Carboxysome Mispositioning Alters Growth, Morphology, and Rubisco Level of the Cyanobacterium Synechococcus elongatus PCC 7942

Rillema, Rees; Hoang, Y; MacCready, Joshua S.; Vecchiarelli, Anthony G.

doi:10.1128/mbio.02696-20

Cited by 13 publications

(12 citation statements)

References 76 publications

(100 reference statements)

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“…Overall, McdA mutant strains predicted to be defective for ATP-binding and dimerization displayed a cell elongation phenotype, and possessed few and irregularly-spaced carboxysome aggregates. These phenotypes match what we have previously observed in the mcdA deletion strain (Rillema et al, 2021), which suggests a complete loss of function in carboxysome positioning when McdA cannot bind ATP and dimerize.…”

Section: Strategy For Trapping and Imaging Mcda At Specific Steps Of Its Atpase Cyclesupporting

confidence: 89%

“…Heterotrophic bacteria have been shown to undergo cell elongation as a carbon-limitation response (Rangarajan et al, 2020). We also recently proposed that carboxysome aggregation results in decreased carbon-fixation efficiency, and that cell elongation is a response triggered by the resulting carbon limitation in this photoautotroph (Rillema et al, 2021). Since the phenotype of these predicted McdA trap mutants mirror the mcdA deletion strain, our findings suggest a complete loss of function in carboxysome positioning when McdA cannot bind ATP, dimerize, and adopt its nucleoid-binding conformation.…”

Section: Discussionmentioning

confidence: 99%

“…These mutant strains also displayed cell elongation. We have recently shown that mcdA and mcdB deletion strains also elongate (Rillema et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

“…We have previously shown that mNG-McdA is fully functional for carboxysome positioning when expressed as the only copy of McdA at its native locus (MacCready et al, 2018). Finally, we also performed phase contrast imaging to monitor for changes in cell morphology, as we have recently shown that carboxysome mispositioning in mcdA or mcdB deletion strains triggers cell elongation, which we proposed is a response to carbon limitation in this obligate photoautotroph (Rillema et al, 2021).…”

Section: Strategy For Trapping and Imaging Mcda At Specific Steps Of Its Atpase Cyclementioning

confidence: 98%

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Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution

Hakim

Hoang

Vecchiarelli

2021

MBoC

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Carboxysomes, the most prevalent and well-studied anabolic bacterial microcompartment, play a central role in efficient carbon fixation by cyanobacteria and proteobacteria. In previous studies, we identified the two-component system called McdAB that spatially distributes carboxysomes across the bacterial nucleoid. McdA, a ParA-like ATPase, forms a dynamic oscillating gradient on the nucleoid in response to carboxysome-localized McdB. As McdB stimulates McdA ATPase activity, McdA is removed from the nucleoid in the vicinity of carboxysomes, propelling these proteinaceous cargos toward regions of highest McdA concentration via a Brownian-ratchet mechanism. How the ATPase cycle of McdA governs its in vivo dynamics and carboxysome positioning remains unresolved. Here, by strategically introducing amino acid substitutions in the ATP-binding region of McdA, we sequentially trap McdA at specific steps in its ATP cycle. We map out critical events in the ATPase cycle of McdA that allows the protein to bind ATP, dimerize, change its conformation into a DNA-binding state, interact with McdB-bound carboxysomes, hydrolyze ATP and release from the nucleoid. We also find that McdA is a member of a previously unstudied subset of ParA family ATPases, harboring unique interactions with ATP and the nucleoid for trafficking their cognate intracellular cargos. [Media: see text] [Media: see text] [Media: see text]

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Section: Strategy For Trapping and Imaging Mcda At Specific Steps Of Its Atpase Cyclesupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

“…These mutant strains also displayed cell elongation. We have recently shown that mcdA and mcdB deletion strains also elongate (Rillema et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

Section: Strategy For Trapping and Imaging Mcda At Specific Steps Of Its Atpase Cyclementioning

confidence: 98%

See 2 more Smart Citations

Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution

Hakim

Hoang

Vecchiarelli

2021

MBoC

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“…However, it is attractive to speculate that coating carboxysomes with a viscous, pH-sensitive McdB phase may influence the diffusivity of carboxysome shells to protons, which is an important aspect of current models for an efficient CCM (68, 69). Indeed, in a previous study, we have shown that cells deleted for McdB show additional growth deficits compared to cells lacking McdA, suggesting that McdB plays additional roles in the CCM beyond spatially regulating carboxysomes with McdA (71). Moreover, a separate study has shown that the shells of purified carboxysomes, which lack McdB, are freely permeable to protons (72).…”

Section: Discussionmentioning

confidence: 95%

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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Responding to light signals: a comprehensive update on photomorphogenesis in cyanobacteria

Gupta,

Pandey,

Gupta

et al. 2023

Physiol Mol Biol Plants

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Carboxysome Mispositioning Alters Growth, Morphology, and Rubisco Level of the Cyanobacterium Synechococcus elongatus PCC 7942

Cited by 13 publications

References 76 publications

Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution

Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution

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

Responding to light signals: a comprehensive update on photomorphogenesis in cyanobacteria

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