Positioning the Model Bacterial Organelle, the Carboxysome

MacCready, Joshua S.; Vecchiarelli, Anthony G.

doi:10.1128/mbio.02519-19

Cited by 20 publications

(19 citation statements)

References 148 publications

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“…In both eukaryotes and prokaryotes, LLPS has been demonstrated as an underlying self-organizing mechanism that allows for the spatiotemporal concentration of certain biomolecules under specific environmental conditions (15,16). How McdB is recruited to carboxysomes has yet to be determined, but its LLPS activity has been suggested to be a contributing factor (17).…”

Section: Introductionmentioning

confidence: 99%

“…In both eukaryotes and prokaryotes, phase separation has been demonstrated as an underlying self-organizing mechanism that allows for the spatiotemporal concentration of certain biomolecules under specific environmental conditions (15, 16). How McdB is recruited to carboxysomes has yet to be determined, but its phase separation activity has been suggested to be a contributing factor (17). Furthermore, characterizing the underlying chemistries for protein phase separation has led to the development of phase separation as a synthetic tool to engineer cytoplasmic organization (18, 19, 20, 21).…”

Section: Introductionmentioning

confidence: 99%

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Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB

Basalla

Mak

Limcaoco

et al. 2022

Preprint

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Carboxysomes are protein-based organelles essential for efficient CO2-fixation in cyanobacteria and some chemoautotrophic bacteria. We recently identified the two-component system responsible for spatially regulating carboxysomes, consisting of the proteins McdA and McdB. McdA is a member of the ParA/MinD-family of ATPases, which position a variety of cellular cargos across bacteria. McdB, however, represents a widespread but unstudied class of proteins. We previously found that McdB forms a hexamer and undergoes robust Liquid-Liquid Phase Separation (LLPS) in vitro, but the sequence and structural determinants underlying these properties are unknown. Here we define the domain architecture for McdB from the model cyanobacterium S. elongatus PCC 7942 which we use to dissect McdB oligomerization and LLPS. We identify an N-terminal Intrinsically Disordered Region (IDR), a central Q-rich dimerizing domain, and a C-terminal domain that trimerizes McdB dimers. Intriguingly, all three domains contributed to McdB LLPS. The Q-rich domain drove LLPS, the IDR tuned solubility, and the C-terminal domain provided further oligomerization to achieve full-length LLPS activity. We also identified critical basic residues in the IDR that modulate McdB LLPS, which we mutate to fine-tune condensate solubility both in vitro and in vivo. Our findings show that IDRs are not always drivers of LLPS, but can play secondary roles in modulating condensate solubility. Finally, we provide in silico evidence suggesting the N-terminal IDR of McdB acts as a MoRF, folding upon interaction with McdA. The data advance our understanding and application of carboxysomes, their positioning system, and the molecular grammar governing protein phase separation.SIGNIFICANCEThe recently characterized Maintenance of Carboxysome Distribution (Mcd) system is responsible for spatially regulating carbon-fixing organelles in bacteria called carboxysomes. Although an understanding of the Mcd system would advance our application of carboxysomes to help engineer carbon-fixing organisms and combat the climate crisis, one of its two essential components, McdB, has only recently been identified and is poorly understood. Here, we provide a thorough biochemical characterization of McdB from the model cyanobacterium S. elognatus. We define a structural model for McdB and identify how specific domains and residues contribute to its oligomerization and phase separation. Notably, we saw that a disordered region of McdB regulates phase separation in response to pH; impactful to both carboxysome regulation and protein phase separation.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB

Basalla

Mak

Limcaoco

et al. 2022

Preprint

Self Cite

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“…Even though disintegration occurs less often at increased PomY concentration (Schumacher et al, 2017), it is unlikely to be caused by dilution of PomY because total cell volumes do not change during division, as opposed to the sudden volume increase during nuclear envelope breakdown in eukaryotes that results in disintegration of the nucleolus condensates (Lafontaine et al, 2021). We speculate that (Babl et al, 2022;Guilhas et al, 2020;MacCready and Vecchiarelli, 2021)…”

Section: Thus Pomy Condensates Concentrate Ftsz In Vitromentioning

confidence: 74%

A phase-separated biomolecular condensate nucleates polymerization of the tubulin homolog FtsZ to spatiotemporally regulate bacterial cell division

Ramm

Schumacher

Harms

et al. 2022

Preprint

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SummaryCell division is spatiotemporally precisely regulated, but the underlying mechanisms are incompletely understood. In the social, predatory bacterium Myxococcus xanthus, the PomX/PomY/PomZ proteins form a single large megadalton-sized complex that directly positions and stimulates cytokinetic ring formation by the tubulin homolog FtsZ. Here, we studied the structure and mechanism of this complex in vitro and in vivo. We demonstrate that PomY forms liquid-like biomolecular condensates by phase separation, while PomX self-assembles into filaments generating a single large cellular structure. The PomX structure enriches PomY, thereby guaranteeing the formation of precisely one PomY condensate per cell through surface-assisted condensation. In vitro, PomY condensates selectively enrich FtsZ and nucleate GTP-dependent FtsZ polymerization, suggesting a novel cell division site positioning mechanism in which the single PomY condensate enriches FtsZ to guide FtsZ-ring formation and division. PomY-nucleated FtsZ polymerization shares features with microtubule nucleation by biomolecular condensates in eukaryotes, supporting this mechanism’s ancient origin.

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“…Overall, the data appears to be reminiscent of liquid droplets that display the tendency to fuse and also separate (or drip), except that the sizes here are ‘sub-micron’ whereas liquid droplets formed by proteins in vitro typically have diameters measured in microns. Given that sub-micron-sized phase-separated droplets have previously been reported by other authors, 39 in particular, in respect of carboxysomes within bacteria, 40 and given the possibility that micron-sized (or larger) droplets could be formed by the same entities in vitro , we proceeded to examine whether recombinantly-expressed forms of HU cause the formation, and maintenance, of a phase separated state of DNA.…”

Section: Resultsmentioning

confidence: 96%

Nucleoid-Associated Proteins Undergo Liquid-Liquid Phase Separation with DNA into Multiphasic Condensates Resembling Bacterial Nucleoids

Gupta

Joshi

Arora

et al. 2022

Preprint

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Liquid-liquid phase separation offers unique spatiotemporal control over myriad complex intracellular biochemical processes through compartmentalization of biomolecules into highly dynamic, liquid-like condensates known as membrane-less organelles. The bacterial nucleoid is thought to be one such phase-separated condensate; however, its formation, regulation, and biophysical characteristics are poorly understood. Our super-resolution imaging data suggests that nucleoids are dynamic assemblages of sub-micron-sized liquid-like droplets. We demonstrate that non-sequence-specific Nucleoid-Associated Proteins (NAPs) such as HU-A, HU-B and Dps, accrete nucleic acids and spontaneously condense with them into liquid-like, multicomponent, multiphasic, heterotypic phase-separated droplets. Upon mixing of HU-B, DNA and Dps, HU-B-enriched droplets are seen to contain demixed Dps-enriched droplets. Our findings indicate scope for the possible existence of multiphasic liquid-like compartments within nucleoids, providing insights into bacterial growth phase-dependent variations in the levels of different NAPs.

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Positioning the Model Bacterial Organelle, the Carboxysome

Cited by 20 publications

References 148 publications

Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB

Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB

A phase-separated biomolecular condensate nucleates polymerization of the tubulin homolog FtsZ to spatiotemporally regulate bacterial cell division

Nucleoid-Associated Proteins Undergo Liquid-Liquid Phase Separation with DNA into Multiphasic Condensates Resembling Bacterial Nucleoids

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