Functional Characterization and Assembly Studies of Carboxysomes in \u3ci\u3eHalothiobacillus neapolitanus\u3c/i\u3e

Functional characterization and assembly studies of carboxysomes in Halothiobacillus neapolitanus were pursued in order to understand the roles of carboxysomes in the carbon metabolism in H. neapolitanus and the assembly of carboxysomes in vitro and in vivo. Previously, a low abundance H. neapolitanus carboxysomal protein, CsoSCA was identified as a novel carboxysomal shell-bound carbonic anhydrase. The enzyme is thought to dehydrate the cytosolic bicarbonate to CO2, the substrate of the RuBisCO packaged within the carboxysome [14, 21]. In this study, the carboxysomal shell was identified as a diffusion barrier for CO2 and O2 molecules. The shell-bound CsoSCA protein facilitates the diffusion of CO2 molecules into the carboxysomes and enhances the catalytic efficiency of the encapsulated RuBisCO. The discrimination between CO2 and O2 molecules by the shell makes the sequestered RuBisCO favor the carboxylation over the oxygenation reaction at low O2 concentration. A second substrate, ribulose 1,5-bisphosphate (RuBP), and the carboxylation reaction product, 3-phosphoglycerate (PGA), are negatively charged molecules and need to be transported into and out of the carboxysomes. Bioinformatics analysis of CsoS2 protein revealed that CsoS2 protein carries a positive charge in the cytoplasm. The protein may interact with these negatively charged molecules due to its high isoelectric point (approximately 9.2). Full length CsoS2 and an N-terminally truncated CsoS2 protein were expressed in E.coli to permit three dimensional structure determination. A sequential peptide affinity (SPA) tag was added at the C-terminus of CsoS2 protein to determine its location in the carboxysomes and trap the intermediates of in vitro carboxysome shell assembly. Finally, the cso operon was heterologously expressed in E. coli and some functional carboxysome-like structures were isolated and analyzed. The assembly of carboxysome-like structures in E. coli will guide the in vitro assembly of carboxysomes with recombinant carboxysomal proteins