Halothiobacillus neapolitanus Carboxysomes Sequester Heterologous and Chimeric RubisCO Species

Abstract

BackgroundThe carboxysome is a bacterial microcompartment that consists of a polyhedral protein shell filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme that catalyzes the first step of CO2 fixation via the Calvin-Benson-Bassham cycle.Methodology/Principal FindingsTo analyze the role of RubisCO in carboxysome biogenesis in vivo we have created a series of Halothiobacillus neapolitanus RubisCO mutants. We identified the large subunit of the enzyme as an important determinant for its sequestration into α-carboxysomes and found that the carboxysomes of H. neapolitanus readily incorporate chimeric and heterologous RubisCO species. Intriguingly, a mutant lacking carboxysomal RubisCO assembles empty carboxysome shells of apparently normal shape and composition.Conclusions/SignificanceThese results indicate that carboxysome shell architecture is not determined by the enzyme they normally sequester. Our study provides, for the first time, clear evidence that carboxysome contents can be manipulated and suggests future nanotechnological applications that are based upon engineered protein microcompartments.

Addressing microbial organelles: A short peptide directs enzymes to the interior

“…However, that same study provided strong evidence for specific interactions between payload and shell proteins by identifying the large subunit of the enzyme as the factor that determines whether a particular RubisCO species can be packaged into the carboxysome. Contrary to the N-terminal peptide that is identified by Fan et al (5) on some enzymes of the Pdu BMC, sequence comparisons between carboxysomal and noncarboxysomal large subunits of RubisCO from a variety of autotrophic bacteria did not reveal an obvious packaging signal (12 identified the short C-terminal extension on those DyP and Flp homologues that are packaged into the polyhedral encapsulin nanocompartment as necessary and sufficient for targeting. Previous reports had also noted that two subunits (PduD and PduE) of the Pdu BMC-associated diol dehydratase contain N-terminal segments that are not required for enzymatic activity but were speculated to play a role in BMC assembly (14).…”

“…The other model posits the existence of a preassembled (partial) shell that directs the packaging of RubisCO into the BMC interior (11). Clearly, carboxysome shell formation is not dependent on the presence of RubisCO, as was shown for a Halothiobacillus neapolitanus RubisCO deletion mutant that forms empty BMC shells of otherwise normal shape and composition (12). However, that same study provided strong evidence for specific interactions between payload and shell proteins by identifying the large subunit of the enzyme as the factor that determines whether a particular RubisCO species can be packaged into the carboxysome.…”

Short N-terminal sequences package proteins into bacterial microcompartments

“…With regard to biotechnology, it has been suggested that bacterial MCPs might find application as specialized reaction chambers for the production of industrial chemicals (13,(43)(44)(45)(46)(47). A major challenge in engineering pathways for the production of biofuels, green chemicals, and pharmaceuticals is balancing enzyme activities to limit the accumulation of pathway intermediates, particularly those that may be toxic to host cells.…”

Bacterial Microcompartments

“…Some mutants, selected from a population of chemically mutagenized cyanobacteria that display a high CO 2 requiring (hcr) phenotype, lack or form aberrantly shaped carboxysomes, suggesting that functional carboxysomes are needed for efficient growth at ambient CO 2 levels (48-50). After the carboxysome operon (cso operon) was identified, targeted mutagenesis of individual genes could be correlated with changes in carboxysome appearance or their disappearance, and the hcr phenotype (6,12,22,24,42).…”