Oligomerization-mediated phase separation in the nucleoid-associated sensory protein H-NS is controlled by ambient...

H-NS, a nucleoid-associated protein (NAP) from enterobacteria, regulates gene expression by dynamically transducing environmental cues to conformational assembly and DNA binding. In this work, we show that H-NS from Escherichia coli, which can assemble into octameric and tetrameric oligomerization states, forms spontaneous micron-sized liquid-like condensates with DNA at sub-physiological concentrations in vitro. The heterotypic condensates are metastable at 298 K, partially solubilizing with time, while still retaining their liquid-like properties. The condensates display UCST-like phase behavior solubilizing at higher temperatures, but with a large decrease in droplet-assembly propensities at 310 K and at higher ionic strength. Condensate formation can be tuned in a cyclic manner between 298 and 310 K with the extent of reversibility determined by the incubation time, highlighting strong hysteresis. An engineered phospho-mimetic variant of H-NS (Y61E), which is dimeric and only weakly binds DNA, is unable to form condensates. The Y61E mutant solubilizes pre-formed H-NS condensates with DNA in a few minutes with nearly an order of magnitude speed-up in droplet dissolution at 310 K relative to 298 K, demonstrating rapid molecular transport between dilute and condensed phases. Our results establish that the oligomerization of H-NS is intrinsically tied not only to DNA binding but also its phase-separation tendencies, while showcasing the regulatable and programmable nature of heterotypic condensates formed by an archetypal NAP via multiple cues and their lifetimes.