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

We have earlier shown that HU, a nucleoid-associated protein, uses its DNA-binding surfaces to bind to bacterial outer-membrane lipopolysachharide (LPS), with this explaining how HU act as a potential glue for the adherence of bacteria to DNA, e.g., in biofilms. We have also earlier shown that HU and DNA together condense into a state of liquid-liquid phase separation (LLPS) both within, and outside, bacterial cells. Here, we report that HU and free LPS also condense into a state of phase separation, with coacervates of HU, DNA and free LPS being less liquid-like than condensates of HU and DNA alone. E. coli cells bearing surface LPS and also shedding LPS, are shown to adhere to (as well as enter into) condensates of HU and DNA. HU thus appears to play a role in maintaining both an intracellular state of phase separation involving genomic nucleoids that are phase-separated from the cytoplasm, and an extracellular state of phase separation involving coacervates of extracellular DNA, HU and LPS, in the extracellular polymeric substances (EPS) of biofilms, in which LPS content is shown to modulate liquidity. ImportanceUnderstanding biofilm genesis and nature are crucial to understanding how to deal with the bacterial resistance to antibiotics that develops eventually in persistent biofilms. This study, together with two other recent landmark studies from our group, elucidates a novel aspect of the extracellular polymeric substance (EPS) of an Escherichia coli biofilm, by creating a simulacrum of the EPS and demonstrating that its formation involves liquid-liquid phase separation (LLPS) by its component HU, DNA, and lipopolysaccharide (LPS), with LPS determining the liquidity of the EPS simulacrum. The findings provide insight into the physical nature of biofilms but also suggests that the interplay of HU, DNA, and LPS facilitates the structural integrity and functional dynamics of biofilms. These findings are a stepping stone to the eventual development of strategies to disrupt biofilm.