Identity and function of an essential nitrogen ligand of the nitrogenase cofactor biosynthesis protein NifB.

NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. Previously, a nitrogen ligand was shown to be involved in coupling a pair of [Fe4S4] clusters (designated K1 and K2) concomitant with carbide insertion into an [Fe8S9C] cofactor core (designated L) on NifB. However, the identity and function of this ligand remain elusive. Here, we use combined mutagenesis and pulse electron paramagnetic resonance analyses to establish histidine-43 of Methanosarcina acetivorans NifB (MaNifB) as the nitrogen ligand for K1. Biochemical and continuous wave electron paramagnetic resonance data demonstrate the inability of MaNifB to serve as a source for cofactor maturation upon substitution of histidine-43 with alanine; whereas x-ray absorption spectroscopy/extended x-ray fine structure experiments further suggest formation of an intermediate that lacks the cofactor core arrangement in this MaNifB variant. These results point to dual functions of histidine-43 in structurally assisting the proper coupling between K1 and K2 and concurrently facilitating carbide formation via deprotonation of the initial carbon radical

Cascading the use of Web 2.0 technology in secondary schools in the United Kingdom: identifying the barriers beyond pre-service training

This paper reports on research that took place at Nottingham Trent University and Sheffield Hallam University, United Kingdom, over two years. The research focuses on the use of Web 2.0 technology, specifically web logs, with pre-service teachers, both during their university programme and the first year of teaching as full-time newly qualified teachers (NQTs). The purpose of this research was to add a developing body of knowledge by identifying whether technology used by pre-service teachers during their training course can be cascaded into their practice once qualified. Key findings identify a number of enablers and barriers to cascading technology in the classroom; these include curriculum time, pupil skills and support. The research concludes that early professional support and development should be on-going and assumptions about new teachers as champions of cascading innovative use of Web 2 technologies into their practice as NQTs may be over optimisti

In Vivo Structures of the Helicobacter pylori cag Type IV Secretion System

The type IV secretion system (T4SS) is a versatile nanomachine that translocates diverse effector molecules between microbes and into eukaryotic cells. Here, using electron cryotomography, we reveal the molecular architecture of the Helicobacter pylori cag T4SS. Although most components are unique to H. pylori, the cag T4SS exhibits remarkable architectural similarity to other T4SSs. Our images revealed that, when H. pylori encounters host cells, the bacterium elaborates membranous tubes perforated by lateral ports. Sub-tomogram averaging of the cag T4SS machinery revealed periplasmic densities associated with the outer membrane, a central stalk, and peripheral wing-like densities. Additionally, we resolved pilus-like rod structures extending from the cag T4SS into the inner membrane, as well as densities within the cytoplasmic apparatus corresponding to a short central barrel surrounded by four longer barrels. Collectively, these studies reveal the structure of a dynamic molecular machine that evolved to function in the human gastric niche

\u3cem\u3eIn Vivo\u3c/em\u3e Structures of the \u3cem\u3eHelicobacter pylori cag\u3c/em\u3e Type IV Secretion System

The type IV secretion system (T4SS) is a versatile nanomachine that translocates diverse effector molecules between microbes and into eukaryotic cells. Here, using electron cryotomography, we reveal the molecular architecture of the Helicobacter pylori cag T4SS. Although most components are unique to H. pylori, the cag T4SS exhibits remarkable architectural similarity to other T4SSs. Our images revealed that, when H. pylori encounters host cells, the bacterium elaborates membranous tubes perforated by lateral ports. Sub-tomogram averaging of the cag T4SS machinery revealed periplasmic densities associated with the outer membrane, a central stalk, and peripheral wing-like densities. Additionally, we resolved pilus-like rod structures extending from the cag T4SS into the inner membrane, as well as densities within the cytoplasmic apparatus corresponding to a short central barrel surrounded by four longer barrels. Collectively, these studies reveal the structure of a dynamic molecular machine that evolved to function in the human gastric niche

From Mars analogue environments to space: ground data evaluation of the survivability of Buttiauxella sp. MASE-IM-9 and Salinisphaera shabanensis

Mars analogues environments are some of the most extreme locations on Earth. Their unique combination of multiple extremes (e.g. high salinity, anoxia, and low nutrient availability) make them a valuable source of new polyextremophilic microbes in general, and for exploring the limits of life. These are seen as vital sources of information for Astrobiology, with implications for planetary protection and the search for life outside our planet. Despite this well-recognized relevance, current knowledge on the capability of (facultative) anaerobic microbes as single strains or in communities to withstand extraterrestrial conditions is still very sparse. Addressing this knowledge gap is one of the main goals of the project MEXEM (Mars EXposed Extremophiles Mixture), which is in preparation at the moment. As part of MEXEM, selected model organisms from all three domains of Life, will be exposed in a 3-month passive experiment with exposure to space conditions under anoxia followed by evaluation after their arrival back on Earth. The launch to the International Space Station is currently foreseen for 2024, and implies a series of preliminary tests and data collection on some of the selected strains. Here, we report on the survivability of Salinisphaera shabanensis, isolated from a deep-sea brine pool within the Red Sea, and of Buttiauxella sp. MASE-IM-9 isolated from a German sulphidic spring after exposure to Mars relevant stress factors (like desiccation and UV-radiation under anoxic conditions). Both organisms showed survival after anoxic desiccation for up to three months but this could be further extended by adding low amounts of artificial Mars regolith (MGS- 1S; 0.5 % wt/vol) and sucrose (0.1 M). The addition of these two components resulted in an elevation of the survival rate after desiccation of up to three orders of magnitude. Survival after desiccation could even be reproduced, if the cells were mixed, as an artificial community, before desiccation treatment. The presence of these two components also positively influenced survival after exposure to polychromatic UV (200 - 400 nm) up to 12 kJ/m2 in liquid and in a desiccated form

Architecture of the type IVa pilus machine

Many bacteria, including important pathogens, move by projecting grappling-hook–like extensions called type IV pili from their cell bodies. After these pili attach to other cells or objects in their environment, the bacteria retract the pili to pull themselves forward. Chang et al. used electron cryotomography of intact cells to image the protein machines that extend and retract the pili, revealing where each protein component resides. Putting the known structures of the individual proteins in place like pieces of a three-dimensional puzzle revealed insights into how the machine works, including evidence that ATP hydrolysis by cytoplasmic motors rotates a membrane-embedded adaptor that slips pilin subunits back and forth from the membrane onto the pilus

Seasonal and diurnal variations in Martian surface ultraviolet irradiation: biological and chemical implications for the Martian regolith

The issue of the variation of the surface ultraviolet (UV) environment on Mars was investigated with particular emphasis being placed on the interpretation of data in a biological context. A UV model has been developed to yield the surface UV irradiance at any time and place over the Martian year. Seasonal and diurnal variations were calculated and dose rates evaluated. Biological interpretation of UV doses is performed through the calculation of DNA damage effects upon phage T7 and Uracil, used as examples for biological dosimeters. A solar UV 'hotspot' was revealed towards perihelion in the southern hemisphere, with a significant damaging effect upon these species. Diurnal profiles of UV irradiance are also seen to vary markedly between aphelion and perihelion. The effect of UV dose is also discussed in terms of the chemical environment of the Martian regolith, since UV irradiance can reach high enough levels so as to have a significant effect upon the soil chemistry. We show, by assuming that H2O is the main source of hydrogen in the Martian atmosphere, that the stoichiometrically desirable ratio of 2:1 for atmospheric H and O loss rates to space are not maintained and at present the ratio is about 20:1. A large planetary oxygen surface sink is therefore necessary, in contrast with escape to space. This surface oxygen sink has important implications for the oxidation potential and the toxicology of the Martian soil. UV-induced adsorption of {\rm O}_{2}^{-} super-radicals plays an important role in the oxidative environment of the Martian surface, and the biologically damaging areas found in this study are also shown to be regions of high subsurface oxidation. Furthermore, we briefly cover the astrobiological implications for landing sites that are planned for future Mars missions

An Architectural Approach to Autonomics and Self-management of Automotive Embedded Electronic Systems

International audienceEmbedded electronic systems in vehicles are of rapidly increasing commercial importance for the automotive industry. While current vehicular embedded systems are extremely limited and static, a more dynamic configurable system would greatly simplify the integration work and increase quality of vehicular systems. This brings in features like separation of concerns, customised software configuration for individual vehicles, seamless connectivity, and plug-and-play capability. Furthermore, such a system can also contribute to increased dependability and resource optimization due to its inherent ability to adjust itself dynamically to changes in software, hardware resources, and environment condition. This paper describes the architectural approach to achieving the goals of dynamically self-configuring automotive embedded electronic systems by the EU research project DySCAS. The architecture solution outlined in this paper captures the application and operational contexts, expected features, middleware services, functions and behaviours, as well as the basic mechanisms and technologies. The paper also covers the architecture conceptualization by presenting the rationale, concerning the architecture structuring, control principles, and deployment concept. In this paper, we also present the adopted architecture V&V strategy and discuss some open issues in regards to the industrial acceptance

Structural Analysis of a Nitrogenase Iron Protein from Methanosarcina acetivorans: Implications for CO2 Capture by a Surface-Exposed [Fe4S4] Cluster.

Nitrogenase iron (Fe) proteins reduce CO2 to CO and/or hydrocarbons under ambient conditions. Here, we report a 2.4-Å crystal structure of the Fe protein from Methanosarcina acetivorans (MaNifH), which is generated in the presence of a reductant, dithionite, and an alternative CO2 source, bicarbonate. Structural analysis of this methanogen Fe protein species suggests that CO2 is possibly captured in an unactivated, linear conformation near the [Fe4S4] cluster of MaNifH by a conserved arginine (Arg) pair in a concerted and, possibly, asymmetric manner. Density functional theory calculations and mutational analyses provide further support for the capture of CO2 on MaNifH while suggesting a possible role of Arg in the initial coordination of CO2 via hydrogen bonding and electrostatic interactions. These results provide a useful framework for further mechanistic investigations of CO2 activation by a surface-exposed [Fe4S4] cluster, which may facilitate future development of FeS catalysts for ambient conversion of CO2 into valuable chemical commodities.IMPORTANCE This work reports the crystal structure of a previously uncharacterized Fe protein from a methanogenic organism, which provides important insights into the structural properties of the less-characterized, yet highly interesting archaeal nitrogenase enzymes. Moreover, the structure-derived implications for CO2 capture by a surface-exposed [Fe4S4] cluster point to the possibility of developing novel strategies for CO2 sequestration while providing the initial insights into the unique mechanism of FeS-based CO2 activation