Membraneless organelles play a key role in the subcellular organization and regulation of bacterial cells. In particular, the bacterial genome is highly compacted into a membraneless organelle: the nucleoid. The structure and accessibility of the nucleoid significantly influences gene expression, and this morphology varies with cell growth phase and under various environmental conditions. Therefore, revealing how the DNA meshwork changes as a function of the bacterial growth phase will deepen our understanding of how bacteria regulate transcription in response to stress. To realize this goal, we applied single-particle tracking (SPT) in <i>E. coli</i> and super-resolved the dynamics of an extrinsic 25-nm protein assembly with a well-defined dodecahedron structure: GFP-nanocages. By super-resolving the GFP-nanocage positions at different stages from log phase to late stationary phase, we observed an increasingly obvious exclusion of the probe by nucleoid, which indicates that the chromosome is compacting and becoming less accessible for gene expression. Our preliminary experiments also found a novel observation: the nucleoid size and compaction are not necessarily correlated. We hypothesize that at the intermediate stage of compaction, cells with small nucleoids may have a relatively low global compaction level because DNA chains are dragged by the force generated by DNA-NAP complex formation, leading to compacted rigid cores surrounded by a loosened DNA meshwork. By measuring the single-molecule step sizes at the periphery of the nucleoid, we understand the rigidity and smoothness of nucleoid surface at various phases. Furthermore, by analyzing the angle distribution within single-molecule trajectories, we quantify the orientation of interactions with the nucleoid. The gene expression levels differ with cell growth stage. Our research provides a novel way of understanding how subcellular structure and gene expression are related.

Revealing the accessibility and surface morphology of the <i>Escherichia coli</i> nucleoid by genetically encoded live-cell single-particle tracking

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Revealing the accessibility and surface morphology of the Escherichia coli nucleoid by genetically encoded live-cell single-particle tracking