E. coli cells advance into phase-separated (biofilm-simulating) extracellular polymeric substance containing DNA, HU, and lipopolysaccharide.

We have previously shown that the nucleoid-associated protein, HU, uses its DNA-binding surfaces to bind to bacterial outer-membrane lipopolysaccharide (LPS), causing HU to act as a glue aiding the adherence of DNA to bacteria, e.g., in biofilms. We have also previously shown that HU and DNA coacervate into a state of liquid-liquid phase separation (LLPS), within bacterial cells and also in vitro. Here, we show that HU and free LPS (which is ordinarily shed by bacteria) also condense into a state of phase separation. Coacervates of HU, DNA, and free LPS are less liquid-like than coacervates of HU and DNA. Escherichia coli cells bearing LPS on their surfaces are shown to adhere to (as well as advance into) coacervates of HU and DNA. HU appears to play a role, therefore, in maintaining both intracellular and extracellular states of phase separation with DNA that are compatible with LPS and LPS-bearing E. coli, with LPS determining the liquidity of the biofilm-simulating coacervate.

Importance: Understanding the constitution and behavior of biofilms is crucial to understanding how to deal with persistent biofilms. This study, together with other recent studies from our group, elucidates a novel aspect of the extracellular polymeric substance (EPS) of Escherichia coli biofilms, by creating a simulacrum of the EPS and then demonstrating that its formation involves liquid-liquid phase separation (LLPS) of HU, DNA, and lipopolysaccharide (LPS) components, with LPS determining the liquidity of this EPS simulacrum. The findings provide insight into the nature of biofilms and into how the interplay of HU, DNA, and LPS could modulate the structural integrity and functional dynamics of biofilms.