Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution

Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (K_cat) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5‐fold increased specific activity (15°C) and over 100 times prolonged half‐life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity‐altering amino acid substitutions were from non‐charged to non‐charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions. Biotechnol. Bioeng. 2013; 110: 711–720. © 2012 Wiley Periodicals, Inc.     

Land use intensification in grasslands: higher trophic levels are more negatively affected than lower trophic levels

Increasing land use intensity and human influence are leading to a reduction in plant and animal species diversity. However, little is known about how these changes may affect higher trophic levels, apart from simply reducing species numbers. Here we investigated, over 3 years, the influence of different land practices on a tritrophic system in grassland habitats. The system consisted of the host plant Plantago lanceolata L. (Plantaginaceae), two monophagous weevils, Mecinus labilis Herbst and Mecinus pascuorum Gyllenhal (Coleoptera: Curculionidae), and their parasitoid Mesopolobus incultus Walker (Hymenoptera: Pteromalidae). At over 70 sites across three geographic regions in Germany, we measured plant species diversity and vegetation structure, as well as abundance of P. lanceolata, the two weevils, and the parasitoid. Land use intensity (fertilization) and type (mowing vs. grazing) negatively affected not only plant species richness but also the occurrence of the two specialized herbivores and their parasitoid. In contrast, land use had a mostly positive effect on host plant size, vegetation structure, and parasitization rate. This study reveals that intensification of land use influences higher trophic organisms even without affecting the availability of the host plant. The observed relationships between land use, vegetation complexity, and the tritrophic system are not restricted locally; rather they are measureable along a broad range of environmental conditions and years throughout Germany. Our findings may have important implications for the conservation of insect species of nutrient‐poor grasslands.     

Membrane adsorption,folding, insertion and translocation of synthetic trans-membrane peptides

Spontaneous membrane adsorption, folding and insertion of the synthetic WALP16 and KALP16 peptides was studied by computer simulations starting from completely extended conformations. The peptides were simulated using an unmodified all-atom force field in combination with an efficient Monte Carlo sampling algorithm. The membrane is represented implicitly as a hydrophobic zone inside a continuum solvent modelled using the generalized Born theory of solvation. The method was previously parameterized to match insertion energies of hydrophobic side chain analogs into cyclohexane and no parameters were optimized for the present simulations. Both peptides rapidly precipitate out of bulk solution and adsorb to the membrane surface. Interfacial folding into a helical conformation is followed by membrane insertion. Both the peptide conformations and their location in the membrane are strongly temperature dependent. The temperature dependent behaviour can be summarized by fitting to a four-state model, separating the system into folded and unfolded conformers, which are either inserted into the membrane or located at the interfaces. As the temperature is lowered the dominant peptide conformation of the system changes from unfolded surface bound configurations to folded surface bound states. Folded trans-membrane conformers represent the dominant configuration at low temperatures. The analysis allows direct estimates of the free energies of peptide folding and membrane insertion. In the case of WALP the quality of the fit is excellent and the thermodynamic behaviour is in good agreement with expected theoretical consideration. For KALP the fit is more problematic due to the large solvation energies of the charged lysine residues.     

Intraspecific phenotypic variation in a fish predator affects multitrophic lake metacommunity structure

Contemporary insights from evolutionary ecology suggest that population divergence in ecologically important traits within predators can generate diversifying ecological selection on local community structure. Many studies acknowledging these effects of intraspecific variation assume that local populations are situated in communities that are unconnected to similar communities within a shared region. Recent work from metacommunity ecology suggests that species dispersal among communities can also influence species diversity and composition but can depend upon the relative importance of the local environment. Here, we study the relative effects of intraspecific phenotypic variation in a fish predator and spatial processes related to plankton species dispersal on multitrophic lake plankton metacommunity structure. Intraspecific diversification in foraging traits and residence time of the planktivorous fish alewife (Alosa pseudoharengus) among coastal lakes yields lake metacommunities supporting three lake types which differ in the phenotype and incidence of alewife: lakes with anadromous, landlocked, or no alewives. In coastal lakes, plankton community composition was attributed to dispersal versus local environmental predictors, including intraspecific variation in alewives. Local and beta diversity of zooplankton and phytoplankton was additionally measured in response to intraspecific variation in alewives. Zooplankton communities were structured by species sorting, with a strong influence of intraspecific variation in A. pseudoharengus. Intraspecific variation altered zooplankton species richness and beta diversity, where lake communities with landlocked alewives exhibited intermediate richness between lakes with anadromous alewives and without alewives, and greater community similarity. Phytoplankton diversity, in contrast, was highest in lakes with landlocked alewives. The results indicate that plankton dispersal in the region supplied a migrant pool that was strongly structured by intraspecific variation in alewives. This is one of the first studies to demonstrate that intraspecific phenotypic variation in a predator can maintain contrasting patterns of multitrophic diversity in metacommunities.     

The Wobbler Mouse Model of Amyotrophic Lateral Sclerosis (ALS) Displays Hippocampal Hyperexcitability,and Reduced Number of Interneurons,but No Presynaptic Vesicle Release Impairments

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease. It is a fatal degenerative disease, best recognized for its debilitating neuromuscular effects. ALS however also induces cognitive impairments in as many as 50% of affected individuals. Moreover, many ALS patients demonstrate cortical hyperexcitability, which has been shown to precede the onset of clinical symptoms. The wobbler mouse is a model of ALS, and like ALS patients the wobbler mouse displays cortical hyperexcitability. Here we investigated if the neocortical aberrations of the wobbler mouse also occur in the hippocampus. Consequently, we performed extracellular field excitatory postsynaptic potential recordings in the CA1 region of the hippocampus on acute brain slices from symptomatic (P45-P60) and presymptomatic (P17-P21) wobbler mice. Significant increased excitation of hippocampal synapses was revealed by leftward shifted input/output-curves in both symptomatic and presymptomatic wobbler mice, and substantiated by population spike occurrence analyses, demonstrating that the increased synaptic excitation precedes the onset of visible phenotypic symptoms in the mouse. Synaptic facilitation tested by paired-pulse facilitation and trains in wobbler and control mice showed no differences, suggesting the absence of presynaptic defects. Immunohistochemical staining revealed that symptomatic wobbler mice have a lower number of parvalbumin positive interneurons when compared to controls and presymptomatic mice. This study reveals that the wobbler mouse model of ALS exhibits hippocampal hyperexcitability. We suggest that the hyperexcitability could be caused by increased excitatory synaptic transmission and a concomitant reduced inhibition due to a decreased number of parvalbumin positive interneurons. Thus we substantiate that wobbler brain impairments are not confined to the motor cortex, but extend to the hippocampus. Importantly, we have revealed more details of the early pathophysiology in asymptomatic animals, and studies like the present may facilitate the development of novel treatment strategies for earlier intervention in ALS patients in the future.     

N2-fixation,ammonium release and N-transfer to the microbial and classical food web within a plankton community

We investigated the role of N₂-fixation by the colony-forming cyanobacterium, Aphanizomenon spp., for the plankton community and N-budget of the N-limited Baltic Sea during summer by using stable isotope tracers combined with novel secondary ion mass spectrometry, conventional mass spectrometry and nutrient analysis. When incubated with ¹⁵N₂, Aphanizomenon spp. showed a strong ¹⁵N-enrichment implying substantial ¹⁵N₂-fixation. Intriguingly, Aphanizomenon did not assimilate tracers of ¹⁵NH₄⁺ from the surrounding water. These findings are in line with model calculations that confirmed a negligible N-source by diffusion-limited NH₄⁺ fluxes to Aphanizomenon colonies at low bulk concentrations (<250 nm) as compared with N₂-fixation within colonies. No N₂-fixation was detected in autotrophic microorganisms <5 μm, which relied on NH₄⁺ uptake from the surrounding water. Aphanizomenon released about 50% of its newly fixed N₂ as NH₄⁺. However, NH₄⁺ did not accumulate in the water but was transferred to heterotrophic and autotrophic microorganisms as well as to diatoms (Chaetoceros sp.) and copepods with a turnover time of ~5 h. We provide direct quantitative evidence that colony-forming Aphanizomenon releases about half of its recently fixed N₂ as NH₄⁺, which is transferred to the prokaryotic and eukaryotic plankton forming the basis of the food web in the plankton community. Transfer of newly fixed nitrogen to diatoms and copepods furthermore implies a fast export to shallow sediments via fast-sinking fecal pellets and aggregates. Hence, N₂-fixing colony-forming cyanobacteria can have profound impact on ecosystem productivity and biogeochemical processes at shorter time scales (hours to days) than previously thought.     

Cell‐to‐cell heterogeneity emerges as consequence of metabolic cooperation in a synthetic yeast community

Cells that grow together respond heterogeneously to stress even when they are genetically similar. Metabolism, a key determinant of cellular stress tolerance, may be one source of this phenotypic heterogeneity, however, this relationship is largely unclear. We used self‐establishing metabolically cooperating (SeMeCo) yeast communities, in which metabolic cooperation can be followed on the basis of genotype, as a model to dissect the role of metabolic cooperation in single‐cell heterogeneity. Cells within SeMeCo communities showed to be highly heterogeneous in their stress tolerance, while the survival of each cell under heat or oxidative stress, was strongly determined by its metabolic specialization. This heterogeneity emerged for all metabolite exchange interactions studied (histidine, leucine, uracil, and methionine) as well as oxidant (H₂O₂, diamide) and heat stress treatments. In contrast, the SeMeCo community collectively showed to be similarly tolerant to stress as wild‐type populations. Moreover, stress heterogeneity did not establish as sole consequence of metabolic genotype (auxotrophic background) of the single cell, but was observed only for cells that cooperated according to their metabolic capacity. We therefore conclude that phenotypic heterogeneity and cell to cell differences in stress tolerance are emergent properties when cells cooperate in metabolism.