Connecting a Bacterial Organelle to Its Positioning System

Abstract

In bacteria, protein-based organelles called bacterial microcompartments (BMCs) are a widespread strategy for subcellular organization. BMCs encapsulate enzymes within a selectively permeable protein shell to drive unfavorable reactions and sequester intermediates. Through this mechanism, BMCs regulate a diversity of metabolisms across bacteria, including human pathogens. The study of BMC biology is therefore important for understanding aspects of bacterial cell biology and metabolism, its consequences to human health, and the development of in vivo encapsulation biotechnologies. The model BMC is the carboxysome, which helps drive carbon-fixation in cyanobacteria and some chemoautotrophs. Carboxysomes are a paradigm for understanding fundamental aspects of BMC biology, including their spatial organization in the cell. Carboxysome spatial organization results in their uniform distribution in the cell, and disruptions to this organization cause carboxysome aggregation, decreased carbon-fixation, and slower cell growth. Thus, understanding how cells spatially distribute carboxysomes is necessary for our understanding and application of functional and efficient BMCs. Our lab recently identified the two-protein system which spatially organizes carboxysomes, named the maintenance of carboxysome distribution (Mcd) system, consisting of the proteins McdA and McdB. McdA is the positioning ATPase that drives active carboxysome distribution, but does not interact directly with carboxysomes. Instead, McdB associates with carboxysomes, acting as an adaptor to link carboxysomes to the positioning ATPase, McdA. We now know that McdAB systems are widespread in BMC-containing bacteria, yet how different McdB proteins associate with their respective BMCs remains to be determined. McdB thus represents a novel, widespread, but unstudied class of proteins. To address this gap, my thesis work began with a biochemical characterization of McdB proteins from several carboxysome-containing bacteria. Intriguingly, all purified McdB proteins formed condensates in vitro. Condensates are the result of molecules undergoing a density transition to form two coexisting phases: a dense, solvent-poor condensate phase and a dilute soluble phase. Condensate formation has now been implicated in a diversity of biological processes across eukaryotes and prokaryotes. My thesis work addresses two important questions critical to our understanding of the mechanisms governing the spatial organization of carboxysomes, and BMCs in general: (1) What is the functional consequence of McdB condensate formation in the spatial organization of carboxysomes and (2) What are the molecular features of McdB proteins that specify their interactions with a specific BMC-type. To answer these questions, I first dissected the condensate formation and oligomerization activities of McdB, and provided evidence suggesting that McdB condensation plays a role in its association with carboxysomes. Second, I identified C-terminal motifs containing an invariant tryptophan necessary for McdB proteins to associate with their respective carboxysomes. Substituting this tryptophan with other aromatic residues reveals a gradient of carboxysome colocalization by McdB, and a corresponding gradient in carboxysome positioning activity in vivo. Intriguingly, these activity gradients correlated with the ability of McdB to form condensates in vitro. Together, my thesis reveals a common mechanism underlying adaptor protein binding for carboxysomes, and possibly for other BMCs that use McdAB-like systems for their active positioning. My findings advance our understanding of subcellular organization in bacteria and, more specifically, the mechanisms governing the spatial regulation of protein-based organelles across the bacterial world.