BPS2026 – Fluorescence imaging of gene loci in Escherichia coli reveals marker size-dependent dynamics and spatially heterogeneous chromosome confinement

Understanding the spatial-temporal dynamics of gene loci in living bacteria is crucial for unraveling the principles of chromosome organization and gene regulation. In this study, we employed fluorescence time-lapse imaging to track gene loci in Escherichia coli and systematically examined how marker size affects chromosomal dynamics. We found that large fluorescent markers, commonly favored for their brightness and comprising over 100 copies of DNA-binding proteins, artificially displaced loci from the nucleoid and distorted native loci dynamics. Specifically, large markers produced an anomalous diffusion exponent of α∼0.4, distinct from α∼0.25 observed with small markers, leading to potential misinterpretations of chromosomal dynamics. Using the optimized small marker system, we further investigated loci across different macrodomains of the chromosome. We observed distinct subcellular positions and loci dynamics, suggesting spatial heterogeneity in chromosomal confinement effects. These findings highlight the importance of minimally perturbative labeling strategies for gene loci tracking and offer new insights into the biophysical principles governing chromosome organization in living bacteria.