Exploring the Relationship Between Gene Expression and the Three-Dimensional Locus Positioning : A Microfluidics-based Study on the Escherichia coli Chromosome

Understanding how chromosome structure influences function is a central question across alldomains of life. In eukaryotes, heterochromatin, which is tightly packed and transcriptionally inactive, differs from more often transcribed euchromatin. However, such a division has notbeen observed in prokaryotic chromosomes. The three-dimensional organization of prokaryotic chromosomes and its functional implications remain poorly understood, despite the relative simplicity of bacterial genomes. In this project, we aimed to analyze the relationship betweengene expression and the three-dimensional locus position over the cell cycle in the Escherichia coli chromosome through two approaches; (1) inducing high expression at several different loci across the chromosome to analyze the three-dimensional locus position pre- and post induction, and (2) investigating the locus-dependent expression by introducing a gene expression reporterat loci with different distributions. Tracking of chromosomal loci was performed using a microfluidic system and fluorescence microscope, to get single-cell resolution. The results demonstrated that genomic position where gene expression was induced affected both the spatial organization of the chromosomal loci and the physiological response of the cells. Furthermore, these effects varied between different loci. Additionally, introducing a gene expression reporter at different loci showed that expression levels vary depending on the position of the locus, implying locus-dependent expression. The findings of this project demonstrated that gene expression in E. coli is influenced not only by regulatory elements suchas promoters and transcription factors, but also by the genomic- and spatial position of the gene. These findings could improve our ability to engineer bacteria for biotechnological applications or improve the development of antimicrobial strategies that disrupt gene expression by targeting chromosomal organization.