Several species of cyanobacteria biomineralizing intracellular amorphous calcium carbonates (ACC) were recently discovered. However, the mechanisms involved in this biomineralization process and the determinants discriminating species forming intracellular ACC from those not forming intracellular ACC remain unknown. Recently, it was hypothesized that the intensity of Ca uptake (i.e., how much Ca was scavenged from the extracellular solution) might be a major parameter controlling the capability of a cyanobacterium to form intracellular ACC. Here, we tested this hypothesis by systematically measuring the Ca uptake by a set of 52 cyanobacterial strains cultured in the same growth medium. The results evidenced a dichotomy among cyanobacteria regarding Ca sequestration capabilities, with all strains forming intracellular ACC incorporating significantly more calcium than strains not forming ACC. Moreover, Ca provided at a concentration of 50 μM in BG‐11 was shown to be limiting for the growth of some of the strains forming intracellular ACC, suggesting an overlooked quantitative role of Ca for these strains. All cyanobacteria forming intracellular ACC contained at least one gene coding for a mechanosensitive channel, which might be involved in Ca influx, as well as at least one gene coding for a Ca2+/H+ exchanger and membrane proteins of the UPF0016 family, which might be involved in active Ca transport either from the cytosol to the extracellular solution or the cytosol toward an intracellular compartment. Overall, massive Ca sequestration may have an indirect role by allowing the formation of intracellular ACC. The latter may be beneficial to the growth of the cells as a storage of inorganic C and/or a buffer of intracellular pH. Moreover, high Ca scavenging by cyanobacteria biomineralizing intracellular ACC, a trait shared with endolithic cyanobacteria, suggests that these cyanobacteria should be considered as potentially significant geochemical reservoirs of Ca.
Cyanobacteria have massively contributed to carbonate deposition over the geological history. They are traditionally thought to biomineralize CaCO3 extracellularly as an indirect byproduct of photosynthesis. However, the recent discovery of freshwater cyanobacteria forming intracellular amorphous calcium carbonates (iACC) challenges this view. Despite the geochemical interest of such a biomineralization process, its molecular mechanisms and evolutionary history remain elusive. Here, using comparative genomics, we identify a new gene (ccyA) and protein family (calcyanin) possibly associated with cyanobacterial iACC biomineralization. Proteins of the calcyanin family are composed of a conserved C-terminal domain, which likely adopts an original fold, and a variable N-terminal domain whose structure allows differentiating 4 major types among the 35 known calcyanin homologs. Calcyanin lacks detectable full-length homologs with known function. The overexpression of ccyA in iACC-lacking cyanobacteria resulted in an increased intracellular Ca content. Moreover, ccyA presence was correlated and/or co-localized with genes involved in Ca or HCO3- transport and homeostasis, supporting the hypothesis of a functional role of calcyanin in iACC biomineralization. Whatever its function, ccyA appears as diagnostic of intracellular calcification in cyanobacteria. By searching for ccyA in publicly available genomes, we identified 13 additional cyanobacterial strains forming iACC, as confirmed by microscopy. This extends our knowledge about the phylogenetic and environmental distribution of cyanobacterial iACC biomineralization, especially with the detection of multicellular genera as well as a marine species. Moreover, ccyA was probably present in ancient cyanobacteria, with independent losses in various lineages that resulted in a broad but patchy distribution across modern cyanobacteria.
Microbial phosphatase activity can trigger the precipitation of metal-phosphate minerals, a process called phosphatogenesis with global geochemical and environmental implications. An increasing diversity of phosphatases expressed by diverse microorganisms has been evidenced in various environments. However, it is challenging to link the functional properties of genomic repertoires of phosphatases with the phosphatogenesis capabilities of microorganisms. Here, we studied the betaproteobacterium Ramlibacter tataouinensis (Rta), known to biomineralize Ca-phosphates in the environment and the laboratory. We investigated the functional repertoire of this biomineralization process at the cell, genome and molecular level. Based on a mineralization assay, Rta is shown to hydrolyse the phosphoester bonds of a wide range of organic P molecules. Accordingly, its genome has an unusually high diversity of phosphatases: five genes belonging to two non-homologous families, phoD and phoX, were detected. These genes showed diverse predicted cis-regulatory elements. Moreover, they encoded proteins with diverse structural properties according to molecular models. Heterologously expressed PhoD and PhoX in Escherichia coli had different profiles of substrate hydrolysis. As evidenced for Rta cells, recombinant E. coli cells induced the precipitation of Ca-phosphate mineral phases, identified as poorly crystalline hydroxyapatite. The phosphatase genomic repertoire of Rta (containing phosphatases of both the PhoD and PhoX families) was previously evidenced as prevalent in marine oligotrophic environments. Interestingly, the Tataouine sand from which Rta was isolated showed similar P-depleted, but Ca-rich conditions. Overall, the diversity of phosphatases in Rta allows the hydrolysis of a broad range of organic P substrates and therefore the release of orthophosphates (inorganic phosphate) under diverse trophic conditions. Since the release of orthophosphates is key to the achievement of high saturation levels with respect to hydroxyapatite and the induction of phosphatogenesis, Rta appears as a particularly efficient driver of this process as shown experimentally.
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