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

Abstract

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 HU-PAmCherry in living Escherichia coli cells in different growth phases, and we further examined how two NAPs, Dps and H-NS, impact HU dynamics. HU mobility varies with growth phase: In exponential phase, HU 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 HU binding or confinement within compacted DNA. Deleting dps increases HU 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 hns deletion leads to nucleoid compaction, faster HU diffusion, and a third population of very slow HU molecules in these cells. In stationary phase, deleting hns increases these stably bound HU molecules. Our results show that growth-phase-dependent nucleoid reorganization by Dps and H-NS influences the behavior and function of other NAPs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/695591v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@16d8f9forg.highwire.dtl.DTLVardef@1f03355org.highwire.dtl.DTLVardef@ba1fc7org.highwire.dtl.DTLVardef@17c5de2_HPS_FORMAT_FIGEXP M_FIG C_FIG The nucleoid-associated proteins Dps, H-NS, and HU shape the bacterial chromosome in the deep stationary phase through their interactions with the nucleoid.