In Vitro Assembly of Diverse Bacterial Microcompartment Shell Architectures

Abstract

Bacterial Microcompartments (BMCs) are organelles composed of a selectively permeable protein shell that encapsulates enzymes involved in CO2 fixation (carboxysomes) or carbon catabolism (metabolosomes). Confinement of sequential reactions by the BMC shell presumably increases the efficiency of the pathway by reducing the crosstalk of metabolites, release of toxic intermediates, and accumulation of inhibitory products. Because BMCs are composed entirely of protein and self-assemble, they are an emerging platform for engineering nanoreactors and molecular scaffolds. However, testing designs for assembly and function through in vivo expression is labor-intensive and has limited the potential of BMCs in bioengineering. Here, we developed a new method for in vitro assembly of defined nanoscale BMC architectures: shells and nanotubes. By inserting a “protecting group”, a short ubiquitin-like modifier (SUMO) domain, self-assembly of shell proteins in vivo was thwarted, enabling preparation of concentrates of shell building blocks. Addition of the cognate protease removes the SUMO domain and subsequent mixing of the constituent shell proteins in vitro results in the self-assembly of three types of supramolecular architectures: a metabolosome shell, a carboxysome shell, and a BMC protein-based nanotube. We next applied our method to generate a metabolosome shell engineered with a hyper-basic luminal surface, allowing for the encapsulation of biotic or abiotic cargos functionalized with an acidic accessory group. This is the first demonstration of using charge complementarity to encapsulate diverse cargos in BMC shells. Collectively, our work provides a generally applicable method for in vitro assembly of natural and engineered BMC-based architectures.

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment

“…CcmM in the β-carboxysome appears as two isoforms, a 35-kDa truncated CcmM35 and a full-length 58-kDa CcmM58 (Long et al, 2007;Long et al, 2010;Long et al, 2011). CcmM35 contains three Rubisco small subunit-like (SSU) domains that interact with Rubisco (Hagen et al, 2018b;Wang et al, 2019), whereas CcmM58 has an N-terminal γ-CA-like domain in addition to the SSU domains and recruits CcaA to the shell. RbcX is recognized as a chaperonin-like protein for Rubisco assembly (Emlyn-Jones et al, 2006;Saschenbrecker et al, 2007;Occhialini et al, 2016); it has been recently revealed to serve as one component of the carboxysome and play roles in mediating carboxysome assembly and subcellular distribution (Huang et al, 2019).…”

Occurrence and stability of hetero-hexamer associations formed by β-carboxysome CcmK shell components

“…Yet, the formation of hexamers combining CsoS1 and CcmK subunits should not be excluded. Indeed, considering that the YFP domain attached to CsoS1 monomers is considerably bulkier than SUMO domains exploited by the same authors to prevent BMC-H component assembly in vitro [45], the resulting CsoS1-YFP hexamers would be expected to be assembly-incompetent. If these arguments hold true, fluorescence puncta observed in vivo should result from the integration on shells of CsoS1-YFP/CcmK hexamers.…”

Functionalization of Bacterial Microcompartment Shell Proteins With Covalently Attached Heme

“…We therefore examined if shell-cytochrome H 5 retained the capability to form the reported compartment assemblies when combined with wildtype Haliangium ochraceum (HO) BMC shell proteins (Sutter et al, 2017). We performed a recently-described in vitro BMC assembly (Hagen A. R. et al, 2018), where three HO shell proteins (BMC-H, BMC-P [pentamer], and BMC-T [trimer]) are used to assemble a spherical, hollow protein compartment (Sutter et al, 2017). When H 5 shell-cytochrome was substituted for native BMC-H in these assemblies, we found it associated with native BMC-P and BMC-T proteins, although the strength of the association was greatly enhanced by including wild-type BMC-H in the reactions (Supplementary Figure 8A).…”

“…When H 5 shell-cytochrome was substituted for native BMC-H in these assemblies, we found it associated with native BMC-P and BMC-T proteins, although the strength of the association was greatly enhanced by including wild-type BMC-H in the reactions (Supplementary Figure 8A). Assembly reactions containing only H 5 with native BMC-P and BMC-T, contained higher-order structures resembling the size and shape of HO compartments (Figure 4D), although these structures appeared less robust than the published compartments using WT BMC-H proteins (Sutter et al, 2017;Hagen A. R. et al, 2018;Ferlez et al, 2019). Indeed, H 5 -based assemblies frequently appeared as compartments with "broken shells" or as clusters of partially-assembled shell fragments (Supplementary Figure 8B).…”