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ABSTRACT Themaintenance ofcarboxysomedistribution (Mcd) system comprises the proteins McdA and McdB, which spatially organize carboxysomes to promote efficient carbon fixation and ensure their equal inheritance during cell division. McdA, a member of the ParA/MinD family of ATPases, forms dynamic gradients on the nucleoid that position McdB-bound carboxysomes. McdB belongs to a widespread but poorly characterized class of ParA/MinD partner proteins, and the molecular basis of its interaction with McdA remains unclear. Here, we demonstrate that the N-terminal 20 residues ofH. neapolitanusMcdB are both necessary and sufficient for interaction with McdA. Within this region, we identify three lysine residues whose individual substitution modulates McdA binding and leads to distinct carboxysome organization phenotypes. Notably, lysine 7 (K7) is critical for McdA interaction: substitutions at this site result in the formation of a single carboxysome aggregate positioned at mid-nucleoid. This phenotype contrasts with that of an McdB deletion, in which carboxysome aggregates lose their nucleoid association and become sequestered at the cell poles. These findings suggest that weakened McdA–McdB interactions are sufficient to maintain carboxysome aggregates on the nucleoid but inadequate for partitioning individual carboxysomes across it. We propose that, within the ParA/MinD family of ATPases, cargo positioning and partitioning are mechanistically separable: weak interactions with the cognate partner can mediate positioning, whereas effective partitioning requires stronger interactions capable of overcoming cargo self-association forces. 

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.