High resolution linkage maps of the model organism Petunia reveal substantial synteny decay with the related genome of tomato

Two linkage maps were constructed for the model plant Petunia. Mapping populations were obtained by crossing the wild species Petunia axillaris subsp. axillaris with Petunia inflata, and Petunia axillaris subsp. parodii with Petunia exserta. Both maps cover the seven chromosomes of Petunia, and span 970 centimorgans (cM) and 700 cM of the genomes, respectively. In total, 207 markers were mapped. Of these, 28 are multilocus amplified fragment length polymorphism (AFLP) markers and 179 are gene-derived markers. For the first time we report on the development and mapping of 83 Petunia microsatellites. The two maps retain the same marker order, but display significant differences of recombination frequencies at orthologous mapping intervals. A complex pattern of genomic rearrangements was detected with the related genome of tomato (Solanum lycopersicum), indicating that synteny between Petunia and other Solanaceae crops has been considerably disrupted. The newly developed markers will facilitate the genetic characterization of mutants and ecological studies on genetic diversity and speciation within the genus Petunia. The maps will provide a powerful tool to link genetic and genomic information and will be useful to support sequence assembly of the Petunia genome.     

Structure-function relationship in an archaebacterial methionine sulphoxide reductase B

Oxidation of methionine to methionine sulphoxide (MetSO) may lead to loss of molecular integrity and function. This oxidation can be 'repaired' by methionine sulphoxide reductases (MSRs), which reduce MetSO back to methionine. Two structurally unrelated classes of MSRs, MSRA and MSRB, show stereoselectivity towards the S and the R enantiomer of the sulphoxide respectively. Interestingly, these enzymes were even maintained throughout evolution in anaerobic organisms. Here, the activity and the nuclear magnetic resonance (NMR) structure of MTH711, a zinc containing MSRB from the thermophilic, methanogenic archaebacterium Methanothermobacter thermoautotrophicus, are described. The structure appears more rigid as compared with similar MSRBs from aerobic and mesophilic organisms. No significant structural differences between the oxidized and the reduced MTH711 state can be deduced from our NMR data. A stable sulphenic acid is formed at the catalytic Cys residue upon oxidation of the enzyme with MetSO. The two non-zinc-binding cysteines outside the catalytic centre are not necessary for activity of MTH711 and are not situated close enough to the active-site cysteine to serve in regenerating the active centre via the formation of an intramolecular disulphide bond. These findings imply a reaction cycle that differs from that observed for other MSRBs.     

The human Cas1 protein: a sialic acid-specific O-acetyltransferase?

Sialic acids are important sugars at the reducing end of glycoproteins and glycolipids. They are among many other functions involved in cell-cell interactions, host-pathogen recognition and the regulation of serum half-life of glycoproteins. An important modification of sialic acids is O-acetylation, which can alter or mask the biological properties of the parent sialic acid molecule. The nature of mammalian sialate-O-acetyltransferases (EC 2.3.1.45) involved in their biosynthesis is still unknown. We have identified the human CasD1 (capsule structure1 domain containing 1) gene as a candidate to encode the elusive enzyme. The human CasD1 gene encodes a protein with a serine-glycine-asparagine-histidine hydrolase domain and a hydrophobic transmembrane domain. Expression of the Cas1 protein tagged with enhanced green fluorescent protein in mammalian and insect cells directed the protein to the medial and trans-cisternae of the Golgi. Overexpression of the Cas1 protein in combination with α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (GD3 synthase) resulted in an up to 40% increased biosynthesis of 7-O-acetylated ganglioside GD3. By quantitative real-time polymerase chain reaction, we found up to 5-fold increase in CasD1 mRNA in tumor cells overexpressing O-Ac-GD3. CasD1-specific small interfering RNA reduced O-acetylation in tumor cells. These results suggest that the human Cas1 protein is directly involved in O-acetylation of α2-8-linked sialic acids.     

Linear approaches to intramolecular Förster resonance energy transfer probe measurements for quantitative modeling

Numerous unimolecular, genetically-encoded F?rster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find that FRET measured by standard intensity-based, ratiometric methods is inherently non-linear with respect to the fraction of probes undergoing FRET. Alternatively, we find that quantifying FRET either via (1) fluorescence lifetime imaging (FLIM) or (2) ratiometric methods where the donor emission intensity is divided by the directly-excited acceptor emission intensity (denoted R(alt)) is linear with respect to the fraction of probes undergoing FRET. This linearity property allows one to calculate the fraction of active probes based on the FRET measurement. Thus, our results suggest that either FLIM or ratiometric methods based on R(alt) are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purposes.     

Single dose novel Salmonella vaccine enhances resistance against visceralizing L. major and L. donovani infection in susceptible BALB/c mice

Visceral leishmaniasis is a major neglected tropical disease, with an estimated 500,000 new cases and more than 50,000 deaths attributable to this disease every year. Drug therapy is available but costly and resistance against several drug classes has evolved. Despite all efforts, no commercial, let alone affordable, vaccine is available to date. Thus, the development of cost effective, needle-independent vaccines is a high priority. Here, we have continued efforts to develop live vaccine carriers based on recombinant Salmonella. We used an in silico approach to select novel Leishmania parasite antigens from proteomic data sets, with selection criteria based on protein abundance, conservation across Leishmania species and low homology to host species. Five chosen antigens were differentially expressed on the surface or in the cytosol of Salmonella typhimurium SL3261. A two-step procedure was developed to select optimal Salmonella vaccine strains for each antigen, based on bacterial fitness and antigen expression levels. We show that vaccine strains of Salmonella expressing the novel Leishmania antigens LinJ08.1190 and LinJ23.0410 significantly reduced visceralisation of L. major and enhanced systemic resistance against L. donovani in susceptible BALB/c mice. The results show that Salmonella are valid vaccine carriers for inducing resistance against visceral leishmaniasis but that their use may not be suitable for all antigens.     

Substrate preference and phosphatidylinositol monophosphate inhibition of the catalytic domain of the Per-Arnt-Sim domain kinase PASKIN

The Per-Arnt-Sim (PAS) domain serine/threonine kinase PASKIN, or PAS kinase, links energy flux and protein synthesis in yeast, regulates glycogen synthesis and protein translation in mammals, and might be involved in insulin regulation in the pancreas. According to the current model, binding of a putative ligand to the PAS domain disinhibits the kinase domain, leading to PASKIN autophosphorylation and increased kinase activity. To date, only synthetic but no endogenous PASKIN ligands have been reported. In the present study, we identified a number of novel PASKIN kinase targets, including ribosomal protein S6. Together with our previous identification of eukaryotic elongation factor 1A1, this suggests a role for PASKIN in the regulation of mammalian protein translation. When searching for endogenous PASKIN ligands, we found that various phospholipids can bind PASKIN and stimulate its autophosphorylation. Interestingly, the strongest binding and autophosphorylation was achieved with monophosphorylated phosphatidylinositols. However, stimulated PASKIN autophosphorylation did not correlate with ribosomal protein S6 and eukaryotic elongation factor 1A1 target phosphorylation. Although autophosphorylation was enhanced by monophosphorylated phosphatidylinositols, di- and tri-phosphorylated phosphatidylinositols inhibited autophosphorylation. By contrast, target phosphorylation was always inhibited, with the highest efficiency for di- and tri-phosphorylated phosphatidylinositols. Because phosphatidylinositol monophosphates were found to interact with the kinase rather than with the PAS domain, these data suggest a multiligand regulation of PASKIN activity, including a still unknown PAS domain binding/activating ligand and kinase domain binding modulatory phosphatidylinositol phosphates.     

Linear Approaches to Intramolecular F?rster Resonance Energy Transfer Probe Measurements for Quantitative Modeling

Numerous unimolecular, genetically-encoded Förster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find that FRET measured by standard intensity-based, ratiometric methods is inherently non-linear with respect to the fraction of probes undergoing FRET. Alternatively, we find that quantifying FRET either via (1) fluorescence lifetime imaging (FLIM) or (2) ratiometric methods where the donor emission intensity is divided by the directly-excited acceptor emission intensity (denoted R_alt) is linear with respect to the fraction of probes undergoing FRET. This linearity property allows one to calculate the fraction of active probes based on the FRET measurement. Thus, our results suggest that either FLIM or ratiometric methods based on R_alt are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purposes.     

Fine‐scale patterns of species and phylogenetic turnover in a global biodiversity hotspot: Implications for climate change vulnerability

Question: What is the relative importance of environmental and spatial factors for species compositional and phylogenetic turnover? Location: High‐rainfall zone of the Southwest Australian Floristic Region (SWAFR). Methods: Correlates of species compositional turnover were assessed using quadrat‐based floristic data, and establishing relationships with environmental and spatial factors using canonical correspondence analyses and Mantel tests. Between‐quadrat phylogenetic distance measures were computed and examined for correlations with environmental and spatial attributes. Processes structuring pa2t2terns of beta diversity were also evaluated within four broad floristic assemblages defined a priori. Results: Floristic diversity was strongly related to environmental attributes. A low significance of spatial variables on assemblage patterns suggested no evident effect of dispersal limitations. Species compositional turnover was especially high within the swamp and outcrop assemblage. Phylogenetic turnover was closely coupled to species compositional turnover, implying the occurrence of many locally endemic and phylogenetically relict taxa. Beta diversity patterns within assemblages were also significantly correlated with the local environment, and relevant correlates differed between floristic assemblage types. Conclusion: Phylogenetic diversity in the SWAFR high‐rainfall zone is clustered within edaphic microhabitats in a generally subdued landscape. A clustered rather than dispersed distribution of phylogenetic diversity increases the probability of significant plant diversity loss during periods of climate change. Climate change susceptibility of the region's flora is accordingly estimated to be high. We highlight the conservation significance of swamp and outcrops that are characterized by distinct hydrological properties and may provide refugial habitat for plant diversity during periods of moderate climate change.     

Linking carbon balance to establishment patterns: comparison of whitebark pine and Engelmann spruce seedlings along an herb cover exposure gradient at treeline

There is increasing evidence that landscape vegetation patterns near species?? range limits are associated with positive biotic interactions, such as in the alpine-treeline ecotone. In the northern Rocky Mountains, whitebark pine (Pinus albicaulis) is considered an early-successional species, able to establish in exposed microsites, while late-successional species such as Engelmann spruce (Picea engelmannii) are more dependent on neighboring vegetation to facilitate establishment. We compared ecophysiological traits associated with carbon balance of newly germinated seedlings of whitebark pine and Engelmann spruce along an herb cover gradient to (1) infer which ecophysiological properties explain the establishment success of seedlings, and (2) to assess differences in establishment patterns with respect to distance from neighboring vegetation. We measured survival over 2 years, and concurrently measured gas exchange and water relations (photosynthesis, respiration, and transpiration), morphology [specific leaf area (SLA)], and biochemistry [chlorophyll fluorescence (F _v /F _m) and nonstructural carbohydrates]. Both species initially established in the most exposed microsites away from vegetation during their first growing season, but only pine persisted in exposed microsites to the end of the second growing season. Pine exhibited phenotypic traits to increase stress tolerance (e.g., higher soluble sugar concentrations, lower SLA) and improve carbon balance (e.g., greater water use efficiency, lower respiration, higher F _v /F _m) compared to spruce in exposed sites, but had lower carbon balance under herb cover. Superior establishment success of pine in exposed microsites at treeline could thus be attributed to a suite of intrinsic physiological advantages that are apparent at the earliest stage of development.     

Dissection of a type I interferon pathway in controlling bacterial intracellular infection in mice

Defence mechanisms against intracellular bacterial pathogens are incompletely understood. Our study characterizes a type I IFN-dependent cell-autonomous defence pathway directed against Legionella pneumophila, an intracellular model organism and frequent cause of pneumonia. We show that macrophages infected with L. pneumophila produced IFNβ in a STING- and IRF3- dependent manner. Paracrine type I IFNs stimulated upregulation of IFN-stimulated genes and a cell-autonomous defence pathway acting on replicating and non-replicating Legionella within their specialized vacuole. Our infection experiments in mice lacking receptors for type I and/or II IFNs show that type I IFNs contribute to expression of IFN-stimulated genes and to bacterial clearance as well as resistance in L. pneumophila pneumonia in addition to type II IFN. Overall, our study shows that paracrine type I IFNs mediate defence against L. pneumophila, and demonstrates a protective role of type I IFNs in in vivo infections with intracellular bacteria.