In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary in vitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yet allows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles.

Live cell imaging is a powerful tool to understand how nano-sized objects, such as drug carriers used for nanomedicine applications, are taken up and trafficked by cells. Here we visualized...

In all organisms, DNA molecules are tightly compacted into a dynamic 3D nucleoprotein complex. In bacteria, this compaction is governed by the family of nucleoid-associated proteins (NAPs). Under conditions of stress and starvation, an NAP called Dps (DNAbinding protein from starved cells) becomes highly up-regulated and can massively reorganize the bacterial chromosome. Although static structures of Dps-DNA complexes have been documented, little is known about the dynamics of their assembly. Here, we use fluorescence microscopy and magnetic-tweezers measurements to resolve the process of DNA compaction by Dps. Real-time in vitro studies demonstrated a highly cooperative process of Dps binding characterized by an abrupt collapse of the DNA extension, even under applied tension. Surprisingly, we also discovered a reproducible hysteresis in the process of compaction and decompaction of the Dps-DNA complex. This hysteresis is extremely stable over hour-long timescales despite the rapid binding and dissociation rates of Dps. A modified Ising model is successfully applied to fit these kinetic features. We find that long-lived hysteresis arises naturally as a consequence of protein cooperativity in large complexes and provides a useful mechanism for cells to adopt unique epigenetic states.DNA condensation | Dps | cooperativity | hysteresis | Ising model P urified DNA behaves as an entropic spring with a radius of gyration that scales as a function of the contour length (1). In contrast, DNA in vivo is highly organized and condensed. In bacteria, this condensation is caused by nucleoid-associated proteins (NAPs) that collectively shape the chromosome (2, 3). NAPs are capable of binding genomic DNA and in doing so alter its shape, control the transcriptional expression of genes, and remodel the structure of the nucleoid in response to external stimuli (2, 3).DNA-binding protein from starved cells (Dps) is an NAP structurally related to ferritins and associated with the response to stress. Dps is highly expressed in stationary phase (4-7) and is also involved in the cellular response to oxidative (4, 8-10), UV (8, 11), thermal (8), and pH shocks (8). In addition, Dps has been implicated in biofilm formation and tolerance to bacteriophage attacks (12). Dps monomers have a molecular mass of 19 kDa and assemble into a dodecameric shell (Fig. S1A) (13). The resulting complex binds to both supercoiled and linear DNA to form a dense biocrystal structure (4,7,9,14).Although the crystal structure of the Dps dodecamer has been solved (13), no atomic-scale structure of Dps-DNA assemblies currently exists and little is known about complex formation. The affinity of Dps for DNA is very sensitive to buffer conditions. Like many DNA-binding proteins, Dps binds DNA more weakly in the presence of higher salt concentrations. Less typically, divalent cations such as Mg 2+ can substantially weaken the affinity of Dps for DNA (9, 15). It has been proposed that fluctuations in divalent cation concentrations act as a trigger for biocrystal as...

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