Analysis and Reconstitution of a Bacterial CO2-Concentrating Mechanism

Ribulose Bisphosphate Carboxylase/Oxygenase (Rubisco) is the central enzyme of the Calvin-Benson-Bassham (CBB) cycle and the most abundant enzyme on Earth. Nearly all carbon enters the biosphere via Rubisco carboxylation, yet no Rubisco has a maximum carboxylation rate above 15 /s and all Rubiscos catalyze a competing oxygenation of their five-carbon substrate. Evolution seems to have overcome this problem by selecting for CO2 concentrating mechanisms (CCMs) that place Rubisco in compartments where CO2 is highly concentrated. Distinct families of CCMs are found among bacteria, algae and plants, but they all function by elevating CO2 to promote carboxylation and inhibit oxygenation by Rubisco./nCyanobacteria are the ancestors of all green photosynthetic lineages, including plant chloroplasts, and have a CCM that requires a large (> 200 MDa) proteinaceous organelle called the carboxysome. Carboxysomes are composed of 10,000 proteins and encapsulate 2000 Rubisco active sites. Although present-day atmosphere is rich in O2 (21%) and CO2-poor (0.04%), cyanobacteria use the CCM to grow robustly in air. Mutations to the CCM abrogate air growth and are only rescued by markedly elevated CO2. By contrast, few plant species have CCMs. Land plants with CCMs, such as maize, are particularly productive, but no plant CCMs resemble those in bacteria. These considerations raise the prospect that engineering plants to express bacterial CCMs might improve their growth. Motivated by these questions, I use complementary experimental, informatic and modeling approaches to study the evolution and function of the bacterial CCM./nI first perform a meta-analysis of the kinetic properties of 300 distinct Rubiscos and show that carboxylation and oxygenation are inextricably linked: increasing carboxylation efficiency entails an equal increase to oxygenation efficiency, suggesting that oxygenation cannot be avoided by mutating Rubisco itself. Second, by mathematical modeling I show that the 20 known components of the bacterial CCM are sufficient to concentrate CO2 and promote fast carboxylation in silico. As pH has wide-ranging effects on inorganic carbon chemistry, I also demonstrate that pH must be considered for CCM models to produce plausible results matching physiological measurements of cyanobacteria./nTo confirm that the CCM is formed of a small number of genes, I designed a high-throughput genetic screen in the chemotrophic bacterium H. neapolitanus. This screen identified 20 genes in 3 operons directly involved in the H. neapolitanus CCM, including a novel class of inorganic carbon transporters I term "DABs." To test whether these genes are sufficient for CCM function, I developed a Rubisco-dependent E. coli strain, CCMB1, whose growth reports on CCM function in vivo. CCMB1 only grows in minimal media when Rubisco is expressed, and only under elevated CO2 (> 20x ambient). I found that expression of the H. neapolitanus CCM genes permits CCMB1 growth in ambient air, thereby establishing a facile system for testing the contributions of individual genes to the functioning of the bacterial CCM.

Advisor: Savage, David F