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 (LLPS) condensates in vitro. Upon mixing with nucleic acids, HU-A and HU-B form (a) bi-phasic heterotypic mixed condensates in which HU-B helps to lower the Csat of HU-A; and also (b) multi-phasic heterotypic condensates, with Dps, in which de-mixed 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 amongst NAPs in nucleoids, involving local and global variations in the relative abundances of the different NAPs, especially in de-mixed sub-domains 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.