K-077. Studies of Carboxysomes Assembly in vitro and in vivo

Z. Dou1, S. Heinhorst1, J. M. Shively1,2, G. C. Cannon1;
1The Univ. of Southern Mississippi, Hattiesburg, MS, 2Clemson Univ., Clemson, SC.

Carboxysomes are polyhedral microcompartments with a diameter of approximately 100 nm. They exist in many autotrophic bacteria and contain multiple copies of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) encapsulated in a thin proteinaceous shell. Sequestration of RuBisCO into carboxysomes seems to enhance the catalytic efficiency of this rather poor biocatalyst by providing it with its substrate, CO2, through the action of a carboxysome shell-associated carbonic anhydrase. Although great strides have been made in advancing our understanding of carboxysome structure and function, very little is known about the pathway that leads to the assembly of the microcompartment. To develop a tool that can aid in elucidating carboxysome assembly, we have fused the gene for the large carboxysomal shell protein, CsoS2, from Halothiobacillus neapolitanus with an affinity tag and replaced the wild type csoS2 gene with the tagged mutant version in the genome of this bacterium. In the resulting mutant, tagged CsoS2 protein was expressed and incorporated into intact carboxysomes. Purified tagged carboxysomes were disrupted chemically and subsequently dialyzed to permit re-assembly to take place. In vitro assembled, tagged carboxysome protein complexes were trapped on an affinity column and their polypeptide composition analyzed by denaturing gel electrophoresis. Preliminary evidence suggests that the trapped complexes contain all shell proteins but less RuBisCO than native carboxysomes. Transmission electron microscopy and dynamic light scattering analysis revealed that the trapped protein complexes consist of particles with sizes and shapes similar to those of intact carboxysomes. Assembled particles were also obtained when the entire H. neapolitanus cso operon was expressed in E.coli. The irregular shapes and sizes of the protein assemblies, however, suggested that the microcompartment shells were frequently misassembled in this bacterium.