Colocalization of distant chromosomal loci in space in <i>E. coli</i>: a bacterial nucleolus

Gaál, Tamás; Bratton, Benjamin P.; Sanchez-Vazquez, Patricia; Sliwicki, Alexander; Sliwicki, Kristine; Vegel, Andrew; Pannu, Rachel; Gourse, Richard L.

doi:10.1101/gad.290312.116

Cited by 57 publications

(68 citation statements)

References 52 publications

(76 reference statements)

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“…RNAP is distributed throughout the nucleoid in cells grown in minimal media, but concentrates into distinct clusters in cells grown in rich media (31,32). This nutrient-dependent localization has been well-characterized (33,34), but the mechanism(s) that govern RNAP clustering remains unclear (35,36). Here, we show that clusters of bacterial RNAP are biomolecular condensates.…”

Section: Introductionmentioning

confidence: 66%

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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Section: Introductionmentioning

confidence: 66%

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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“…One possible explanation derives from the very different spatial distributions of actively transcribing RNA polymerase copies in the two conditions. In fast growth, most transcription occurs on the seven rrn operons, six of which have been shown to cluster with each other (Gaal et al, 2016). These 'transcription foci' (Jin et al, 2013) tend to locate at the periphery of the nucleoid (Jin et al, 2013;Stracy et al, 2015), perhaps due to excluded volume effects (Mondal et al, 2011).…”

Section: Degree Of Nucleoid Compaction In Fast and Slow Growthmentioning

confidence: 99%

Functional mapping of the E. coli translational machinery using single‐molecule tracking

Mohapatra

Weisshaar

2018

Molecular Microbiology

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The organization of the chromosomal DNA and ribosomes in living Escherichia coli is compared under two growth conditions: 'fast' (50 min doubling time) and 'slow' (147 min doubling time). Superresolution fluorescence microscopy reveals strong DNA-ribosome segregation in both cases. In both fast and slow growth, free ribosomal subunits evidently must circulate between the nucleoid (where they initiate co-transcriptional translation) and ribosome-rich regions (where most translation occurs). Single-molecule diffusive behavior dissects the ribosome copies into translating 70S polysomes and free 30S subunits, providing separate spatial distributions for each. In slow growth, ~21,000 total 30S copies/cell comprise ~65% translating 70S ribosomes and ~35% free 30S subunits. The ratio of 70S ribosomes to free 30S subunits is ~2.5 outside the nucleoid and ~0.50 inside the nucleoid. This new level of quantitative detail may motivate development of comprehensive, three-dimensional reaction-diffusion models of ribosome, DNA, mRNA and RNAP spatial distributions and dynamics within the E. coli cytoplasm.

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“…Taken together, these results argued strongly against the hypothesis that rRNA transcription activity from multiple rrn operons contributes significantly to the spatial organization of RNAP. Consistent with this notion, a recent study reported that multiple rrn operons could colocalize with each other independent of rRNA transcription activity 34 .…”

Section: Discussionmentioning

confidence: 63%

“…Next, we reasoned that while rRNA transcription activity was diminished in cells treated with rifampicin or serine hydroxamate, it is possible that RNAP remained associated with multiple rrn operons that spatially colocalize with each other 34 , despite the lack of high transcription activity from these operons. To examine this possibility, we used a Δ 6rrn strain, in which six out of seven rrn operons (except for rrnC) were removed from the chromosome 16 .…”

Section: Resultsmentioning

confidence: 99%

“…Alternatively, it is also possible that the two pre-rRNA clusters reflected two groups of transcribing rrn operons on the same copy of chromosome that are spatially distinguishble from each other under our resolutions. A recent study found that, except for rrnC , all the rrn operons are within a spatial distance of ~ 80 – 130 nm (median of distributions) to each other 34 , but it remains unknown whether these rrn operons indeed co-occupy the same area in the nucleoid and whether they could be accommodated in one pre-rRNA cluster (radius of ~ 130 nm). Because of the nearly identical pre-rRNA sequences of all the rrn operons, we could not design a probe with high confidence to distinguish the transcription activity of individual rrn operons, and hence further investigation is required to address whether each pre-rRNA cluster reflects the transcription activity from individual or a collection of rrn operons.…”

Section: Discussionmentioning

confidence: 99%

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RNA polymerase organizes into distinct spatial clusters independent of ribosomal RNA transcription inE. coli

Weng

Bohrer

Bettridge

et al. 2018

Preprint

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Recent studies have shown that RNA polymerase (RNAP) is spatially organized into distinct clusters in E. coli and B. subtilis cells. Spatially organized molecular components in prokaryotic systems imply compartmentalization without the use of membranes, which may offer new insights into pertinent functions and regulations. However, the function of RNAP clusters and whether its formation is driven by active ribosomal RNA (rRNA) transcription remain elusive. In this work, we investigated the spatial organization of RNAP in E. coli cells using quantitative superresolution imaging. We observed that RNAP formed large, distinct clusters under a rich medium growth condition and preferentially located in the center of the nucleoid. Two-color superresolution colocalization imaging showed that under the rich medium growth condition, nearly all RNAP clusters were active in synthesizing rRNA, suggesting that rRNA synthesis may be spatially separated from mRNA synthesis that most likely occurs at the nucleoid periphery. Surprisingly, a large fraction of RNAP clusters persisted under conditions in which rRNA synthesis was reduced or abolished, or when only one out of the seven rRNA operons (rrn) remained. Furthermore, when gyrase activity was inhibited, we observed a similar rRNA synthesis level, but multiple dispersed, smaller rRNA and RNAP clusters occupying not only the center but also the periphery of the nucleoid, comparable to an expanded nucleoid. These results suggested that RNAP was organized into active transcription centers for rRNA synthesis under the rich medium growth condition; their presence and spatial organization, however, were independent of rRNA synthesis activity under the conditions used but were instead influenced by the structure and characteristics of the underlying nucleoid. Our work opens the door for further investigations of the function and molecular nature of RNAP clusters and points to a potentially new mechanism of transcription regulation by the spatial organization of individual molecular components.

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Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus

Cited by 57 publications

References 52 publications

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

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

Functional mapping of the E. coli translational machinery using single‐molecule tracking

RNA polymerase organizes into distinct spatial clusters independent of ribosomal RNA transcription inE. coli

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