Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria

Abstract

Cyanobacteria have played a significant role in the formation of past and modern carbonate deposits at the surface of the Earth using a biomineralization process that has been almost systematically considered induced and extracellular. Recently, a deep-branching cyanobacterial species, Candidatus Gloeomargarita lithophora, was reported to form intracellular amorphous Ca-rich carbonates. However, the significance and diversity of the cyanobacteria in which intracellular biomineralization occurs remain unknown. Here, we searched for intracellular Ca-carbonate inclusions in 68 cyanobacterial strains distributed throughout the phylogenetic tree of cyanobacteria. We discovered that diverse unicellular cyanobacterial taxa form intracellular amorphous Ca-carbonates with at least two different distribution patterns, suggesting the existence of at least two distinct mechanisms of biomineralization: (i) one with Ca-carbonate inclusions scattered within the cell cytoplasm such as in Ca. G. lithophora, and (ii) another one observed in strains belonging to the Thermosynechococcus elongatus BP-1 lineage, in which Ca-carbonate inclusions lie at the cell poles. This pattern seems to be linked with the nucleation of the inclusions at the septum of the cells, showing an intricate and original connection between cell division and biomineralization. These findings indicate that intracellular Ca-carbonate biomineralization by cyanobacteria has been overlooked by past studies and open new perspectives on the mechanisms and the evolutionary history of intra- and extracellular Ca-carbonate biomineralization by cyanobacteria.

Keywords: amorphous calcium carbonate; calcification; polyphosphate.

Fig. 1.

Bayesian phylogenetic tree of 16S rRNA gene sequences of the cyanobacterial strains observed by electron microscopy. Strains forming intracellular Ca-carbonates are shown in color (green for those with Ca-carbonate inclusions at the cell poles and red for those with inclusions scattered in the cytoplasm). The tree is based on 1,292 conserved sites; numbers at branches are posterior probabilities (only those >0.75 are shown).

Fig. 2.

Electron microscopy images of cyanobacteria forming intracellular carbonates scattered throughout the cells. (A and B) SEM images in AsB detection mode of Chroococcidiopsis thermalis PCC 7203. (B) A baeocyte filled with Ca-carbonate inclusions. (C) STEM-EDX map of a vegetative cell of PCC 7203. (D and E) STEM-HAADF images of Cyanothece sp. PCC 7425. Ca-carbonate inclusions appear as bright round-shaped objects. PolyP granules are darker, sometimes shapeless forms (arrows). (F) STEM-EDX map of PCC 7425. (G and H) STEM-HAADF images of Ca. G. lithophora strain D10. Ca-carbonates appear as brighter round-shaped inclusions, whereas PolyP granules are darker, sometimes bigger globules. PolyP granules sometimes seem to partly surround Ca-carbonate inclusions (arrows). (I) STEM-EDX map of D10. The color code is the same for all STEM-EDX maps (C, F, and I): calcium, green; phosphorus, red; carbon, blue. As a result, Ca-carbonates appear in green and PolyP granules in red.

Fig. 3.

Electron microscopy images of Synechococcus sp., Ca. S. calcipolaris, and Thermosynechococcus elongatus strains forming intracellular carbonates; all of them form intracellular Ca-carbonates (brightest spots in STEM-HAADF images) located at the septum and the poles of the cells. (A) STEM-HAADF image and (B) EDX map of Synechococcus sp. PCC 6716. Four aligned PolyP granules measuring ∼500 nm in diameter can be observed in the top cell (arrows). Clusters of 20–30 Ca-carbonate inclusions can be observed at both poles of the cell. (C) STEM-HAADF image and (D) STEM-EDX map of strain PCC 6717 cell. (E) STEM-HAADF image and (F) STEM-EDX map of Ca. S. calcipolaris G9 (Lake Alchichica). (G) STEM-HAADF image and (H) STEM-EDX map of Thermosynechococcus elongatus BP1. The color code is the same for all STEM-EDX maps: calcium, green; phosphorus, red; carbon, blue.

Fig. 4.

STEM-HAADF images of Synechococcus sp. PCC 6312. Ca-carbonate inclusions were always observed at the poles of the cells and sometimes at the same location where septation occurs (arrows). Inclusions at the septum are usually smaller than at the poles. Division as observed in A, B, and D seems to partition the Ca-carbonate inclusions between the daughter cells, resulting in the polar distribution observed in all of the cells. STEM-EDX map is shown in C. Calcium, green; phosphorus, red; carbon, blue.