Modularity of a carbon-fixing protein organelle

Abstract

Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO(2)-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO(2). Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO(2) in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures.

Fig. 1.

Physical and genetic organization of the carboxysome. (A) Molecular surface representation of a partial model of the carboxysome showing RuBisCO (green), carbon anhydrase (orange), CsoS1ABC (blue), and CsoS4AB (red). (B) Schematic of the carbon concentrating mechanism adapted from Kaplan and Reinhold (14). (C) Genomic organization of the carboxysome operon in H. neapolitanus. Colors match the structural model in A.

Fig. 2.

Electron microscopy of carboxysomes in vivo. (A) Expression of HnCB at 0 μM IPTG. (B) Same as A but at 50 μM IPTG. (Inset) Icosahedral structures. (C) Same as A, but at 200 μM IPTG. (Inset) Inclusion body. (D) Appearance of filaments (arrow) under high induction conditions. (Scale bars: 500 nm.)

Fig. 3.

Carboxysomes assemble and are functional in vivo. (A–D) Microscopy images of cells expressing fluorescently labeled carboxysome components in the presence and absence of pHnCB. (Scale bar: 2.5 μm.) Controls are shown in Fig. S2. The arrow in B highlights a likely CB particle. (E) Schematic of metabolic complementation strategy. (F) Representative growth curves of WT, pPRK, and pPRK/pHnCB strains showing complementation via carbon fixation.

Fig. 4.

Characterization of purified carboxysomes. (A) Representative electron microscopy image of purified carboxysomes expressed using the pHnCB plasmid. The arrow indicates a low-density lumen area, the asterisk indicates a shell defect, and the box highlights the RuBisCO octomer. (Scale bar: 100 nm.) (B) Same as A, but of purified carboxysomes expressed using the pHnCBS1D plasmid. (C) SDS/PAGE gel of purified carboxysomes from B with annotated proteomics data (arrows). (D) Carbon fixation activity of preparations imaged in A and B as a function of d-ribulose 1,5-bisphosphate concentration. Error bars represent SD.