Inside of a cell, some biomolecules cluster together to behave as a liquid. These liquids can reversibly form distinct phases, like the classic example of lava lamps, where mineral oil blobs slip and slide off the water-and-alcohol mix. This is liquid-liquid phase separation (LLPS), a protean dynamo of a physical phenomenon. It gives rise to a whole menagerie of organelles, from familiar faces like the nucleolus, to more unexpected recent discoveries like organelles that promote cancer metastasis. LLPS is hot stuff in eukaryotic cell biology these days.
But what about the bacterial cell? First, to quickly address the elephant in the room, here's Elio's recent pithy statement: Prokaryotic organelles. Yes, there are such things. Bacterial cells, once thought to lack much internal organization, are also compartmentalized. It turns out that the bacterial cytoplasm is partitioned into both membranous and membrane-less organelles, like its eukaryotic counterpart.
LLPS is the reason membrane-less organelles exist. For both formation and maintenance of membrane-less internal structures, LLPS is often driven by multivalent interactions between nucleic acids and proteins. On the protein side of things, low-complexity domains (runs of repeating amino acids) and intrinsically disordered regions (floppy regions without a favored 3D conformation) both contribute to condensation. As Roberto previously covered here on the blog, phase separation may underlie the Dps-DNA complexes in nutrient-starved bacteria.
It appears that the life of an RNA molecule can be traced from start to finish through the lens of LLPS, even in bacteria.
We can start with polymerase, the mother of all RNA. In eukaryotes, the intrinsically disordered C-terminal domain of RNA polymerase II drives clustering into dense foci, which act as transcriptional powerhouses. In bacteria, too, the clustering of RNA polymerase is a dynamic response to environmental conditions. For example, bacterial polymerases cluster when cells are moved from minimal to rich media and form phase-separated intracellular compartments during the lag-to-exponential transition. These foci disappear – reversibly, a defining trait of LLPS – upon addition of hexanediol, which dissolves LLPS condensates. During the stringent response the distribution of RNA polymerase in E. coli becomes diffuse. Along with the loss of stable RNA synthesis, the RNA-polymerase condensates dissolve.
Onward to RNA's liminal "middle years." Up first: sRNAs, those multi-talented factotums of the cell. Many bacterial sRNAs are reliant upon a chaperone protein partner, the most famous of which is Hfq, found in at least 50% of bacterial genomes. Kannaiah et al. (2019) reported that the hexameric protein donut assembles into foci at the poles of E. coli cells under osmotic stress, complexed with stress-related mRNAs and sRNAs. Then McQuail et al. (2020) reported that Hfq assembles into a single focus in E. coli under nitrogen-starvation conditions, and then disassembles once nitrogen is plentiful again. Déjà vu? Reminiscent of eukaryotic stress granules, these foci also appear to be LLPS-dependent compartments formed in response to stressors. McQuail et al. speculated that they could be ribonucleoprotein complexes that degrade unneeded RNA or ribosomes to free up much-needed nitrogen.
Interesting links to LLPS can also be found in RNA folding. Amid the array of intermediate states, the energy landscape of RNA folding is often riddled with many peaks and troughs. With so many thermodynamic pitfalls, RNA molecules sometimes need help folding into the right shape. Just as metal ions can screen RNA's negative charge to counter electrostatic repulsion, so can proteins that bind RNA. Holmstrom et al. (2019) discovered this property in a viral core protein: the positively charged disordered protein functions as a counterion to allow faster RNA-folding into compact structures. Perhaps other RNA-binding proteins in bacteria with intrinsically disordered regions (looking at you, Hfq and friends) also serve as molecular counter-charges? Electrostatic interactions can contribute to LLPS, and indeed some engineered positively "supercharged" proteins form condensates with RNA transcripts.
At the end of the lifespan, RNA turnover, too, invokes LLPS. In eukaryotes, stress granule RNA-protein condensates act as hubs of degradation. In bacteria, the intrinsically disordered C-terminal domain of RNase E is the key for the formation of reversible, phase-separated, mRNA-bound "BR-bodies" that are on the same size scale as the stress granules. Like their eukaryotic doppelgängers, these condensates crop up under stressors like ethanol or hydrogen peroxide exposure. It's possible that these RNase hubs speed up mRNA degradation so that cells can adapt faster via changes in RNA processing. Indeed, mutant cells lacking the RNase's disordered C-terminus were out of luck, incapable of forming BR-bodies and thus becoming less stress-resistant.
But it's not just the intracellular landscape taking part in this shape-shifting, it's at the level of the whole cells as well. Motility Induced Phase Separation (MIPS) is a form of phase separation that takes place when faster moving particles separate from slower moving ones. These particles can be swarming bacteria. Such swarming bacteria can separate into clusters of different cell density, which in turn can promote the formation of biofilms. Grobas et al. (2021) discovered that swarms of B. subtilis transition by a MIPS-like process into biofilms.
Among the hustle and bustle of bacteria – both within and around cells – phase separation seems to be a bit of a dark horse.