Single-molecule imaging of the McdA ATPase reveals mechanistic details of carboxysome trafficking.

Abstract

Unlike eukaryotes, which facilitate intracellular transport and organization with linear motor proteins, bacteria use the ParA family of ATPases to spatially organize various cellular cargoes. For example, carboxysomes are bacterial microcompartments (BMCs) found in cyanobacteria and proteobacteria and account for approximately 30% of global carbon fixation. Recently, a ParA-like ATPase termed maintenance of carboxysome distribution A (McdA) and its partner protein, McdB, were revealed to position carboxysomes. Like many other ParA ATPases, McdA non-specifically interacts with DNA and associates with its cargo via its partner protein. It has been proposed that McdA positions carboxysomes using a Brownian-ratchet diffusion mechanism, akin to that of DNA segregation by ParA. Yet, there is little in vivo evidence supporting a Brownian ratchet-based positioning for carboxysomes. Using Halothiobacillus neapolitanus as a model organism, we measured how the ATPase activity of McdA governs carboxysome positioning on the nucleoid. Using single-molecule tracking, we found that the motion of McdA is consistent with its non-specific DNA-binding activity and that this interaction depends on ATP-binding. Moreover, we found based on deletion of function mutants that ATP hydrolysis is necessary for McdA release from the nucleoid. Finally, we found that mcdB deletion results in an enrichment of nucleoid-associated McdA molecules, consistent with the stimulation of ATP-hydrolysis and DNA release of McdA by McdB in vitro. Our results support the biochemical details of Brownian ratchet-based positioning by McdA, which constantly updates the contact points between the nucleoid and carboxysomes via its ATPase activity and upon stimulation by McdB. We anticipate that in addition to providing key details about BMC trafficking, our in vivo single-molecule experiments will provide a foundation towards understanding similarities and differences among the seven ParA-like ATPases encoded in H. neapolitanus which independently position cellular cargo.