The biophysical properties of the bacterial nucleoid are dynamic, heterogeneous, and responsive to perturbations of cellular processes.

Biophysical properties play central roles in cellular function by controlling the diffusion and spatial organization of biomolecules. Because bacteria lack a nuclear membrane, biophysical measurements are often averaged over the entire cell. However, the nucleoid environment is distinct from that of the surrounding cytoplasm, and averaging also ignores local characteristics within the nucleoid. Here, we developed a microrheology framework to quantitatively characterize the bacterial nucleoid and investigate the interplay of its physical properties with cellular processes. We combined single-particle tracking of a genetically encoded protein probe and three-dimensional (3D) Brownian dynamics simulations to separate the nucleoid from the cytoplasm and specifically measure nucleoid accessibility and viscosity. We found that the nucleoid viscosity is 2.5-fold higher than the cytoplasmic viscosity, and that both viscosity and accessibility change systematically across growth phases and the cell cycle. Inhibiting transcription or translation produces opposite changes in nucleoid viscosity in exponential versus stationary phase cells, indicating that the regulation of nucleoid viscosity is sensitive to the underlying biomolecular composition, crowding, and spatial organization. Using Hi-C assays, we further show that changes in nucleoid viscosity may occur without detectable alterations in genome organization, which suggests that nucleoid mechanics provide an independent regulatory mechanism. Spatially, viscosity differences are more pronounced between the nucleoid core and periphery than between genomic locations, and the periphery-core contrast correlates with the coupling of transcription, translation, and membrane insertion. Together, these results indicate that the bacterial nucleoid is a dynamic, heterogeneous viscoelastic environment in which actively regulated biophysical properties may help coordinate multiple cellular processes and provide a physical layer of control that complements canonical biochemical regulation.