项研究项目旨在确定复制和细胞分裂之间的联系，以促进低温下的生长和小细胞尺寸。更广泛的影响包括了解细菌在低温下生存的重要性，因为我们在保存和销售食品时使用了冷藏。此外，该项目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8摄氏度。细胞通过增加细胞分裂从而缩小细胞大小来适应低温。与37摄氏度下形成的大杆状细菌相比，野生型大肠杆菌在略高于8摄氏度的温度下形成较小的杆状细菌，在低于最低生长温度的温度下形成丝状细菌棒。晚期细胞分裂蛋白FtsN和DedD刺激隔膜肽聚糖的合成和隔膜，是低温下生长和形成小棒所必需的。小棒的生长和生产也需要一种能够增强DNA复制的类核相关蛋白。该项目旨在测试复制和细胞分裂错综复杂地联系在一起的假设，促进较小的细胞尺寸并使其能够在低温下生长。这一假设将使用两种方法进行检验。对不同的冷敏感突变体进行流式细胞仪和类核染色分析，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。将进行基因筛选，以确定冷敏感突变的抑制子，从而阐明在低温下连接DNA复制和细胞分裂所需的蛋白质。这项研究项目应该对细胞周期进程随温度变化的不同要求提供新的见解。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.