Ferritin-like Protein Research Articles

Metabolite phosphatase from anhydrobiotic tardigrades.

Terrestrial organisms have systems to escape from desiccation stresses. For example, tardigrades (also known as water bears) can survive severe dried and other extreme environments by anhydrobiosis. Although their extraordinary ability has enchanted people, little is known about the detailed molecular mechanisms of anhydrobiosis. Here, we focused on the tardigrade Ramazzottius varieornatus, one of the toughest animals on Earth. A transcriptome database of R. varieornatus shows that genes encoding a Ferritin-like protein are upregulated during desiccation or ultraviolet radiation. This protein shows sequence similarity to enigmatic proteins in desiccation-tolerant bacteria and plants, which are hypothesized to be desiccation-related. However, because these proteins lack detailed biological information, their functions are relatively unknown. We determined an atomic (1.05 Å) resolution crystal structure of a Ferritin-like protein from R. varieornatus. The structure revealed a dinuclear metal binding site, and we showed that this Ferritin-like protein has phosphatase activity toward several metabolite compounds including unusual nucleotide phosphates produced by oxidative or radiation damage. We also found that a homologous protein from a desiccation- and ultraviolet-tolerant bacterium Deinococcus radiodurans is a metabolite phosphatase. Our results indicate that through cleaning up damaged metabolites or regulation of metabolite levels, this phosphatase family can contribute to stress tolerances. This study provides a clue to one of the universal molecular bases of desiccation-stress tolerance.

Encapsulated Ferritin-like Proteins: A Structural Perspective.

Encapsulins are self-assembling nano-compartments that naturally occur in bacteria and archaea. These nano-compartments encapsulate cargo proteins that bind to the shell's interior through specific recognition sequences and perform various metabolic processes. Encapsulation enables organisms to perform chemical reactions without exposing the rest of the cell to potentially harmful substances while shielding cargo molecules from degradation and other adverse effects of the surrounding environment. One particular type of cargo protein, the ferritin-like protein (FLP), is the focus of this review. Encapsulated FLPs are members of the ferritin-like protein superfamily, and they play a crucial role in converting ferrous iron (Fe+2) to ferric iron (Fe+3), which is then stored inside the encapsulin in mineralized form. As such, FLPs regulate iron homeostasis and protect organisms against oxidative stress. Recent studies have demonstrated that FLPs have tremendous potential as biosensors and bioreactors because of their ability to catalyze the oxidation of ferrous iron with high specificity and efficiency. Moreover, they have been investigated as potential targets for therapeutic intervention in cancer drug development and bacterial pathogenesis. Further research will likely lead to new insights and applications for these remarkable proteins in biomedicine and biotechnology.

Synergy between polypyrrol and benzoic acid against antibiotic-resistant Salmonella spp.

The purpose was to characterize Salmonella Heidelberg (SH) and Minnesota (SM) isolates in terms of their resistance and persistence profile and to assess the antimicrobial effect of benzoic acid (BA) and polypyrrole (PPy). The 20 isolates from broiler litter drag swabs were submitted to antibiogram and efflux pump expression. The minimum inhibitory/bactericidal concentration (MIC/MBC) of the compounds, synergistic activity, time kill, biofilm production, presence of related genes, and molecular docking between compounds and bacterial target sites were evaluated. All isolates showed multidrug resistance (MDR) and BA and PPy showed mean MIC (1750 and 342 µg ml-1) and MBC (3167 and 1000 µg ml-1), respectively. None of the isolates expressed an efflux pump. The compounds showed synergism against an SH isolate and reduced the count by 3 logs in the presence of the compounds after 4 h. Most isolates (16/20) produced weak to moderate biofilm and 17 showed genes related to biofilm. The compounds interacted with two essential proteins, 3,4-dihydroxy-2-butanone 4-phosphate synthase proteins and ferritin-like domain-containing protein, in bacterial metabolism at different target sites. It can be concluded that BA and PPy showed activity on SH and SM, MDR, and biofilm producers, with a potential synergistic effect.

Localization of the E. coli Dps protein molecules in a silicon wires matrix according to scanning electron microscopy and X-ray photoelectron spectroscopy

The work is related to the study of the morphological features of silicon wires arrays combined with a nanomaterial of natural origin, a bacterial ferritin-like protein Dps, and their relationship with the composition of the surface and interior. A silicon wires array was formed by metal-assisted wet chemical etching. To obtain recombinant protein, Escherichia coli BL21*(DE3) cells were used as producers, and purification was carried out by the chromatography method. The combination of silicon wires with protein molecules was carried out by layering under laboratory conditions, followed by drying. The resulting hybrid material was studied by scanning electron microscopy and X-ray photoelectron spectroscopy. The initial silicon wires array had sharp boundaries on the surface. The diameter of the silicon wires was about 100 nm, while the distances between the wires can vary widely, reaching several hundred nanometres or be less than 100 nanometres, depending on the formation conditions, in the absence of noticeable transition layers. The pores formed in this way are available for filling with protein during deposition. The effectiveness of using the scanning electron microscopy method to study the morphology of the hybrid material “silicon wires – bacterial protein Dps” as well as X-ray photoelectron spectroscopy method together with ion etching for the investigation of the composition and physico-chemical of the hybrid material was demonstrated. Complementary results have shown that the molecular culture, which is a solution of oligomers of the recombinant Dps protein of E.coli bacterial cells, can penetrate deep into the pores of the silicon wires array with an extremely developed surface. The possibility of the control of the filling of silicon wires arrays by varying the pore morphology and other modes of formation of structuresand their surface has been demonstrated. The obtained data can be used to study the possibilities of the functionalization of the developed surface of silicon wires by their driven coating with controlled delivery of biohybrid material.

Charge Transport across Proteins inside Proteins: Tunneling across Encapsulin Protein Cages and the Effect of Cargo Proteins.

Charge transport across proteins can be surprisingly efficient over long distances-so-called long-range tunneling-but it is still unclear as to why and under which conditions (e.g., presence of co-factors, type of cargo) the long-range tunneling regime can be accessed. This paper describes molecular tunneling junctions based on an encapsulin (Enc), which is a large protein cage with a diameter of 24 nm that can be loaded with various types of (small) proteins, also referred to as "cargo". We demonstrate with dynamic light scattering, transmission electron microscopy, and atomic force microscopy that Enc, with and without cargo, can be made stable in solution and immobilized on metal electrodes without aggregation. We investigated the electronic properties of Enc in EGaIn-based tunnel junctions (EGaIn = eutectic alloy of Ga and In that is widely used to contact (bio)molecular monolayers) by measuring the current density for a large range of applied bias of ±2.5 V. The encapsulated cargo has an important effect on the electrical properties of the junctions. The measured current densities are higher for junctions with Enc loaded with redox-active cargo (ferritin-like protein) than those junctions without cargo or redox-inactive cargo (green fluorescent protein). These findings open the door to charge transport studies across complex biomolecular hierarchical structures.

Abstract 2428: Structural investigations of rubrerythrin from B. pseudomallei: Metals, domain swapping, and other interesting features of this ferritin-like superfamily protein

Abstract 2428: Structural investigations of rubrerythrin from B. pseudomallei: Metals, domain swapping, and other interesting features of this ferritin-like superfamily protein

Formation of Iron Oxide Nanoparticles in the Internal Cavity of Ferritin-Like Dps Protein: Studies by Anomalous X-Ray Scattering

DNA-binding protein from starved cells (Dps) takes a special place among dodecamer mini-ferritins. Its most important function is protection of bacterial genome from various types of destructive external factors via in cellulo Dps–DNA co-crystallization. This protective response results in the emergence of bacterial resistance to antibiotics and other drugs. The protective properties of Dps have attracted a significant attention of researchers. However, Dps has another equally important functional role. Being a ferritin-like protein, Dps acts as an iron depot and protects bacterial cells from the oxidative damage initiated by the excess of iron. Here we investigated formation of iron oxide nanoparticles in the internal cavity of the Dps dodecamer. We used anomalous small-angle X-ray scattering as the main research technique, which allows to examine the structure of metal-containing biological macromolecules and to analyze the size distribution of metal nanoparticles formed in them. The contributions of protein and metal components to total scattering were distinguished by varying the energy of the incident X-ray radiation near the edge of the metal atom absorption band (the K-band for iron). We examined Dps specimens containing 50, 500, and 2000 iron atoms per protein dodecamer. Analysis of the particle size distribution showed that, depending on the iron content in the solution, the size of the nanoparticles formed inside the protein molecule was 2 to 4 nm and the growth of metal nanoparticles was limited by the size of the protein inner cavity. We also found some amount of iron ions in the Dps surface layer. This layer is very important for the protein to perform its protective functions, since the surface-located N-terminal domains determine the nature of interactions between Dps and DNA. In general, the results obtained in this work can be useful for the next step in studying the Dps phenomenon, as well as in creating biocompatible and solution-stabilized metal nanoparticles.

Structural Insights into Iron Ions Accumulation in Dps Nanocage.

Dps (DNA-binding protein from starved cells) is well known for the structural protection of bacterial DNA by the formation of highly ordered intracellular assemblies under stress conditions. Moreover, this ferritin-like protein can perform fast oxidation of ferrous ions and subsequently accumulate clusters of ferric ions in its nanocages, thus providing the bacterium with physical and chemical protection. Here, cryo-electron microscopy was used to study the accumulation of iron ions in the nanocage of a Dps protein from Escherichia coli. We demonstrate that Fe2+ concentration in the solution and incubation time have an insignificant effect on the volume and the morphology of iron minerals formed in Dps nanocages. However, an increase in the Fe2+ level leads to an increase in the proportion of larger clusters and the clusters themselves are composed of discrete ~1–1.5 nm subunits.

Comparative structural analysis provides new insights into the function of R2‐like ligand‐binding oxidase

R2‐like ligand‐binding oxidase (R2lox) is a ferritin‐like protein that harbours a heterodinuclear manganese–iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine–valine ether cross‐link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry‐based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora erythraea and Sulfolobus acidocaldarius, at 1.38 Å and 2.26 Å resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzyme.

TEM and XPS studies of bio-nanohybrid material based on bacterial ferritin-like protein Dps

TEM and XPS studies of bio-nanohybrid material based on bacterial ferritin-like protein Dps

The encapsulin from Thermotoga maritima is a flavoprotein with a symmetry matched ferritin-like cargo protein

Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based ‘organelles’ found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a ~ 25–50 nm shell surrounding a specific cargo enzyme. Compartmentalization is thought to create a unique chemical environment to facilitate catalysis and isolate toxic intermediates. Many questions regarding nanocompartment structure–function remain unanswered, including how shell symmetry dictates cargo loading and to what extent the shell facilitates enzymatic activity. Here, we explore these questions using the model Thermotoga maritima nanocompartment known to encapsulate a redox-active ferritin-like protein. Biochemical analysis revealed the encapsulin shell to possess a flavin binding site located at the interface between capsomere subunits, suggesting the shell may play a direct and active role in the function of the encapsulated cargo. Furthermore, we used cryo-EM to show that cargo proteins use a form of symmetry-matching to facilitate encapsulation and define stoichiometry. In the case of the Thermotoga maritima encapsulin, the decameric cargo protein with fivefold symmetry preferentially binds to the pentameric-axis of the icosahedral shell. Taken together, these observations suggest the shell is not simply a passive barrier—it also plays a significant role in the structure and function of the cargo enzyme.

Time-Resolved Studies of Ytterbium Distribution at Interfacial Surfaces of Ferritin-like Dps Protein Demonstrate Metal Uptake and Storage Pathways.

Cage-shaped protein (CSP) complexes are frequently used in bionanotechnology, and they have a variety of different architectures and sizes. The smallest cage-shaped protein, Dps (DNA binding protein from starved cells), can naturally form iron oxide biominerals in a multistep process of ion attraction, translocation, oxidation, and nucleation. The structural basis of this biomineralization mechanism is still unclear. The aim of this paper is to further develop understanding of this topic. Time-resolved metal translocation of Yb3+ ions has been investigated on Dps surfaces using X-ray crystallography. The results reveal that the soak time of protein crystals with Yb3+ ions strongly affects metal positions during metal translocation, in particular, around and inside the ion translocation pore. We have trapped a dynamic state with ongoing translocation events and compared this to a static state, which is reached when the cavity of Dps is entirely filled by metal ions and translocation is therefore blocked. By comparison with La3+ and Co2+ datasets, the time-dependence together with the coordination sphere chemistry primarily determine metal−protein interactions. Our data can allow structure-based protein engineering to generate CSPs for the production of tailored nanoparticles.

Cryo-EM structure of a thermophilic encapsulin offers clues to its functions.

Wiryaman & Toor [IUCrJ (2021). 8, 342-350] report the cryo-EM structure of a Thermotoga maritima encapsulin, a nanocompartment that encapsulates a ferritin-like protein cargo. The 2 Å resolution structure offers insights into the active role of this thermostable encapsulin in regulating iron homeostasis to reduce oxidative stress.

His-Mediated Reversible Self-Assembly of Ferritin Nanocages through Two Different Switches for Encapsulation of Cargo Molecules.

Protein nanocages represent a class of nanovehicles for a variety of applications. However, precise manipulation of self-assembly behavior of these protein nanocages in response to multiple external stimuli for custom-tailored applications remains challenging. Herein, we established a simple but effective strategy for controlling protein nanocage self-assembly that combines a dual property of His motifs (their significantly pH-dependent protonation state and their capacity to coordinate with transition metals) with its high symmetry. With this strategy, we enabled two different ferritin nanocages to disassemble into protein tetramers under neutral solution by introducing His6 motifs at the 4-fold channel interfaces. Notably, these tetramers are able to self-assemble into ferritin-like protein nanocages in response to multiple external stimuli such as transition metal ions and pH, and vice versa, indicative of a reversible self-assembly process. Furthermore, such His-mediated reversible protein self-assembly has been explored for encapsulation of bioactive cargo molecules within these reconstituted protein nanocages with higher loading efficiency under milder conditions as compared to the reported acid denaturation encapsulation method for ferritin.

Global transcriptomic and proteomics analysis of Lactobacillus plantarum Y44 response to 2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH) stress

Our previous study demonstrated that Lactobacillus plantarum Y44 exhibited antioxidant activity. However, the physiological characteristics of L. plantarum Y44 exposure to oxidative stress was not clear. In this research, the differentially expressed proteins and genes in L. plantarum Y44 under 2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH) stress at different concentrations were studied by using integrated transcriptomic and proteomic methods. Under 100 mM AAPH stress condition, 1139 differentially expressed genes (DEGs, 546 up-regulated and 593 down-regulated) and 329 differentially expressed proteins (DEPs, 127 up-regulated and 202 down-regulated) were observed. Under 200 mM AAPH stress condition, 1526 DEGs (751 up-regulated and 775 down-regulated) and 382 DEPs (139 up-regulated and 243 down-regulated) were observed. Overall, we found that L. plantarum Y44 fought against AAPH induced oxidative stress by up-regulating antioxidant enzymes and DNA repair proteins, such as ATP-dependent DNA helicase RuvA, adenine DNA glycosylase, single-strand DNA-binding protein SSB, DNA-binding ferritin-like protein DPS, thioredoxin reductase, protein-methionine-S-oxide reductase and glutathione peroxidase. Additionally, cell envelope composition of L. plantarum Y44 was highly remodeled by accelerating peptidoglycan and teichoic-acid (LTA) biosynthesis and modulating the fatty acids (FA) composition to achieve a higher ratio of unsaturated/saturated fatty acids (UFAs/SFAs) against AAPH stress. Moreover, metabolism processes including carbohydrate metabolism, amino acid biosynthesis, and nucleotide metabolism altered to respond to AAPH-induced damage. Altogether, our findings allow us to facilitate a better understanding of L. plantarum Y44 against oxidative stress. SignificanceThis study represents an integrated proteomic and transcriptomic analysis of Lactobacillus plantarum Y44 response to 2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH) stress. Differentially expressed proteins and genes were identified between the proteome and transcriptome of L. plantarum Y44 under different AAPH stress. AAPH-induced response of L. plantarum Y44 appears to be primarily based on ROS scavenging, DNA repair, highly remodeled cell surface and specific metabolic processes. The knowledge about these proteomes and transcriptomes provides significant insights into the oxidative stress response of Lactobacillus plantarum.

16-Mer ferritin-like protein templated gold nanoclusters for bioimaging detection of methylmercury in the brain of living mice

Methylmercury (MeHg+) as one of the most potent neurotoxins is mainly accumulated in brain, so in vivo imaging detection of MeHg+ in brain is of crucial importance. Herein, we reported a photoluminescent nanosensor for MeHg+ detection in brain by integrating the bioimaging of gold nanoclusters (Au NCs), the fluorescence of Au NCs quenched by MeHg+, and the brain targeting feature of our recently constructed 16-mer shell-like protein (7A). First, Au NCs with 7A as a biotemplate (7A-Au NCs) by a facile and green method in water are fabricated for the first time, the fluorescence of which (∼650 nm) can be quenched by MeHg+ in a dose-dependent manner in vitro. Second, the as-prepared 7A-Au NCs are not only suitable for bioimaging of BBB endothelial cells, but also are an effective probe for bioimaging MeHg+ detection in a brain-specific manner. These findings open a door for MeHg+ detection in the brain of living subjects.

Metal Positions and Translocation Pathways of the Dodecameric Ferritin-like Protein Dps.

Iron storage in biology is carried out by cage-shaped proteins of the ferritin superfamily, one of which is the dodecameric protein Dps. In Dps, four distinct steps lead to the formation of metal nanoparticles: attraction of ion-aquo complexes to the protein matrix, passage of these complexes through translocation pores, oxidation of these complexes at ferroxidase centers, and, ultimately, nanoparticle formation. In this study, we investigated Dps from Listeria innocua to structurally characterize these steps for Co2+, Zn2+, and La3+ ions. The structures reveal that differences in their ion coordination chemistry determine alternative metal ion-binding sites on the areas of the surface surrounding the translocation pore that captures nine La3+, three Co2+, or three Zn2+ ions as aquo clusters and passes them on for translocation. Inside these pores, ion-selective conformational changes at key residues occur before a gating residue to actively move ions through the constriction zone. Ions upstream of the Asp130 gate residue are typically hydrated, while ions downstream directly interact with the protein matrix. Inside the cavity, ions move along negatively charged residues to the ferroxidase center, where seven main residues adapt to the three different ions by dynamically changing their conformations. In total, we observed more than 20 metal-binding sites per Dps monomer, which clearly highlights the metal-binding capacity of this protein family. Collectively, our results provide a detailed structural description of the preparative steps for amino acid-assisted biomineralization in Dps proteins, demonstrating unexpected protein matrix plasticity.

Bacterioferritin of Magnetospirillum gryphiswaldense Is a Heterotetraeicosameric Complex Composed of Functionally Distinct Subunits but Is Not Involved in Magnetite Biomineralization.

The biomineralization pathway of magnetite in magnetotactic bacteria is still poorly understood and a matter of intense debates. In particular, the existence, nature, and location of possible mineral precursors of magnetite are not clear. One possible precursor has been suggested to be ferritin-bound ferrihydrite. To clarify its role for magnetite biomineralization, we analyzed and characterized ferritin-like proteins from the magnetotactic alphaproteobacterium Magnetospirillum gryphiswaldense MSR-1, employing genetic, biochemical, and spectroscopic techniques. Transmission Mössbauer spectroscopy of the wild type (WT) and a bacterioferritin (bfr) deletion strain uncovered that the presence of ferrihydrite in cells is coupled to the presence of Bfr. However, bfr and dps deletion mutants, encoding another ferritin-like protein, or even mutants with their codeletion had no impact on magnetite formation in MSR-1. Thus, ferritin-like proteins are not involved in magnetite biomineralization and Bfr-bound ferrihydrite is not a precursor of magnetite biosynthesis. Using transmission electron microscopy and bacterial two-hybrid and electrophoretic methods, we also show that MSR-1 Bfr is an atypical representative of the Bfr subfamily, as it forms tetraeicosameric complexes from two distinct subunits. Furthermore, our analyses revealed that these subunits are functionally divergent, with Bfr1 harboring a ferroxidase activity while only Bfr2 contributes to heme binding. Because of this functional differentiation and the poor formation of homooligomeric Bfr1 complexes, only heterooligomeric Bfr protects cells from oxidative stress in vivo. In summary, our results not only provide novel insights into the biomineralization of magnetite but also reveal the unique properties of so-far-uncharacterized heterooligomeric bacterioferritins.IMPORTANCE Magnetotactic bacteria like Magnetospirillum gryphiswaldense are able to orient along magnetic field lines due to the intracellular formation of magnetite nanoparticles. Biomineralization of magnetite has been suggested to require a yet-unknown ferritin-like ferrihydrite component. Here, we report the identification of a bacterioferritin as the source of ferrihydrite in M. gryphiswaldense and show that, contrary to previous reports, bacterioferritin is not involved in magnetite biomineralization but required for oxidative stress resistance. Additionally, we show that bacterioferritin of M. gryphiswaldense is an unusual member of the bacterioferritin subfamily as it is composed of two functionally distinct subunits. Thus, our findings extend our understanding of the bacterioferritin subfamily and also solve a longstanding question about the magnetite biomineralization pathway.

A Mutation Directs the Structural Switch of DNA Binding Proteins under Starvation to a Ferritin-like Protein Cage

Proteins of the ferritin family are ubiquitous in living organisms. With their spherical cage-like structures they are the iron storehouses in cells. Subfamilies of ferritins include 24-meric ferritins and bacterioferritins (maxiferritins), and 12-meric Dps (miniferritins). Dps safeguards DNA by direct binding, affording physical protection and safeguards from free radical-mediated damage by sequestering iron in its core. The maxiferritins can oxidize and store iron but cannot bind DNA. Here we show that a mutation at a critical interface in Dps alters its assembly from the canonical 12-mer to a ferritin-like 24-mer under crystallization. This structural switch was attributed to theconformational alteration of a highly conserved helical loop and rearrangement of the C-terminus. Our results demonstrate a novel concept of mutational switch between related protein subfamilies and corroborate the popular model for evolution by which subtle substitutions in an amino acid sequence lead to diversification among proteins.

Identification and Differential Expression of Biomineralization Genes in the Mantle of Pearl Oyster Pinctada fucata.

A series of proteins are involved in shell formation of the pearl oyster Pinctada fucata, but the involved mechanisms and the relative expression levels of these proteins have not been elucidated. In this study, we sequenced and characterized the transcriptome of P. fucata mantle tissue. A total of 100,679 unique transcripts were assembled, 43687 Unigenes were annotated, and 48654 CDSs were determined. Of these, GO annotated 16353 Unigenes, COG defined 11585 unigenes into 25 categories, and KEGG sorted 25053 unigenes into 258 pathways. In total, 67 biomineralization-related genes were identified, of which 23 genes were newly described in P. fucata. These genes included ones that expressed shell matrix proteins, regulatory factors, and uncharacterized genes. Differential expression of these 67 genes and 9 other biomineralization-related genes was confirmed using qPCR. Of the 8 nacreous layer-related genes, MSI60 (774.00) was expressed at a much higher level than the others. KRMP2-4 and MSI31 were the most highly expressed of the 13 prismatic layer-related genes and KRMP2 was expressed at nearly 10000 times of the level of the 18S gene. For genes related to both layers, shematrin 2 (3977.84), nacrein (2404.75), PFMG 10 (2113.93), and PFMG 4 (1015.89) were highly expressed, and ferritin-like protein (877.54) and PFMG 8 (516.48) were highly expressed among the 16 undefined genes. The expression levels of regulation factors were generally low, and the highest level was 324.09 (EF-hand) and the lowest occurred in the BMP and wnt families. The expression levels of the prismatic matrix proteins were much higher than those of nacreous ones, consistent with a thicker prismatic layer. MSI60 and nacrein are likely the main components of the nacreous layer, and KRMP2-4, MSI31, shematrin 2, and PFMG 10 gene products are the main components of the prismatic layer. This is the first report of transient expression levels of a large number of biomineralization-related genes at the same time in mantle tissue of P. fucata. These findings provide a novel perspective to understand the molecular mechanisms of shell formation and will be beneficial to genetic improvement of P. fucata for the production of high-quality pearls as well.