BPS2026 – Measuring the physical properties of the bacterial chromosome in living cells

The physical properties of cellular components play critical roles in fundamental life processes, including gene regulation and disease, protein synthesis and degradation, and cell replication and division. In bacteria, previous studies have shown that the cytoplasm has glass-like properties and is fluidized by metabolic activities. However, due to its small size, confined geometry, and membraneless nature, much less is known about the physical properties of the bacterial chromosome. Given the heterogeneity of cellular components and the central role of the chromosome in core processes, measuring the physical properties of the nucleoid is essential for advancing our understanding of bacterial regulation with a model encompassing more than just biochemical interactions. To address this gap, we developed a passive microrheology framework that integrates single-particle tracking with Monte Carlo simulations. By resolving the positions and dynamics of a 25-nm protein nanocage in living Escherichia coli cells and correcting for geometrical effects in 2D experiments using 3D Brownian dynamics simulations, we isolate nanocage motion specifically within the nucleoid, which enables us to characterize the accessibility and viscosity of the E. coli chromosome under various conditions. Our results show that nucleoid viscosity is significantly higher than that of the cytoplasm and that the chromosomal physical properties evolve with growth phases and the cell cycle. We further examined the effects of transcription and translation and found that the response of nucleoid viscosity to perturbations can be opposite during exponential and stationary phases. By incorporating Hi-C data to probe the chromosome structure, we also discovered that nucleoid viscosity can vary independently of genome organization. This work enhances our understanding of how physical properties are regulated within membraneless organelles and provides valuable insight for synthetic biology and the design of artificial cells.