In corals, biocalcification is a major function that may be drastically affected by ocean acidification (OA). Scleractinian corals grow by building up aragonitic exoskeletons that provide support and protection for soft tissues. Although this process has been extensively studied, the molecular basis of biocalcification is poorly understood. Notably lacking is a comprehensive catalog of the skeleton-occluded proteins—the skeletal organic matrix proteins (SOMPs) that are thought to regulate the mineral deposition. Using a combination of proteomics and transcriptomics, we report the first survey of such proteins in the staghorn coral Acropora millepora. The organic matrix (OM) extracted from the coral skeleton was analyzed by mass spectrometry and bioinformatics, enabling the identification of 36 SOMPs. These results provide novel insights into the molecular basis of coral calcification and the macroevolution of metazoan calcifying systems, whereas establishing a platform for studying the impact of OA at molecular level. Besides secreted proteins, extracellular regions of transmembrane proteins are also present, suggesting a close control of aragonite deposition by the calicoblastic epithelium. In addition to the expected SOMPs (Asp/Glu-rich, galaxins), the skeletal repertoire included several proteins containing known extracellular matrix domains. From an evolutionary perspective, the number of coral-specific proteins is low, many SOMPs having counterparts in the noncalcifying cnidarians. Extending the comparison with the skeletal OM proteomes of other metazoans allowed the identification of a pool of functional domains shared between phyla. These data suggest that co-option and domain shuffling may be general mechanisms by which the trait of calcification has evolved.

Proteins that are occluded within the molluscan shell, the so-called shell matrix proteins (SMPs), are an assemblage of biomolecules attractive to study for several reasons. They increase the fracture resistance of the shell by several orders of magnitude, determine the polymorph of CaCO 3 deposited, and regulate crystal nucleation, growth initiation and termination. In addition, they are thought to control the shell microstructures. Understanding how these proteins have evolved is also likely to provide deep insight into events that supported the diversification and expansion of metazoan life during the Cambrian radiation 543 million years ago. Here, we present an analysis of SMPs isolated form the CaCO 3 shell of the limpet Lottia gigantea, a gastropod that constructs an aragonitic cross-lamellar shell. We identified 39 SMPs by combining proteomic analysis with genomic and transcriptomic database interrogations. Among these proteins are various low-complexity domain-containing proteins, enzymes such as peroxidases, carbonic anhydrases and chitinases, acidic calcium-binding proteins and protease inhibitors. This list is likely to contain the most abundant SMPs of the shell matrix. It reveals the presence of both highly conserved and lineage-specific biomineralizing proteins. This mosaic evolutionary pattern suggests that there may be an ancestral molluscan SMP set upon which different conchiferan lineages have elaborated to produce the diversity of shell microstructures we observe nowadays.

The carbonic anhydrase (CA; EC 4.2.1.1) superfamily is a class of ubiquitous metallo-enzymes that catalyse the reversible hydration of carbon dioxide. The α-CA family, present in all metazoan clades, is a key enzyme involved in a wide range of physiological functions including pH regulation, respiration, photosynthesis, and biocalcification. This paper reviews the evolution of the α-CA family, with an emphasis on metazoan α-CA members involved in biocalcification. Phylogenetic analyses reveal a complex evolutionary history of α-CAs, and suggest α-CA was independently co-opted into a variety of skeleton forming roles (e.g. as a provider of HCO 3 − ions, a structural protein, a nucleation activator, etc.) in multiple metazoan lineages. This evolutionary history is most likely the result of multiple gene duplications coupled with the insertion of repetitive or non-repetitive low-complexity domains (RLCDs/LCDs). These domains, of largely unknown function, appear to be lineage-specific, and provide further support for the hypothesis of independent recruitment of α-CAs to diverse metazoan biocalcification processes. An analysis of α-CA sequences associated with biocalcification processes indicates that the domains involved in the activity and conformation of the active site are extremely conserved among metazoans.

Bacteria of the Firmicutes phylum are able to enter a developmental pathway that culminates with the formation of highly resistant, dormant endospores. Endospores allow environmental persistence, dissemination and for pathogens, are also infection vehicles. In both the model Bacillus subtilis, an aerobic organism, and in the intestinal pathogen Clostridioides difficile, an obligate anaerobe, sporulation mobilizes hundreds of genes. Their expression is coordinated between the forespore and the mother cell, the two cells that participate in the process, and is kept in close register with the course of morphogenesis. The evolutionary mechanisms by which sporulation emerged and evolved in these two species, and more broadly across Firmicutes, remain largely unknown. Here, we trace the origin and evolution of sporulation using the genes known to be involved in the process in B. subtilis and C. difficile, and estimating their gain-loss dynamics in a comprehensive bacterial macroevolutionary framework. We show that sporulation evolution was driven by two major gene gain events, the first at the base of the Firmicutes and the second at the base of the B. subtilis group and within the Peptostreptococcaceae family, which includes C. difficile. We also show that early and late sporulation regulons have been coevolving and that sporulation genes entail greater innovation in B. subtilis with many Bacilli lineage-restricted genes. In contrast, C. difficile more often recruits new sporulation genes by horizontal gene transfer, which reflects both its highly mobile genome, the complexity of the gut microbiota, and an adjustment of sporulation to the gut ecosystem.

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The scleractinian coral Acropora millepora is one of the most studied species from the Great Barrier Reef. This species has been used to understand evolutionary, immune and developmental processes in cnidarians. It has also been subject of several ecological studies in order to elucidate reef responses to environmental changes such as temperature rise and ocean acidification (OA). In these contexts, several nucleic acid resources were made available. When combined to a recent proteomic analysis of the coral skeletal organic matrix (SOM), they enabled the identification of several skeletal matrix proteins, making A. millepora into an emerging model for biomineralization studies. Here we describe the skeletal microstructure of A. millepora skeleton, together with a functional and biochemical characterization of its occluded SOM that focuses on the protein and saccharidic moieties. The skeletal matrix proteins show a large range of isoelectric points, compositional patterns and signatures. Besides secreted proteins, there are a significant number of proteins with membrane attachment sites such as transmembrane domains and GPI anchors as well as proteins with integrin binding sites. These features show that the skeletal proteins must have strong adhesion properties in order to function in the calcifying space. Moreover this data suggest a molecular connection between the calcifying epithelium and the skeletal tissue during biocalcification. In terms of sugar moieties, the enrichment of the SOM in arabinose is striking, and the monosaccharide composition exhibits the same signature as that of mucus of acroporid corals. Finally, we observe that the interaction of the acetic acid soluble SOM on the morphology of in vitro grown CaCO3 crystals is very pronounced when compared with the calcifying matrices of some mollusks. In light of these results, we wish to commend Acropora millepora as a model for biocalcification studies in scleractinians, from molecular and structural viewpoints.

The formation of the molluskan shell is regulated by an array of extracellular proteins secreted by the calcifying epithelial cells of the mantle. These proteins remain occluded within the recently formed biominerals. To date, many shell proteins have been retrieved, but only a few of them, such as nacreins, have clearly identified functions. In this particular case, by combining molecular biology and biochemical approaches, we performed the molecular characterization of a novel protein that we named Upsalin, associated with the nacreous shell of the freshwater mussel Unio pictorum. The full sequence of the upsalin transcript was obtained by RT‐PCR and 5′/3′ RACE, and the expression pattern of the transcript was studied by PCR and qPCR. Upsalin is a 12 kDa protein with a basic theoretical pI. The presence of Upsalin in the shell was demonstrated by extraction of the acetic‐acid‐soluble nacre matrix, purification of a shell protein fraction by mono‐dimensional preparative SDS‐PAGE, and by submitting this fraction, after trypsic digestion, to nano‐LC‐MS/MS. In vitro experiments with the purified protein showed that it interferes poorly with the precipitation of calcium carbonate. Homology searches also could not affiliate Upsalin to any other protein of known function, leaving open the question of its exact role in shell formation. An antibody raised against an immunogenic peptide of Upsalin was found to be specific to this protein and was subsequently assayed for immunogold localization of the target protein in the shell, revealing the ubiquitous presence of Upsalin in the nacreous and prismatic layers. Recently, with the application of high‐throughput proteomic studies to shells, the number of candidate proteins without clear functions has been increasing exponentially. The Upsalin example highlights the crucial need, for the scientific community dealing with biomineralization in general, to dedicate the coming years to designing experimental approaches, such as gene silencing, that focus on the functions of mineral‐associated proteins.

In a recent editorial (Proc. Natl. Acad. Sci., 2013 and elsewhere, questions have been raised regarding the experimental practices in relation to the proteomic analysis of organic matrices associated to the biomineralized CaCO 3 skeletons of metazoans such as molluscan shells and coral skeletons. Indeed, although the use of new high sensitivity MS technology potentially allows to identify a greater number of proteins, it is also equally (or even more) sensitive to contamination of residual proteins from soft tissues, which are in close contact with the biomineral. Based on our own past and present experimental know-howobservations that are reproducible and coherent with the current understanding of extracellular biomineralization processes-we are convinced that a careful and appropriate cleaning of biominerals prior to any analysis is crucial for accurate proteomic investigations and subsequent pertinent interpretation of the results. Our goal is to alert the scientific community about the associated bias that definitely should be avoided, and to provide critical recommendations on sample preparation and experimental design, in order to better take advantage of the aptness of proteomic approaches aiming at improving our understanding of the molecular mechanisms in biomineralization.