The bacterial nucleoid-associated proteins, HU and Dps, condense DNA into context-dependent biphasic or multiphasic complex coacervates.

The bacterial chromosome, known as its nucleoid, is an amorphous assemblage of globular nucleoprotein domains. It exists in a state of phase separation from the cell's cytoplasm, as an irregularly-shaped, membrane-less, intracellular compartment. This state (the nature of which remains largely unknown) is maintained through bacterial generations ad infinitum. Here, we show that HU and Dps, two of the most abundant nucleoid-associated proteins (NAPs) of Escherichia coli, undergo spontaneous complex coacervation with different forms of DNA/RNA, both individually and in each other's presence, to cause accretion and compaction of DNA/RNA into liquid-liquid phase separated condensates in vitro. Upon mixing with nucleic acids, HU-A and HU-B form (a) biphasic heterotypic mixed condensates in which HU-B helps to lower the C of HU-A and also (b) multiphasic heterotypic condensates, with Dps, in which demixed domains display different contents of HU and Dps. We believe that these modes of complex coacervation that are seen in vitro can serve as models for the in vivo relationships among NAPs in nucleoids, involving local and global variations in the relative abundances of the different NAPs, especially in demixed subdomains that are characterized by differing grades of phase separation. Our results clearly demonstrate some quantitative, and some qualitative, differences in the coacervating abilities of different NAPs with DNA, potentially explaining (i) why E. coli has two isoforms of HU, and (ii) why changes in the abundances of HU and Dps facilitate the lag, logarithmic, and stationary phases of E. coli growth.