How tobacco (Nicotiana tabacum) BY-2 cells cope with Eu(III) - a microspectroscopic study.

The extensive use of lanthanides in science, industry and high-technology products is accompanied by an anthropogenic input of rare earth elements into the environment. Knowledge of a metal's environmental fate is essential for reasonable risk assessment and remediation approaches. In the present study, Eu(III) was representatively used as a luminescent probe to study the chemical environment and to elucidate the molecular interactions of lanthanides with a suspension cell culture of Nicotiana tabacum BY-2. Biochemical methods were combined with luminescence spectroscopy, two-dimensional microspectroscopic mappings, and data deconvolution methods to resolve the bioassociation behavior and spatial distribution of Eu(III) in plant cells. BY-2 cells were found to gradually take up the metal after exposure to 100 µM Eu(III) without significant loss of viability. Time-resolved luminescence measurements were used to specify the occurrence of Eu(III) species as a function of time, revealing the transformation of an initial Eu(III) species into another after 24 h exposure. Chemical microscopy and subsequent iterative factor analysis reveal the presence of four distinct Eu(III) species located at different cellular compartments, e.g., the cell nucleus, nucleolus and cell walls, which could be assigned to intracellular binding motifs. In addition, a special type of bioaccumulation occurs through the formation of a Eu(III)-containing oxalate biomineral, which is already formed within the first 24 hours after metal exposure. Oxalate crystals were also obtained in analogous experiments with Gd and Sm. These results indicate that tobacco BY-2 cells induce the precipitation of metal oxalate biominerals for detoxification of lanthanides, although they also bind to other cellular ligands at the same time.

Spatially resolved Eu(III) environments by chemical microscopy.

Chemical microscopy combines high-resolution emission spectra with Abbe-limited spatial resolution and is used for studies of inhomogeneous samples at the (sub-)micronscale. The spatial distinction of multiple Eu(III) coordination sites allows for a comprehensive understanding of environmental samples and highlights the applicability of Eu(III) as a molecular probe in medicine and biology.

Curium(III) speciation in the presence of microbial cell wall components.

Trivalent actinides such as Cm(III) are able to strongly interact with microbes and especially with bacterial cell walls. However, detailed knowledge of the influence of different cell wall components is somewhat lacking. For this investigation, we studied the formation of aqueous Cm(III) complexes with cell wall components (e.g., lipopolysaccharide, peptidoglycan, and plasma membranes) using time-resolved laser-induced fluorescence spectroscopy (TRLFS). For all systems, two specific Cm(III) complexes with the biomacromolecules were observed as a function of pH. Specifically, Cm(III) was found to bind to phosphate and carboxyl groups present in the structure of the biomacromolecules. Stability constants and luminescence parameters of the specific Cm(III) complexes were determined and are presented. The pH of the surrounding aqueous solution, the plasma membrane concentration, and proteins included in the crude plasma membrane fraction were found to significantly impact the complexation of Cm(III). The Cm(III) luminescence spectra with plasma membranes, cell wall polymers, as well as Gram-negative (Sporomusa sp. MT-2.99 and Pseudomonas fluorescens) and Gram-positive (Paenibacillus sp. MT-2.2) bacteria will be explained by linear combination fitting using the investigated components.

Detecting Bacterial Cell Viability in Few µL Solutions from Impedance Measurements on Silicon-Based Biochips.

Using two different types of impedance biochips (PS5 and BS5) with ring top electrodes, a distinct change of measured impedance has been detected after adding 1-5 µL (with dead or live Gram-positive Lysinibacillus sphaericus JG-A12 cells to 20 µL DI water inside the ring top electrode. We relate observed change of measured impedance to change of membrane potential of L. sphaericus JG-A12 cells. In contrast to impedance measurements, optical density (OD) measurements cannot be used to distinguish between dead and live cells. Dead L. sphaericus JG-A12 cells have been obtained by adding 0.02 mg/mL of the antibiotics tetracycline and 0.1 mg/mL chloramphenicol to a batch with OD0.5 and by incubation for 24 h, 30 °C, 120 rpm in the dark. For impedance measurements, we have used batches with a cell density of 25.5 × 108 cells/mL (OD8.5) and 270.0 × 108 cells/mL (OD90.0). The impedance biochip PS5 can be used to detect the more resistive and less capacitive live L. sphaericus JG-A12 cells. Also, the impedance biochip BS5 can be used to detect the less resistive and more capacitive dead L. sphaericus JG-A12 cells. An outlook on the application of the impedance biochips for high-throughput drug screening, e.g., against multi-drug-resistant Gram-positive bacteria, is given.

How Do Actinyls Interact with Hyperphosphorylated Yolk Protein Phosvitin?

The development of the nuclear industry has raised multiple questions about its impact on the biotope and humans. Proteins are key biomolecules in cell machinery and essential in deciphering toxicological processes. Phosvitin was chosen as a relevant model for phosphorylated proteins because of its important role as an iron, calcium, and magnesium storage protein in egg yolk. A multitechnique spectroscopic investigation was performed to reveal the coordination geometry of two oxocations of the actinide family (actinyl UVI , NpV ) in speciation with phosvitin. IR spectroscopy revealed phosphoryl groups as the main functional groups interacting with UVI . This was confirmed through laser luminescence spectroscopy (U) and UV/Vis absorption spectroscopy (Np). For UVI , X-ray absorption spectroscopy at the LIII edge revealed a small contribution of bidentate binding present, along with predominantly monodentate binding of phosphoryl groups; for NpV , uniquely bidentate binding was revealed. As a perspective to this work, X-ray absorption spectroscopy speciation of UVI and NpV in the extracted yolk of living eggs of the dogfish Scyliorhinus canicula was determined; this corroborated the binding of phosphorous together with a reduction of the actinyl moiety. Such data are essential to pinpoint the mechanisms of heavy metals (actinyls) accumulation and toxicity in oviparous organisms, and therefore, contribute to a shift from descriptive approaches to predictive toxicology.

Cm3+/Eu3+ induced structural, mechanistic and functional implications for calmodulin.

Trivalent actinides and their lanthanide homologues are being scrutinized for their potential health risk when ingested as a result of a range of industrial activities such as mining. Importantly, these ions are known to exhibit high affinity towards calmodulin (CaM). In case of their inadvertent uptake, the holoproteins that are occupied by these cations may block signal transduction pathways or increase the concentration of these ions in intact cells, which could lead to accumulation in human organs. Accordingly, this investigation employed spectroscopy, computational chemistry, calorimetry, and biochemistry to study the results of metal ion substitution on the protein structure, enzymatic activity and chemo- and cytotoxicity of An3+/Ln3+ ions. As will be demonstrated herein, our data confirm the higher affinity of Cm3+ and Eu3+ compared to Ca2+ to all 4 binding sites of CaM, with one site differing from the remaining three. This higher-affinity site will complex Eu3+ in an exothermic fashion; in contrast, ion binding to the three lower-affinity EF-hands was found to be endothermic. The overall endothermic binding process is ascribed to the loss of the hydration shells of the trivalent ions upon protein binding. These findings are supported by extensive quantum chemical calculations of full holo-CaM, which were performed at the MP2 level using the fragment molecular orbital method. The exceptional binding site (EF-hand 3) features fewer negatively charged residues compared to the other EF-hands, thereby allowing Eu3+ and Cm3+ to carry one or two additional waters compared to Ca2+-CaM, while also causing the structure of Cm3+/Eu3+-CaM to become slightly disordered. Moreover, the enzymatic activity decreases somewhat in comparison to Ca2+-CaM. By utilizing a combination of techniques, we were able to generate a comprehensive picture of the CaM-actinide/lanthanide system from the molecular level to its functional impact. Such knowledge could also be applied to other metal-binding proteins.

Analysis of self-assembly of S-layer protein slp-B53 from Lysinibacillus sphaericus.

The formation of stable and functional surface layers (S-layers) via self-assembly of surface-layer proteins on the cell surface is a dynamic and complex process. S-layers facilitate a number of important biological functions, e.g., providing protection and mediating selective exchange of molecules and thereby functioning as molecular sieves. Furthermore, S-layers selectively bind several metal ions including uranium, palladium, gold, and europium, some of them with high affinity. Most current research on surface layers focuses on investigating crystalline arrays of protein subunits in Archaea and bacteria. In this work, several complementary analytical techniques and methods have been applied to examine structure-function relationships and dynamics for assembly of S-layer protein slp-B53 from Lysinibacillus sphaericus: (1) The secondary structure of the S-layer protein was analyzed by circular dichroism spectroscopy; (2) Small-angle X-ray scattering was applied to gain insights into the three-dimensional structure in solution; (3) The interaction with bivalent cations was followed by differential scanning calorimetry; (4) The dynamics and time-dependent assembly of S-layers were followed by applying dynamic light scattering; (5) The two-dimensional structure of the paracrystalline S-layer lattice was examined by atomic force microscopy. The data obtained provide essential structural insights into the mechanism of S-layer self-assembly, particularly with respect to binding of bivalent cations, i.e., Mg2+ and Ca2+. Furthermore, the results obtained highlight potential applications of S-layers in the fields of micromaterials and nanobiotechnology by providing engineered or individual symmetric thin protein layers, e.g., for protective, antimicrobial, or otherwise functionalized surfaces.

Speciation Studies of Metals in Trace Concentrations: The Mononuclear Uranyl(VI) Hydroxo Complexes.

A direct luminescence spectroscopic experimental setup for the determination of complex stability constants of mononuclear uranyl(VI) hydrolysis species is presented. The occurrence of polynuclear species is prevented by using a low uranyl(VI) concentration of 10­8 M (2.4 ppb). Time-resolved laser-induced fluorescence spectra were recorded in the pH range from 3 to 10.5. Deconvolution with parallel factor analysis (PARAFAC) resulted in three hydrolysis complexes. A tentative assignment was based on thermodynamic calculations: UO22+, UO2(OH)+, UO2(OH)2, UO2(OH)3­. An implementation of a Newton­Raphson algorithm into PARAFAC allowed a direct extraction of complex stability constants during deconvolution yielding log(ß1M,1°C)1:1 = −4.6, log(ß1M,1°C)1:2 = −12.2, log(ß1M,1°C)1:3 = −22.3. Extrapolation to standard conditions gave log(ß0)1:1 = −3.9, log(ß0)1:2 = −10.9, and log(ß0)1:3 = −20.7. Luminescence characteristics (band position, lifetime) of the individual mononuclear hydroxo species were derived to serve as a reference data set for further investigations. A correlation of luminescence spectroscopic features with Raman frequencies was demonstrated for the mononuclear uranyl(VI) hydroxo complexes for the first time. Thereby a signal-to-structure correlation was achieved and the complex assignment validated.

S-Layer-Based Nanocomposites for Industrial Applications.

This chapter covers the fundamental aspects of bacterial S-layers: what are S-layers, what is known about them, and what are their main features that makes them so interesting for the production of nanostructures. After a detailed introduction of the paracrystalline protein lattices formed by S-layer systems in nature the chapter explores the engineering of S-layer-based materials. How can S-layers be used to produce "industry-ready" nanoscale bio-composite materials, and which kinds of nanomaterials are possible (e.g., nanoparticle synthesis, nanoparticle immobilization, and multifunctional coatings)? What are the advantages and disadvantages of S-layer-based composite materials? Finally, the chapter highlights the potential of these innovative bacterial biomolecules for future technologies in the fields of metal filtration, catalysis, and bio-functionalization.

Nanopatterning of Magnetic CrNi Prussian Blue Nanoparticles Using a Bacterial S-Layer as a Biotemplate.

We have developed a simple process to fabricate on a bioplatform patterns of nanoparticles of a molecule-based magnet. Nanoparticles of the ferromagnetic Prussian blue derivative CsxNi[Cr(CN)6] were orderly deposited onto S-layers of Lysinibacillus sphaericus, forming a dense carpet of nanoparticles following the square lattice (p4) pattern of the biotemplate. These results are encouraging to extend this approach by focusing on molecule-based magnets patterned into domains with controlled shapes and positions on a biosurface.

Synthesis of S-layer conjugates and evaluation of their modifiability as a tool for the functionalization and patterning of technical surfaces.

Chemical functional groups of surface layer (S-layer) proteins were chemically modified in order to evaluate the potential of S-layer proteins for the introduction of functional molecules. S-layer proteins are structure proteins that self-assemble into regular arrays on surfaces. One general feature of S-layer proteins is their high amount of carboxylic and amino groups. These groups are potential targets for linking functional molecules, thus producing reactive surfaces. In this work, these groups were conjugated with the amino acid tryptophan. In another approach, SH-groups were chemically inserted in order to extend the spectrum of modifiable groups. The amount of modifiable carboxylic groups was further evaluated by potentiometric titration in order to evaluate the potential efficiency of S-layer proteins to work as matrix for bioconjugations. The results proved that S-layer proteins can work as effective matrices for the conjugation of different molecules. The advantage of using chemical modification methods over genetic methods lies in its versatile usage enabling the attachment of biomolecules, as well as fluorescent dyes and inorganic molecules. Together with their self-assembling properties, S-layer proteins are suitable as targets for bioconjugates, thus enabling a nanostructuring and bio-functionalization of surfaces, which can be used for different applications like biosensors, filter materials, or (bio)catalytic surfaces.

Investigation of metal sorption behavior of Slp1 from Lysinibacillus sphaericus JG-B53: a combined study using QCM-D, ICP-MS and AFM.

Surface layer proteins (S-layer) of Lysinibacillus sphaericus JG-B53 are biological compounds with several bio-based technical applications such as biosorptive materials for metal removal or rare metals recovery from the environment. Despite their well-described applications, a deeper understanding of their metal sorption behavior still remains challenging. The metal sorption ability of Au(3+), Pd(2+), Pt(2+) and Eu(3+) was investigated by ICP-MS, AFM and QCM-D which enables the sorption detection in real-time during in situ experiments. Results indicate a high binding of Pd, followed by Au, Eu and Pt to the proteins. The comparison between different methods allowed a deeper understanding of the metal sorption of isolated S-layer either frees in liquid, adsorbed forming a protein layer or as the bacteria surface.

Interaction of U(VI) with Schizophyllum commune studied by microscopic and spectroscopic methods.

Biosorption of actinides like uranium by fungal cells can play an important role in the mobilization or immobilization of these elements in nature. Sorption experiments of U(VI) with Schizophyllum commune at different initial uranium concentrations and varying metal speciation showed high uranium sorption capacities in the pH range of 4­7. A combination of high angle annular dark-field and scanning transmission electron microscopy analysis (HAADF-STEM) showed that living mycelium cells accumulate uranium at the cell wall and intracellular. For the first time the fluorescence properties of uranium accumulates were investigated by means of time-resolved laser-induced fluorescence spectroscopy (TRLFS) beside the determination of corresponding structural parameters using X-ray absorption fine structure spectroscopy (EXAFS). While the oxidation state of uranium remained unchanged during sorption, uranium speciation changed significantly. Extra and intracellular phosphate groups are mainly responsible for uranium binding. TRLFS spectra clearly show differences between the emission properties of dissolved species in the initial mineral medium and of uranium species on fungi. The latter were proved to be organic and inorganic uranyl phosphates formed depending on the uranyl initial concentration and in some cases on pH.

Biostimulation of indigenous microbes for uranium bioremediation in former U mine water: multidisciplinary approach assessment.

Characterizing uranium (U) mine water is necessary to understand and design an effective bioremediation strategy. In this study, water samples from two former U-mines in East Germany were analysed. The U and sulphate (SO42-) concentrations of Schlema-Alberoda mine water (U: 1 mg/L; SO42-: 335 mg/L) were 2 and 3 order of magnitude higher than those of the Pöhla sample (U: 0.01 mg/L; SO42-: 0.5 mg/L). U and SO42- seemed to influence the microbial diversity of the two water samples. Microbial diversity analysis identified U(VI)-reducing bacteria (e.g. Desulfurivibrio) and wood-degrading fungi (e.g. Cadophora) providing as electron donors for the growth of U-reducers. U-bioreduction experiments were performed to screen electron donors (glycerol, vanillic acid, and gluconic acid) for Schlema-Alberoda U-mine water bioremediation purpose. Thermodynamic speciation calculations show that under experimental conditions, U(VI) is not coordinated to the amended electron donors. Glycerol was the best-studied electron donor as it effectively removed 99% of soluble U, 95% of Fe, and 58% of SO42- from the mine water, probably by biostimulation of indigenous microbes. Vanillic acid removed 90% of U, and no U removal occurred using gluconic acid.

Identification of multiple putative S-layer genes partly expressed by Lysinibacillus sphaericus JG-B53.

Lysinibacillus sphaericus JG-B53 was isolated from the uranium mining waste pile Haberland near Johanngeorgenstadt, Germany. Previous studies have shown that many bacteria that have been isolated from these heavy metal contaminated environments possess surface layer (S-layer) proteins that enable the bacteria to survive by binding metals with high affinity. Conversely, essential trace elements are able to cross the filter layer and reach the interior of the cell. This is especially true of the S-layer of L. sphaericus JG-B53, which possesses outstanding recrystallization and metal-binding properties. In this study, S-layer protein gene sequences encoded in the genome of L. sphaericus JG-B53 were identified using next-generation sequencing technology followed by bioinformatic analyses. The genome of L. sphaericus JG-B53 encodes at least eight putative S-layer protein genes with distinct differences. Using mRNA analysis the expression of the putative S-layer protein genes was studied. The functional S-layer protein B53 Slp1 was identified as the dominantly expressed S-layer protein in L. sphaericus JG-B53 by mRNA studies, SDS-PAGE and N-terminal sequencing. B53 Slp1 is characterized by square lattice symmetry and a molecular mass of 116 kDa. The S-layer protein B53 Slp1 shows a high similarity to the functional S-layer protein of L. sphaericus JG-A12, which was isolated from the same uranium mining waste pile Haberland and has been described by previous research. These similarities indicate horizontal gene transfer and DNA rearrangements between these bacteria. The presence of multiple S-layer gene copies may enable the bacterial strains to quickly adapt to changing environments.

Development of functionalised polyelectrolyte capsules using filamentous Escherichia coli cells.

BACKGROUND: Escherichia coli is one of the best studied microorganisms and finds multiple applications especially as tool in the heterologous production of interesting proteins of other organisms. The heterologous expression of special surface (S-) layer proteins caused the formation of extremely long E. coli cells which leave transparent tubes when they divide into single E. coli cells. Such natural structures are of high value as bio-templates for the development of bio-inorganic composites for many applications. In this study we used genetically modified filamentous Escherichia coli cells as template for the design of polyelectrolyte tubes that can be used as carrier for functional molecules or particles. Diversity of structures of biogenic materials has the potential to be used to construct inorganic or polymeric superior hybrid materials that reflect the form of the bio-template. Such bio-inspired materials are of great interest in diverse scientific fields like Biology, Chemistry and Material Science and can find application for the construction of functional materials or the bio-inspired synthesis of inorganic nanoparticles. RESULTS: Genetically modified filamentous E. coli cells were fixed in 2% glutaraldehyde and coated with alternating six layers of the polyanion polyelectrolyte poly(sodium-4styrenesulfonate) (PSS) and polycation polyelectrolyte poly(allylamine-hydrochloride) (PAH). Afterwards we dissolved the E. coli cells with 1.2% sodium hypochlorite, thus obtaining hollow polyelectrolyte tubes of 0.7 µm in diameter and 5-50 µm in length. For functionalisation the polyelectrolyte tubes were coated with S-layer protein polymers followed by metallisation with Pd(0) particles. These assemblies were analysed with light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy and transmission electron microscopy. CONCLUSION: The thus constructed new material offers possibilities for diverse applications like novel catalysts or metal nanowires for electrical devices. The novelty of this work is the use of filamentous E. coli templates and the use of S-layer proteins in a new material construct.

Speciation and spatial distribution of Eu(III) in fungal mycelium.

Europium, as an easy-to-study analog of the trivalent actinides, is of particular importance for studying the behavior of lanthanides and actinides in the environment. Since different soil organisms can influence the migration behavior of these elements, a detailed knowledge of these interaction mechanisms is important. The aim of this study was to investigate the interaction of mycelia of selected wood-inhabiting (S. commune, P. ostreatus, L. tigrinus) and soil-inhabiting fungi (L. naucinus) with Eu(III). In addition to determining the Eu(III) complexes in the sorption solution, the formed Eu(III) fungal species were characterized using scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy, chemical microscopy in combination with the time-resolved laser-induced fluorescence spectroscopy. Our data show that S. commune exhibited significantly higher Eu(III) binding capacity in comparison to the other fungi. Depending on fungal strain, the metal was immobilized on the cell surface, in the cell membranes, and within the membranes of various organelles, or in the cytoplasm in some cases. During the bioassociation process two different Eu(III) fungal species were formed in all investigated fungal strain. The phosphate groups of organic ligands were identified as being important functional groups to bind Eu(III) and thus immobilize the metal in the fungal matrix. The information obtained contributes to a better understanding of the role of fungi in migration, removal or retention mechanisms of rare earth elements and trivalent actinides in the environment.

Peptidoglycan as major binding motif for Uranium bioassociation on Magnetospirillum magneticum AMB-1 in contaminated waters.

The U(VI) bioassociation on Magnetospirillum magneticum AMB-1 cells was investigated using a multidisciplinary approach combining wet chemistry, microscopy, and spectroscopy methods to provide deeper insight into the interaction of U(VI) with bioligands of Gram-negative bacteria for a better molecular understanding. Our findings suggest that the cell wall plays a prominent role in the bioassociation of U(VI). In time-dependent bioassociation studies, up to 95 % of the initial U(VI) was removed from the suspension and probably bound on the cell wall within the first hours due to the high removal capacity of predominantly alive Magnetospirillum magneticum AMB-1 cells. PARAFAC analysis of TRLFS data highlights that peptidoglycan is the most important ligand involved, showing a stable immobilization of U(VI) over a wide pH range with the formation of three characteristic species. In addition, in-situ ATR FT-IR reveals the predominant strong binding to carboxylic functionalities. At higher pH polynuclear species seem to play an important role. This comprehensive molecular study may initiate in future new remediation strategies on effective immobilization of U(VI). In combination with the magnetic properties of the bacteria, a simple technical water purification process could be realized not only for U(VI), but probably also for other heavy metals.

Uranium(VI) bioassociation by different fungi - a comparative study into molecular processes.

After the Chernobyl and Fukushima incidents it has become clear that fungi can take up and accumulate large quantities of radionuclides and heavy metals, but the underlying processes are not well understood yet. For this study, the molecular interactions of uranium(VI) with the white-rot fungi, Schizophyllum commune and Pleurotus ostreatus, and the soil-living fungus, Leucoagaricus naucinus, were investigated. First, the uranium concentration in the biomass was determined by time-dependent bioassociation experiments. To characterize the molecular interactions, uranium was localized in the biomass by transmission electron microscopy analysis. Second, the formed uranyl complexes in both biomass and supernatant were determined by fluorescence spectroscopy. Additionally, possible bioligands in the supernatant were identified. The results show that the discernible interactions between metals and fungi are similar, namely biosorption, accumulation, and subsequent crystallization. But at the same time, the underlying biochemical mechanisms are different and specific to the fungal species. In addition, Schizophyllum commune was found to be the only fungus that, under the chosen experimental conditions, released tryptophan and other indole derivatives in the presence of uranium(VI) as determined by nuclear magnetic resonance spectroscopy. These released substances most likely act as messenger molecules rather than serving the direct detoxification of uranium(VI).

Survival of the basidiomycete Schizophyllum commune in soil under hostile environmental conditions in the Chernobyl Exclusion Zone.

Radioactive contamination resulting from major nuclear accidents presents harsh environmental conditions. Inside the Chernobyl exclusion zone, even more than 30 years after the accident, the resulting contamination levels still does not allow land-use or human dwellings. To study the potential of basidiomycete fungi to survive the conditions, a field trial was set up 5 km south-south-west of the destroyed reactor unit. A model basidiomycete, the lignicolous fungus Schizophyllum commune, was inoculated and survival in the soil could be verified. Indeed, one year after inoculation, the fungus was still observed using DNA-dependent techniques. Growth led to spread at a high rate, with approximately 8 mm per day. This shows that also white-rot basidiomycetes can survive the harsh conditions in soil inside the Chernobyl exclusion zone. The unadapted fungal strain showed the ability to grow and thrive in the contaminated soil where both stress from radiation and heavy metals were present.