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.

蛋白质自组装是一种常见且必不可少的生物学现象，细菌微区隔提供了一个有前途的模型系统来研究这一过程。细菌微区室是大型的，基于蛋白质的细胞器，其天然执行对于蓝细菌中的碳固定和肠细菌的存活很重要的过程。这些结构由于其作为纳米生物反应器或药物输送工具的潜在用途而越来越受到生物工程师的欢迎。但是，对这些细菌微区室的组装机制的了解有限，阻碍了将它们重新用于非天然功能的努力。在这里，我们全面调查参与沙门氏菌1,2-丙二醇利用细菌微隔室组装的蛋白质。血清型鼠伤寒LT2，是研究最广泛的微隔室系统之一。我们首先证明了两种壳蛋白，PduA和PduJ，在过表达时具有很高的自组装倾向，并且我们提供了一种自组装定量的新方法。使用基因组敲除和敲入，我们系统地显示这两种蛋白质在细菌微区室装配中起着至关重要的作用，而其他壳蛋白无法补偿这些作用。必须存在两种蛋白质PduA和PduJ中的至少一种，才能组装细菌微隔室壳。我们还证明了这些蛋白质的装配缺陷型变体无法挽救微隔室的形成，从而突出了这种装配特性的重要性。