Characterizing the Impact of Nucleoid-Associated Proteins on HU-DNA Interactions by Live-Cell Single-Molecule Tracking

The bacterial nucleoid undergoes extensive structural reorganization during growth, influenced by nucleoid-associated proteins (NAPs) whose interactions and effects on nucleoid organization remain unclear. We investigated these interactions by tracking single molecules of the NAP HUα-PAmCherry in living <i>Escherichia coli</i> cells in different growth phases, and we further examined how two NAPs, Dps and H-NS, impact HUα dynamics. HUα mobility varies with growth phase: In exponential phase, HUα has two distinct mobility states: a fast-diffusing state and a slower, interacting state. In stationary phase, we observed a third population of very slow molecules, suggesting stable HUα binding or confinement within compacted DNA. Deleting <i>dps</i> increases HUα mobility in stationary phase, consistent with findings that Dps promotes short-range DNA contacts and nucleoid compaction in deep stationary phase. We measured in exponential phase that <i>hns</i> deletion leads to nucleoid compaction, faster HUα diffusion, and a third population of very slow HUα molecules in these cells. In stationary phase, deleting <i>hns</i> increases these stably bound HUα molecules. Our results show that growth-phase-dependent nucleoid reorganization by Dps and H-NS influences the behavior and function of other NAPs.