Development of an Efficient FRET-Based Ratiometric Uranium Biosensor.

The dispersion of uranium in the environment can pose a problem for the health of humans and other living organisms. It is therefore important to monitor the bioavailable and hence toxic fraction of uranium in the environment, but no efficient measurement methods exist for this. Our study aims to fill this gap by developing a genetically encoded FRET-based ratiometric uranium biosensor. This biosensor was constructed by grafting two fluorescent proteins to both ends of calmodulin, a protein that binds four calcium ions. By modifying the metal-binding sites and the fluorescent proteins, several versions of the biosensor were generated and characterized in vitro. The best combination results in a biosensor that is affine and selective for uranium compared to metals such as calcium or other environmental compounds (sodium, magnesium, chlorine). It has a good dynamic range and should be robust to environmental conditions. In addition, its detection limit is below the uranium limit concentration in drinking water defined by the World Health Organization. This genetically encoded biosensor is a promising tool to develop a uranium whole-cell biosensor. This would make it possible to monitor the bioavailable fraction of uranium in the environment, even in calcium-rich waters.

Optimized Coordination of Uranyl in Engineered Calmodulin Site 1 Provides a Subnanomolar Affinity for Uranyl and a Strong Uranyl versus Calcium Selectivity.

As an alpha emitter and chemical toxicant, uranium toxicity in living organisms is driven by its molecular interactions. It is therefore essential to identify main determinants of uranium affinity for proteins. Others and we showed that introducing a phosphoryl group in the coordination sphere of uranyl confers a strong affinity of proteins for uranyl. In this work, using calmodulin site 1 as a template, we modulate the structural organization of a metal-binding loop comprising carboxylate and/or carbonyl ligands and reach affinities for uranyl comparable to that provided by introducing a strong phosphoryl ligand. Shortening the metal binding loop of calmodulin site 1 from 12 to 10 amino acids in CaMΔ increases the uranyl-binding affinity by about 2 orders of magnitude to log KpH7 = 9.55 ± 0.11 (KdpH7 = 280 ± 60 pM). Structural analysis by FTIR, XAS, and molecular dynamics simulations suggests an optimized coordination of the CaMΔ-uranyl complex involving bidentate and monodentate carboxylate groups in the uranyl equatorial plane. The main role of this coordination sphere in reaching subnanomolar dissociation constants for uranyl is supported by similar uranyl affinities obtained in a cyclic peptide reproducing CaMΔ binding loop. In addition, CaMΔ presents a uranyl/calcium selectivity of 107 that is even higher in the cyclic peptide.

Determination of drug efficacy to dissolve cobalt oxide particles in cellular models: Towards a therapeutic approach to decrease pulmonary retention.

Following accidental inhalation of radioactive cobalt particles, the poorly soluble and highly radioactive Co3O4 particles are retained for long periods in lungs. To decrease their retention time is of crucial importance to minimize radiation-induced damage. As dissolved cobalt is quickly transferred to blood and eliminated by urinary excretion, enhancing the dissolution of particles would favor 60Co elimination. We evaluated the ability of ascorbic acid alone or associated with the chelating agents DTPA1, DFOB2 or EDTA3 to enhance dissolution of cobalt particles after macrophage engulfment, and the drug effects on the translocation of the soluble species CoCl2 through an epithelial barrier. We exposed differentiated THP-1 macrophage-like cells and Calu-3 lung epithelial cells cultured in a bicameral system to cobalt and selected molecules up to 7 days. DTPA, the recommended treatment in man, used alone showed no effect, whereas ascorbic acid significantly increased dissolution of Co3O4 particles. An additional efficacy in intracellular particles dissolution was observed for combinations of ascorbic acid with DTPA and EDTA. Except for DFOB, treatments did not significantly modify translocation of dissolved cobalt across the epithelial lung barrier. Our study provides new insights for decorporating strategies following radioactive cobalt particle intake.

Discovery and characterization of UipA, a uranium- and iron-binding PepSY protein involved in uranium tolerance by soil bacteria.

Uranium is a naturally occurring radionuclide. Its redistribution, primarily due to human activities, can have adverse effects on human and non-human biota, which poses environmental concerns. The molecular mechanisms of uranium tolerance and the cellular response induced by uranium exposure in bacteria are not yet fully understood. Here, we carried out a comparative analysis of four actinobacterial strains isolated from metal and radionuclide-rich soils that display contrasted uranium tolerance phenotypes. Comparative proteogenomics showed that uranyl exposure affects 39-47% of the total proteins, with an impact on phosphate and iron metabolisms and membrane proteins. This approach highlighted a protein of unknown function, named UipA, that is specific to the uranium-tolerant strains and that had the highest positive fold-change upon uranium exposure. UipA is a single-pass transmembrane protein and its large C-terminal soluble domain displayed a specific, nanomolar binding affinity for UO22+ and Fe3+. ATR-FTIR and XAS-spectroscopy showed that mono and bidentate carboxylate groups of the protein coordinated both metals. The crystal structure of UipA, solved in its apo state and bound to uranium, revealed a tandem of PepSY domains in a swapped dimer, with a negatively charged face where uranium is bound through a set of conserved residues. This work reveals the importance of UipA and its PepSY domains in metal binding and radionuclide tolerance.

In vitro assessment of cobalt oxide particle dissolution in simulated lung fluids for identification of new decorporating agents.

Inhalation of 60Co3O4 particles may occur at the work place in nuclear industry. Their low solubility may result in chronic lung exposure to γ rays. Our strategy for an improved therapeutic approach is to enhance particle dissolution to facilitate cobalt excretion, as the dissolved fraction is rapidly eliminated, mainly in urine. In vitro dissolution of Co3O4 particles was assessed with two complementary assays in lung fluid surrogates to mimic a pulmonary contamination scenario. Twenty-one molecules and eleven combinations were selected through an extensive search in the literature, based on dissolution studies of other metal oxides (Fe, Mn, Cu) and tested for dissolution enhancement of cobalt particles after 1-28 days of incubation. DTPA, the recommended treatment following cobalt contamination did not enhance 60Co3O4 particles dissolution when used alone. However, by combining molecules with different properties, such as redox potential and chelating ability, we greatly improved the efficacy of each drug used alone, leading for the highest efficacy, to a 2.7 fold increased dissolution as compared to controls. These results suggest that destabilization of the particle surface is an important initiating event for a good efficacy of chelating drugs, and open new perspectives for the identification of new therapeutic strategies.

Uranium Uptake in Paracentrotus lividus Sea Urchin, Accumulation and Speciation.

Uranium speciation and bioaccumulation were investigated in the sea urchin Paracentrotus lividus. Through accumulation experiments in a well-controlled aquarium followed by ICP-OES analysis, the quantification of uranium in the different compartments of the sea urchin was performed. Uranium is mainly distributed in the test (skeletal components), as it is the major constituent of the sea urchin, but in terms of quantity of uranium per gram of compartment, the following rating: intestinal tract > gonads ≫ test, was obtained. Combining both extended X-ray Absorption Spectroscopy and time-resolved laser-induced fluorescence spectroscopic analysis, it was possible to identify two different forms of uranium in the sea urchin, one in the test, as a carbonato-calcium complex, and the second one in the gonads and intestinal tract, as a protein complex. Toposome is a major calcium-binding transferrin-like protein contained within the sea urchin. EXAFS data fitting of both contaminated organs in vivo and the uranium-toposome complex from protein purified out of the gonads revealed that it is suspected to complex uranium in gonads and intestinal tract. This hypothesis is also supported by the results from two imaging techniques, i.e., Transmission Electron Microscopy and Scanning Transmission X-ray Microscopy. This thorough investigation of uranium uptake in sea urchin is one of the few attempts to assess the speciation in a living marine organism in vivo.

Proteogenomic insights into uranium tolerance of a Chernobyl's Microbacterium bacterial isolate.

Microbacterium oleivorans A9 is a uranium-tolerant actinobacteria isolated from the trench T22 located near the Chernobyl nuclear power plant. This site is contaminated with different radionuclides including uranium. To observe the molecular changes at the proteome level occurring in this strain upon uranyl exposure and understand molecular mechanisms explaining its uranium tolerance, we established its draft genome and used this raw information to perform an in-depth proteogenomics study. High-throughput proteomics were performed on cells exposed or not to 10µM uranyl nitrate sampled at three previously identified phases of uranyl tolerance. We experimentally detected and annotated 1532 proteins and highlighted a total of 591 proteins for which abundances were significantly differing between conditions. Notably, proteins involved in phosphate and iron metabolisms show high dynamics. A large ratio of proteins more abundant upon uranyl stress, are distant from functionally-annotated known proteins, highlighting the lack of fundamental knowledge regarding numerous key molecular players from soil bacteria. BIOLOGICAL SIGNIFICANCE: Microbacterium oleivorans A9 is an interesting environmental model to understand biological processes engaged in tolerance to radionuclides. Using an innovative proteogenomics approach, we explored its molecular mechanisms involved in uranium tolerance. We sequenced its genome, interpreted high-throughput proteomic data against a six-reading frame ORF database deduced from the draft genome, annotated the identified proteins and compared protein abundances from cells exposed or not to uranyl stress after a cascade search. These data show that a complex cellular response to uranium occurs in Microbacterium oleivorans A9, where one third of the experimental proteome is modified. In particular, the uranyl stress perturbed the phosphate and iron metabolic pathways. Furthermore, several transporters have been identified to be specifically associated to uranyl stress, paving the way to the development of biotechnological tools for uranium decontamination.

Structural Analysis of Uranyl Complexation by the EF-Hand Motif of Calmodulin: Effect of Phosphorylation.

Better understanding of uranyl-protein interactions is a prerequisite to predict uranium chemical toxicity in cells. The EF-hand motif of the calmodulin site I is about thousand times more affine for uranyl than for calcium, and threonine phosphorylation increases the uranyl affinity by two orders of magnitude at pH 7. In this study, we confront X-ray absorption spectroscopy with Fourier transform infrared (FTIR) spectroscopy, time-resolved laser-induced fluorescence spectroscopy (TRLFS), and structural models obtained by molecular dynamics simulations to analyze the uranyl coordination in the native and phosphorylated calmodulin site I. For the native site I, extended X-ray absorption fine structure (EXAFS) data evidence a short U-Oeq distance, in addition to distances compatible with mono- and bidentate coordination by carboxylate groups. Further analysis of uranyl speciation by TRLFS and thorough investigation of the fluorescence decay kinetics strongly support the presence of a hydroxide uranyl ligand. For a phosphorylated site I, the EXAFS and FTIR data support a monodentate uranyl coordination by the phosphoryl group and strong interaction with mono- and bidentate carboxylate ligands. This study confirms the important role of a phosphoryl ligand in the stability of uranyl-protein interactions. By evidencing a hydroxide uranyl ligand in calmodulin site I, this study also highlights the possible role of less studied ligands as water or hydroxide ions in the stability of protein-uranyl complexes.

Use of combined microscopic and spectroscopic techniques to reveal interactions between uranium and Microbacterium sp. A9, a strain isolated from the Chernobyl exclusion zone.

Although uranium (U) is naturally found in the environment, soil remediation programs will become increasingly important in light of certain human activities. This work aimed to identify U(VI) detoxification mechanisms employed by a bacteria strain isolated from a Chernobyl soil sample, and to distinguish its active from passive mechanisms of interaction. The ability of the Microbacterium sp. A9 strain to remove U(VI) from aqueous solutions at 4 °C and 25 °C was evaluated, as well as its survival capacity upon U(VI) exposure. The subcellular localisation of U was determined by TEM/EDX microscopy, while functional groups involved in the interaction with U were further evaluated by FTIR; finally, the speciation of U was analysed by TRLFS. We have revealed, for the first time, an active mechanism promoting metal efflux from the cells, during the early steps following U(VI) exposure at 25 °C. The Microbacterium sp. A9 strain also stores U intracellularly, as needle-like structures that have been identified as an autunite group mineral. Taken together, our results demonstrate that this strain exhibits a high U(VI) tolerance based on multiple detoxification mechanisms. These findings support the potential role of the genus Microbacterium in the remediation of aqueous environments contaminated with U(VI) under aerobic conditions.

Microbacterium lemovicicum sp. nov., a bacterium isolated from a natural uranium-rich soil.

An actinobacterial strain, designated ViU22(T), was isolated from a natural uranium-rich soil and was studied using a polyphasic approach. Cells formed orange-pigmented colonies, were rod-shaped, Gram-positive (non-staining method), non-motile and non-spore-forming. This organism grew in 0-4.5 % (w/v) NaCl and at 15-37 °C, with optimal growth occurring in 0.5 % (w/v) NaCl and at 30 °C. Comparative 16S rRNA gene sequence analysis revealed that the strain ViU22(T) belonged to the genus Microbacterium. It exhibited highest 16S rRNA gene sequence similarity with the type strains of Microbacterium testaceum (98.14 %) and Microbacterium binotii (98.02 %). The DNA-DNA relatedness of strains ViU22(T) with the most closely related type strains Microbacterium testaceum and Microbacterium binotii DSM 19164(T) was 20.10 % (± 0.70) and 28.05 % (± 0.35), respectively. Strain ViU22(T) possessed a type B2ß peptidoglycan with partial substitution of glutamic acid by 3-hydroxy glutamic acid. The major menaquinones were MK-11 and MK-12. Major polar lipids detected in the strain ViU22(T) were diphosphatidylglycerol, phosphatidylglycerol, an unknown phospholipid and unknown glycolipids. The predominant fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0, a pattern reported for other Microbacterium species. The major cell-wall sugars were galactose, xylose and mannose and the DNA G+C content was 71 mol%. Together, the DNA-DNA hybridization results and the differentiating phenotypic characteristics, showed that strain ViU22(T) should be classified as the type strain of a novel species within the genus Microbacterium, for which the name Microbacterium lemovicicum sp. nov. is proposed. The type strain is ViU22(T) ( = ATCC BAA-2396(T) = CCUG 62198(T) = DSM 25044(T)).

Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T(9)TKE(12) sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K(d) = 25±6 nM to K(d) = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K(d) = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν(as)(P-O) and ν(s)(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν(as)(UO(2))(2+) vibration (from 923 cm(-1) to 908 cm(-1)) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.

Influence of uranium on bacterial communities: a comparison of natural uranium-rich soils with controls.

This study investigated the influence of uranium on the indigenous bacterial community structure in natural soils with high uranium content. Radioactive soil samples exhibiting 0.26% - 25.5% U in mass were analyzed and compared with nearby control soils containing trace uranium. EXAFS and XRD analyses of soils revealed the presence of U(VI) and uranium-phosphate mineral phases, identified as sabugalite and meta-autunite. A comparative analysis of bacterial community fingerprints using denaturing gradient gel electrophoresis (DGGE) revealed the presence of a complex population in both control and uranium-rich samples. However, bacterial communities inhabiting uraniferous soils exhibited specific fingerprints that were remarkably stable over time, in contrast to populations from nearby control samples. Representatives of Acidobacteria, Proteobacteria, and seven others phyla were detected in DGGE bands specific to uraniferous samples. In particular, sequences related to iron-reducing bacteria such as Geobacter and Geothrix were identified concomitantly with iron-oxidizing species such as Gallionella and Sideroxydans. All together, our results demonstrate that uranium exerts a permanent high pressure on soil bacterial communities and suggest the existence of a uranium redox cycle mediated by bacteria in the soil.

Structural consequences of binding of UO2(2+) to apotransferrin: can this protein account for entry of uranium into human cells?

It has been established that transferrin binds a variety of metals. These include toxic uranyl ions which form rather stable uranyl-transferrin derivatives. We determined the extent to which the iron binding sites might accommodate the peculiar topographic profile of the uranyl ion and the consequences of its binding on protein conformation. Indeed, metal intake via endocytosis of the transferrin/transferrin receptor depends on the adequate coordination of the metal in its site, which controls protein conformation and receptor binding. Using UV-vis and Fourier transform infrared difference spectroscopy coupled to a microdialysis system, we showed that at both metal binding sites two tyrosines are uranyl ligands, while histidine does not participate with its coordination sphere. Analysis by circular dichroism and differential scanning calorimetry (DSC) showed major differences between structural changes associated with interactions of iron or uranyl with apotransferrin. Uranyl coordination reduces the level of protein stabilization compared to iron, but this may be simply related to partial lobe closure. The lack of interaction between uranyl-TF and its receptor was shown by flow cytometry using Alexa 488-labeled holotransferrin. We propose a structural model summarizing our conclusion that the uranyl-TF complex adopts an open conformation that is not appropriate for optimal binding to the transferrin receptor.