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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. 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. 

Cyanobacteria are tiny organisms that can harness the energy of the sun to power their cells. Many of the tools required for this complex photosynthetic process are packaged into small compartments inside the cell, the carboxysomes. In Synechococcus elongatus, a cyanobacterium that is shaped like a rod, the carboxysomes are positioned at regular intervals along the length of the cell. This ensures that, when the bacterium splits itself in half to reproduce, both daughter cells have the same number of carboxysomes. Researchers know that, in S. elongatus, a protein called McdA can oscillate from one end of the cell to the other. This protein is responsible for the carboxysomes being in the right place, and some scientists believe that it helps to create an internal skeleton that anchors and drags the compartments into position. Here, MacCready et al. propose another mechanism and, by combining various approaches, identify a new partner for McdA. This protein, called McdB, is present on the carboxysomes. McdB also binds to McdA, which itself attaches to the nucleoid – the region in the cell that contains the DNA. McdB forces McdA to release itself from DNA, causing the protein to reposition itself along the nucleoid. Because McdB attaches to McdA, the carboxysomes then follow suit, constantly seeking the highest concentrations of McdA bound to nearby DNA. Instead of relying on a cellular skeleton, these two proteins can organize themselves on their own using the nucleoid as a scaffold; in turn, they distribute carboxysomes evenly along the length of a cell. Plants also obtain their energy from the sun via photosynthesis, but they do not carry carboxysomes. Scientists have tried to introduce these compartments inside plant cells, hoping that it could generate crops with higher yields. Knowing how carboxysomes are organized so they can be passed down from one generation to the next could be important for these experiments. 

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.