Johannes Raff

Complexation of Uranium by Cells and S-Layer Sheets of Bacillus sphaericus JG-A12

ABSTRACT 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.

EXAFS investigation of uranium(VI) complexes formed at Bacillus cereus and Bacillus sphaericus surfaces

Uranium(VI) complex formation at vegetative cells and spores of Bacillus cereus and Bacillus sphaericus was studied using uranium LII-edge and LIII-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.

Complexation of U(VI) with highly phosphorylated protein, phosvitin: A vibrational spectroscopic approach

The complexation of uranium(VI) to variant functional groups of the highly phosphorylated protein phosvitin in aqueous solution was investigated by attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy. For the verification of the affinity of the actinyl ions to carboxyl and phosphate groups of the amino acid side chains, samples with different phosphate to uranium(VI) (P/U) ratios were investigated under denaturing conditions as well as in aqueous medium. From a comparative study with other heavy metal ions, i.e. Ba(2+) and Pb(2+), a strong coordination of U(VI) to carboxyl and phosphoryl groups can be derived. Furthermore, with increasing P/U ratios, a preferential binding of U(VI) to phosphoryl groups is indicated by the spectra of the batch samples. These findings are confirmed by spectra of aqueous U(VI)-phosvitin complexes reflecting an explicit coordination of the uranyl ions to phosphate groups at a high P/U ratio. Our study provides a deeper insight into the molecular interactions between actinyl ions and protein, and can be conferred to other basic biomolecules such as polysaccharides and nucleic acids.

Bacterial communities in uranium mining waste piles and their interaction with heavy metals

High diversity and significant differences were found in the structures of bacterial communities present in several U mill tailings and U mining waste piles. Many bacterial strains were successfully cultured from those uranium wastes, most of which are unusually effective in different biotransformations of U. The molecular basis for the selective and reversible binding of U and some other toxic metals by one of the natural bacterial isolates was found to be a novel kind of S-layer protein. Our analysis indicates that uranium wastes are a valuable reservoir for unusual microorganisms prospective for bacteria-based bioremediation.

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.

Colorimetric As (V) detection based on S-layer functionalized gold nanoparticles.

Herein, we present simple and rapid colorimetric and UV/VIS spectroscopic methods for detecting anionic arsenic (V) complexes in aqueous media. The methods exploit the aggregation of S-layer-functionalized spherical gold nanoparticles of sizes between 20 and 50 nm in the presence of arsenic species. The gold nanoparticles were functionalized with oligomers of the S-layer protein of Lysinibacillus sphaericus JG-A12. The aggregation of the nanoparticles results in a color change from burgundy-red for widely dispersed nanoparticles to blue for aggregated nanoparticles. A detailed signal analysis was achieved by measuring the shift of the particle plasmon resonance signal with UV/VIS spectroscopy. To further improve signal sensitivity, the influence of larger nanoparticles was tested. In the case of 50 nm gold nanoparticles, a concentration of the anionic arsenic (V) complex lower than 24 ppb was detectable.

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 Au3+, Pd2+, Pt2+ and Eu3+ 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.

S‐layer proteins as an immobilization matrix for aptamers on different sensor surfaces

S‐layer proteins provide a biocompatible environment with different kinds of functional groups, perfect for the sequential coupling of any kind of biofunctional molecule. In addition, their nanostructure and their ability to crystallize on surfaces in a nanometer‐thick monolayer ensure a regular arrangement of these molecules on solid supports. In this work, a thrombin‐binding aptamer and an ofloxacin‐binding aptamer were coupled with different chemical crosslinkers to S‐layer proteins using them for defined immobilization. S‐layer protein monomers and paracrystalline S‐layers were successfully modified with the thrombin‐binding aptamer. However, S‐layer protein monomers were not able to crystallize after aptamer modification and showed no thrombin binding during random surface attachment. In contrast, aptamers linked to an intact S‐layer in suspension or an S‐layer coating were still functional. The modification rate of S‐layers with the thrombin‐binding aptamer was estimated with one aptamer to two unit cells (unit cell = four monomers). Verification of the functionality of both aptamers through target binding after S‐layer‐immobilization on solid supports was proven by laser‐induced fluorescence spectroscopy (LIFS), resonant mirror sensor (IAsys), and quartz crystal microbalance with dissipation monitoring (QCM‐D), respectively. Hence, this study presents S‐layer proteins as an interesting alternative to existing immobilization matrices for recognition biomolecules.

E. coli filament formation induced by heterologous S-layer expression

Escherichia coli is a rod-shaped intestinal bacterium which has a size of 1.1-1.5 µm x 2.0-6.0 µm. The fast cell division process and the uncomplicated living conditions have turned E. coli into a widely used host in genetic engineering and into one of the best studied microorganisms of all. We used E. coli BL21(DE3) as host for heterologous expression of S-layer proteins of Lysinibacillus sphaericus JG-A12 in order to enable a fast and high efficient protein production. The S-layer expression induced in E. coli an unusual elongation of the cells, thus producing filaments of >100 µm in length. In the stationary growth phase, E. coli filaments develop tube-like structures that contain E. coli single cells. Fluorescence microscopic analyses of S-layer expressing E. coli cells that were stained with membrane stain FM® 5-95 verify the membrane origin of the tubes. Analyses of DAPI stained GFP-S-layer expressing E. coli support the assumption of a disordered cell division that is induced by the huge amount of recombinant S-layer proteins. However, the underlying mechanism is still not characterized in detail. These results describe the occurrence of a novel stable cell form of E. coli as a result of a disordered cell division process.

Heterologous expression of the surface-layer-like protein SllB induces the formation of long filaments of Escherichia coli consisting of protein-stabilized outer membrane

Escherichia coli is one of the best studied micro-organisms and is the most widely used host in genetic engineering. The Gram-negative single cells are rod-shaped, and filaments are usually not found. Here, we describe the reproducible formation of elongated E. coli cells. During heterologous expression of the silent surface (S)-layer protein gene sllB from Lysinibacillus sphaericus JG-A12 in E. coli BL21(DE3), the cells were arranged as long chains which were surrounded by highly stable sheaths. These filaments had a length of >100 μm. In the stationary growth phase, microscopic analyses demonstrated the formation of unusually long transparent tube-like structures which were enclosing separate single cells. The tube-like structures were isolated and analysed by SDS-PAGE, infrared-spectroscopy and different microscopic methods in order to identify their unusual composition and structure. The tube-like structures were found to be like outer membranes, containing high levels of proteins and to which the recombinant S-layer proteins were attached. Despite the entire structure being indicative of a disordered cell division, the bacterial cells were highly viable and stable. To our knowledge, this is the first time that the induction of drastic morphological changes in E. coli by the expression of a foreign protein has been reported.