Katrin Pollmann

Construction of an S-layer protein exhibiting modified self-assembling properties and enhanced metal binding capacities

The functional S-layer protein gene slfB of the uranium mining waste pile isolate Bacillus sphaericus JG-A12 was cloned as a polymerase chain reaction product into the expression vector pET Lic/Ek 30 and heterologously expressed in Escherichia coli Bl21(DE3). The addition of His tags to the N and C termini enabled the purification of the recombinant protein by Ni-chelating chromatography. The Ni binding capacity of the His-tagged recombinant S-layer protein was compared with that of the wild-type S layer. The inductively coupled plasma mass spectrometry analyses demonstrate a significantly enhanced Ni binding capability of the recombinant protein. In addition, the self-assembling properties of the purified modified S-layer proteins were studied by light microscopy and scanning electron microscopy. Whereas the wild-type S-layer proteins re-assembled into regular cylindric structures, the recombinant S-layer proteins reassembled into regular sheets that formed globular agglomerating structures. The nanoporous structure of the protein meshwork, together with its enhanced Ni binding capacity, makes the recombinant S-layer attractive as a novel self-assembling biological template for the fabrication of metal nanoclusters and construction of nanomaterials that are of technical interest.

Chloromethylmuconolactones as Critical Metabolites in the Degradation of Chloromethylcatechols: Recalcitrance of 2-Chlorotoluene

To elucidate possible reasons for the recalcitrance of 2-chlorotoluene, the metabolism of chloromethylcatechols, formed after dioxygenation and dehydrogenation by Ralstonia sp. strain PS12 tetrachlorobenzene dioxygenase and chlorobenzene dihydrodiol dehydrogenase, was monitored using chlorocatechol dioxygenases and chloromuconate cycloisomerases partly purified from Ralstonia sp. strain PS12 and Wautersia eutropha JMP134. Two chloromethylcatechols, 3-chloro-4-methylcatechol and 4-chloro-3-methylcatechol, were formed from 2-chlorotoluene. 3-Chloro-4-methylcatechol was transformed into 5-chloro-4-methylmuconolactone and 2-chloro-3-methylmuconolactone. For mechanistic reasons neither of these cycloisomerization products can be dehalogenated by chloromuconate cycloisomerases, with the result that 3-chloro-4-methylcatechol cannot be mineralized by reaction sequences related to catechol ortho-cleavage pathways known thus far. 4-Chloro-3-methylcatechol is only poorly dehalogenated during enzymatic processing due to the kinetic properties of the chloromuconate cycloisomerases. Thus, degradation of 2-chlorotoluene via a dioxygenolytic pathway is evidently problematic. In contrast, 5-chloro-3-methylcatechol, the major dioxygenation product formed from 3-chlorotoluene, is subject to quantitative dehalogenation after successive transformation by chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase, resulting in the formation of 2-methyldienelactone. 3-Chloro-5-methylcatechol is transformed to 2-chloro-4-methylmuconolactone.

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.

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.

Leaching of rare earth elements from fluorescent powder using the tea fungus Kombucha

In most modern technologies such as flat screens, highly effective magnets and lasers, as well as luminescence phosphors, Rare Earth Elements (REE) are used. Unfortunately no environmentally friendly recycling process exists so far. In comparison to other elements the interaction of microorganisms with REE has been studied to a less extent. However, as REE are ubiquitously present in nature it can be assumed that microorganisms play an important role in the biogeochemistry of REE. This study investigates the potential of organic acid-producing microbes for extracting REE from industrial waste. In Germany, 175 tons of fluorescent phosphor (FP) are collected per year as a distinct fraction from the recycling of compact fluorescent lamps. Because the FP contains about 10% of REE-oxides bound in the so-called triband dyes it is a readily accessible secondary resource of REE. Using the symbiotic mixed culture Kombucha, consisting of yeasts and acetic acid bacteria, REE were leached at a significant rate. The highest leaching-rates were observed in shake cultures using the entire Kombucha-consortium or its supernatant as leaching agent compared to experiments using the isolates Zygosaccharomyces lentus and Komagataeibacter hansenii as leaching organisms. During the cultivation, the pH decreased as a result of organic acid production (mainly acetic and gluconic acid). Thus, the underlying mechanism of the triband dye solubilisation is probably linked to the carboxyl-functionality or a proton excess. In accordance with the higher solubility of REE-oxides compared to REE-phosphates and -aluminates, the red dye Y2O3:Eu2+ containing relatively expensive REE was shown to be preferentially solubilized. These results show that it is possible to dissolve the REE-compounds of FP with the help of microbial processes. Moreover, they provide the basis for the development of an eco-friendly alternative to the currently applied methods that use strong inorganic acids or toxic chemicals.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding. The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Immobilization of microorganisms for AFM studies in liquids

In this paper a new sample preparation method is described that allows for the in vivo AFM imaging of a wide range of different microorganisms. The primary focus of this work was the immobilization of fixed and living cells of various microorganisms on substrates. The tested organisms of interest were Gram-negative and Gram-positive bacteria, yeast, and algae. The immobilization of the biological samples on a sample holder is crucial for AFM. Lateral forces of the probe tip can alter or remove sample material during scanning. This effect occurs especially on soft biological samples, which causes artifacts within the imaging and leads to a loss in quality and structural information. For the immobilization organisms were deposited on polyelectrolyte coated surfaces by centrifugation. Microorganisms were imaged without the use of any drying steps including either living or with glutaraldehyde fixation. Glutaraldehyde fixation enables long time scans that cover wide areas or the investigation of organisms in special growth stages, such as cell division or budding. Skipping fixation steps allows in vivo imaging to investigate living organisms and cellular processes under physiological conditions. A method for the reliable and efficient immobilization of microorganisms has been demonstrated by imaging the proteinaceous surface layer (S-layer) of living Lysinibacillus sphaericus and Viridibacilli arvi cells. In additional experiments, cell division of E. coli was successfully imaged. During repeated wide area scans, fixed sample material was not removed by the AFM tip, proving the suitability of these methods for AFM analyses. Ultimately, this method can be easily applied for the immobilization of a wide range of microorganisms and in vivo imaging of whole cells and cell ultrastructure.