Distinct Effects of Chemical Toxicity and Radioactivity on Metabolic Heat of Cultured Cells Revealed by "Isotope-Editing".

Studying the toxicity of chemical compounds using isothermal microcalorimetry (IMC), which monitors the metabolic heat from living microorganisms, is a rapidly expanding field. The unprecedented sensitivity of IMC is particularly attractive for studies at low levels of stressors, where lethality-based data are inadequate. We have revealed via IMC the effect of low dose rates from radioactive ß--decay on bacterial metabolism. The low dose rate regime (<400 µGyh-1) is typical of radioactively contaminated environmental sites, where chemical toxicity and radioactivity-mediated effects coexist without a predominance or specific characteristic of either of them. We found that IMC allows distinguishing the two sources of metabolic interference on the basis of "isotope-editing" and advanced thermogram analyses. The stable and radioactive europium isotopes 153Eu and 152Eu, respectively, were employed in monitoring Lactococcus lactis cultures via IMC. ß--emission (electrons) was found to increase initial culture growth by increased nutrient uptake efficiency, which compensates for a reduced maximal cell division rate. Direct adsorption of the radionuclide to the biomass, revealed by mass spectrometry, is critical for both the initial stress response and the "dilution" of radioactivity-mediated damage at later culture stages, which are dominated by the chemical toxicity of Eu.

Chromatopanning for the identification of gallium binding peptides.

This study is concerned with a chromatography-based approach (Immobilized Metal Ion Affinity Chromatography) for the recovery of gallium binding peptide sequences from a recombinant phage display library. The here described methods apply the fundamental knowledge and methods of separation science and meet thereby the key requirement of the phage display technique of precise separation of target-binding bacteriophage clones from non-interacting bacteriophage during the biopanning. During the chromatopanning called process, a total of 101 bacteriophage clones were identified of which in subsequent binding experiments, phage clones expressing the peptide sequences TMHHAAIAHPPH, SQALSTSRQDLR and HTQHIQSDDHLA were characterized to bind >10 fold better to a target that presents immobilized gallium ions than control phage, displaying no peptide sequence. The performance of biopanning experiments in chromatographic systems is particularly suitable for demanding targets such as trivalent metal ions. We found, that the selection process benefits immensely from the stable immobilization of the target metal ions during the entire biopanning process as well as the complete recovery of well interacting bacteriophage clones. Among others, this was possible due to an enhanced monitoring of process conditions and fractionation of eluates.

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.

Phylogenetic diversity of archaea and the archaeal ammonia monooxygenase gene in uranium mining-impacted locations in Bulgaria.

Uranium mining and milling activities adversely affect the microbial populations of impacted sites. The negative effects of uranium on soil bacteria and fungi are well studied, but little is known about the effects of radionuclides and heavy metals on archaea. The composition and diversity of archaeal communities inhabiting the waste pile of the Sliven uranium mine and the soil of the Buhovo uranium mine were investigated using 16S rRNA gene retrieval. A total of 355 archaeal clones were selected, and their 16S rDNA inserts were analysed by restriction fragment length polymorphism (RFLP) discriminating 14 different RFLP types. All evaluated archaeal 16S rRNA gene sequences belong to the 1.1b/Nitrososphaera cluster of Crenarchaeota. The composition of the archaeal community is distinct for each site of interest and dependent on environmental characteristics, including pollution levels. Since the members of 1.1b/Nitrososphaera cluster have been implicated in the nitrogen cycle, the archaeal communities from these sites were probed for the presence of the ammonia monooxygenase gene (amoA). Our data indicate that amoA gene sequences are distributed in a similar manner as in Crenarchaeota, suggesting that archaeal nitrification processes in uranium mining-impacted locations are under the control of the same key factors controlling archaeal diversity.

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.