The emergence of phase separation as an organizing principle in bacteria

Azaldegui, Christopher A.; Vecchiarelli, Anthony G.; Biteen, Julie S.

doi:10.1101/2020.08.05.239012

Cited by 35 publications

(41 citation statements)

References 139 publications

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“…Bacteria display many mechanisms to control and position precisely and specifically macromolecular complexes in their cellular environment. Some of these mechanisms use the nucleoid as a matrix [1,2] like, e.g., the system PomXYZ (cell division site positioning [3]) or McdAB (carboxysomes positioning [4]). Here, we investigate the case of the ParABS system essential for the stable inheritance of most chromosomes and low-copy-number plasmids [5,6].…”

Section: Introductionmentioning

confidence: 99%

Supercoiled DNA and non-equilibrium formation of protein complexes: A quantitative model of the nucleoprotein ParBS partition complex

et al. 2021

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ParABS, the most widespread bacterial DNA segregation system, is composed of a centromeric sequence, parS, and two proteins, the ParA ATPase and the ParB DNA binding proteins. Hundreds of ParB proteins assemble dynamically to form nucleoprotein parS-anchored complexes that serve as substrates for ParA molecules to catalyze positioning and segregation events. The exact nature of this ParBS complex has remained elusive, what we address here by revisiting the Stochastic Binding model (SBM) introduced to explain the non-specific binding profile of ParB in the vicinity of parS. In the SBM, DNA loops stochastically bring loci inside a sharp cluster of ParB. However, previous SBM versions did not include the negative supercoiling of bacterial DNA, leading to use unphysically small DNA persistences to explain the ParB binding profiles. In addition, recent super-resolution microscopy experiments have revealed a ParB cluster that is significantly smaller than previous estimations and suggest that it results from a liquid-liquid like phase separation. Here, by simulating the folding of long (≥ 30 kb) supercoiled DNA molecules calibrated with realistic DNA parameters and by considering different possibilities for the physics of the ParB cluster assembly, we show that the SBM can quantitatively explain the ChIP-seq ParB binding profiles without any fitting parameter, aside from the supercoiling density of DNA, which, remarkably, is in accord with independent measurements. We also predict that ParB assembly results from a non-equilibrium, stationary balance between an influx of produced proteins and an outflux of excess proteins, i.e., ParB clusters behave like liquid-like protein condensates with unconventional “leaky” boundaries.

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Section: Introductionmentioning

confidence: 99%

Supercoiled DNA and non-equilibrium formation of protein complexes: A quantitative model of the nucleoprotein ParBS partition complex

et al. 2021

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“…However, as sensitivity to HEX is not a definitive test for LLPS (Alberti et al ., 2019), we considered whether the Hfq foci (and thus H-bodies) conform with other known properties of LLPS condensates. A comprehensive review by Azaldegui et al (Azaldegui et al ., 2021), proposed the following criteria for determining whether a structure is formed by LLPS: (i) condensates are spherical in nature due to surface tension, (ii) condensate droplets can fuse upon contact, (iii) molecules within condensates remain mobile and (iv) are able to diffuse across the condensate boundary, and (v) condensate formation occurs in a concentration dependent manner. It seems that the Hfq foci meet three ( i, iii and iv ) of these five criteria: As seen in figures 1-4, the Hfq foci, i.e., the H-body, is consistently spherical-shaped in 2D space and are therefore likely to be spherical in 3D space.…”

Section: Resultsmentioning

confidence: 99%

The association between Hfq and RNase E in long-term nitrogen starvedEscherichia coli

McQuail

Carpousis

Wigneshweraraj

2021

Preprint

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The regulation of bacterial gene expression is underpinned by the synthesis and degradation of mRNA. In Escherichia coli, RNase E is the central enzyme involved in RNA degradation and serves as a scaffold for the assembly of the multiprotein complex known as the RNA degradosome. The activity of RNase E against specific mRNAs can also be regulated by the action of small RNAs (sRNA). The ubiquitous bacterial chaperone Hfq bound to sRNAs interacts with the RNA degradosome for the sRNA guided degradation of target mRNAs. The association between RNase E and Hfq has never been observed in live bacteria. We now show that in long-term nitrogen starved E. coli, both RNase E and Hfq co-localise in a single, large focus. This subcellular assembly, which we refer to as the H-body, also includes components of the RNA degradosome, namely, the helicase RhlB and the exoribonuclease polynucleotide phosphorylase. We further show that H-bodies are important for E. coli to optimally survive sustained nitrogen starvation. Collectively, the properties and features of the H-body suggests that it represents a hitherto unreported example of subcellular compartmentalisation of a process(s) associated with RNA management in stressed bacteria.

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“… 163 Furthermore, transcriptional hubs in eukaryotes display properties of liquid condensates, where multiple components have been implicated with the phase separation, namely transcription factors, 27 , 28 coactivators, 30 , 31 the enhancer sequence, 29 and RNA polymerase. 27 , 28 , 30 , 33 Lastly, phase separation is also significant for bacterial chromosomes, 164 , 165 for example, in transcriptional hubs surrounding the nucleoid in E. coli ( 32 ) and in ParB protein clusters in B. subtilis . 166 ParB loads the bacterial SMCs onto the DNA, whereupon the SMCs actively proceed along the DNA, wrapping the two chromosome arms together.…”

Section: An Overview Of Chromosome Building Blocksmentioning

confidence: 99%

Genome-in-a-Box: Building a Chromosome from the Bottom Up

Birnie

Dekker

2020

ACS Nano

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Chromosome structure and dynamics are essential for life, as the way that our genomes are spatially organized within cells is crucial for gene expression, differentiation, and genome transfer to daughter cells. There is a wide variety of methods available to study chromosomes, ranging from live-cell studies to single-molecule biophysics, which we briefly review. While these technologies have yielded a wealth of data, such studies still leave a significant gap between top-down experiments on live cells and bottom-up in vitro single-molecule studies of DNA–protein interactions. Here, we introduce “genome-in-a-box” (GenBox) as an alternative in vitro approach to build and study chromosomes, which bridges this gap. The concept is to assemble a chromosome from the bottom up by taking deproteinated genome-sized DNA isolated from live cells and subsequently add purified DNA-organizing elements, followed by encapsulation in cell-sized containers using microfluidics. Grounded in the rationale of synthetic cell research, the approach would enable to experimentally study emergent effects at the global genome level that arise from the collective action of local DNA-structuring elements. We review the various DNA-structuring elements present in nature, from nucleoid-associated proteins and SMC complexes to phase separation and macromolecular crowders. Finally, we discuss how GenBox can contribute to several open questions on chromosome structure and dynamics.

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The emergence of phase separation as an organizing principle in bacteria

Cited by 35 publications

References 139 publications

Supercoiled DNA and non-equilibrium formation of protein complexes: A quantitative model of the nucleoprotein ParBS partition complex

Supercoiled DNA and non-equilibrium formation of protein complexes: A quantitative model of the nucleoprotein ParBS partition complex

The association between Hfq and RNase E in long-term nitrogen starvedEscherichia coli

Genome-in-a-Box: Building a Chromosome from the Bottom Up

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