Structure and mutational analysis of the archaeal GTP:AdoCbi-P guanylyltransferase (CobY) from Methanocaldococcus jannaschii: insights into GTP binding and dimerization

In archaea and bacteria, the late steps in adenosylcobalamin (AdoCbl) biosynthesis are collectively known as the nucleotide loop assembly (NLA) pathway. In the archaeal and bacterial NLA pathways, two different guanylyltransferases catalyze the activation of the corrinoid. Structural and functional studies of the bifunctional bacterial guanylyltransferase that catalyze both ATP-dependent corrinoid phosphorylation and GTP-dependent guanylylation are available, but similar studies of the monofunctional archaeal enzyme that catalyzes only GTP-dependent guanylylation are not. Herein, the three-dimensional crystal structure of the guanylyltransferase (CobY) enzyme from the archaeon Methanocaldococcus jannaschii (MjCobY) in complex with GTP is reported. The model identifies the location of the active site. An extensive mutational analysis was performed, and the functionality of the variant proteins was assessed in vivo and in vitro. Substitutions of residues Gly8, Gly153, or Asn177 resulted in ≥94% loss of catalytic activity; thus, variant proteins failed to support AdoCbl synthesis in vivo. Results from isothermal titration calorimetry experiments showed that MjCobY(G153D) had 10-fold higher affinity for GTP than MjCobY(WT) but failed to bind the corrinoid substrate. Results from Western blot analyses suggested that the above-mentioned substitutions render the protein unstable and prone to degradation; possible explanations for the observed instability of the variants are discussed within the framework of the three-dimensional crystal structure of MjCobY(G153D) in complex with GTP. The fold of MjCobY is strikingly similar to that of the N-terminal domain of Mycobacterium tuberculosis GlmU (MtbGlmU), a bifunctional acetyltransferase/uridyltransferase that catalyzes the formation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc).     

Dynamic chromatin modifications characterise the first cell cycle in mouse embryos

On fertilisation, gametes undergo epigenetic reorganisation and re-establish totipotency. Here, we investigate links between chromatin remodelling and asymmetric maintenance of DNA methylation in the early mouse embryo. Using antibodies for lysine specific H3 methylation reveals that the male pronucleus is negative for di- and trimethyl H3-K9 yet the female is positive for these residues. However, the male is positive for monomethyl H3-K9 and H3-K27 and these signals increase during pronuclear maturation. Non-histone chromatin proteins of the Polycomb group are found in the paternal compartment as early as sperm decondensation. However, trimethyl H3-K27 is not observed in the male until the completion of DNA replication. Heterochromatin protein 1 beta (HP1beta) is abundant in the male pronucleus, despite the absence of di- and trimethyl H3-K9, and co-localises with monomethyl H3-K9. Recent evidence identifies monomethyl H3-K9 as the preferred substrate of Suvar39h, the histone methyl transferase (HMT) responsible for heterochromatic H3-K9 trimethylation. The association of HP1beta with monomethyl H3-K9 may assist in preventing further modification of H3-K9. Association of dimethylation but not trimethylation of H3-K9 with DNA methylation, in the female pronucleus, suggests a mechanistically significant link. These differences begin to provide a chromatin based explanation for paternal-specific active DNA demethylation and maternal specific protection in the mouse.     

Associations between attributes of live poultry trade and HPAI H5N1 outbreaks: a descriptive and network analysis study in northern Vietnam

Background  

The structure of contact between individuals plays an important role in the incursion and spread of contagious diseases in both human and animal populations. In the case of avian influenza, the movement of live birds is a well known risk factor for the geographic dissemination of the virus among poultry flocks. Live bird markets (LBM's) contribute to the epidemiology of avian influenza due to their demographic characteristics and the presence of HPAI H5N1 virus lineages. The relationship between poultry producers and live poultry traders (LPT's) that operate in LBM's has not been adequately documented in HPAI H5N1-affected SE Asian countries. The aims of this study were to document and study the flow of live poultry in a poultry trade network in northern Vietnam, and explore its potential role in the risk for HPAI H5N1 during 2003 to 2006.     

Quantifying the climatic niche of symbiont partners in a lichen symbiosis indicates mutualist‐mediated niche expansions

The large distributional areas and ecological niches of many lichenized fungi may in part be due to the plasticity in interactions between the fungus (mycobiont) and its algal or cyanobacterial partners (photobionts). On the one hand, broad‐scale phylogenetic analyses show that partner compatibility in lichens is rather constrained and shaped by reciprocal selection pressures and codiversification independent of ecological drivers. On the other hand, sub‐species‐level associations among lichen symbionts appear to be environmentally structured rather than phylogenetically constrained. In particular, switching between photobiont ecotypes with distinct environmental preferences has been hypothesized as an adaptive strategy for lichen‐forming fungi to broaden their ecological niche. The extent and direction of photobiont‐mediated range expansions in lichens, however, have not been examined comprehensively at a broad geographic scale. Here we investigate the population genetic structure of Lasallia pustulata symbionts at sub‐species‐level resolution across the mycobiont's Europe‐wide range, using fungal MCM7 and algal ITS rDNA sequence markers. We show that variance in occurrence probabilities in the geographic distribution of genetic diversity in mycobiont‐photobiont interactions is closely related to changes in climatic niches. Quantification of niche extent and overlap based on species distribution modeling and construction of Hutchinsonian climatic hypervolumes revealed that combinations of fungal–algal interactions change at the sub‐species level along latitudinal temperature gradients and in Mediterranean climate zones. Our study provides evidence for symbiont‐mediated niche expansion in lichens. We discuss our results in the light of symbiont polymorphism and partner switching as potential mechanisms of environmental adaptation and niche evolution in mutualisms.     

Distribution and effects of tree leaf litter on vegetation composition and biomass in a forest-grassland ecotone

Aims After abandonment of grasslands, secondary succession leads to the invasion by woody species. This process begins with the accumulation of tree litter in the forest–grassland ecotone. Our objectives were to determine the relationships between litter amounts and vegetation composition and cover along natural forest–grassland ecotones and to experimentally study the initial effects of tree litter accumulation on grassland vegetation and on microsite conditions.Methods We established 11 transects varying from 12 to 15 m in length in different forest–grassland ecotones in the Lahn-Dill highlands, Germany, and measured the mass and cover of tree litter and the cover and composition of vegetation at five sequential positions along each transect by using 1 m 2 plots with five replications. In a field experiment, we established plots subjected to different litter amounts (0, 200 and 600g m ?2) and evaluated changes in grassland vegetation, soil temperature and soil nutrient availability below the litter layer.Important findings Tree litter amounts decrease from 650 to 65g m ?2 across the forest–grassland ecotone. Vegetation changed from shrubs and annual species (adapted to more stressful conditions) in the forests edge to grasses, rosettes and hemirosette species (with higher competitive abilities) in the grassland. These anthropogenic forest–grassland ecotones showed abrupt edges, and the two adjacent ecosystems were characterized by different species pools and functional groups. In the field experiment, the presence of a litter layer reduced vegetation biomass and cover; the species richness was only reduced in the treatment with high litter (600g m ?2). Additionally, adding litter on top of vegetation also reduced thermal amplitude and the number of frost days, while increasing the availability of some nutrients, such as nitrogen and aluminium, the latter being an indicator of soil acidification. Adding a tree litter layer of 600g m ?2 in grassland areas had strong effects on the composition and diversity of grassland vegetation by reducing the cover of several key grassland species. In, or near, forest edges, litter accumulation rapidly changes established vegetation, microsite conditions and soil nutrients.     

Transferability of miRNA-technology to bioprocessing: Influence of cultivation mode and media

The biopharmaceutical industry strives for improvement of their production processes. In recent years, miRNAs have been shown to positively impact the production capacity of recombinant CHO cells, especially with regard to difficult to express proteins. Effective and reliable gene regulation of process relevant target genes by miRNAs is a prerequisite for integrating them into the toolbox of industrial cell engineering strategies. However, most studies rely on transient transfection of miRNA mimics; there is low standardization in evaluation of miRNA function and little knowledge on transferability of effects found during transient expression to stable expression during industry relevant fed-batch cultivation. In order to provide more insight into this topic, we used the pcDNA6.2 vector for stable miRNA overexpression during batch and fed-batch cultivation in CHO DG44 cells, optimized the vector, and compared the miRNA levels and effects with those achieved by transfection of miRNA mimics. We found that miR-1 downregulated TWF1 mRNA in different recombinant CHO DG44 clones in a dose-dependent manner during transient batch cultivation. Cells stably overexpressing miR-1 also showed a TWF1 mRNA downregulation when cultivated in batch mode using in-house medium 1. However, when the cells stably overexpressing miR-1 were cultivated in fed-batch mode using in-house medium 2. Consequently, a change of cultivation mode and medium seems to have an impact on target gene regulation by miRNA. Taken together, our findings highlight the importance to standardize miRNA evaluations and test miRNAs in the final application environment.     

Phenotypic convergence in a natural Daphnia population acclimated to low temperature

Fluidity of a given membrane decreases at lower ambient temperatures, whereas it rises at increasing temperatures, which is achieved through changes in membrane lipid composition. In consistence with homeoviscous adaptation theory, lower temperatures result in increased tissue concentrations of polyunsaturated fatty acids (PUFAs) in Daphnia magna, suggesting a higher PUFA requirement at lower temperatures. However, so far homeoviscous adaptation has been suggested for single or geographically separated Daphnia genotypes only. Here, we investigated changes in relative fatty acid (FA) tissue concentrations in response to a lower temperature (15°C) within a D. magna population. We determined juvenile growth rates (JGR) and FA patterns of 14 genotypes that were grown on Chlamydomonas klinobasis at 15°C and 20°C. We report significant differences of JGR and the relative body content of various FAs between genotypes at either temperature and between temperatures. Based on slopes of reaction norms, we found genotype‐specific changes in FA profiles between temperatures suggesting that genotypes have different strategies to cope with changing temperatures. In a hierarchical clustering analysis, we grouped genotypes according to differences in direction and magnitude of changes in relative FA content, which resulted in three clusters of genotypes following different patterns of changes in FA composition. These patterns suggest a lower importance of the PUFA eicosapentaenoic acid (EPA, C20:5ω3) than previously assumed. We calculated an unsaturation index (UI) as a proxy for membrane fluidity at 15°C, and we neither found significant differences for this UI nor for fitness, measured as JGR, between the three genotype clusters. We conclude that these three genotype clusters represent different physiological solutions to temperature changes by altering the relative share of different FAs, but that their phenotypes converge with respect to membrane fluidity and JGR. These clusters will be subjected to different degrees of PUFA limitation when sharing the same diet.