Proteins and Water Control the Stability of the Intracellular Amorphous Calcium Carbonate Inclusions Formed by Cyanobacteria

The formation of stable intracellular amorphous calcium carbonate (ACC) inclusions is widespread among bacteria, yet the mechanisms underlying their long-term stabilization remain unresolved. In this study, we apply multinuclear solid-state NMR (ssNMR) spectroscopy to dried cells of the ACC-forming cyanobacterium Cyanothece sp. PCC 7425 to probe the molecular environment surrounding cyanobacterial ACC and its role in promoting ACC stability. Our ¹³C and ⁴³Ca ssNMR analyses confirm that the intracellular carbonate phase is composed exclusively of hydrated ACC, with no detectable crystalline CaCO₃ polymorphs. The ¹H-¹³C HETCOR correlations reveal short-range proximity between ACC and water molecules but no direct interactions with organic molecules. The ¹³C-¹³C DARR long-range correlations detect organic signals consistent with proteins that interact with ACC indirectly, possibly via water molecules. This finding was further supported by the absence of organic components in the vicinity of ACC in short-range ¹³C-¹³C POST-C7 experiments. Taken together, these results are compatible with a model in which cyanobacterial hydrated ACC granules are stabilized by a proteinaceous shell, with some water molecules serving as molecular bridges. This configuration likely restricts water and ion mobility, contributing to the kinetic stabilization of the cyanobacterial ACC.