(531b) Novel High-Throughput Protein Assembly Screens Reveal Essential Molecular Interactions in Bacterial Organelle Assembly

Spatial organization of processes within the cell is essential across all facets of life. This is true even in the simplest life forms, bacteria, where proteinaceous organelles called bacterial microcompartments (MCPs) aid in bacterial metabolism of niche carbon sources. MCPs are delimited by a protein shell that encases an enzymatic core responsible for carrying out enzymatic reactions that support proliferation. A multitude of protein-protein interactions (PPIs) between different proteins act in concert to assemble a complete MCP; however, detailed knowledge of the specific PPIs involved in MCP formation was previously limited by both the low throughput of existing MCP characterization techniques and an incomplete knowledge of which proteins were essential for MCP assembly.

Recently, we identified the key proteins required for formation of the 1,2-propanediol utilization (Pdu) MCP from the model pathogen Salmonella enterica serovar Typhimurium LT2. Namely, self-assembling proteins PduA and PduJ initiate assembly, PduB recruits enzymatic cargo, and PduN mediates bending of the shell that results in closed compartment structures. Importantly, in addition to the importance of these proteins in MCP assembly, we found that we could screen for the assembly competency of key components PduA, PduJ, and PduN with a phenotype detectable using either microscopy or flow cytometry, an example of which is shown, for PduN. These screens provide a novel method for assaying properties relevant to MCP formation with unprecedented throughput. We leveraged this phenotype-assembly link to generate assembly fitness landscapes for PduA, PduJ, and PduN, three of the four essential proteins involved in Pdu MCP assembly. Subsequently, we compared these results to conservation data and were able to thereby differentiate between residues in these key proteins that are essential for assembly and residues that must be conserved for some other orthogonal function. Because this high-throughput screen for MCP closure reports on the basic structural requirements for a functional MCP, its application space is as broad as MCP engineering, encompassing antibiotic discovery efforts and repurposing of MCPs for applications in metabolic engineering and drug delivery.