Bacterial microcompartments (MCPs) are protein-based bacterial organelles that enclose metabolic pathways within a protein shell. MCPs help improve metabolic efficiency, providing an advantage for microbes in resource-limited environments. Metabolic engineers have attempted to repurpose MCPs to improve yields of industrially relevant pathways of interest. However, the mechanisms for MCP assembly and function remain unclear. Here, we describe the role of three critical protein components of the MCP shells from <i>Salmonella</i>, providing insight into the assembly mechanism of a model MCP. Specifically, we demonstrate the protein-protein interactions necessary for building the MCP shell and then linking the shell to the enzymatic core. First, we demonstrate that two proteins, PduA and PduJ, have a unique propensity towards self-assembly and that this property allows these two proteins to stimulate and drive MCP biogenesis. We also developed a novel, high-throughput method that enables rapid analysis of MCP shell protein variants, which yielded exquisite detail on the role of these proteins in MCP shell assembly and the specific molecular interactions necessary for this function. These results provide the first demonstration of the essential yet partially redundant role that PduA and PduJ play in MCP shell formation. Furthermore, we demonstrate that a third MCP shell protein, PduB, is responsible for linking the MCP shell to the enzymatic core via a conserved protein-protein interaction mechanism. Our work demonstrates that MCP shells lacking PduB are smaller and empty, lacking the encapsulated enzymatic core, implying that PduB is important for enzyme encapsulation but not MCP shell assembly. Overall, these findings support a mechanism where PduA and PduJ drive MCP shell assembly. PduB then bridges the gap between the MCP shell and enzymatic core via interactions with PduA and PduJ. These are critical findings for those seeking to re-engineer MCPs for non-native functions. An understanding of the essential components of MCPs, as well as their function in the MCP assembly process, will be necessary to repurpose or design compartments with novel functionalities.

Interactions between three proteins are essential for the assembly and function of bacterial microcompartments

ACS Spring 2022

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