Structure of Micrococcus cryophilus after freeze-etching

Summary Principle. Nitrate reductases A and B use methyl viologen as an electron donor. This oxidation-reduction indicator is reduced, in anaerobic conditions, by an excess of dithionite. The nitrite produced is estimated by colorimetry. Enzyme A. The activity is stable for at least the first 30 min of the reaction and it is proportional to enzyme concentration. Enzyme B. The activity decreases rapidly and continuously from the start of the reaction. Conclusion. The colorimetric method can be used to measure the activity of enzyme A. But it is inapplicable to enzyme B.

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Les nitrate-réductases bactériennesVII. Mesure de l'activité des enzymes A et B par une méthode colorimétrique

Résumé Principe. Les nitrate-réductases A et B utilisent le méthyl-viologène comme donneur d'électrons. Cet indicateur d'oxydoréduction est réduit, en anaérobiose, par un excès d'hydrosulfite. Le nitrite produit est dosé par colorimétrie. Enzyme A. L'activité demeure constante pendant 30 min au moins après le début de la réaction et elle varie proportionnellement à la concentration d'enzyme. Enzyme B. L'activité diminue rapidement et de façon continue dès le début de la réaction. Conclusion. La méthode colorimétrique perment de mesurer l'activité de l'enzyme A. Par contre, elle est inapplicable à l'enzyme B.

     

Serpentine ecotypic differentiation in a polyploid plant complex: shared tolerance to Mg and Ni stress among di- and tetraploid serpentine populations of <Emphasis Type="Italic">Knautia arvensis</Emphasis> (Dipsacaceae)

Background and aims

Serpentine soils impose limits on plant growth and survival and thus provide an ideal model for studying plant adaptation under environmental stress. Despite the increasing amount of data on serpentine ecotypic differentiation, no study has assessed the potential role of polyploidy. We tested for links between polyploidy and the response to serpentine stress in Knautia arvensis, a diploid-tetraploid, edaphically differentiated complex.

Methods

Variation in growth, biomass yield and tissue Mg and Ni accumulation in response to high Mg and Ni concentrations were experimentally tested using hydroponic cultivation of seedlings from eight populations of different ploidy and edaphic origin.

Results

Regardless of ploidy level, serpentine populations exhibited higher tolerance to both Mg and Ni stress than their non-serpentine counterparts, suggesting an adaptive character of these traits in K. arvensis. The effect of ploidy was rather weak and confined to a slightly better response of serpentine tetraploids to Mg stress and to higher biomass yields in tetraploids from both soil types.

Conclusions

The similar response of diploid and tetraploid serpentine populations to edaphic stress corresponded with their previously described genetic proximity. This suggests that serpentine tolerance might have been transmitted during the local autopolyploid origin of serpentine tetraploids.

     

Clearance rates of the bdelloid rotifer, <Emphasis Type="Italic">Habrotrocha thienemanni</Emphasis>, a tree-hole inhabitant

Bdelloid rotifers are basal consumers in aquatic and limnoterrestrial communities that feed primarily on small bacteria. Unfortunately, we know only a little of the role they play in the trophic dynamics in some unusual habitats they inhabit. Habrotrocha thienemanni is a typical example; it is a typical tree-hole inhabitant, commonly achieving dense populations. Filtering rates of H. thienemanni were estimated using fluorescent microspheres of a size close to natural bacterial community (0.5 μm in diameter) at two temperatures (15 and 20°C). This microspheres artificial food had been coated with BSA protein. Mean clearance rates of this rotifer varied between 1.65 and 3.79 μl ind⁻¹ h⁻¹ under different temperatures. Uptake of particles coated with protein was significantly higher than that on uncoated particles (t = 2.85; P = 0.005). Particle uptake also was correlated to the body size of the animal (r = 0.44; P = 0.004,). The clearance rate of the natural H. thienemanni population (56,800 ind l⁻¹) ranged from 981 to 5170 ml l⁻¹ d⁻¹.     

Insights into the Effect of Soil pH on N2O and N2 Emissions and Denitrifier Community Size and Activity

The objective of this study was to investigate how changes in soil pH affect the N₂O and N₂ emissions, denitrification activity, and size of a denitrifier community. We established a field experiment, situated in a grassland area, which consisted of three treatments which were repeatedly amended with a KOH solution (alkaline soil), an H₂SO₄ solution (acidic soil), or water (natural pH soil) over 10 months. At the site, we determined field N₂O and N₂ emissions using the ¹⁵N gas flux method and collected soil samples for the measurement of potential denitrification activity and quantification of the size of the denitrifying community by quantitative PCR of the narG, napA, nirS, nirK, and nosZ denitrification genes. Overall, our results indicate that soil pH is of importance in determining the nature of denitrification end products. Thus, we found that the N₂O/(N₂O + N₂) ratio increased with decreasing pH due to changes in the total denitrification activity, while no changes in N₂O production were observed. Denitrification activity and N₂O emissions measured under laboratory conditions were correlated with N fluxes in situ and therefore reflected treatment differences in the field. The size of the denitrifying community was uncoupled from in situ N fluxes, but potential denitrification was correlated with the count of NirS denitrifiers. Significant relationships were observed between nirS, napA, and narG gene copy numbers and the N₂O/(N₂O + N₂) ratio, which are difficult to explain. However, this highlights the need for further studies combining analysis of denitrifier ecology and quantification of denitrification end products for a comprehensive understanding of the regulation of N fluxes by denitrification.Denitrification is the microbial reduction of NO₃⁻ via NO₂⁻ to gaseous NO, N₂O, and N₂, which are then lost into the atmosphere (36). It therefore results in considerable loss of nitrogen, one of the most limiting nutrients for crop production in agriculture (20). Denitrification is also of environmental concern since, together with nitrification, it is the main biological process responsible for N₂O emissions (7). N₂O is a potent greenhouse gas which has a global warming potential about 320 times greater than that of CO₂ and has a lifetime of approximately 120 years (32). In the stratosphere, N₂O can also react with O₂ to produce NO, which induces the destruction of stratospheric ozone (8). N₂O can be released into the atmosphere by incomplete denitrification due to the effect of environmental conditions on the regulation of the different denitrification reductases (14, 41, 51), but it has recently been suggested that it could also be due to lack of nitrous oxide reductase in some denitrifiers (19, 41). Since N₂O is an intermediate in the denitrification pathway, both the amount of N₂O produced and the N₂O/(N₂O + N₂) ratio are important in understanding and predicting N₂O fluxes from soils.The main environmental factors known to influence the N₂O/(N₂O + N₂) ratio are pH, organic carbon and NO₃⁻ availability, water content, and O₂ partial pressure (50). Soil pH is one of the most important factors influencing both denitrification and N₂O production (43). In general, the denitrification rate increases with increasing pH values (up to the optimum pH) while, in contrast, the N₂O/(N₂O + N₂) ratio decreases (50). This relationship has been characterized in laboratory experiments (9, 45), but it is not clear whether the same relationships exist in the field because of methodological limitations of in situ measurement of N₂ emissions (16). Nevertheless, ¹⁵N tracing experiments based on the addition of a labeled denitrification substrate to soil offer a useful tool to quantify emissions of both N₂O and N₂ in situ (47, 49). Soil pH is also an important factor influencing denitrifier community composition (35, 39), which can be an important driver of denitrification activity and N₂O emissions (5, 21). A recent study reported a negative relationship between the proportion of bacteria genetically capable of reducing N₂O within the total bacterial community and the N₂O/(N₂O + N₂) ratio, with both being strongly correlated with soil pH (38).The objective of the present study was to explore the effect of changes in soil pH on in situ N₂O and N₂ emissions, denitrifying enzyme activity (DEA), and potential N₂O production. In addition, we also investigated whether differences in N fluxes could be related to changes in the size of the microbial community possessing the different denitrification genes. A field experiment was conducted using replicated grassland plots in which the soil pH was modified by addition of either acid or hydroxide to the soil. A ¹⁵N tracer method was used to provide information on N emissions. In addition to measuring potential denitrification activity, the size of the denitrifier community was determined by real-time PCR quantification of the denitrification genes.     

The double-blind sham-controlled study of high-frequency rTMS (20 Hz) for negative symptoms in schizophrenia: negative results

The high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) over the prefrontal cortex is a promising method for the treatment of negative symptoms of schizophrenia. Using double-blind sham-controlled parallel design, we evaluated the effect of HF-rTMS over the left dorsolateral prefrontal cortex (DLPFC) on negative symptoms in patients with schizophrenia. Sixteen schizophrenia patients with predominantly negative symptoms on stable antipsychotic medication were treated with 20 Hz rTMS (90% of motor threshold, 2000 stimuli per session) over ten days within 2 weeks with six weeks follow-up. The effect was assessed using PANSS, CGI, MADRS and neuropsychological tests. We failed to find any significant effect of active rTMS. Sham rTMS showed a trend for improvement over time on positive and negative subscales of PANSS and MADRS. Between-group comparisons failed to reveal any significant differences on any rating scales except a positive subscale of PANSS after 8 weeks. Results from our study did not confirm that HF-rTMS over the left DLPCF affects the negative symptoms of schizophrenia and alternative rTMS approaches are discussed.     

Purification and characterization of a nitrilase from Aspergillus niger K10

Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg^-1) at 45°C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of d-sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.     

Ectopic over-expression of the maize beta-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco

The activity of the phytohormone cytokinin depends on a complex interplay of factors such as its metabolism, transport, stability, and cellular/tissue localization. O-glucosides of zeatin-type cytokinins are postulated to be storage and/or transport forms, and are readily deglucosylated. Transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants were constructed over-expressing Zm-p60.1, a maize beta-glucosidase capable of releasing active cytokinins from O- and N3-glucosides, to analyse its potential to perturb zeatin metabolism in planta. Zm-p60.1 in chloroplasts isolated from transgenic leaves has an apparent K(m) more than 10-fold lower than the purified enzyme in vitro. Adult transgenic plants grown in the absence of exogenous zeatin were morphologically indistinguishable from the wild type although differences in phytohormone levels were observed. When grown on medium containing zeatin, inhibition of root elongation was apparent in all seedlings 14 d after sowing (DAS). Between 14 and 21 DAS, the transgenic seedlings accumulated fresh weight leading later (28-32 DAS) to ectopic growths at the base of the hypocotyl. The development of ectopic structures correlated with the presence of the enzyme as demonstrated by histochemical staining. Cytokinin quantification showed that transgenic seedlings grown on medium containing zeatin accumulate active metabolites like zeatin riboside and zeatin riboside phosphate and this might lead to the observed changes. The presence of the enzyme around the base of the hypocotyl and later, in the ectopic structures themselves, suggests that the development of these structures is due to the perturbance in zeatin metabolism caused by the ectopic presence of Zm-p60.1.