Phase separation and clustering of an ABC transporter in
            <i>Mycobacterium tuberculosis</i>

Heinkel, Florian; Abraham, Libin; Ko, Mary; Chao, Joseph D.; Bach, Horacio; Hui, Lok Tin; Li, Haoran; Zhu, Mang; Ling, Maggie; Rogalski, Jason C.; Scurll, Joshua M; Bui, Jennifer M.; Mayor, Thibault; Gold, Michael R.; Chou, Kuang-Chao; McIntosh, Lawrence P.; Gsponer, Jörg

doi:10.1073/pnas.1820683116

Cited by 59 publications

(70 citation statements)

References 33 publications

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“…All in all, along with the few recently discovered cases of bacterial LLPS (Al-Husini et al, 2018;Heinkel et al, 2019;Monterroso et al, 2019), our findings highlight that LLPS is a fundamental, universally exploited mechanism that is governed by similar molecular mechanisms in bacteria and eukaryotes. The LLPS propensity predicted for human SSB homologs hSSB1/SOSB1 and hSSB2/SOSB2 suggests broad evolutionary conservation of this feature (Fig.…”

Section: Discussionsupporting

confidence: 59%

“…Whereas LLPS seems to be a heavily exploited process in eukaryotes and viruses (Heinrich et al, 2018;Nikolic et al, 2017Nikolic et al, , 2018Zhou et al, 2019), up until today only a few bacterial proteins were reported to undergo LLPS. These include Caulobacter crescentus Ribonuclease E (RNase E) that orchestrates RNA degradosomes (Al-Husini et al, 2018), Escherichia coli (E. coli) FtsZ that forms the division ring during cell divisions (Monterroso et al, 2019) and Mycobacterium tuberculosis Rv1747 that is a membrane transporter of cell wall biosynthesis intermediates (Heinkel et al, 2019). All in all, the hitherto accumulated observations imply that LLPS is a fundamental mechanism for the effective spatiotemporal organization of cellular space, with emerging but hardly explored functions in bacteria.…”

Section: Introductionmentioning

confidence: 99%

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Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Harami

Kovács

Pancsa

et al. 2019

Preprint

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Bacterial single stranded (ss) DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL) and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein-protein interactions with DNA-metabolizing proteins. Here we show that the E. coli SSB protein forms liquid-liquid phase separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA and SSBinteracting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage. RESULTS SSB forms dynamic LLPS condensates in physiologically relevant conditions in vitroDue to the fact that SSB shares several features with hitherto described LLPS drivers, we sought to determine if LLPS could be a yet undiscovered capability of SSB facilitating its multifaceted roles in genome maintenance. We performed a bioinformatics analysis that revealed that E. coli SSB, in particular its IDL region, shows high propensity for LLPS, according to multiple dedicated sequencebased prediction methods (Bolognesi et al., 2016;Lancaster et al., 2014;Vernon et al., 2018) (Fig. 1B-D). This feature of SSB is universally conserved among bacteria, as the majority of analyzed SSBs (72.1 %) harbored by representative bacterial species from 15 large phylogenetic groups show similar LLPS propensities (Tables S1-2; Fig. 1E).In line with its predicted LLPS propensity, purified E. coli SSB (30 µM; SSB concentrations are expressed as those of tetramer molecules throughout the paper) forms an opaque, turbid solution at low NaCl concentration (50 mM) in vitro at room temperature and also at the native temperature of E. coli cells (37°C) ( Fig. 2A-B). The process is reversible as elevation of NaCl concentration to 200 mM leads to immediate loss of turbidity. The Clconcentration in E. coli cells depends on their environment (Schultz et al., 1962); however, while the exact value is not known, it can be assumed to be in a similar range (5-100 mM) as that measured for the cells of mammalian E. coli hosts, whereas the major metabolic anion in bacterial cells is glutamate (Glu; 100 mM) (Bennett et al., 2009). Strikingly, SSB forms a turbid solution even in 200 mM NaGlu ( Fig. 2A-B). (It must be noted that experiments using NaGlu always contained a fixed amount of 20 mM NaCl, for technical reasons.) Investigation of the protein solution by differential interference contrast (DIC) microscopy after diluting SSB into low-salt buffer revealed the formation of regular spherical drop...

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Section: Discussionsupporting

confidence: 59%

Section: Introductionmentioning

confidence: 99%

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Harami

Kovács

Pancsa

et al. 2019

Preprint

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“…In stationary phase, Dps compacts the E. coli nucleoid into a dense crystalline-like structure that excludes restriction enzymes yet permits access to RNAP (109). LLPS may also occur on the bacterial membrane, as the ABC transporter Rv1747 phase separates in vitro and forms clusters in Mycobacterium (110). There is also in vitro evidence that FtsZ and SlmA may form condensates that regulate the positioning of Z-ring assembly during E. coli cell division (85).…”

Section: Biomolecular Condensates In Bacteriamentioning

confidence: 99%

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

Ladouceur

Parmar

Biedzinski

et al. 2020

Preprint

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Once described as mere "bags of enzymes", bacterial cells are in fact highly organized, with many macromolecules exhibiting non-uniform 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 E. coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrientrich 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 suggests that this process serves as a universal mechanism for intracellular organization across the tree of life. SignificanceBacterial cells are small and were long thought to have little to no internal structure. However, advances in microscopy have revealed that bacteria do indeed contain subcellular compartments. But how these compartments form has remained a mystery. Recent progress in larger, more complex eukaryotic cells has identified a novel mechanism for intracellular organization known as liquid-liquid phase separation. This process causes certain types of molecules to concentrate within distinct compartments inside the cell. Here, we demonstrate that the same process also occurs in bacteria. This work, together with a growing body of literature, suggests that liquidliquid phase separation is a universal mechanism for intracellular organization that extends across the tree of life.

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“…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).…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

The emergence of phase separation as an organizing principle in bacteria

Azaldegui

Vecchiarelli

Biteen

2020

Preprint

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Recent investigations in bacteria suggest that membraneless organelles play a crucial role in the subcellular organization of bacterial cells. However, the biochemical functions and assembly mechanisms of these compartments have not yet been completely characterized. This Review assesses the current methodologies used in the study of membraneless organelles in bacteria, highlights the limitations in determining the phase of complexes in cells that are typically an order of magnitude smaller than a eukaryotic cell, and identifies gaps in our current knowledge about the functional role of membraneless organelles in bacteria. Liquid-liquid phase separation (LLPS) is one proposed mechanism for membraneless organelle assembly. Overall, we outline the framework to evaluate LLPS in vivo in bacteria, we describe the bacterial systems with proposed LLPS activity, and we comment on the general role LLPS plays in bacteria and how it may regulate cellular function. Lastly, we provide an outlook for super-resolution microscopy and single-molecule tracking as tools to assess condensates in bacteria.Statement of SignificanceThough membraneless organelles appear to play a crucial role in the subcellular organization and regulation of bacterial cells, the biochemical functions and assembly mechanisms of these compartments have not yet been completely characterized. Furthermore, liquid-liquid phase separation (LLPS) is one proposed mechanism for membraneless organelle assembly, but it is difficult to determine subcellular phases in tiny bacterial cells. Thus, we outline the framework to evaluate LLPS in vivo in bacteria and we describe the bacterial systems with proposed LLPS activity in the context of these criteria.

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Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis

Cited by 59 publications

References 33 publications

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

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

The emergence of phase separation as an organizing principle in bacteria

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