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

This project uses a diversity of methods and approaches making it interdisciplinary. A first series of approach uses diverse tools of bioinformatics to assess the distribution of calcyanin within the diversity of cyanobacteria. This assessment also allows to evaluate the sequence diversity among calcyanins as well as conserved motifs. Additional tools allow to predict calcyanin structures. We combine with these bioinformatics approaches, experimental approaches which include heterologous expression and purification of proteins and which allow producing protein samples which are subsequently used for determining protein structures either after crystallization and x-ray cristallography analyses or by transmission electron microscopy. Purified proteins are also used for analyses of calcyanin chemical properties, including its affinity for earth alkaline elements. These analyses combine fluorescence spectroscopy measurements and mass spectrometry.
A second series of approaches uses genetics techniques. A first method couples deletion and complementation of the gene coding for calcyanin. A second method aims at inserting and expressing this gene in cyanobacteria not forming intracellular CaCO3. Localization of the protein within the cells will be determined by the use of recombinant proteins fused with a fluorescing protein such as GFP. Phenotypes of all produced mutants will be analyzed by diversed approaches including (i) aqueous geochemistry to quantiy sequestration of earth alkaline elements by the strains, and (ii) electron and x-ray microscopy and spectromicroscopy techniques allowing to quantifiy and localize calcium within cells.
Last, random mutagenesis approaches on one hand and co-immunoprecipitation experiments will be conducted to identify additional proteins possibly involved together with calcyanin in the biomineralization of intracellular CaCO3

Up to now, we have obtaiend several striking results.
- We have set a bioinformatics pipeline allowing automatic detection and expert annotation of calcyanine among genomes found in databses. This allowed us to dramatically increase the list of existing calcyanins in cyanobacteria and their diversity
- Moreover, this allowed us to mke several structural prediction about calcyanin, including a 2-domain based model of the protein with the identification of several folds in the protein. We also established a structural model for a new domain, found in some calcyanins and affiliated to the superfamily of HMA domains.
- We could build an evolutive scenaio for calcyanin suggesting an ancient origin of this protein, then a vertical transfer, mutliple lossess in many branches of the cyanobacteria tree of life and few cases of horizontal transfers of the gene.
- experimentally, we built 11 constructions to express different calcyanins in E. coli plus 3 constructions to expression domains of calcyanin in E. coli.
- For genetics, we built deletion cassettes of calcyanin in 2 strains forming intracellular CaCO3. In parallel, we built mutants of strains forming intracellular CaCO3 to overexpress calcyanin. Some of these mutants express a calcyanin fused with the gfp protein. Last we have built mutants of strains not forming intracellular CaCO3 but overexpressing calcyanin.
- The phenotypic analysis of the different mutants is in progress but there are already some results: an impact of the overexpression of calcynine on the growth of mutants of strains forming intracellular CaCO3. Moreover, we set an approach using synchrotron-based x-ray absorption microspectroscopy at the L2,3 edges of calcium to quantify Ca and its disitrbution in cells. With this approach we have shown that mutants of strains not forming intracellular CaCO3 but overexpressing calcyanin have increased Ca contents.

Our perspectives stay in line with those established at the beginning of the project.
- Regarding bioinformatics, we will also analyze environmental metagenomes to further enrich our knowledge about the diversity of calcyanins. We will also look for the domains of calcyanin separately in the genomes in order to assess with which other proteins they can be associated. Last, we will continue the development of structural models with new tools.
- Concerning biochemistry, we continue to improve purification protocols and tackle the challenge of the protein stability. Our main objective is still to obtain the structure of one or several calcyanins. The production of polyclonal antibodies is about to be achieved and should help make several great progresses. Now the hiring of a non permanent staff done in Cadarache, we will start the analyses of chemical properties of calcyanins.
- For genetics, the construction of deletion mutants is in progress. Several mutants are still to be phenotypically analyzed.
-Once all these objectives broadly advanced, we will tackle the search of additional proteins that may be involved in the biomineralization of intracellular CaCO3.

Up to now, we published a review on the molecular mechanisms of CaCO3 biomineralization by bacteria. And one paper showing the possibility to genetically modify a cyanobacterium capable of intracellular CaCO3 formation.
Additional publications are submitted or in preparation about the diversity of calcyanins among cyanobacteria and the measurement of Ca concentration at the nanoscale in bacteria using STXM.
Part of our work has been presented in a keynote talk at the international Goldchmidt conference


1. Sigrid Görgen, Karim Benzerara, Fériel Skouri-Panet, Muriel Gugger, Franck Chauvat, Corinne Cassier-Chauvat (2021) The diversity of molecular mechanisms of carbonate biomineralization by bacteria. Discover Materials 1:2.
2. Chenebault C, Diaz-Santos E, Kammerscheit X, Görgen S, Ilioaia C, Streckaite S, Gall A, Robert B, Marcon E, Buisson DA, Benzerara K, Sassi JF, Cassier-Chauvat C, Chauvat F. A (2020) Genetic Toolbox for the New Model Cyanobacterium Cyanothece PCC 7425: A Case Study for the Photosynthetic Production of Limonene. Front Microbiol. 11:586601.
1. Keynote, Goldschmidt Conference. “Biomineralization of intracelllar amorphous calcium carbonates (ACC) by bacteria: molecular mechanisms, evolutionary history and environmental significance” Benzerara K, Bitard-Feildel T, Bolzoni R, Cassier-Chauvat C, Chauvat F, Dezi M, Duprat Elodie, Görgen S, Lefevre C, Lopez-Garcia P, Menguy M, Monteil C, Moreira David, Skouri-Panet F, Callebaut I.

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.