ABSTRACT

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.

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