Paläogenetik der Pest

Paleogenetics of the plague Plague has been part of our history since ancient times. Introduced again and again to Europe over centuries, the 'pestilences' killed millions of people before disappearing from the continent in 19^th Century. In the Middle Ages, bacteria were unknown and often the transmission routes of plague remained unrecognized. Plague was considered as a punishment by God disseminated by Miasma. With modern methods, microbiologists and palaeogenetists have now successfully shown that all the three historical recognized plague pandemics indeed were caused by the bacterium Yersinia pestis. In addition, important information has been obtained about the evolution of the bacterium and its origin. New data from ongoing studies on victims of the past epidemics help to shed new light on the past and perhaps even on the present of plague.     

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.     

Site-specific basicities regulate molecular recognition in receptor binding: in silico docking of thyroid hormones

Interactions between thyroid hormone α and β receptors and the eight protonation microspecies of each of the main thyroid hormones (thyroxine, liothyronine, and reverse liothyronine) were investigated and quantitated by molecular modeling. Flexible docking of the various protonation forms of thyroid hormones and high-affinity thyromimetics to the two thyroid receptors was carried out. In this method the role of the ionization state of each basic site could be studied in the composite process of molecular recognition. Our results quantitate at the molecular level how the ionization state and the charge distribution influence the protein binding. The anionic form of the carboxyl group (i.e., carboxylate site) is essential for protein binding, whereas the protonated form of amino group worsens the binding. The protonation state of the phenolate plays a less important role in the receptor affinity; its protonation, however, alters the electron density and the concomitant stacking propensity of the aromatic rings, resulting in a different binding score. The combined results of docking and microspeciation studies show that microspecies with the highest concentration at the pH of blood are not the strongest binding ones. The calculated binding free energy values can be well interpreted in terms of the interactions between the actual sites of the microspecies and the receptor amino acids. Our docking results were validated and compared with biological data from the literature. Since the thyroid hormone receptors influence several physiologic functions, such as metabolic rate, cholesterol and triglyceride levels, and heart frequency, our binding results provide a molecular basis for drug design and development in related therapeutic indications.     

Abatement of odorant compounds in one- and two-phase biotrickling filters under steady and transient conditions

The removal of hydrophobic volatile organic compounds (VOCs) still remains the main restriction in the biological treatment of odorous emissions due to mass transfer limitations. The addition of a non-aqueous phase to conventional biotrickling filters (BTF) may overcome this limitation by enhancing VOCs transport from the gas to the microorganisms. This study compared the long-term and transient performance of a one- (1P) and two-liquid phase (2P; with silicone oil as non-aqueous phase) BTFs for the removal of four VOCs (butanone, toluene, alpha-pinene, and hexane) at empty bed residence times (EBRT) ranging from 47 to 6 s. Removal efficiencies (RE) >96 % were obtained for butanone, toluene, and alpha-pinene in both bioreactors regardless of the EBRT, while higher hexane REs were recorded in the 2P-BTF (81–92 %) compared to the 1P-BTF (60–97 %). The two-phase system always showed a more consistent performance, being able to better withstand step VOC concentration increases and starvation periods, although it was more affected by liquid recycling shutdowns due to a reduced VOC mass transfer. The analysis of the microbial communities showed a high biodiversity and richness despite the low C source spectrum and high community evenness and richness. In this context, the presence of silicone oil mediated the development of a highly different phylogenetic composition of the communities.     

Changing distributions of ticks: causes and consequences

Today, we are witnessing changes in the spatial distribution and abundance of many species, including ticks and their associated pathogens. Evidence that these changes are primarily due to climate change, habitat modifications, and the globalisation of human activities are accumulating. Changes in the distribution of ticks and their invasion into new regions can have numerous consequences including modifications in their ecological characteristics and those of endemic species, impacts on the dynamics of local host populations and the emergence of human and livestock disease. Here, we review the principal causes for distributional shifts in tick populations and their consequences in terms of the ecological attributes of the species in question (i.e. phenotypic and genetic responses), pathogen transmission and disease epidemiology. We also describe different methodological approaches currently used to assess and predict such changes and their consequences. We finish with a discussion of new research avenues to develop in order to improve our understanding of these host–vector–pathogen interactions in the context of a changing world.     

Molecular dynamics of water in the neighborhood of aquaporins

Present knowledge obtained by molecular dynamics (MD) simulation studies regarding the dynamics of water, both in the vicinity of biological membranes and within the proteinaceous water channels, also known as aquaporins (AQPs), is reviewed. A brief general summary of the water models most extensively employed in MD simulations (SPC, SPC/E, TIP3P, TIP4P), indicating their most relevant pros and cons, is likewise provided. Structural considerations of water are also discussed, based on different order parameters, which can be extracted from MD simulations as well as from experiments. Secondly, the behaviour of water in the neighbourhood of membranes by means of molecular dynamics simulations is addressed. Consequently, the comparison with previous experimental evidence is pointed out. In living cells, water is transported across the plasma membrane through the lipid bilayer and the aforementioned AQPs, which motivates this review to focus mostly on MD simulation studies of water within AQPs. Relevant contributions explaining peculiar properties of these channels are discussed, such as selectivity and gating. Water models used in these studies are also summarised. Finally, based on the information presented here, further MD studies are encouraged.     

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

The rate of rotation of the rotor in the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or steady parts of the enzyme, is estimated in native vacuolar membrane vesicles from Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are formed spontaneously after exposing purified yeast vacuoles to osmotic shock. The fraction of total ATPase activity originating from the V-ATPase is determined by using the potent and specific inhibitor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during ten min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit of the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data are incompatible with models assuming multiple binding sites), to the inhibitor titration curve determines the concentration of the enzyme. Combining this with the known ATP/rotation stoichiometry of the V-ATPase and the assayed concentration of inorganic phosphate liberated by the V-ATPase, leads to an average rate of ~10 Hz for full 360° rotation (and a range of 6–32 Hz, considering the ± standard deviation of the enzyme concentration), which, from the time-dependence of the activity, extrapolates to ~14 Hz (8–48 Hz) at the beginning of the reaction. These are lower-limit estimates. To our knowledge, this is the first report of the rotation rate in a V-ATPase that is not subjected to genetic or chemical modification and is not fixed to a solid support; instead it is functioning in its native membrane environment.