On Oct. 20th, 2021, the Fan Bai Group from Biomedical Pioneering Innovation Center (BIOPIC) and Beijing Advanced Innovation Center for Genomics (ICG) in Peking University, collaborating with Mark C. Leake group at the University of York, published a paper entitled “Membraneless organelles formed by liquid-liquid phase separation increase bacterial fitness” in Science Advances . The research group exploits rapid super-resolved single-molecule tracking and multiscale modeling in tandem to determine the molecular biophysics of their spatial and temporal control to deduce their crucial role in bacterial fitness.

　　Liquid-liquid phase separation (LLPS) drives the formation of membrane-less compartments in eukaryotic cells, such as P granules, nucleoli, heterochromatin, and stress granules, enabling concentrations of associated biomolecules to increase biochemical reaction efficiency and protection of mRNA or proteins to promote cell survival under stress. The characteristic feature of reversibility of LLPS, which is often highly sensitive to several microenvironmental factors, offers potential benefits to cells in terms of dynamic compartmentalization of their complex cytoplasmic components in response to changes in cellular physiology in the absence of membranes. However, the function of LLPS in bacteria, which, generally lack membrane-bound compartments, remains less clear.

　　Fig. 1. Aggresome formation in bacteria.

　　Bai group had published a paper in Molecular Cell in 2018 entitled ATP-dependent dynamic protein aggregation regulates bacterial dormancy depth critical for antibiotic tolerance, reporting the existence of bacterial aggresomes, which are subcellular collections of endogenous proteins that appear as dark foci. Unlike protein aggregates formed by misfolded proteins, as reported previously, aggresomes are dynamic, reversible structures that form under stressed conditions and disassemble when cells experience fresh growth media. In this paper, to further investigate the mechanism of aggresome formation, the research group initially screened three proteins (HslU, Kbl, and AcnB) as biomarkers, according to the previous analysis of their abundance in aggresomes, and labeled each with fluorescent protein reporters at chromosome. By overexpressing HokB protein, a toxin causing cellular ATP depletion, aggresome formation was triggered in E. coli. Monitoring the proportion of cells containing distinct fluorescent foci from time-lapse epifluorescence microscopy indicated different clustering dynamics of the three proteins with respect to ATP depletion (Fig. 1). HslU is the fastest to incorporate into aggrsomes, followed by Kbl, and then AcnB.

　　Fig. 2. Aggresome formation occurs through LLPS.

　　Since HslU showed the highest abundance and rate of response following ATP depletion, it was used as the biomarker to explore spatiotemporal features of aggresome formation with rapid super-resolved single-molecule tracking. To investigate the patterns of mobility for HslU in the cytoplasm, the Bai Group photobleached the majority of HslU-EGFP and tracked the movement of fluorescent foci with only a few HslU molecules (Fig. 2). The result shows that a single HslU-EGFP inside aggresomes diffuses faster than immobilized EGFP at all stages. HslU molecules are thus definitively mobile inside aggresomes, consistent with a liquid state. The fusion of two aggresomes was also captured, supporting the hypothesis that LLPS drives bacterial aggresome formation. To further test the liquid nature of aggresomes, the Bai Group implemented FRAP measurements. By focusing a laser laterally offset approximately 0.5 µm from an aggresome center, it was possible to photobleach EGFP content within approximately one spatial half of the aggresome while leaving the other half intact. Fluorescence in the bleached region recovered with a half time 50 ± 9 sec (±SD) to approximately 35% of the initial intensity at steady-state, revealing that HslU-EGFP undergoes dynamic turnover within the aggresome over a timescale of tens of seconds.

　　Fig. 3. Simulating aggresomes using an Individual-Protein-Based Model.

　　Bai Group developed an Individual-Protein-Based Model (IPBM) to interpret the experimental observations by simulating collective dynamics of aggresome formation, protein turnover, and dynamics as LLPS under thermal equilibrium (Fig. 3). The straightforward theoretical framework demonstrates that relatively simple protein physics – energetic interaction of diffusing proteins under local thermal equilibrium characterized by a simple and coarse-grained pairwise interaction energy – accounts not only for the conditions of aggresome formation in the first place but also for complex, emergent biological features in their spatial localization and their dynamics and kinetics of molecular turnover.

　　Fig. 4. Aggresome formation promotes cellular survival under fierce stresses.

　　Finally, the aggresomes are not unique to E. coli; by prolonged stationary culturing, the Bai Group observed aggresome formation in multiple Gram-negative species. To investigate the biological function of aggresomes, the Bai Group used chemical inhibitors and knockout to block the formation of aggresomes. The researchers tested the survival ratio wild type, knockout strains, and wild type supplemented with chemical inhibitor after stationary culturing under antibiotic and phage attack. The results show that the wild type strain with successful aggresome formation showed a significantly higher survival ratio in comparison to strains in which aggresome formation was blocked (Fig. 4). Taken together, the aggresome formation through LLPS promotes bacterial survival under a range of fierce stresses.

　　The formation of certain small biological condensates in bacteria has recently been ascribed to LLPS. This study has established that aggresomes are membraneless liquid droplets hundreds of nm in diameter that phase separate following clustering of diffusing proteins under thermal equilibrium in the bacterial cytoplasm. The modeling indicates no requirement of external free energy for aggresome formation, but a dependence on ATP cellular concentration. A possible explanation could lie in recent work, which demonstrated that ATP not only energizes processes inside cells but also acts as a hydrotrope to increase specific protein solubility; decreasing cellular ATP may thus act to favor LLPS. This method of regulation using ATP might be an important and recurring theme in several other biological processes that utilize LLPS.

　　Xin Jin from Biomedical Pioneering Innovation Center (BIOPIC), School of Life Sciences, Peking University, Ji-Eun Lee from Department of Physics, University of York, York, United Kingdom, Charley Schaefer from Department of Physics, University of York, York, United Kingdom, Xinwei Luo from Biomedical Pioneering Innovation Center (BIOPIC), School of Life Sciences, Peking University are co-first authors of this work. Fan Bai from Peking University and Mark C.Leake from University of York are co-corresponding authors. This research was supported by the National Natural Science Foundation of China, the Beijing Future Genetic Diagnostics High Precision Innovation Center, and the National Center for Protein Science of Peking University.

　　Link: https://www.science.org/doi/10.1126/sciadv.abh2929