PROJECT SUMMARY Chromosomes must be highly packaged to fit with cells. Modern tools like 3D chromosome capture allow for visualization of chromosome organization but are limited in describing gene-level packaging and cannot reveal the mechanisms that underlie packaging. Understanding these mechanisms is critical to understand genome function and to identify chromosome vulnerabilities as targets for future therapeutics. We propose to attack two major challenges limiting our understanding of chromosome organization in bacteria: understanding supercoiling and structuring protein function. First, supercoiling represents the DNA winding about itself, with “positive” supercoiling (+SC) describing an overwound structure, which is refractory to DNA duplex melting. +SC is generated as a byproduct of replication and must be removed by topoisomerase enzymes or replication will cease, leading to cell death. Prior to my work, a lack of tools to map +SC and a lack of knowledge of topoisomerase regulators meant that our cellular understanding of +SC was limited. Second, bacterial chromosomes are structured by nucleoid-associated proteins (NAPs). NAPs are a group of sequence-diverse, functionally heterogeneous, and poorly understood proteins. Our discoveries in Caulobacter crescentus of an essential NAP and +SC regulator called GapR has empowered us to begin untangling chromosome packaging. We initially showed that GapR binds +SC in a sequence-independent manner to stimulate topoisomerase activity and promote replication. We then developed GapR into technology that allows us to “see” where +SC occurs. While GapR homologs are ubiquitous in ⍺-proteobacteria, analogous NAPs that regulate topoisomerase activity in other bacteria have not been identified. Our preliminary data suggests that bacterial viruses (phages) encode GapR homologs that hijack bacterial GapR to promote infection, uncovering a beneficial role for phage hijacking of bacterial NAPs during infection. We have also developed technology to capture topoisomerase regulators in bacteria. In this proposal, we will 1) elucidate GapR mechanism as a model for understanding how bacteria and phage control +SC to proliferate, 2) mine bacteria and phage genomes to characterize additional topoisomerase regulators, and 3) examine NAP hijacking during phage infection. These projects will describe fundamental paradigms of bacterial chromosome organization and improve our understanding of phage-bacterial warfare. Further, topoisomerases are important antibacterial targets while phages are potential antibiotic alternatives, thus the findings of this proposal will identify vulnerabilities in bacterial chromosome regulation as future antibiotic targets and improve our understanding of phage infection for phage therapy.