Nucleoid-Associated Proteins Undergo Liquid-Liquid Phase Separation with DNA into Multiphasic Condensates Resembling Bacterial Nucleoids

Liquid-liquid phase separation offers unique spatiotemporal control over myriad complex intracellular biochemical processes through compartmentalization of biomolecules into highly dynamic, liquid-like condensates known as membrane-less organelles. The bacterial nucleoid is thought to be one such phase-separated condensate; however, its formation, regulation, and biophysical characteristics are poorly understood. Our super-resolution imaging data suggests that nucleoids are dynamic assemblages of sub-micron-sized liquid-like droplets. We demonstrate that non-sequence-specific N ucleoid- A ssociated P roteins (NAPs) such as HU-A, HU-B and Dps, accrete nucleic acids and spontaneously condense with them into liquid-like, multicomponent, multiphasic, heterotypic phase-separated droplets. Upon mixing of HU-B, DNA and Dps, HU-B-enriched droplets are seen to contain demixed Dps-enriched droplets. Our findings indicate scope for the possible existence of multiphasic liquid-like compartments within nucleoids, providing insights into bacterial growth phase-dependent variations in the levels of different NAPs.