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Abstract Carboxysomes are protein-based organelles that are essential for allowing cyanobacteria to fix CO2. Previously, we identified a two-component system, McdAB, responsible for equidistantly positioning carboxysomes in the model cyanobacterium Synechococcus elongatus PCC 7942 (MacCready JS, Hakim P, Young EJ, Hu L, Liu J, Osteryoung KW, Vecchiarelli AG, Ducat DC. 2018. Protein gradients on the nucleoid position the carbon-fixing organelles of cyanobacteria. eLife 7:pii:e39723). McdA, a ParA-type ATPase, nonspecifically binds the nucleoid in the presence of ATP. McdB, a novel factor that directly binds carboxysomes, displaces McdA from the nucleoid. Removal of McdA from the nucleoid in the vicinity of carboxysomes by McdB causes a global break in McdA symmetry, and carboxysome motion occurs via a Brownian-ratchet-based mechanism toward the highest concentration of McdA. Despite the importance for cyanobacteria to properly position their carboxysomes, whether the McdAB system is widespread among cyanobacteria remains an open question. Here, we show that the McdAB system is widespread among β-cyanobacteria, often clustering with carboxysome-related components, and is absent in α-cyanobacteria. Moreover, we show that two distinct McdAB systems exist in β-cyanobacteria, with Type 2 systems being the most ancestral and abundant, and Type 1 systems, like that of S. elongatus, possibly being acquired more recently. Lastly, all McdB proteins share the sequence signatures of a protein capable of undergoing liquid–liquid phase separation. Indeed, we find that representatives of both McdB types undergo liquid–liquid phase separation in vitro, the first example of a ParA-type ATPase partner protein to exhibit this behavior. Our results have broader implications for understanding carboxysome evolution, biogenesis, homeostasis, and positioning in cyanobacteria.
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Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution
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. Maintenance of carboxysome distribution protein A (McdA), a partition protein A (ParA)-like ATPase, forms a dynamic oscillating gradient on the nucleoid in response to the carboxysome-localized Maintenance of carboxysome distribution protein B (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.
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- PAR ID:
- 10298477
- Editor(s):
- Goley, Erin
- Date Published:
- Journal Name:
- Molecular Biology of the Cell
- Volume:
- 32
- Issue:
- 20
- ISSN:
- 1059-1524
- Page Range / eLocation ID:
- ar11
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Carboxysomes are protein‐based organelles essential for carbon fixation in cyanobacteria and proteobacteria. Previously, we showed that the cyanobacterial nucleoid is used to equally space out β‐carboxysomes across cell lengths by a two‐component system (McdAB) in the model cyanobacteriumSynechococcus elongatusPCC 7942. More recently, we found that McdAB systems are widespread among β‐cyanobacteria, which possess β‐carboxysomes, but are absent in α‐cyanobacteria, which possess structurally and phyletically distinct α‐carboxysomes. Cyanobacterial α‐carboxysomes are thought to have arisen in proteobacteria and then horizontally transferred into cyanobacteria, which suggests that α‐carboxysomes in proteobacteria may also lack the McdAB system. Here, using the model chemoautotrophic proteobacteriumHalothiobacillus neapolitanus, we show that a McdAB system distinct from that of β‐cyanobacteria operates to position α‐carboxysomes across cell lengths. We further show that this system is widespread among α‐carboxysome‐containing proteobacteria and that cyanobacteria likely inherited an α‐carboxysome operon from a proteobacterium lacking themcdABlocus. These results demonstrate that McdAB is a cross‐phylum two‐component system necessary for positioning both α‐ and β‐carboxysomes. The findings have further implications for understanding the positioning of other protein‐based bacterial organelles involved in diverse metabolic processes. Plain language summaryCyanobacteria are well known to fix atmospheric CO2into sugars using the enzyme Rubisco. Less appreciated are the carbon‐fixing abilities of proteobacteria with diverse metabolisms. Bacterial Rubisco is housed within organelles called carboxysomes that increase enzymatic efficiency. Here we show that proteobacterial carboxysomes are distributed in the cell by two proteins, McdA and McdB. McdA on the nucleoid interacts with McdB on carboxysomes to equidistantly space carboxysomes from one another, ensuring metabolic homeostasis and a proper inheritance of carboxysomes following cell division. This study illuminates how widespread carboxysome positioning systems are among diverse bacteria. Carboxysomes significantly contribute to global carbon fixation; therefore, understanding the spatial organization mechanism shared across the bacterial world is of great interest.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1091/mbc.E21-03-0151
