Two pathways of sphingolipid biosynthesis are separated in the yeast Pichia pastoris

Although the yeast Saccharomyces cerevisiae has only one sphingolipid class with a head group based on phosphoinositol, the yeast Pichia pastoris as well as many other fungi have a second class, glucosylceramide, which has a glucose head group. These two sphingolipid classes are in addition distinguished by a characteristic structure of their ceramide backbones. Here, we investigate the mechanisms controlling substrate entry into the glucosylceramide branch of the pathway. By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris. By preferring dihydroxy sphingoid bases and C(16)/C(18) acyl-coenzyme A as substrates, Bar1p produces a structurally well defined group of ceramide species, which is the exclusive precursor for glucosylceramide biosynthesis. Correlating with the absence of glucosylceramide in this yeast, a gene encoding Bar1p is missing in S. cerevisiae. We could not successfully investigate the second ceramide synthase in P. pastoris that is orthologous to S. cerevisiae Lag1p/Lac1p. By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid Δ4-desaturase and fatty acid α-hydroxylase, before the final addition of the glucose head group by the glucosylceramide synthase. Together, this set of four enzymes specifically defines the pathway leading to glucosylceramide biosynthesis.     

Identification of enzyme activity quantitative trait loci in a Solanum lycopersicum x Solanum pennellii introgression line population

Activities of 28 enzymes from central carbon metabolism were measured in pericarp tissue of ripe tomato fruits from field trials with an introgression line (IL) population generated by introgressing segments of the genome of the wild relative Solanum pennellii (LA0716) into the modern tomato cultivar Solanum lycopersicum M82. Enzyme activities were determined using a robotized platform in optimized conditions, where the activities largely reflect the level of the corresponding proteins. Two experiments were analyzed from years with markedly different climate conditions. A total of 27 quantitative trait loci were shared in both experiments. Most resulted in increased enzyme activity when a portion of the S. lycopersicum genome was substituted with the corresponding portion of the genome of S. pennellii. This reflects the change in activity between the two parental genotypes. The mode of inheritance was studied in a heterozygote IL population. A similar proportion of quantitative trait loci (approximately 30%) showed additive, recessive, and dominant modes of inheritance, with only 5% showing overdominance. Comparison with the location of putative genes for the corresponding proteins indicates a large role of trans-regulatory mechanisms. These results point to the genetic control of individual enzyme activities being under the control of a complex program that is dominated by a network of trans-acting genes.     

Intraguild interactions among generalist predator functional groups drive impact on herbivore and decomposer prey

Different functional groups of generalist predators may complement each other in controlling prey populations; but intraguild interactions, common among generalist predators, may also reduce the strength of top–down control. In natural communities greater alterations to ecosystem function are expected if a whole functional group declines in abundance or is lost. Therefore studying functional group diversity is important for predicting effects of predator loss. We studied the top–down impact of web‐building spiders, hunting spiders and ants, which are highly abundant generalist predators in most terrestrial ecosystems, on prey from the herbivore and decomposer system of a grassland food web. The density of the three predator groups was manipulated by continuous removal in a three‐factorial designed field experiment, which was carried out for two years. We found no positive effect of increasing predator functional group richness on prey control. However there was evidence for strong composition effects between the functional groups. The presence of ants in predator assemblages reduced the prey suppression through mostly trait‐mediated intraguild interactions, while hunting and web‐building spiders contributed additively to prey suppression and reduced the density of herbivore and decomposer prey by 50–60%. A trophic cascade on plant biomass triggered by web‐builders and hunting spiders was diminished at levels of higher predator group diversity. In conclusion, our experiments showed that intraguild interactions strongly influence the strength of top–down control by generalist predators. Among spiders there was evidence for a positive relation between functional group richness and prey suppression but the overall outcome strongly depended on the occurrence of interference, driven by trait‐mediated indirect interactions.     

Elimination kinetics of dexamethasone in bovine urine, hair and feces following single administration of dexamethasone acetate and phosphate esters

Corticosteroids are hormonal substances widely used in human and veterinary medicine for their anti-inflammatory properties. Among the numerous existing artificial corticosteroids, dexamethasone remains the most commonly used, mainly throughout esterified forms such as acetate or phosphate. An experimental study was designed to assess its drug residue levels in urine and feces, as well as its fixation in bovine hair following a single administration of 0.15 mg/kg b.w. dexamethasone acetate and 0.12 mg/kg b.w. dexamethasone sodium phosphate. Different analytical methods based on GC-MS or LC-MS/MS were used for measuring dexamethasone and its esterified forms, which were implemented in 3 different European laboratories in the field that collaborated for this study. The obtained results confirmed the high and rapid urinary excretion rate of dexamethasone, with a maximal concentration (267 μg/L) measured one day after administration and 98% elimination within 3 days. The concentrations obtained with the GC-NCI-MS procedure (using chemical oxidation as derivatization) were found significantly higher than the ones obtained with LC-ESI-MS/MS, indicating a possible contribution of dexamethasone phase I and/or II metabolites to the monitored signal. Fecal elimination was also found rapid (95% elimination within 3 days) with a maximum concentration level (28.5 μg/kg) observed one day after administration. Detectable levels of dexamethasone in hair appeared on day 2 (11.5 μg/kg), reached a maximum around one week, and could be identified until 22 days upon treatment, establishing the suitability of hair as a biological matrix for medium to long-term residue controls of dexamethasone.     

Volatile-mediated killing of Arabidopsis thaliana by bacteria is mainly due to hydrogen cyanide

The volatile-mediated impact of bacteria on plant growth is well documented, and contrasting effects have been reported ranging from 6-fold plant promotion to plant killing. However, very little is known about the identity of the compounds responsible for these effects or the mechanisms involved in plant growth alteration. We hypothesized that hydrogen cyanide (HCN) is a major factor accounting for the observed volatile-mediated toxicity of some strains. Using a collection of environmental and clinical strains differing in cyanogenesis, as well as a defined HCN-negative mutant, we demonstrate that bacterial HCN accounts to a significant extent for the deleterious effects observed when growing Arabidopsis thaliana in the presence of certain bacterial volatiles. The environmental strain Pseudomonas aeruginosa PUPa3 was less cyanogenic and less plant growth inhibiting than the clinical strain P. aeruginosa PAO1. Quorum-sensing deficient mutants of C. violaceum CV0, P. aeruginosa PAO1, and P. aeruginosa PUPa3 showed not only diminished HCN production but also strongly reduced volatile-mediated phytotoxicity. The double treatment of providing plants with reactive oxygen species scavenging compounds and overexpressing the alternative oxidase AOX1a led to a significant reduction of volatile-mediated toxicity. This indicates that oxidative stress is a key process in the physiological changes leading to plant death upon exposure to toxic bacterial volatiles.     

Ralstonia solanacearum ��PGI-1 Strain KZR-5 Is Affected in Growth, Response to Cold Stress and Invasion of Tomato

The survival and persistence of Ralstonia solanacearum biovar 2 in temperate climates is still poorly understood. To assess whether genomic variants of the organism show adaptation to local conditions, we compared the behaviour of environmental strain KZR-5, which underwent a deletion of the 17.6?kb genomic island PGI-1, with that of environmental strain KZR-1 and potato-derived strains 1609 and 715. PGI-1 harbours two genes of potential ecological relevance, i.e. one encoding a hypothetical protein with a RelA/SpoT domain and one a putative cellobiohydrolase. We thus assessed bacterial fate under conditions of amino acid starvation, during growth, upon incubation at low temperature and invasion of tomato plants. In contrast to the other strains, environmental strain KZR-5 did not grow on media that induce amino acid starvation. In addition, its maximum growth rate at 28°C in rich medium was significantly reduced. On the other hand, long-term survival at 4°C was significantly enhanced as compared to that of strains 1609, 715 and KZR-1. Although strain KZR-5 showed growth rates (at 28°C) in two different media, which were similar to those of strains 1609 and 715, its ability to compete with these strains under these conditions was reduced. In singly inoculated tomato plants, no significant differences in invasiveness were observed among strains KZR-5, KZR-1, 1609 and 715. However, reduced competitiveness of strain KZR-5 was found in experiments on tomato plant colonisation and wilting when using 1:1 or 5:1 mixtures of strains. The potential role of PGI-1 in plant invasion, response to stress and growth in competition at high and moderate temperatures is discussed.     

The effect of area on local and regional elevational patterns of species richness

Aim To calculate the degree to which differences between local and regional elevational species richness patterns can be accounted for by the effects of regional area. Location Five elevational transects in Costa Rica, Ecuador, La Réunion, Mexico and Tanzania. Methods We sampled ferns in standardized field plots and collated regional species lists based on herbarium and literature data. We then used the Arrhenius function S = cA^z to correct regional species richness (S) for the effect of area (A) using three slightly different approaches, and compared the concordance of local and regional patterns prior to and after accounting for the effect of area on regional richness using linear regression analyses. Results We found a better concordance between local and regional elevational species richness after including the effect of area in the majority of cases. In several cases, local and regional patterns are very similar after accounting for area. In most of the cases, the maximum regional richness shifted to a higher elevation after accounting for area. Different approaches to correct for area resulted in qualitatively similar results. Main conclusions The differences between local and regional elevational richness patterns can at least partly be accounted for by area effects, suggesting that the underlying causes of elevational richness patterns might be the same at both spatial scales. Values used to account for the effect of area differ among the different study locations, showing that there is no generally applicable elevational species–area relationship.