Unlike eukaryotes, which utilize linear motor proteins to facilitate intracellular transport and organization, bacteria use the ParAB family of proteins to spatially organize protein cargoes involved in diverse aspects of bacterial physiology. For example, carboxysomes are Bacterial Microcompartments (BMCs) crucial for carbon fixation in cyanobacteria and proteobacteria. Recently, a ParA-like ATPase, termed Maintenance of carboxysome distribution A (McdA) and its partner protein, McdB, were revealed to position carboxysomes. Like many ParAB systems, McdA non-specifically interacts with DNA and uses McdB to associate with carboxysomes. Here, we use the α-carboxysome/McdAB system in Halothiobacillus neapolitanus as a model to understand the mechanism for high-copy cargo trafficking in bacteria. Towards this goal, we implemented single-molecule fluorescence imaging in live H. neapolitanus to track the motions of fluorescently labeled McdA and McdB. We used nonparametric Bayesian inference to analyze the single-molecule trajectories and found that McdA and McdB exhibit heterogeneous dynamics consistent with their distinct roles in nucleoid and carboxysome association, respectively. Furthermore, we introduced point mutations in the ATP-binding pocket of McdA to assign these biophysical mobility states to the biochemical states in its ATPase cycle. Our results suggest a short-lived association between McdA and McdB, consistent with the high ATPase activity of β-McdA revealed in vitro. Moreover, McdB exhibits two mobility states: one highly populated slow state and a sparse fast state, indicative of its carboxysome-bound and unbound states, respectively. Our single-molecule investigations of the in vivo McdAB system dynamics will provide the biochemical details needed to understand how carboxysomes and other BMCs are positioned in the cell.

Probing a positioning system for high-copy cargoes at the single-molecule level

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.