Functional and Mechanistic Characterization of Heterochromatin-like Domains in Bacteria
Amemiya, Haley
2021
Abstract
Every organism faces the challenge of organizing immense amounts of genetic information into a small physical space that is the cell. In eukaryotes, this process is facilitated by histones that wrap DNA into small units. While it has been historically assumed that bacteria do not have an organized genome, increasing evidence implicates a robust structure that enables bacteria to quickly cope with a variety of environmental pressures. The organization and regulation of DNA is incredibly important to engineer bacteria for biotechnological purposes and to understand bacteria that cause disease. However, while bacteria impact almost every aspect of human life, we do not fully understand their genomes. In this thesis I investigated genome organization with a specific focus in bacteria. To improve our understanding of bacterial genomes, I helped design a high-throughput tool, in-vivo protein occupancy display at high-resolution (IPOD-HR), that allows resolution of how proteins bind across the whole length of a bacterial chromosome. By applying this tool to a number of different bacteria, we discovered conserved areas of the genome that are densely bound by proteins but are transcriptionally silent – similarly to heterochromatin in eukaryotes. I show that these regions, termed extended protein occupancy domains (EPODs), have functional roles in bacteria that enable them to use new carbon sources for energy and provide an immune defense against viruses in Escherichia coli (E. coli). I show that EPODs are occupied by nucleoid associated proteins (NAPs). By performing deletions of single NAPs, I identified the key NAPs that bind to specific regulons. In E. coli, I find that EPODs silence a number of metabolic pathways and toxic prophages. I induced changes of particular EPODs by exposing cells to exotic carbon sources and find that EPODs mediate a transcriptional memory affect, where upon a second exposure to an exotic carbon source mounts a faster growth rate and de-repression of genes required for metabolism. In addition, I show one essential role of the formation of EPODs by NAPs in E. coli is to silence harmful genetic elements that have integrated into the genome, such as mobile elements and prophages, that can be potentially toxic to the cell. I define novel prophage silencers, Hfq and Fis that are required for silencing specific prophages. In collaboration with the Jakob Lab, I employed biochemistry, genetics, and bioinformatics and discovered that Hfq binds with a poly-anion, polyphosphate (polyP), to DNA to silence prophages. Biochemical results suggest a model in which polyphosphate acts as an Hfq chaperone in order to permit appropriate silencing at EPODs. These results provided the first evidence that polyP might act in DNA damage control by either directly or indirectly suppressing the expression of genetic mobile elements and prophages, and a mechanism by which bacterial heterochromatin enables regulation during times of stress. Ultimately, my work defines the importance of genome organization in bacteria and provides a scaffold for further investigation into mechanisms underlying the establishment heterochromatin-like domains in bacteria.Deep Blue DOI
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Functional and Mechanistic Characterization of Heterochromatin-like Domains in Bacteria
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