Chromosomal dynamics as a driver of subcellular organization in a bacterial cell

这个项目的目的是揭示一些基本原则，支配组织的细菌细胞质。这个细胞内空间的一个关键特征是类核，一种独特的无膜细胞器，容纳细菌DNA。该项目旨在了解毫米长的DNA分子是如何被压缩在微米大小的类核中，而类核没有核膜。预计这种压实会影响DNA复制、分离、转录，并通过转录影响大多数细胞过程。此外，该项目旨在确定在细胞分裂过程中染色体DNA如何在两个子细胞之间分裂，这是细胞繁殖和细菌感染性的关键过程。除了提供对基本生物过程的见解之外，该项目还将开发可用于细菌和无细胞酶生产的不同研究的微流体装置。该项目将为博士和本科生提供研究机会，包括来自田纳西大学VolsTeach项目的学生，该项目旨在培养STEM学科的高中教师。这些pi将指导VolsTeach学生的研究方法课程，并提供暑期研究实习机会。这两项活动都将有助于增强下一代科学教师的能力，为他们提供宝贵的实践经验，使他们在开始教学时能够从中吸取经验。研究人员和他们的研究生还将在当地的初中和高中进行研究报告，以普及科学教育和就业。一个细胞要繁殖，它的DNA必须在两个新的子细胞之间复制和分裂。虽然DNA复制的过程是众所周知的，但新合成的染色体分离和分裂成子细胞的机制却知之甚少。没有证据支持有丝分裂纺锤体样装置参与任何细菌物种的染色体分离。相反，有人假设DNA合成产生的多余自由能驱动分离，而不需要专用的蛋白质机器。本项目目的1将探讨构型熵在分离两个子染色体中的作用。目的2侧重于分裂方面，并将确定激活DNA转位酶FtsK的机制，FtsK在间隔关闭期间将DNA从分裂面抽离。虽然FtsK的DNA泵送已经被证实，但我们的初步数据表明，即使没有FtsK，细胞也可以分裂它们的染色体。因此，我们也将测试这种运动是由染色体和闭合隔膜之间的空间相互作用造成的假设。这种立体诱导的运动，因为它不依赖于特定的蛋白质，可能是早期原始细胞的工作方式，也可能存在于细菌以外的生物体中。该项目的目的3是确定不同大小的大分子在类核相和细胞质溶胶的其余部分之间是如何分布的。这种分布影响蛋白质合成和细胞生长的速率。它也是类核压实的关键决定因素。大肠杆菌模型的实验工作将通过多学科方法完成，包括遗传学、生物化学、高分辨率和超分辨率光学显微镜以及微流体学。实验结果将辅以理论和建模方法，使用聚合物物理学和统计力学的概念。这些努力的目的是开发一个预测模型如何原核染色体DNA组织自己和它的细胞质环境。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project aims to uncover some of the fundamental principles that govern organization of the bacterial cytosol. A key feature of this intracellular space is the nucleoid, a distinct membrane-less organelle that houses bacterial DNA. The project seeks to understand how the millimeter-long DNA molecule is compacted within the micron-sized nucleoid, which lacks a nuclear membrane. This compaction is expected to affect DNA replication, segregation, transcription, and, via transcription, most cellular processes. Additionally, the project aims to determine how chromosomal DNA is partitioned between two daughter cells during cell division, a crucial process for cell propagation and bacterial infectivity. Beyond offering insights into basic biological processes, the project will develop microfluidic devices that could be used in different studies of bacteria and the cell-free production of enzymes. The project will provide research opportunities for Ph.D. and undergraduate students, including those from the University of Tennessee VolsTeach program, which prepares high school teachers in STEM disciplines. The PIs will supervise VolsTeach students in their Research Methods course and offer internships for summer research. Both activities will help empower the next generation of science teachers by providing them with valuable hands-on experience they will be able to draw from when they start teaching. The researchers and their graduate students will also give presentations on their research in local middle and high schools to popularize science education and careers.For a cell to propagate, its DNA must be replicated and partitioned between two new daughter cells. While processes involved in DNA replication are well-known, the mechanisms by which newly synthesized chromosomes segregate and partition into daughter cells are poorly understood. No evidence exists that supports the involvement of a mitotic spindle-like apparatus in segregating chromosomes in any bacterial species. Instead, it has been hypothesized that excess free energy created from DNA synthesis drives the segregation without a need for dedicated protein machinery. Objective 1 of this project will investigate the role that configurational entropy plays in segregating two daughter chromosomes. Objective 2 focuses on the partitioning aspect and will determine the mechanism that activates DNA translocase FtsK, which pumps DNA away from the division plane during septal closure. While DNA pumping by FtsK has been demonstrated, our preliminary data indicate that even without FtsK, cells can partition their chromosomes. Thus, we will also test the hypothesis that this movement results from steric interactions between chromosomes and the closing septum. Such sterically induced movement, as it does not rely on specific proteins, may have been the modus operandi of early protocells and may also be present in organisms beyond bacteria. Objective 3 of the project is to determine how differently-sized macromolecules are distributed between the nucleoid phase and the remainder of the cytosol. This distribution impacts the rate of protein synthesis and cell growth. It is also a key determinant in the compaction of the nucleoid. The experimental work in the Escherichia coli model will be accomplished via a multidisciplinary approach that includes genetics, biochemistry, high- and super-resolution optical microscopy, and microfluidics. Experimental results will be complemented with theoretical and modeling approaches using concepts from polymer physics and statistical mechanics. These efforts aim to develop a predictive model of how prokaryotic chromosomal DNA organizes itself and its cytosolic environment.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.