Probing α-carboxysome biogenesis and modularity with bacterial microcompartment counterparts
Abstract
Bacterial microcompartments (BMCs) are protein-based organelles in prokaryotes that optimize metabolic pathways by confining specific enzymatic reactions within selectively permeable shells, playing critical roles in processes such as CO2 fixation, pathogenesis, and ecological adaptation. Among them, carboxysomes (CBs) are paradigmatic BMCs found in cyanobacteria and some chemoautotrophs, in which they encapsulate Rubisco and carbonic anhydrase to enhance CO2 fixation. However, the molecular principles underlying α- carboxysome (α-CB) self-assembly, biogenesis, organisation, and intracellular diffusive dynamics remain poorly understood. In this work, I systematically dissected the roles of individual building blocks in α-CB assembly and organisation in Halothiobacillus neapolitanus. By constructing a series of gene-deletion strains and combining growth assays, electron microscopy, live-cell fluorescence imaging, and immunoblotting, I revealed functional diversification among CsoS1 and CsoS4 homologs and demonstrated that cargo proteins are dispensable for shell biogenesis but required for efficient encapsulation. To further dissect the de novo assembly pathway, I developed an inducible α-CB expression system combined with dual-fluorescence labelling, enabling spatiotemporally controlled induction and real-time visualization of α-CB biogenesis. I showed that α-CBs assemble predominantly via a “concomitant assembly” pathway, with a smaller fraction of “shell-first” assembly. Moreover, assembled α-CBs exhibited constrained, non-Brownian motion, and their proper localization and dynamic behaviour depended on the McdAB positioning system. Building upon these insights, I explored BMC modularity and cross-compatibility by coexpressing the native 1,2-propanediol utilisation (Pdu) BMC with synthetic α-CBs in Salmonella enterica LT2. Shell proteins from the two systems interchanged to form hybrid BMCs, while the internal cargo proteins remained largely segregated. These hybrid compartments displayed altered mobility and enzymatic activity. Collectively, these findings provide insights into the assembly, organisation, and dynamic behaviour of α-CBs, highlight the structural modularity and evolutionary adaptability of BMC systems, and establish a versatile platform for the rational design and engineering of synthetic organelles for applications in synthetic biology and metabolic engineering.