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Across bacteria, protein-based organelles called bacterial microcompartments (BMCs) encapsulate key enzymes to regulate their activities. The model BMC is the carboxysome that encapsulates enzymes for CO2fixation to increase efficiency and is found in many autotrophic bacteria, such as cyanobacteria. Despite their importance in the global carbon cycle, little is known about how carboxysomes are spatially regulated. We recently identified the two-factor system required for the maintenance of carboxysome distribution (McdAB). McdA drives the equal spacing of carboxysomes via interactions with McdB, which associates with carboxysomes. McdA is a ParA/MinD ATPase, a protein family well studied in positioning diverse cellular structures in bacteria. However, the adaptor proteins like McdB that connect these ATPases to their cargos are extremely diverse. In fact, McdB represents a completely unstudied class of proteins. Despite the diversity, many adaptor proteins undergo phase separation, but functional roles remain unclear. Here, we define the domain architecture of McdB from the model cyanobacteriumSynechococcus elongatusPCC 7942, and dissect its mode of biomolecular condensate formation. We identify an N-terminal intrinsically disordered region (IDR) that modulates condensate solubility, a central coiled-coil dimerizing domain that drives condensate formation, and a C-terminal domain that trimerizes McdB dimers and provides increased valency for condensate formation. We then identify critical basic residues in the IDR, which we mutate to glutamines to solubilize condensates. Finally, we find that a condensate-defective mutant of McdB has altered association with carboxysomes and influences carboxysome enzyme content. The results have broad implications for understanding spatial organization of BMCs and the molecular grammar of protein condensates.
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Positioning the Model Bacterial Organelle, the Carboxysome
Bacterial microcompartments (BMCs) confine a diverse array of metabolic reactions within a selectively permeable protein shell, allowing for specialized biochemistry that would be less efficient or altogether impossible without compartmentalization. BMCs play critical roles in carbon fixation, carbon source utilization, and pathogenesis. Despite their prevalence and importance in bacterial metabolism, little is known about BMC “homeostasis,” a term we use here to encompass BMC assembly, composition, size, copy-number, maintenance, turnover, positioning, and ultimately, function in the cell. The carbon-fixing carboxysome is one of the most well-studied BMCs with regard to mechanisms of self-assembly and subcellular organization. In this minireview, we focus on the only known BMC positioning system to date—the maintenance of carboxysome distribution (Mcd) system, which spatially organizes carboxysomes. We describe the two-component McdAB system and its proposed diffusion-ratchet mechanism for carboxysome positioning. We then discuss the prevalence of McdAB systems among carboxysome-containing bacteria and highlight recent evidence suggesting how liquid-liquid phase separation (LLPS) may play critical roles in carboxysome homeostasis. We end with an outline of future work on the carboxysome distribution system and a perspective on how other BMCs may be spatially regulated. We anticipate that a deeper understanding of BMC organization, including nontraditional homeostasis mechanisms involving LLPS and ATP-driven organization, is on the horizon.
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- PAR ID:
- 10252809
- Editor(s):
- Garsin, Danielle A.
- Date Published:
- Journal Name:
- mBio
- ISSN:
- 2150-7511
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Goley, Erin (Ed.)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.more » « less
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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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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/mBio.02519-19
