Tuning the interaction of a ParA-type ATPase with its partner separates bacterial organelle positioning from partitioning.

The maintenance of carboxysome distribution (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 of H. neapolitanus McdB 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.