Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid–liquid phase separation

Abstract

Once described as mere “bags of enzymes,” bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid–liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.

The emergence of phase separation as an organizing principle in bacteria

“…Given that condensate constituents maintain their ability to diffuse inside the compartment, single-molecule tracking provides a quantitative alternative to FRAP measurements. This method has already been implemented in live C. crescentus and E. coli to investigate PopZ microdomains ( Fig 5C) and RNAP clusters, respectively (23,32). These studies revealed differences between the mobility of molecules inside and outside these compartments.…”

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“…(1) actively transcribing RNAP and (2) non-transcribing RNAP (Ladouceur et al, 2020 (40,32). Because the CtrA-P gradient decays away from the swarmercell pole, it results in a skewed inheritance of CtrA-P in the two daughter cells after asymmetric cell division (32,40).…”

“…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.…”

“…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.…”