Secretion and assembly of functional mini-cellulosomes from synthetic chromosomal operons in Clostridium acetobutylicum ATCC 824.

Background: Consolidated bioprocessing (CBP) is reliant on the simultaneous enzyme production, saccharification of biomass, and fermentation of released sugars into valuable products such as butanol. Clostridial species that produce butanol are, however, unable to grow on crystalline cellulose. In contrast, those saccharolytic species that produce predominantly ethanol, such as Clostridium thermocellum and Clostridium cellulolyticum, degrade crystalline cellulose with high efficiency due to their possession of a multienzyme complex termed the cellulosome. This has led to studies directed at endowing butanol-producing species with the genetic potential to produce a cellulosome, albeit by localising the necessary transgenes to unstable autonomous plasmids. Here we have explored the potential of our previously described Allele-Coupled Exchange (ACE) technology for creating strains of the butanol producing species Clostridium acetobutylicum in which the genes encoding the various cellulosome components are stably integrated into the genome. Results: We used BioBrick2 (BB2) standardised parts to assemble a range of synthetic genes encoding C. thermocellum cellulosomal scaffoldin proteins (CipA variants) and glycoside hydrolases (GHs, Cel8A, Cel9B, Cel48S and Cel9K) as well as synthetic cellulosomal operons that direct the synthesis of Cel8A, Cel9B and a truncated form of CipA. All synthetic genes and operons were integrated into the C. acetobutylicum genome using the recently developed ACE technology. Heterologous protein expression levels and mini-cellulosome self-assembly were assayed by western blot and native PAGE analysis. Conclusions: We demonstrate the successful expression, secretion and self-assembly of cellulosomal subunits by the recombinant C. acetobutylicum strains, providing a platform for the construction of novel cellulosomes. © 2013 Kovács et al.; licensee BioMed Central Ltd

Shortest Path versus Multi-Hub Routing in Networks with Uncertain Demand

We study a class of robust network design problems motivated by the need to scale core networks to meet increasingly dynamic capacity demands. Past work has focused on designing the network to support all hose matrices (all matrices not exceeding marginal bounds at the nodes). This model may be too conservative if additional information on traffic patterns is available. Another extreme is the fixed demand model, where one designs the network to support peak point-to-point demands. We introduce a capped hose model to explore a broader range of traffic matrices which includes the above two as special cases. It is known that optimal designs for the hose model are always determined by single-hub routing, and for the fixed- demand model are based on shortest-path routing. We shed light on the wider space of capped hose matrices in order to see which traffic models are more shortest path-like as opposed to hub-like. To address the space in between, we use hierarchical multi-hub routing templates, a generalization of hub and tree routing. In particular, we show that by adding peak capacities into the hose model, the single-hub tree-routing template is no longer cost-effective. This initiates the study of a class of robust network design (RND) problems restricted to these templates. Our empirical analysis is based on a heuristic for this new hierarchical RND problem. We also propose that it is possible to define a routing indicator that accounts for the strengths of the marginals and peak demands and use this information to choose the appropriate routing template. We benchmark our approach against other well-known routing templates, using representative carrier networks and a variety of different capped hose traffic demands, parameterized by the relative importance of their marginals as opposed to their point-to-point peak demands

Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker.

Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms. © 2011 The Author(s)

Teacher Attitudes Toward the Mainstreaming of Exceptional Students: Effects of the Educational Climate

In the United States, PL 94-142 is now ten years old, and for a decade school districts have had the legal responsibility to provide for all exceptional children in the \u27least restrictive environment.\u27 However, the movement toward integrating exceptional children into regular classrooms is not restricted to the United States. Many countries have been exposed to a vigorous advocacy on.behalf of the handicapped for their right to enjoy an existence as close as possible to normal (Mitchell, 1981)

Electronic Raman scattering of Tl-2223 and the symmetry of the supercon- ducting gap

Single crystalline Tl2Ba2Ca2Cu3O10 was studied using electronic Raman scattering. The renormalization of the scattering continuum was investigated as a function of the scattering geometry to determine the superconducting energy gap 2Delta(k). The A1g- and B2g-symmetry component show a linear frequency behaviour of the scattering intensity with a peak related to the energy gap, while the B1g-symmetry component shows a characteristic behaviour at higher frequencies. The observed frequency dependencies are consistent with a dx^2-y^2-wave symmetry of the gap and yield a ratio of 2Delta/k_BT_c=7.4. With the polarization of the scattered and incident light either parallel or perpendicular to the CuO2-planes a strong anisotropy due to the layered structure was detected, which indicates an almost 2 dimensional behaviour of this system.Comment: 2 pages, Postscript-file including 2 figures. Accepted for publication in the Proceedings of the M^2SHTSC IV Conference, Grenoble (France), 5-9 July 1994. Proceedings to be published in Physica C. Contact address: [email protected]

Numerical Investigation of a Mesoscopic Vehicular Traffic Flow Model Based on a Stochastic Acceleration Process

In this paper a spatial homogeneous vehicular traffic flow model based on a stochastic master equation of Boltzmann type in the acceleration variable is solved numerically for a special driver interaction model. The solution is done by a modified direct simulation Monte Carlo method (DSMC) well known in non equilibrium gas kinetic. The velocity and acceleration distribution functions in stochastic equilibrium, mean velocity, traffic density, ACN, velocity scattering and correlations between some of these variables and their car density dependences are discussed.Comment: 23 pages, 10 figure

Optimization of cascaded regenerative links based on phase sensitive amplifiers

We develop an analytical method for optimizing phase sensitive amplifiers for regeneration in multilevel phase encoded transmission systems. The model accurately predicts the optimum transfer function characteristics and identifies operating tolerances for different signal constellations and transmission scenarios. The results demonstrate the scalability of the scheme and show the significance of having simultaneous optimization of the transfer function and the signal alphabet. The model is general and can be applied to any regenerative system

Anisotropic thermal expansion and magnetostriction of YNi2_2B2_2C single crystals

We present results of anisotropic thermal expansion and low temperature magnetostriction measurements on YNi$_2$B$_2$C single crystals grown by high temperature flux and floating zone techniques. Quantum oscillations of magnetostriction were observed at low temperatures for $H \| c$ starting at fields significantly below $H_{c2}$ ($H < 0.7 H_{c2}$). Large irreversible, longitudinal magnetostriction was seen in both, in-plane and along the c-axis, directions of the applied magnetic field in the intermediate superconducting state. Anisotropic uniaxial pressure dependencies of $T_c$ were evaluated using results of zero field, thermal expansion measurements

Adrenaline modulates the global transcriptional profile of Salmonella revealing a role in the antimicrobial peptide and oxidative stress resistance responses

<p>Abstract</p> <p>Background</p> <p>The successful interaction of bacterial pathogens with host tissues requires the sensing of specific chemical and physical cues. The human gut contains a huge number of neurons involved in the secretion and sensing of a class of neuroendocrine hormones called catecholamines. Recently, in <it>Escherichia coli </it>O157:H7, the catecholamines adrenaline and noradrenaline were shown to act synergistically with a bacterial quorum sensing molecule, autoinducer 3 (AI-3), to affect bacterial virulence and motility. We wished to investigate the impact of adrenaline on the biology of <it>Salmonella </it>spp.</p> <p>Results</p> <p>We have determined the effect of adrenaline on the transcriptome of the gut pathogen <it>Salmonella enterica </it>serovar Typhimurium. Addition of adrenaline led to an induction of key metal transport systems within 30 minutes of treatment. The oxidative stress responses employing manganese internalisation were also elicited. Cells lacking the key oxidative stress regulator OxyR showed reduced survival in the presence of adrenaline and complete restoration of growth upon addition of manganese. A significant reduction in the expression of the <it>pmrHFIJKLM </it>antimicrobial peptide resistance operon reduced the ability of <it>Salmonella </it>to survive polymyxin B following addition of adrenaline. Notably, both phenotypes were reversed by the addition of the β-adrenergic blocker propranolol. Our data suggest that the BasSR two component signal transduction system is the likely adrenaline sensor mediating the antimicrobial peptide response.</p> <p>Conclusion</p> <p><it>Salmonella </it>are able to sense adrenaline and downregulate the antimicrobial peptide resistance <it>pmr </it>locus through the BasSR two component signalling system. Through iron transport, adrenaline may affect the oxidative stress balance of the cell requiring OxyR for normal growth. Both adrenaline effects can be inhibited by the addition of the β-adrenergic blocker propranolol. Adrenaline sensing may provide an environmental cue for the induction of the <it>Salmonella </it>stress response in anticipation of imminent host-derived oxidative stress. However, adrenaline may also serve in favour of the host defences by lowering antimicrobial peptide resistance and hence documenting for the first time such a function for a hormone.</p