Spatial organisation and mechanical control of gene expression on the bacterial chromosome

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A crucial condition for life at any level of organisation is the ability to both perceive and correctly respond to a dynamic environment. At the cellular level, complex signalling mechanisms that sense this environment lead ultimately to the regulation of gene expression in the chromosome. The bacterial chromosome is linearly compacted by a factor of approximately 1000 to fit within a region called the nucleoid, supporting gene regulation and correct DNA segregation. Understanding the mechanisms of gene regulation requires the study of the link between chromosome organization and function an open question made challenging by the dynamic nature of the nucleoid.A common assumption is that the sensing and regulation machinery operates on a purely chemical basis. However, evidence is emerging across both eukaryotes and prokaryotes to suggest that chemistry alone is insufficient to fully explain gene regulation hinting at coupling between biochemical and mechanical processes. Building upon existing knowledge and expertise in bacterial systems, the impact and objective of our work will be further study of transcription regulation and organisation, and the role of mechanical forces on it. This work represents a novel collaboration between two Biological Physics research groups in the Department of Physics at the University of Oxford: The Single Molecule "Gene Machines" Group led by Prof. Achillefs Kapanidis, who have extensive expertise in both bacterial transcription and single-molecule super-resolution imaging, both in-vivo and in-vitro.The Nanoscience for Medicine Group, led by Prof. Sonia Contera, who offer expertise in cell mechanics, and nanoscale mechanical characterisation and manipulation of materials. Chromosomal organization in normal conditionsIn this work, we will develop a novel genetic manipulation system based on mutated CRISPR dCas9, working closely with Dr. Hafez El Sayyed from the "Gene Machines" group - this could be used for both fluorescent tagging and biochemical interrogation. The novel system will enable simultaneous multi-colour fluorescent imaging of genetic loci, and will be used with single-molecule super-resolution microscopy to investigate the structure of the chromosome in 3D space. Specifically we will investigate the relative proximity of operons in wild-type species, and of the remaining number of operons in mutant strains; the potential organisation levels of the nucleoid. Another level of organization may be provided by the phenomenon of liquid-liquid phase separation, which has recently been implicated in the formation of membrane-less organelles, most interestingly the eukaryotic nucleolus. Naturally, this raises the possibility of the existence of similar mechanism in the bacterial nucleoid - subject to time constraints; this could also investigated using multi-colour fluorescence imaging. Chromosomal organization under antibiotic-induced stress Imaging chromosomal organization as per Aim 1 also offers the prospect of integrating the work with an ongoing, industry-relevant project on rapid detection of antimicrobial resistance at the single cell level. Here, we will establish antibiotic resistance phenotypes based on changes to nucleoid structure in response to antimicrobial agents, and complement that with species identification from targeted probes - both at the single cell level. Combining that with wide field microscopy offers the prospect of establishing a clinical assay. We will provide proof-of -concept results, working firstly with homogenous samples and moving towards heterogeneous clinical isolates. Collaboration on this initiative is already in place between the Gene Machines group, and the John Radcliffe Hospital in Oxford. Aim 3 The role of mechanical forces in chromosomal organization and regulation Mechanical forces can affect gene regulation in at least two distinct ways. The first is by triggering of mechano-sensitive biochemical pathways a known t