Bacillus sphaericus JG-A12 is a natural isolate recovered from a uranium mining waste pile near the town of Johanngeorgenstadt in Saxony, Germany. The cells of this strain are enveloped by a highly ordered crystalline proteinaceous surface layer (S-layer) possessing an ability to bind uranium and other heavy metals. Purified and recrystallized S-layer proteins were shown to be phosphorylated by phosphoprotein-specific staining, inductive coupled plasma mass spectrometry analysis, and a colorimetric method. We used extended X-ray absorption fine-structure (EXAFS) spectroscopy to determine the structural parameters of the uranium complexes formed by purified and recrystallized S-layer sheets of B. sphaericus JG-A12. In addition, we investigated the complexation of uranium by the vegetative bacterial cells. The EXAFS analysis demonstrated that in all samples studied, the U(VI) is coordinated to carboxyl groups in a bidentate fashion with an average distance between the U atom and the C atom of 2.88 ؎ 0.02 Å and to phosphate groups in a monodentate fashion with an average distance between the U atom and the P atom of 3.62 ؎ 0.02 Å. Transmission electron microscopy showed that the uranium accumulated by the cells of this strain is located in dense deposits at the cell surface.Uranium is a long-lived radionuclide that is an ecological and human health hazard. The mining and processing of uranium for nuclear power plants and nuclear weapons production have resulted in the generation of significant amounts of radioactive wastes. The mobility of this radionuclide is controlled by its interaction with ions, minerals, and microorganisms present in nature. As a consequence of their small size and diverse metabolic activities, bacteria are able to interact intimately with metal ions present in their environment (15). Highly reactive bacterial cell surfaces bind uranium and other metal ions (7). This reactivity arises from the presence of a wide array of ionizable groups, such as carboxylate and phosphate, present in the lipopolysaccharides (LPS) of gram-negative bacterial cell walls (8) and the peptidoglycan, teichuronic acids, and teichoic acids of gram-positive bacteria (9).The bacterial cell wall may be overlayed by a number of surface structures, which can also interact with metal ions. These may be composed primarily of carbohydrate polymers (e.g., capsules) or proteinaceous surface layers (e.g., S-layers) and may occur singly or in combination (15). The crystalline bacterial cell S-layers represent the outermost cell envelope component of many bacteria and archaea (50). S-layers are generally composed of identical protein or glycoprotein subunits, and they completely cover the cell surface during all stages of bacterial growth and division. Most S-layers are 5 to 15 nm thick and possess pores of identical size and morphology in the range of 2 to 6 nm (6). As porous lattices completely covering the cell surface, the S-layers can provide prokaryotic cells with selective advantages by functioning as protective coats, as s...
We report a novel biochemical method based on the sacrificial hydrogen strategy to synthesize bimetallic gold (Au)–palladium (Pd) nanoparticles (NPs) with a core/shell configuration. The ability of Escherichia coli cells supplied with H2 as electron donor to rapidly precipitate Pd(II) ions from solution is used to promote the reduction of soluble Au(III). Pre-coating cells with Pd(0) (bioPd) dramatically accelerated Au(III) reduction, with the Au(III) reduction rate being dependent upon the initial Pd loading by mass on the cells. Following Au(III) addition, the bioPd–Au(III) mixture rapidly turned purple, indicating the formation of colloidal gold. Mapping of bio-NPs by energy dispersive X-ray microanalysis suggested Au-dense core regions and peripheral Pd but only Au was detected by X-ray diffraction (XRD) analysis. However, surface analysis of cleaned NPs by cyclic voltammetry revealed large Pd surface sites, suggesting, since XRD shows no crystalline Pd component, that layers of Pd atoms surround Au NPs. Characterization of the bimetallic particles using X-ray absorption spectroscopy confirmed the existence of Au-rich core and Pd-rich shell type bimetallic biogenic NPs. These showed comparable catalytic activity to chemical counterparts with respect to the oxidation of benzyl alcohol, in air, and at a low temperature (90°C).
The S-layer of Bacillus sphaericus strain JG-A12, isolated from a uranium-mining site, exhibits a high metal-binding capacity, indicating that it may provide a protective function by preventing the cellular uptake of heavy metals and radionuclides. This property has allowed the use of this and other S-layers as self-assembling organic templates for the synthesis of nanosized heavy metal cluster arrays. However, little is known about the molecular basis of the metal-protein interactions and their impact on secondary structure. We have studied the secondary structure, protein stability, and Pd((II)) coordination in S-layers from the B. sphaericus strains JG-A12 and NCTC 9602 to elucidate the molecular basis of their biological function and of the metal nanocluster growth. Fourier transform infrared spectroscopy reveals similar secondary structures, containing approximately 35% beta-sheets and little helical structure. pH-induced infrared absorption changes of the side-chain carboxylates evidence a remarkably low pK < 3 in both strains and a structural stabilization when Pd((II)) is bound. The COO(-)-stretching absorptions reveal a predominant Pd((II)) coordination by chelation/bridging by Asp and Glu residues. This agrees with XANES and EXAFS data revealing oxygens as coordinating atoms to Pd((II)). The additional participation of nitrogen is assigned to side chains rather than to the peptide backbone. The topology of nitrogen- and carboxyl-bearing side chains appears to mediate heavy metal binding to the large number of Asp and Glu in both S-layers at particularly low pH as an adaptation to the environment from which the strain JG-A12 has been isolated. These side chains are thus prime targets for the design of engineered S-layer-based nanoclusters.
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