Molecular Design Principles of Bacterial Carbon Fixation: Investigations into Carboxysome Assembly and Permeability

All life on Earth relies on biological carbon fixation, the process by which organisms convert inorganic carbon, primarily in the form of carbon dioxide (CO2), into longer chain compounds to fuel cellular processes. To enhance the efficiency of CO2 fixation, certain types of bacteria, specifically cyanobacteria and some proteobacteria, evolved specialized proteinaceous microcompartments called carboxysomes. Carboxysomes encapsulate the enzymes carbonic anhydrase and Rubisco inside a polyhedral layer of shell proteins. This molecular architecture serves to concentrate CO2 around Rubisco, allowing it to operate at its maximum catalytic rate./nCorrect carboxysome assembly is essential to the survival of the organism in CO2 concentrations found in today’s atmosphere (~0.04%). In the ⍺-carboxysome lineage, the disordered scaffold protein CsoS2 links Rubisco and shell proteins, and is absolutely required for carboxysome formation and cell growth at ambient CO2 levels. This work examines how the sequence of CsoS2 scales from a disordered amino acid chain to directing the ordered self-assembly of thousands of proteins. It investigates how cells utilize specific chemistries, such as redox reactions, to assist in this assembly pathway. The result of this molecular design and coordinated construction is to build a carboxysome with a precise permeability, yet this permeability has never been measured. Results presented here address these fundamental questions./nI interrogated highly conserved and repetitive residues in CsoS2 to determine their role in carboxysome assembly. Through in vivo mutagenesis and in vitro biochemical assays I discovered that the residues VTG and Y are necessary for carboxysome assembly, and bind weakly yet multivalently to shell proteins. Conserved cysteine doublets, which hinted at a role for redox in assembly, showed no effect when mutated in vivo, but displayed biochemical phenotypes in vitro. In a major step towards reconstituting carboxysomes in vitro, I demonstrated formation of carboxysome-like phase-separated condensates with Rubisco, CsoS2, and shell, thereby showing that key carboxysome proteins can self-associate in a cell-free environment./nOnce assembled correctly, the carboxysome establishes a permeability barrier and selectivity filter, allowing entry of essential metabolites such as ribulose bisphosphate and bicarbonate while restricting leakage of CO2. To measure carboxysome permeability, we developed two parallel methods, one based on a bulk plate assay and one on single-particle microscopy. Both methods utilized the redox sensitive reporter protein roGFP to simultaneously measure both the permeability of reducing agents and the internal carboxysome redox environment. Data from both approaches revealed that purified carboxysomes were permeable to the reducing agent TCEP, which reduced encapsulated roGFP over time./nCarboxysomes are the bacterial domain’s solution to the problem of capturing dilute CO2 from air and water, concentrating it, and converting it into sugars. Carboxysome functionality depends on the robust self-assembly of thousands of proteins, establishment of a specific internal chemical environment, and control over metabolite permeability. Insights from this work augment our understanding of these processes, and will aid future efforts to engineer carboxysomes into alternative organisms or cell-free systems for enhanced biological carbon capture.

Advisor: Savage, David