ATP-Driven Separation of Liquid Phase Condensates in Bacteria

Abstract

Liquid-liquid phase separated (LLPS) states are key to compartmentalise components in the absence of membranes, however it is unclear whether LLPS condensates are actively and specifically organized in the sub-cellular space and by which mechanisms. Here, we address this question by focusing on the ParAB S DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB) and a motor (ParA). We show that parS-ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume, but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favoured by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates and localises non-canonical LLPS condensates in the sub-cellular space.

The emergence of phase separation as an organizing principle in bacteria

“…For a more in-depth review of BR-body function and phylogenetic distribution, see Muthunayake et al (4). (21) In vitro (18) In vitro (18) In vitro (18) In vitro (22) In vitro (22) In vitro (22) In vitro (47) In vitro (23) In vivo In vitro spherical (23,24,26) In situ icosahedral (95)(96)(97)(98) In situ (25,29) In vitro (23) In vivo (91) In vitro (26) In vitro (23,24,26) ND…”

“…The ParABS partition system is responsible for the segregation and faithful inheritance of most chromosomes and plasmids in bacteria (Fig 2I). the F and P1 plasmid partition systems in E. coli (26,75). Interestingly, real-time imaging after…”

“…In In eukaryotes, super-resolution imaging and single-molecule tracking revealed that viral replication compartments are not assembled through LLPS since they form at different concentration levels, internal constituents are not evenly or randomly distributed, and their rate of diffusion inside of the compartment is equivalent to the diffusion rate in the nucleoplasm (127). Accordingly, examinations of protein clusters in bacteria have been applying these methods to quantitatively assess LLPS (23,26,31,32,128). We propose that these implementations need to be expanded and broadly applied to rigorously assess the previously described criteria (Fig 1) to confirm or rule out LLPS as a principal driver of spatial organization in bacteria.…”

“…Additionally, the in vitro concentrations and environmental conditions (salt concentrations, pH, temperature, crowding agents) under which components phase separate have been measured (17,19,(21)(22)(23)(24)(25)27). The liquid-like behavior of condensates has been tested by fluorescence recovery after photobleaching (FRAP) and by observing droplet fusion events (17,21,24,26,28). Finally, (18,21,26,30) and measured the diffusive properties of their molecular components (23,26,31,32).…”

“…The liquid-like behavior of condensates has been tested by fluorescence recovery after photobleaching (FRAP) and by observing droplet fusion events (17,21,24,26,28). Finally, (18,21,26,30) and measured the diffusive properties of their molecular components (23,26,31,32). As the methods improve and rigorous controls are implemented, single-molecule methods are becoming increasingly critical for understanding bacterial subcellular organization due to the very small size of the relevant features in these microscopic organisms.…”