The Formation and Degradation of Carboxysomes During Heterocyst Development in Filamentous Cyanobacteria

Photosynthetic carbon (CO2) fixation and solar-powered nitrogen (N2) fixation are foundational biochemical processes that introduce essential elements into the biosphere. While atmospheric dinitrogen gas (N2) is unusable to most organisms, diazotrophs utilize the nitrogenase enzyme to reduce N2 to ammonia. Because all known nitrogenases are highly susceptible to irreversible oxygen inactivation, N2 fixation is generally incompatible with oxygenic photosynthesis. However, several cyanobacteria are both photosynthetic and diazotrophic, balancing these opposing metabolisms by temporally or spatially separating the respective enzymes. Anabaena sp. PCC 7120 is a model multicellular cyanobacterium that grows in filaments of photosynthetic vegetative cells, which respond to nitrogen starvation by differentiating into microaerobic, N2-fixing cells called heterocysts. The absence of cyanobacterial carboxysomes, proteinaceous microcompartments that encapsulate and saturate the RuBisCO enzyme, is a relatively unexplored feature of mature heterocysts. While the biogenesis and degradation of carboxysomes have been investigated in unicellular cyanobacteria, their removal in heterocysts remains poorly understood. Although it is generally accepted that RuBisCO is similarly nonexistent in heterocysts, inconsistent observations across several studies leave the extent of its depletion unclear. To re-evaluate this claim and further characterize the heterocyst-specific fate of these major photosynthetic proteins, we developed an Anabaena sp. PCC 7120 cargo plasmid harboring a GFP translational fusion to the RuBisCO large subunit, producing an RbcL-GFP chimeric protein. Fluorescence microscopy analysis of this strain under nitrogen-starvation conditions revealed that carboxysomes and RuBisCO are completely absent at all morphologically identifiable stages of heterocyst development. Nitrogen stepdown observations also indicate that carboxysomes and RuBisCO are actively degraded during heterocyst differentiation, which was visualized in collaboratively developed time-lapse microscopy. We subsequently investigated Anabaena sp. PCC 7120 protease genes for potential roles in this process. A list of 33 genes was selected for single-crossover inactivation based on functional annotations and upregulation during nitrogen deprivation. After generating an integrative plasmid library to target each gene, the resulting plasmids were introduced into the RbcL-GFP reporter strain, and single-crossover recombinant mutants were screened for persistent heterocyst RbcL-GFP fluorescence. Although a definitive protease was not identified, our initial observations suggest that two protease genes are involved in carboxysome and RuBisCO degradation during heterocyst differentiation.