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...

No abstract

The sensitive excitation of uranyl(vi) hydrolysis species in the UV (180 to 370 nm) is due to ligand-to-metal charge transfer.

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.

Regular arrays of metallic nanoparticles are formed by these authors using a novel technique involving electron‐beam induced cluster formation in the transmission electron microscope (TEM). Nucleation is initiated in the cavities between crystalline bacterial surface layers deposited on a solid support, as schematically illustrated in the Figure.

Uranium(VI) complex formation at vegetative cells and spores of Bacillus cereus and Bacillus sphaericus was studied using uranium L II -edge and L III -edge extended X-ray absorption fine structure (EXAFS) spectroscopy. A comparison of the measured equatorial U−O distances and other EXAFS structural parameters of uranyl species formed at the Bacillus strains with those of the uranyl structure family indicates that the uranium is predominantly bound as uranyl complexes with phosphoryl residues.

22 25 Uranium / Bacillus isolates / Biosorption / 26 Time-resolved laser fluorescence spectroscopy / Actinides 27 Summary. Accumulation studies with vegetative cells and 28 spores of three Bacillus isolates (JG-A 30, JG-A 12, JG-A 22, 29 classified as Bacillus cereus, Bacillus sphaericus, Bacillus me-30 gaterium) from a uranium mining waste pile (Johanngeorgen-31 stadt, Saxony) and their corresponding reference strains have 32 shown that Bacilli accumulate high amounts of U(VI) in the 33 concentration range examined (11Ϫ214 mg/L). Information on 34 the binding strength and the reversibility were obtained from 35 extraction studies with different extractants. With 0.01 M 36 EDTA solution the uranium bound to the biomass was released 37 almost quantitatively. The characterization of the bacterial-38 UO 2 2ϩ -complexes by time-resolved laser fluorescence spec-39 troscopy (TRLFS) showed the formation of inner-sphere com-40 plexes with phosphate groups of the biomass. The results lead 41 to the conclusion that the cell wall components with phosphate 42 residues e.g., polysaccharides, teichoic and teichuroic acids or 43 phospholipide layers of the membranes are responsible for the 44 uranium binding. The spectroscopic studies of the U(VI)-com-45 plexes with isolated bacterial cell walls and isolated surface-46 layer proteins of the strain Bacillus sphaericus NCTC 9602 47 after cell fractionation have shown that the complexation of 48 U(VI) with intact cells (vegetative cells or spores) is different 49 from the coordination with isolated cell wall components, es-50 pecially with the S-layer proteins. For all Bacillus strains stud-51 ied in this work, a significant contribution of the S-layer pro-52 teins to the binding of uranyl to living cells can be excluded.53

No abstract