[en] The humane transferrin is responsible for the transport of iron in the body and has strong affinity with tetravalent plutonium. Affinity is characterized by a parameter called complexation constant. In 1991 it was concluded that transferrin affinity for plutonium was similar to that for iron but this result needed experimental backing that was impossible to get at that time. The coupling between capillary electrophoresis and mass spectroscopy has shown that the complexation constant between transferrin and plutonium is 10.000 times more important than that between transferrin and iron. (A.C.)

[en] In spite of an increasing number of publications in recent years, information regarding the mechanism of uranium interaction with proteins at the molecular level is limited and few quantitative studies have investigated the binding properties of uranyl with proteins or peptides. It is thus of great interest to better characterize these interactions, and to analyze structural factors governing uranyl binding and thermodynamic stabilization in proteins. Research in this direction will benefit our understanding of the molecular factors governing uranyl toxicity and speciation in cells and will also aid in developing new molecules for selectively binding uranium that could be used for uranium bioremediation purposes. Uranyl coordination properties have similarities with those of calcium, i.e electrostatic interactions preferentially with hard donor oxygen ligands and pentagonal bipyramidal structures. The EF-hand structural motif is the most prevalent Ca2^+-binding site in proteins and is very appealing to analyse uranyl binding properties and develop affine and specific uranyl binding sites for biotechnology approaches. We selected the recombinant N-terminal domain of calmodulin from A. thaliana as a structured template that contains two EF-hand motifs (site I and site II) to analyze its uranyl binding properties and to engineer peptide variants with increased uranyl affinity and specificity. We showed that both site I and site II bind uranyl, with a dissociation constant in the nano-molar range for site I (K_d ≅ 25 nM at pH 6). Using in vitro phosphorylation of a threonine located in the uranyl binding loop, we measured how adding a phosphoryl group affects the calcium and uranium binding affinities. The phosphorylated peptide exhibited a very large affinity for uranyl at pH 7, with a dissociation constant in the sub-nano-molar range K_d = 0.25 ±0.06 nM, and FTIR analysis demonstrated that the phosphoryl group plays a determining role in uranyl binding affinity. We then combined site directed mutagenesis and a modeling approach based on molecular dynamics to increase both the affinity and specificity of the EF-hand motif for uranyl. We could thus obtain a uranyl binding motif with a dissociation constant of 200 pM at pH 6 without phosphorylation and a uranyl/calcium specificity of 10⁷. These results may open new routes for environmental monitoring of uranium based on the use of engineered peptides with high uranyl affinity and specificity. Document available in abstract form only. (authors)

[en] In this study, we explored the microbial diversity of Bacteria and Archaea evolving since 25 years in a radioactive-waste repository trench located in the Chernobyl exclusion zone. To assess the effect of long-term RNs exposure on diversity, microbial assemblages of soil samples highly contaminated with radionuclides (RNs) such as ¹³⁷Cs and uranium were compared with nearby controls using high throughput pyro-sequencing of 16S rRNA genes. The analysis of 690,023 sequences evidenced high diversity in all samples with 34 bacterial and 2 archaeal phylum represented. Chloroflexi, Acidobacteria, Proteobacteria and Verrucomicrobia were the most consistently detected phyla, representing 90% of all sequences.This result demonstrates that a long term exposure did not lead to the decrease of microbial diversity. Furthermore, principal component analysis of pyro-sequencing data showed that microbial communities of RNs contaminated samples differed significantly from that of controls, suggesting the presence of RNs adapted species in the contaminated samples. Several heterotrophic aerobic bacteria have been cultured from the contaminated samples. Among them, the strain Microbacterium sp. A9 exhibited high uranium tolerance. The interaction between this strain and uranium was investigated by a combination of spectroscopic (FTIR and TRLFS) and microscopic (TEM/EDX) approaches. Comparison of data obtained at 4 and 25 deg. C evidenced active and passive mechanisms of uranium uptake and release. We demonstrated that after a first step of uranium and phosphate release via an active efflux mechanism, Microbacterium sp. A9 accumulates U(VI) as intracellular needle-like structures composed of autunite. The functional groups involved in the interactions with uranium were identified. Document available in abstract form only. (authors)

[en] 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 micro-dialysis 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 apo-transferrin. 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 holo-transferrin. 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. (authors)

[en] Complete text of publication follows: Shortly after the Chernobyl accident in 1986, vegetation, contaminated soil and other radioactive debris were buried in situ in trenches. The aims of this work are to analyse the structure of bacterial communities evolving in this environment since 20 years, and to evaluate the potential role of microorganisms in radionuclide migration in soils. Therefore, soil samples exhibiting contrasted radionuclides content were collected in and around the trench number 22. Bacterial communities were examined using a genetic fingerprinting method that allowed a comparative profiling of the samples (DGGE), with universal and group-specific PCR primers. Our results indicate that Chernobyl soil samples host a wide diversity of Bacteria, with stable patterns for Firmicutes and Actinobacteria and more variable for Proteobacteria. A collection of 650 aerobic and anaerobic culturable isolates was also constructed. A phylogenetic analysis of 250 heterotrophic aerobic isolates revealed that 5 phyla are represented: Beta-, Gamma-proteobacteria, Actinobacteria, Bacteroidetes and spore-forming Firmicutes, which is largely dominant. These collection will be screened for the presence of radionuclide-accumulating species in order to estimate the potential influence of microorganisms in radionuclides migration in soils

[en] Complete text of publication follows: Characterization of heavy metals interactions with proteins is fundamental for understanding the molecular factors and mechanisms governing ions toxicity and speciation in cells. This line of research will also help in developing new molecules able to selectively and efficiently bind toxic metal ions, which could find application for bio-detection or bioremediation purposes. We have used the regulatory calcium-binding protein Calmodulin (CaM) from A. thaliana as a structural model and, starting from it, we have designed various mutants by site-directed mutagenesis. We have analysed thermodynamics of uranyl ion binding to both sites I and II of CaM N-terminal domain and we have identified structural factors governing this interaction. Selectivity for uranyl ion has been tested by studying reactions of the investigated peptides with Ca"2"+, in the same conditions used for UO_2"2"+. Spectro-fluorimetric titrations and FTIR analysis have shown that the affinity for uranyl increases by phosphorylation of a threonine in site I, especially approaching the physiological pH, where the phospho-threonine side chain is deprotonated. Based on structural models obtained by Molecular Dynamics, we tested the effect of a two residues deletion on site I properties. We obtained an almost two orders of magnitude increase in affinity for uranyl, with a sub-nanomolar dissociation constant for the uranyl complex with the non phosphorylated peptide, and an improved uranyl/calcium selectivity. Allosteric effects depending on Ca"2"+ and UO_2"2"+ binding have been investigated by comparing thermodynamic parameters obtained for mutants having both sites I and II able to chelate metal ions with those of mutants consisting of just one active site

[en] Actinide elements, all radioactive, are present on Earth for a period several orders of magnitude longer than the life cycle of a human being. Their toxicology is therefore a societal issue, even in the absence of accidental nuclear events. Since the first studies conducted during World War II, actinide toxicologists have studied their retention and excretion rates, their physiological impact in case of exposure and their main biological targets. With the increasing access to unconventional spectroscopic and analytical tools and the growing role of modeling and structural biology, the understanding of the impact of actinides on living organisms at the biomolecular level has improved. The modes of interaction with metabolites and proteins, from a thermodynamic and structural point of view as well as their biological activity, have been better and better described. This also means understanding the mechanisms of complexation and the influence of physicochemical form on affinity, exploring their consequences on the function of proteins/enzymes for certain targets. This article covers twenty years of so-called 'bio-actinide chemistry' with a selection of examples, illustrating the complementarity of the scientific disciplines involved

[en] 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. (authors)

[en] Highlights: • Shotgun proteomics revealed Se detoxification systems in Stenotrophomonas bentonitica. • Proteins for transport, reduction and volatilization of Se(IV) were identified. • A key role of thiol-containing enzymes in Se(IV) reduction is suggested. • Biotransformation from Se(0)NPs to m-Se and t-Se nanostructures was confirmed. • A cellular model for Se(IV) response in Stenotrophomonas bentonitica was proposed. The widespread use of selenium (Se) in technological applications (e.g., solar cells and electronic devices) has led to an accumulation of this metalloid in the environment to toxic levels. The newly described bacterial strain Stenotrophomonas bentonitica BII-R7 has been demonstrated to reduce mobile Se(IV) to Se(0)-nanoparticles (Se(0)NPs) and volatile species. Amorphous Se-nanospheres are reported to aggregate to form crystalline nanostructures and trigonal selenium. We investigated the molecular mechanisms underlying the biotransformation of Se(IV) to less toxic forms using differential shotgun proteomics analysis of S. bentonitica BII-R7 grown with or without sodium selenite for three different time-points. Results showed an increase in the abundance of several proteins involved in Se(IV) reduction and stabilization of Se(0)NPs, such as glutathione reductase, in bacteria grown with Se(IV), in addition to many proteins with transport functions, including RND (resistance-nodulation-division) systems, possibly facilitating Se uptake. Notably proteins involved in oxidative stress defense (e.g., catalase/peroxidase HPI) were also induced by Se exposure. Electron microscopy analyses confirmed the biotransformation of amorphous nanospheres to trigonal Se. Overall, our results highlight the potential of S. bentonitica in reducing the bioavailability of Se, which provides a basis both for the development of bioremediation strategies and the eco-friendly synthesis of biotechnological nanomaterials.

[en] This file shows two complementary parts: one aiming to a better detection of exposure for man and environment and and other one relative to the treatments to be used when there is a contamination. The development of biological captors is a research axis that could be very useful for nuclear toxicologists that wish to dispose of perceptible measurement tools. In the same idea biological markers could be an important help to determine the toxic quantity in organism in case of internal radioactive contamination. About remedial actions, bacteria are able to reduce, to oxide, to capture pollutants and then it is not insane to use them in efficient and low cost remediation for waters or contaminated lands, especially by trace metals or radioactive compounds. Next to them, plants can offer the same service it is the case for sunflower able to treat water loaded in uranium. This file ends with a review of the different treatments known nowadays as therapies for contamination by radioisotopes used in nuclear industry. (N.C.)