Bradyrhizobium japonicum mutants defective in root-nodule bacteroid development and nitrogen fixation

The genome of the slow-growing Bradyrhizobium japonicum (strain 110) was mutagenized with transposon Tn5. A total of 1623 kanamycin/streptomycin resistant derivatives were screened in soybean infection tests for nodulation (Nod) and symbiotic nitrogen fixation (Fix). In this report we describe 14 strains possessing a stable, reproducible Nod⁺Fix^- phenotype. These strains were also grown under microaerobic culture conditions to test them for free-living nitrogen fixation activity (Nif). In addition to strains having reduced Fix and Nif activities, there were also strains that had reduced symbiotic Fix activity but were Nif⁺ ex planta.Analysis of the genomic structure revealed that the majority of the strains had a single Tn5 insertion without any further apparent physical alteration. A few strains had additional insertions (by Tn5 or IS50), or a deletion, or had cointegrated part of the vector used for Tn5 mutagenesis. One of the insertions was found in a known nif gene (nifD) whereas all other mutations seem to affect different, hitherto unknown genes or operons.Several mutant strains had an altered nodulation phenotype, inducing numerous, small, widely distributed nodules. Light and electron microscopy revealed that most of these mutants were defective in different stages of bacteroid development and/or bacteroid persistence. The protein patterns of the mutants were inspected by two-dimensional gel electrophoresis after labelling microaerobic cultures with l-(³⁵S)methionine. Of particular interest were mutants lacking a group of proteins the synthesis of which was known to be under oxygen control. Such strains can be regarded as potential regulatory mutants.     

Molecular evidence for a fourth species within the Isotoma viridis group (Insecta, Collembola)

For identification of single species within the Isotoma viridis group, we present polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) as a fast and efficient DNA-based molecular method. We used five PCR primers amplifying the cytochrome oxidase II (COII) region (760 bp) of the mitochondrial DNA. The sequences clearly separated four species ( I. viridis , I. riparia , I. anglicana and I. caerulea ) out of samples from Norway, Sweden, Germany and Switzerland. Examination of genetic variation and phylogenetic relationship did not support the separation of two colour pattern forms of I. viridis into distinct species. For RFLP, several restriction enzymes were tested for their ability to produce not only species-specific restriction fragment patterns but to discriminate more than one species per enzyme used with as few cleavage sites as possible. Such a design should render a clear fragment pattern when performing a double digest. These demands appear to be fulfilled best by the combination of the restriction enzymes Mfe I, Nci I and one of Aci I, Bst EII, Nde I, or Sfc I. From the enzymes tested in a previous study, Ase I proved to be reliable, whereas Mbo I can no longer be recommended.     

Cloning, functional analysis, and mitochondrial localization of Trypanosoma brucei monothiol glutaredoxin-1

African trypanosomes encode three monothiol glutaredoxins (1-C-Grx1 to 3). 1-C-Grx1 has a putative CAYS active site and Cys181 as single additional cysteine. The recombinant protein forms non-covalent homodimers. As observed for other monothiol glutaredoxins, Trypanosoma brucei 1-C-Grx1 was not active in the glutaredoxin assay with hydroxyethyl disulfide and glutathione nor catalyzed the reduction of insulin disulfide. In addition, it lacked peroxidase activity and did not catalyze protein (de)glutathionylation. Upon oxidation, 1-C-Grx1 forms an intramolecular disulfide bridge and, to a minor degree, covalent dimers. Both disulfide forms are reduced by the parasite trypanothione/tryparedoxin system. 1-C-Grx1 shows mitochondrial localization. The total cellular concentration is at least 5 microm. Thus, 1-C-Grx1 is an abundant protein especially in the rudimentary organelle of the mammalian form of the parasite. Expression of 1-C-Grx1 in Grx5-deficient yeast cells with its authentic presequence targeted the protein to the mitochondria and partially restored the growth phenotype and aconitase activity of the mutant, and conferred resistance against hydroperoxides and diamide. The parasite Grx2 and 3 failed to substitute for Grx5. This is surprising because even bacterial and plant 1-Cys-glutaredoxins efficiently revert the defects, and may be due to the lack of two basic residues conserved in all but the trypanosomatid proteins.     

Opposing Effects of CREBBP Mutations Govern the Phenotype of Rubinstein-Taybi Syndrome and Adult SHH Medulloblastoma

1. Download high-res image (222KB)
2. Download full-size image

     

Metabolism of 1,3-butadiene to toxicologically relevant metabolites in single-exposed mice and rats

1,3-Butadiene (BD) was carcinogenic in rodents. This effect is related to reactive metabolites such as 1,2-epoxy-3-butene (EB) and especially 1,2:3,4-diepoxybutane (DEB). A third mutagenic epoxide, 3,4-epoxy-1,2-butanediol (EBD), can be formed from DEB and from 3-butene-1,2-diol (B-diol), the hydrolysis product of EB. In BD exposed rodents, only blood concentrations of EB and DEB have been published. Direct determinations of EBD and B-diol in blood are missing. In order to investigate the BD-dependent blood burden by all of these metabolites, we exposed male B6C3F1 mice and male Sprague-Dawley rats in closed chambers over 6-8h to constant atmospheric BD concentrations. BD and exhaled EB were measured in chamber atmospheres during the BD exposures. EB blood concentrations were obtained as the product of the atmospheric EB concentration at steady state with the EB blood-to-air partition coefficient. B-diol, EBD, and DEB were determined in blood collected immediately at the end of BD exposures up to 1200 ppm (B-diol, EBD) and 1280 ppm (DEB). Analysis of BD was done by GC/FID, of EB, DEB, and B-diol by GC/MS, and of EBD by LC/MS/MS. EB blood concentrations increased with BD concentrations amounting to 2.6 micromol/l (rat) and 23.5 micromol/l (mouse) at 2000 ppm BD and to 4.6 micromol/l in rats exposed to 10000 ppm BD. DEB (detection limit 0.01 micromol/l) was found only in blood of mice rising to 3.2 micromol/l at 1280 ppm BD. B-diol and EBD were quantitatively predominant in both species. B-diol increased in both species with the BD exposure concentration reaching 60 micromol/l at 1200 ppm BD. EBD reached maximum concentrations of 9.5 micromol/l at 150 ppm BD (rat) and of 42 micromol/l at 300 ppm BD (mouse). At higher BD concentrations EBD blood concentrations decreased again. This picture probably results from a competitive inhibition of the EBD producing CYP450 by BD, which occurs in both species.     

Quantitative assessment of hemolymph metabolites in two physiological states and two populations of the land snail Helix pomatia

Hemolymph metabolite composition in ectothermic species is mainly constrained by trophic and climatic habitat conditions. In temperate regions, ectothermic species have to face subzero temperatures in winter, to which they typically respond with a state of inactivity. With use of ultra-performance liquid chromatography and gas chromatography-mass spectrometry techniques, we investigated the hemolymph metabolite composition of the land snail Helix pomatia with respect to physiological states (activity and hibernation) in a mountain population (800 m above sea level) and a valley population (150 m above sea level) in Germany. The dry masses of active snails as well as the saccharide and amino acid concentrations in active snails were higher in the mountain population than in the valley population. These differences between populations might reflect differences in microhabitat conditions, such as climate and vegetal food, and consequent differences in metabolic activity. Galactose was the most abundant component in hemolymph besides glucose. Both saccharides might indicate glycolytic activity, which could provide energy for locomotion and foraging. In hibernation, glutamate, α-alanine, glycine, aspartate, serine, homoserine, hydroxyproline, glycerol, and triglycerides were accumulated in both populations. The concentrations were correlated with a decrease in body supercooling point. Therefore, these metabolites might have a role in the cold hardiness of H. pomatia that should be further investigated in a functional study.     

Ecological theory meets soil ecotoxicology: Challenge and chance

The degradation of soils due to various anthropogenic stress factors is alarming. Although chemicals are a major reason for soil degradation, most ecologists are not interested in studying such effects. We try to wake their interest by addressing a number of unsolved soil ecotoxicological problems that are related to disturbance ecology, biodiversity, ecosystem functioning and modelling. Features distinguishing chemical from natural stress render promising new aspects in disturbance ecology. Ecotoxicological studies are ideal models of disturbance, particularly regarding frequency, intensity or multitude of stress. Patterns of secondary succession after a major chemical damage can directly be related to the intermediate disturbance hypothesis. More knowledge on altered life history patterns following stress could support both evolutionary ecology and risk assessment. We raise the question if inherent resource competition makes communities more vulnerable to stress. Three aspects of ecotoxicological risk assessment are introduced: (1) exposure and bioavailability, which is directly connected to environmental heterogeneity; (2) tests on ecosystem functioning, suffering from major drawbacks; and (3) modelling. Here, promising approaches exist but need substantial input for being applicable to soils. Ecological modelling should put more emphasis on simulating both natural and chemical disturbances, including behavioural aspects and environmental variability. Finally, research needs for ecological risk assessment in soils are derived such as a simple system to assess the impact of chemicals on soil biodiversity, the inclusion of behavioural changes of keystone species or the consideration of density-dependent effects. Common research efforts of basic ecologists and soil ecotoxicologists could render a lot of mutual benefits.     

Adjustment of metabolite composition in the haemolymph to seasonal variations in the land snail <Emphasis Type="Italic">Helix pomatia</Emphasis>

In temperate regions, land snails are subjected to subzero temperatures in winter and hot temperatures often associated to drought in summer. The response to these environmental factors is usually a state of inactivity, hibernation and aestivation, respectively, in a temperature and humidity buffered refuge, accompanied by physiological adjustments to resist cold or heat stress. We investigated how environmental factors in the microhabitat and body condition influence the metabolite composition of haemolymph of the endangered species Helix pomatia. We used UPLC and GC–MS techniques and analyzed annual biochemical variations in a multivariate model. Hibernation and activity months differed in metabolite composition. Snails used photoperiod as cue for seasonal climatic variations to initiate a physiological state and were also highly sensitive to temperature variations, therefore constantly adjusting their physiological processes. Galactose levels gave evidence for the persistence of metabolic activity with energy expenditure during hibernation and for high reproductive activity in June. Triglycerides accumulated prior to hibernation might act as cryoprotectants or energy reserves. During the last month of hibernation snails activated physiological processes related to arousal. During activity, protein metabolism was reflected by high amino acid level. An exceptional aestivation period was observed in April giving evidence for heat stress responses, like the protection of cells from dehydration by polyols and saccharides, the membrane stabilization by cholesterol and enhanced metabolism using the anaerobic succinic acid pathway to sustain costly stress responses. In conclusion, physiological adjustments to environmental variations in Helix pomatia involve water loss regulation, cryoprotectant or heatprotectant accumulation.