Self-assembling shell proteins PduA and PduJ have essential and redundant roles in bacterial microcompartment assembly

Protein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts. ### Competing Interest Statement The authors have declared no competing interest. * MCP : Bacterial microcompartments BMC : bacterial microcompartment BMV : bacterial microcompartment vertex BMC-H : bacterial microcompartment hexamer BMC-P : bacterial microcompartment pentamer BMC-T : bacterial microcompartment trimer 1,2-PD : 1,2-propanediol Pdu : 1,2-propanediol utilization ssD : signal sequence from PduD ssP : signal sequence from PduP TEM : transmission electron microscopy