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，管理组织的细菌胞质溶胶。这种细胞内空间的一个关键特征是类核，这是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分子如何在微米大小的类核中压缩，而类核缺乏核膜。预期这种压缩会影响DNA复制、分离、转录，并通过转录影响大多数细胞过程。此外，该项目旨在确定细胞分裂期间染色体DNA如何在两个子细胞之间分配，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外，该项目还将开发可用于细菌和酶的无细胞生产的不同研究的微流体设备。该项目将为博士提供研究机会以及本科生，包括田纳西大学VolsTeach项目的学生，该项目为STEM学科的高中教师做准备。PI将监督VolsTeach学生的研究方法课程，并提供暑期研究实习。这两项活动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，使他们能够在开始教学时借鉴。研究人员和他们的研究生还将在当地的中学和高中进行他们的研究报告，以普及科学教育和职业生涯。一个细胞要繁殖，它的DNA必须复制并分配给两个新的子细胞。虽然DNA复制的过程是众所周知的，但新合成的染色体分离并分配到子细胞中的机制却知之甚少。没有证据支持在任何细菌物种中有丝分裂纺锤体样装置参与染色体分离。相反，有人假设DNA合成产生的过量自由能驱动分离，而不需要专门的蛋白质机制。本项目的目的一是研究构型熵在分离两个子染色体中的作用。目的2着重于分配方面，并将确定激活DNA移位酶FtsK的机制，FtsK在隔关闭期间将DNA从分裂平面泵出。虽然已经证明了FtsK的DNA泵送，但我们的初步数据表明，即使没有FtsK，细胞也可以分配它们的染色体。因此，我们也将测试的假设，这种运动的结果从染色体和关闭隔膜之间的空间相互作用。这种空间诱导的运动，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。该项目的目标3是确定不同大小的大分子如何分布在类核相和胞质溶胶的其余部分之间。这种分布影响蛋白质合成和细胞生长的速率。它也是类核致密化的关键决定因素。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和微流体。实验结果将与理论和建模方法使用的概念，从聚合物物理和统计力学的补充。这些努力旨在开发一个原核染色体DNA如何组织自身及其胞质环境的预测模型。该奖项反映了NSF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.