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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. 

Abstract Curli are functional amyloids present on the outer membrane ofE. coli. CsgF is required for the proper assembly of curli. Here, we found that the CsgF phase separates in vitro and that the ability of CsgF variants to phase-separate is tightly correlated with CsgF function during curli biogenesis. Substitution of phenylalanine residues in the CsgF N-terminus both reduced the propensity of CsgF to phase-separate and impaired curli assembly. Exogenous addition of purified CsgF complementedcsgF ⁻cells. This exogenous addition assay was used to assess the ability of CsgF variants to complementcsgF ^‒cells. CsgF on the cell surface modulated the secretion of CsgA, the curli major subunit, to the cell surface. We also found that the CsgB nucleator protein can form SDS-insoluble aggregates within the dynamic CsgF condensate. We propose that these multicomponent CsgF-B condensates form a nucleation-competent complex that templates CsgA amyloid formation on the cell surface.