All cyanobacteria and many chemoautotrophic bacteria contain polyhedral inclusion bodies called carboxysomes. These microcompartments, which consist entirely of protein and are filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), are thought to be part of a carbon concentrating mechanism (CCM) that serves to enhance the CO2 fixation efficiency in organisms that contain them. Although the molecular mechanism by which the carboxysome provides a catalytic enhancement for RubisCO is not certain, it has long been a tenet of the CCM hypothesis that a carboxysomal carbonic anhydrase (CA) rapidly converts cytoplasmic HCO3 - into CO2, the substrate for RubisCO. Recent work from this laboratory established that a protein component of the carboxysomal shell is a novel CA that we have term EPSCOR-CA. Its localization in the carboxysomal shell suggests that carboxysomes function via a mechanism that is more complex than that predicted by the standard CCM model and might indicate an expanded role for the microcompartments in autotrophic carbon assimilation. Biochemical and molecular biological approaches will be combined to elucidate structural features of the carboxysome that form the basis for its function, further characterize the newly discovered carboxysomal CA biochemically and identify its interactions with other proteins in the shell. The obligate chemoautotrophic bacterium Halothiobacillus neapolitanus will serve as a model system for these studies since a) homogeneous preparations of carboxysomes can be isolated in large quantities from the organism, b) its carboxysome operon has been cloned, characterized and expressed in Escherichia coli, c) a substantial number of carboxysome mutants have been created in H. neapolitanus, d) the bacterium can be genetically manipulated, e) gene clusters homologous to the putative operon encoding H. neapolitanus carboxysomes have been observed in both cyanobacteria and other species of chemoautotrophs.
Broader Impacts Given the large contribution of carboxysome-containing organisms towards CO2 fixation and the impact of their metabolism on the global carbon cycle, one broader implication of the work proposed will be the elucidation of one of the most basic and central biochemical processes that control the carbon cycle and influence weather and climate. The PI will be directly involved in conducting key experiments and will be the mentor for graduate and undergraduate student research in various aspects of the research. Students participating in these projects will gain experience in biochemical and molecular biological research techniques.
