Deciphering the mechanisms involved in the hyperaccumulation of alkaline earth metals by cyanobacteria – HARLEY

Cyanobacteria are environmentally important photosynthetic organisms that use solar energy to fix atmospheric CO2 thereby making up a huge biomass that sustains a large part of the food chain of our planet. As recently discovered, some cyanobacteria form intracellular amorphous calcium carbonates (iACC), and accumulate very high intracellular contents of alkaline earth elements (AEEs) such as Ca, Sr, Ba or Ra. While this massive intracellular AEE accumulation questions the peculiarity of AEE homeostasis in these bacteria and offers promising perspectives to remediate radioactive 90Sr and 226Ra pollutions, involved mechanisms remain unknown. Based on a comparative genomics approach, we recently identified one gene with unknown function (hereafter named ccyA) that may contribute to AEE sequestration, as it is shared by iACC-forming cyanobacteria and absent from the other fully-sequenced cyanobacterial genomes. Bioinformatics predictions suggest that ccyA encodes a protein (Calcyanin) which contains two non-homologous domains sharing some similarities and striking differences with already known domains or regions, that may define to a new protein fold. The HARLEY project proposes to decipher the biochemical mechanisms of intracellular hyperaccumulation and homeostasis of AEEs by iACC-forming cyanobacteria and the role of Calcyanin in this process. The Harley project will be carried out by a highly cohesive and interdisciplinary consortium grouping expertise in biochemistry, structural biology, biophysics, biogeochemistry, genetics and physiology of cyanobacteria. Our work program will consist in (1) characterizing structural and chemical properties of Calcyanin in vitro. Heterologous expression will be conducted to produce purified recombinant Calcyanin and its two separate domains. Their structures will be assessed by X-ray crystallography and cryo-transmission electron microscopy (TEM). Their affinities for AEEs such as Ca, Sr and Ba will be measured by spectroscopy and calorimetry. Last, their impact on ACC precipitation will be assessed by in vitro experiments. (2) characterizing the in vivo function of Calcyanin and its role in AEE accumulation. Deletion and complementation of ccyA in iACC-forming strains will be performed to understand how this impacts the phenotype of the cells. In parallel, we will introduce ccyA in genetically well-studied iACC-non-forming cyanobacterial strains to assess the resulting phenotype. The cell localization of Calcyanin will be determined using recombinant protein fused with a green fluorescent protein. The phenotypes of the mutants with respect to AEE homeostasis will be determined by several methods such as solution chemical analyses, TEM and scanning transmission X-ray microscopy. Moreover, free Ca2+ will be measured by confocal laser scanning microscopy within cells modified by the introduction of Ca reporter genes. (3) searching for additional proteins involved in iACC formation. This will be achieved by a 3-fold approach: random mutagenesis using transposon insertion based on a screening method targeting the buoyancy variations resulting from the loss of the iACC formation capability; the extraction and identification of proteins associated with iACC by mass spectrometry; the fishing of partner proteins of Calcyanin by co-immunoprecipitation. The structural and functional annotation of these proteins will be systematically assessed by bioinformatics. In addition to tackling the important issue of AEE homeostasis in these cyanobacteria and the biochemical mechanisms of iACC formation, results by the Harley project will be of great interest to structural biologists by the determination of novel protein folds and associated functions as well as cyanobacteriologists by the production of new genetic models. In the longer term, the Harley project will be crucial for designing new biomimetic or synthetic systems of value for an effective bioremediation of AEEs pollutions.