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, driven in part by nucleoid-associated proteins (NAPs). It is not well understood how NAPs affect one another's binding and diffusion, and hence nucleoid organization, in different growth phases. We addressed this question by tracking single molecules of the non-specific NAP HUα-PAmCherry in living Escherichia coli cells to investigate nucleoid organization and NAP-chromosome interactions. We analyzed the mobility of HUα-PAmCherry in different growth phases and further examined how two NAPs, Dps and H-NS, impact HUα dynamics. We found that HUα mobility depends on the 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, indicative of either stable HUα binding or confinement within densely compacted DNA regions. This result supports reports of chromosome compaction and rigidification in deep stationary phase. We found that deletion of dps increases HUα mobility in stationary phase, consistent with our previous finding that Dps promotes short-range DNA contacts and nucleoid compaction in deep stationary phase. Although hns deletion leads to nucleoid compaction in exponential phase, we measured overall faster HUα diffusion in exponential-phase Δhns cells as well as an additional third population of very slow HUα molecules in these cells. In stationary phase, deleting hns increases the fraction of these stably bound HUα molecules. Together, our results show that growth-phase-dependent nucleoid reorganization by Dps and H-NS influences the behavior and function of other NAPs.