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

染色体动力学作为细菌细胞亚细胞组织的驱动因素

• 批准号: 2313719
• 负责人: Jaan Mannik
• 金额: $ 120万
• 依托单位: University of Tennessee Knoxville
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2313719&HistoricalAwards=false
• 关键词: Chromosomal; dynamics; driver; subcellular; organization

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.

该项目旨在揭示管理细菌细胞质组织组织的一些基本原理。这个细胞内空间的一个关键特征是核苷，核苷，一种无膜细胞器，可容纳细菌DNA。该项目试图了解如何在微米大小的核苷中压实毫米长的DNA分子，该核苷缺乏核膜。预计这种压实会影响DNA复制，分离，转录以及通过转录，大多数细胞过程。此外，该项目旨在确定在细胞分裂过程中如何在两个子细胞之间分配染色体DNA，这是细胞传播和细菌感染性的关键过程。除了提供对基本生物学过程的见解，该项目还将开发微流体设备，这些设备可用于细菌的不同研究和无细胞的酶产生。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大学计划的学生，该计划为STEM学科的高中教师做好准备。 PI将在其研究方法课程中监督Volsteach学生，并为夏季研究提供实习。这两种活动都将通过为他们提供有价值的动手经验来帮助他们从他们开始教学时借鉴，从而有助于增强科学教师的能力。研究人员及其研究生还将在当地中学和高中进行有关研究的研究，以普及科学教育和职业。要使一个细胞传播，必须在两个新的女儿细胞之间进行复制并划分其DNA。尽管与DNA复制相关的过程是众所周知的，但对新合成的染色体分离和分隔为子细胞的机制知之甚少。没有证据表明有丝分裂纺锤样仪器在任何细菌物种中隔离染色体中的参与。取而代之的是，已经假设由DNA合成产生的过量自由能驱动种族隔离而无需专门的蛋白质机械。该项目的目标1将调查构型熵在分离两个女儿染色体中所起的作用。目标2的重点是分区方面，并将确定激活DNA易位酶FTSK的机制，该机制将DNA从分离闭合期间从分裂平面中泵出。尽管已经证明了FTSK的DNA泵送，但我们的初步数据表明，即使没有FTSK，细胞也可以分配其染色体。因此，我们还将检验以下假设：这种运动是由染色体与闭合隔膜之间的空间相互作用引起的。这种空间引起的运动不依赖于特定的蛋白质，可能是早期原子素的作案手法，也可能存在于细菌以外的生物中。该项目的目标3是确定大小尺寸的大分子在核生物相和胞质溶胶其余部分之间的分布方式。这种分布会影响蛋白质合成和细胞生长的速率。它也是核苷压实中的关键决定因素。大肠杆菌模型中的实验工作将通过多学科方法来完成，该方法包括遗传学，生物化学，高分辨率光学显微镜和微流体学。实验结果将使用来自聚合物物理学和统计力学的概念来补充理论和建模方法。这些努力旨在开发一种预测模型，即原核生物染色体DNA如何组织自己及其胞质环境。该奖项反映了NSF的法定任务，并使用基金会的知识分子优点和更广泛的影响审查标准，认为值得通过评估来获得支持。

项目成果

Differentiating the roles of proteins and polysomes in nucleoid size homeostasis in Escherichia coli

• DOI: 10.1016/j.bpj.2023.11.010
• 发表时间: 2024-06-04
• 期刊: BIOPHYSICAL JOURNAL
• 影响因子: 3.4
• 作者: Chang，Mu-Hung;Lavrentovich，Maxim O.;Mannik，Jaan
• 通讯作者: Mannik，Jaan