Amorphous Calcium Carbonate Granules Form Within an Intracellular Compartment in Calcifying Cyanobacteria

The recent discovery of cyanobacteriaforming intracellular amorphous calcium carbonate(ACC) has challenged the former paradigm suggesting that cyanobacteria-mediated carbonatogenesis was exclusively extracellular. Yet, the mechanisms of intracellular biomineralizationin cyanobacteriaand in particular whether this takes place within an intracellularmicrocompartment, remain poorly understood. Here, we analyzed six cyanobacterial strains forming intracellularACC by transmission electron microscopy. We tested two different approaches to preserve as well as possible the intracellularACC inclusions: (i) freeze-substitution followed by epoxy embedding and room-temperature ultramicrotomyand (ii) high-pressure freezing followed by cryo- ultramicrotomy, usually referred to as cryo-electron microscopyof vitreous sections (CEMOVIS). We observed that the first method preserved ACC well in 500-nm-thick sections but not in 70-nm-thick sections. However, cell ultrastructures were difficult to clearly observe in the 500-nm-thick sections. In contrast, CEMOVIS provided a high preservation quality of bacterial ultrastructures, including the intracellularACC inclusions in 50-nm-thick sections. ACC inclusions displayed different textures, suggesting varying brittleness, possibly resulting from different hydration levels. Moreover, an electron dense envelope of ~2.5 nm was systematically observed around ACC granules in all studied cyanobacterial strains. This envelope may be composed of a protein shell or a lipid monolayer, but not a lipid bilayer as usually observed in other bacteria forming intracellularminerals. Overall, this study evidenced that ACC inclusions formed and were stabilized within a previously unidentified bacterial microcompartmentin some species of cyanobacteria.