Form and function of the bacterial nucleoid: structural studies using cryo-ET

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Project Summary/Abstract: All organisms share a common genetic code. From the nucleus of mammalian cells to the nucleoid of the smallest bacterium, we all use DNA to encode the traits and cellular processes that drive the diversity of life on earth. Passing along one’s genome is the most fundamental form of heritability and errors in this process can have dire consequences. It is therefore of great importance that all organisms carry out reliable replication and segregation of DNA during cell division, and that organisms maintain methods to protect nuclear material in the event of cellular stress. To study the form and function of DNA structures, I propose taking advantage of the recent advancements in cryoelectron tomography (cryo-ET) methodology to visualize the bacterial nucleoid, as a model for understanding in vivo chromosome structure and to develop the tools and best practices for the study of the cells most important molecule. Using the resources of the Cryoelectron Microscopy Research Center at UW-Madison, cryo-ET of the model organism Rhodobacter sphaeroides will allow the study of in vivo nuclear structures. Rhodobacter sphaeroides is an ideal organism for studying the nucleoid, not only because its size is amenable to cryo-ET, but also because its two chromosomes require unique replication dynamics. This work seeks to observe the structure and interactions of the nucleoid during the cell cycle of R. sphaeroides. The results of which have a strong potential to provide translational break throughs and will build up molecular tools and methods that will have relevance beyond bacteriology. Additionally, this work will study the compaction of the bacterial nucleoid during physiological stress and antibiotic treatments, preliminary data have been shown. Importantly, studying the compaction of nuclear material following antibiotic stress is expected to generate new knowledge leading to a better understanding of the mechanisms bacteria use to survive and persist during antibiotic treatment. Potentially impacting the study on antibiotic resistance.