A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

BACKGROUND: Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture. RESULTS: Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins. CONCLUSIONS: The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production

Propagating Cell-Membrane Waves Driven by Curved Activators of Actin Polymerization

Cells exhibit propagating membrane waves which involve the actin cytoskeleton. One type of such membranal waves are Circular Dorsal Ruffles (CDR) which are related to endocytosis and receptor internalization. Experimentally, CDRs have been associated with membrane bound activators of actin polymerization of concave shape. We present experimental evidence for the localization of convex membrane proteins in these structures, and their insensitivity to inhibition of myosin II contractility in immortalized mouse embryo fibroblasts cell cultures. These observations lead us to propose a theoretical model which explains the formation of these waves due to the interplay between complexes that contain activators of actin polymerization and membrane-bound curved proteins of both types of curvature (concave and convex). Our model predicts that the activity of both types of curved proteins is essential for sustaining propagating waves, which are abolished when one type of curved activator is removed. Within this model waves are initiated when the level of actin polymerization induced by the curved activators is higher than some threshold value, which allows the cell to control CDR formation. We demonstrate that the model can explain many features of CDRs, and give several testable predictions. This work demonstrates the importance of curved membrane proteins in organizing the actin cytoskeleton and cell shape

The impact of dry atmospheric deposition on the sea-surface microlayer and the sub-surface layer in the SE Mediterranean Sea: an experimental approach

The oligotrophic southeastern Mediterranean Sea (SEMS) is frequently exposed to desert-dust deposition which supplies nutrients, trace metals and a wide array of viable airborne microorganisms. In this study, we experimentally examined the impact of aerosol addition, collected during an intense dust storm event in early September 2015, on the biomass and activity of pico-phytoplankton and heterotrophic bacterial populations at the sea-surface micro layer (SML) relative to the sub surface layer (SSL). Aerosol (1.5 mg L-1) was added to SML and SSL water samples in microcosms (4.5 L) and the water was frequently sampled over a period of 48 hours. While the aerosol amendment triggered a moderate 1.5-2 fold increase in primary production in both the SML and the SSL, bacterial production increased by ~3 and ~7 folds in the SSL and SML, respectively. Concurrently, the abundance and flow-cytometric characteristics (green fluorescence and side scatter signals) of high nucleic acid (HNA) and low nucleic acid (LNA) bacterial cells showed a significant increase in the %HNA, in both SML and SSL samples following aerosol amendment. This shift in nucleic acid content took place at a much faster rate in the SML, suggesting a more active heterotrophic community. These changes were likely a result of higher rates of carbon utilizations in the SML following the dust addition, as assessed by a selected hydrocarbons and saccharides analysis. Additionally, a high absorption rate of hydrocarbons by the aerosol particles was measured following the additions, leaving less than 10% of these molecules available for potential heterotrophic microbial utilization. Our results suggest that the heterotrophic microbial community inhabiting the SML is more efficient in utilizing aerosol associated constituents than the community in the SSL

Conical Diffraction And Gap Solitons In Honeycomb Photonic Lattices

We study wave dynamics in honeycomb photonic lattices, and demonstrate the unique phenomenon of conical diffraction around the singular diabolical (zero-effective-mass) points connecting the first and second bands. This constitutes the prediction and first experimental observation of conical diffraction arising solely from a periodic potential. It is also the first study on k space singularities in photonic lattices. In addition, we demonstrate honeycomb gap solitons residing in the gap between the second and the third bands, reflecting the special properties of these lattices. © 2007 The American Physical Society

Paradoxical effects of low dose MDMA on latent inhibition in the rat

The cognitive effects of MDMA ('Ecstasy') are controversial, particularly in the case of acute administration of low doses. Latent inhibition (LI) refers to the reduction in conditioning to a stimulus that has received non-reinforced pre-exposure, an effect typically abolished by amphetamines and enhanced by antipsychotics. LI enhancement has also been shown using the 5-HT reuptake blocker sertraline. In the present study, the effects of MDMA (6 mg/kg, known to increase 5-HT release) were tested using 10 and 40 pre-exposures to produce weak and strong LI in controls, respectively. MDMA (injected twice, prior to pre-exposure and conditioning) significantly enhanced LI in that the effect was clearly demonstrated after only 10 pre-exposures, when it was absent in the saline controls. On its own such a profile of action would be consistent with a procognitive effect of MDMA mediated by increased availability of 5-HT. However, paradoxically the same MDMA treatment reduced LI in the 40 pre-exposures condition. This component of action is likely attributable to MDMA's actions on catecholaminergic systems and is consistent with other evidence of its adverse effects. Moreover, there were small but significant reductions in 5-HT in medial prefrontal cortex (mPFC) and amygdala assayed 7 days post MDMA administration (2 × 6 mg/kg, 24 h apart)