Enantioselective transformation of alpha-hexachlorocyclohexane by the dehydrochlorinases LinA1 and LinA2 from the soil bacterium Sphingomonas paucimobilis B90A.

Sphingomonas paucimobilis B90A contains two variants, LinA1 and LinA2, of a dehydrochlorinase that catalyzes the first and second steps in the metabolism of hexachlorocyclohexanes (R. Kumari, S. Subudhi, M. Suar, G. Dhingra, V. Raina, C. Dogra, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal, Appl. Environ. Microbiol. 68:6021-6028, 2002). On the amino acid level, LinA1 and LinA2 were 88% identical to each other, and LinA2 was 100% identical to LinA of S. paucimobilis UT26. Incubation of chiral alpha-hexachlorocyclohexane (alpha-HCH) with Escherichia coli BL21 expressing functional LinA1 and LinA2 S-glutathione transferase fusion proteins showed that LinA1 preferentially converted the (+) enantiomer, whereas LinA2 preferred the (-) enantiomer. Concurrent formation and subsequent dissipation of beta-pentachlorocyclohexene enantiomers was also observed in these experiments, indicating that there was enantioselective formation and/or dissipation of these enantiomers. LinA1 preferentially formed (3S,4S,5R,6R)-1,3,4,5,6-pentachlorocyclohexene, and LinA2 preferentially formed (3R,4R,5S,6S)-1,3,4,5,6-pentachlorocyclohexene. Because enantioselectivity was not observed in incubations with whole cells of S. paucimobilis B90A, we concluded that LinA1 and LinA2 are equally active in this organism. The enantioselective transformation of chiral alpha-HCH by LinA1 and LinA2 provides the first evidence of the molecular basis for the changed enantiomer composition of alpha-HCH in many natural environments. Enantioselective degradation may be one of the key processes determining enantiomer composition, especially when strains that contain only one of the linA genes, such as S. paucimobilis UT26, prevail.

Fusarium oxysporum strains as biocontrol agents against Fusarium wilt: effects on soil microbial biomass and activity

Before planning the large-scale use of nonpathogenic strains of Fusarium oxysporum as biocontrol agents of Fusarium wilt, their behaviour and potential impact on soil ecosystems should be carefully studied as part of risk assessment. The aim of this work was to evaluate the effects of antagonistic F. oxysporum strains, genetically manipulated (T26/6) or not (233/1), on soil microbial biomass and activity. The effects were evaluated, in North-western Italy, in two soils from different sites at Albenga, one natural and the other previously solarized, and in a third soil obtained from a 10-year-old poplar stand (Popolus sp.), near Carignano. There were no detectable effects on ATP, fluorescein diacetate hydrolysis, and biomass P that could be attributed to the introduction of the antagonists. A transient increase of carbon dioxide evolution and biomass C was observed in response to the added inoculum. Although the results showed only some transient alterations, further studies are required to evaluate effects on specific microorganism populations.

Deleterious impact of a virulent bacteriophage on survival and biocontrol activity of Pseudomonas fluorescens strain CHAO in natural soil.

Many biotic and abiotic factors affect the persistence and activity of beneficial pseudomonads introduced into soil to suppress plant diseases. One such factor may be the presence of virulent bacteriophages that decimate the population of the introduced bacteria, thereby reducing their beneficial effect. We have isolated a lytic bacteriophage (phi)GP100) that specifically infects the biocontrol bacterium Pseudomonas fluorescens CHA0 and some closely related Pseudomonas strains. phiGP100 was found to be a double-stranded-DNA phage with an icosahedral head, a stubby tail, and a genome size of approximately 50 kb. Replication of phiGP100 was negatively affected at temperatures higher than 25 degrees C. phiGP100 had a negative impact on the population size and the biocontrol activity of P. fluorescens strain CHA0-Rif (a rifampicin-resistant variant of CHA0) in natural soil microcosms. In the presence of phiGP100, the population size of strain CHA0-Rif in soil and on cucumber roots was reduced more than 100-fold. As a consequence, the bacterium's capacity to protect cucumber against a root disease caused by the pathogenic oomycete Pythium ultimum was entirely abolished. In contrast, the phage affected neither root colonization and nor the disease suppressive effect of a phiDGP100-resistant variant of strain CHA0-Rif. To our knowledge, this study is the first to illustrate the potential of phages to impair biocontrol performance of beneficial bacteria released into the natural soil environment.

Synapse formation in adult barrel cortex following naturalistic environmental enrichment.

Environmental enrichment paradigms in adult laboratory animals, consisting of physical, perceptual, and social stimulation, have been shown to affect synapse and cell morphology in sensory cortex and enhance learning ability, whereas enrichment, which is in harmony with the animal's natural habitat may have even greater implications for plasticity. Previous studies in our laboratory have shown that whisker stimulation induced the formation of synapses and spines in the corresponding barrel. In the present study adult C57/Bl6J female laboratory mice at 6 weeks of age were placed during 2 months in a protected enrichment enclosure in a forest clearing at the Chisti Les Biological Station, Tvier, Russia. We analyzed neuropil ultrastructure in the C2 barrel using serial-section electron microscopy on a total of eight mice (n=4 enriched, n=4 standard cagemate controls). Quantitative analyses of volumes of neuropil showed a significant increase in excitatory and inhibitory synapses on spines and excitatory synapses on dendritic shafts in the C2 barrel in the enriched group compared with standard cagemate controls. These results demonstrate that naturalistic experience alters the synaptic circuitry in layer IV of the somatosensory cortex, the first cortical relay of sensory information, leaving a lasting trace that may guide subsequent behavior.

Complex formation by the human RAD51C and XRCC3 recombination repair proteins.

In vertebrates, the RAD51 protein is required for genetic recombination, DNA repair, and cellular proliferation. Five paralogs of RAD51, known as RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3, have been identified and also shown to be required for recombination and genome stability. At the present time, however, very little is known about their biochemical properties or precise biological functions. As a first step toward understanding the roles of the RAD51 paralogs in recombination, the human RAD51C and XRCC3 proteins were overexpressed and purified from baculovirus-infected insect cells. The two proteins copurify as a complex, a property that reflects their endogenous association observed in HeLa cells. Purified RAD51C--XRCC3 complex binds single-stranded, but not duplex DNA, to form protein--DNA networks that have been visualized by electron microscopy.

Survival in soil and detection of co-transformed Trichoderma harzianum by nested PCR

The objective of this work was to evaluate the survival of two Trichoderma harzianum co-transformants, TE 10 and TE 41, carrying genes for green fluorescent protein (egfp) and for resistance to benomyl, during four weeks in a contained soil microcosm. Selective culture media were used to detect viable fungal material, whose identity was confirmed by the observation of the fluorescent phenotype by direct epifluorence microscopy. PCR using two nested primer pairs specific to the egfp gene was also used to detect the transformed fungi. Although it was not possible to reliably detect the egfp gene directly from soil extracts, an enrichment step involving selective culture of soil samples in liquid medium prior to DNA extraction enabled the consistent detection of the T. harzianum co-transformants by nested PCR for the duration of the incubation period.

Fate of Hydrophilic Nanoparticles in Biological Environments

The nanoparticles developed are based on chitosan, a biocompatible and biodegradable polysaccharide. The chitosan nanoparticles are formed in an entirely water-based process by electrostatic interactions with other biocompatible molecules. As a prerequisite to understand the fate of such nanoparticles in cells, comprehensive characterization and stability studies serve to identify quantitatively the impact of the raw material characteristics and preparation conditions on the nanoparticle characteristics. Methods included H-1 NMR spectroscopy, dilution viscometry, particle size analysis and electron microscopy. Cytotoxicity and cell uptake experiments on RAW 264.7 murine macrophages and p23 murine endothelial cells were performed to investigate the correlation with nanoparticle characteristics and effect of surface decoration with alginate. Cytotoxicity was assessed by the MTT survival test; cell uptake was monitored by fluorescent microscopy using labeled polymers.

Regulation of mucoidy in the biological control agent Pseudomonas fluorescens CHA0 and construction of an exopolysaccharide-overproducing variant

Pseudomonas fluorescens CHA0, an effective biological control agent of soilborne plant diseases, is naturally non-mucoid. We have isolated a highly mucoid Tn5 insertion mutant of strain CHA0. The mucoid phenotype was found to be due to the overproduction of exopolysaccharide (EPS), as a result of a mutation in the mucA gene. The wild-type mucA gene was cloned by a two-step, Tn5-dependent cloning procedure previously described and the deduced amino acid sequence showed 71% identity with MucA of P. aeruginosa, a negative regulator of the alternative sigma factor AlgU (=s22, sE). As in P. aeruginosa, mucA is preceded by the algU gene encoding s22 (91% identity at the amino acid sequence level). A mucA in-frame deletion mutant of CHA0 overproduced EPS and formed mucoid colonies, whereas an algU in-frame deletion mutant showed a non-mucoid phenotype. Pyoluteorin, an antibiotic produced by P. fluorescens, was found to be entrapped in EPS of a mucoid mutant. In natural soil, mucoidy negatively affected survival of the bacteria, suggesting that under these conditions the potential to produce abundant EPS does not confer a selective advantage on the bacteria.

Two novel MvaT-like global regulators control exoproduct formation and biocontrol activity in root-associated Pseudomonas fluorescens CHA0.

Pseudomonas fluorescens CHA0 protects various crop plants against root diseases caused by pathogenic fungi. Among a range of exoproducts excreted by strain CHA0, the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) are particularly relevant to the strain's biocontrol potential. Here, we report on the characterization of MvaT and MvaV as novel regulators of biocontrol activity in strain CHA0. We establish the two proteins as further members of an emerging family of MvaT-like regulators in pseudomonads that are structurally and functionally related to the DNA-binding protein H-NS. In mvaT and mvaV in frame-deletion mutants of strain CHA0, PLT production was enhanced about four- and 1.5-fold, respectively, whereas DAPG production remained at wild-type levels. Remarkably, PLT production was increased up to 20-fold in an mvaT mvaV double mutant. DAPG biosynthesis was almost completely repressed in this mutant. The effects on antibiotic production could be confirmed by following expression of gfp-based reporter fusions to the corresponding biosynthetic genes. MvaT and MvaV also influenced levels of other exoproducts, motility, and physicochemical cell-surface properties to various extents. Compared with the wild type, mvaT and mvaV mutants had an about 20% reduced capacity (in terms of plant fresh weight) to protect cucumber from a root rot caused by Pythium ultimum. Biocontrol activity was nearly completely abolished in the double mutant Our findings indicate that MvaT and MvaV act together as further global regulatory elements in the complex network controlling expression of biocontrol traits in plant-beneficial pseudomonads.

DNA barcodes for soil animal taxonomy

The biodiversity of soil communities remains very poorly known and understood. Soil biological sciences are strongly affected by the taxonomic crisis, and most groups of animals in that biota suffer from a strong taxonomic impediment. The objective of this work was to investigate how DNA barcoding - a novel method using a microgenomic tag for species identification and discrimination - permits better evaluation of the taxonomy of soil biota. A total of 1,152 barcode sequences were analyzed for two major groups of animals, collembolans and earthworms, which presented broad taxonomic and geographic sampling. Besides strongly reflecting the taxonomic impediment for both groups, with a large number of species-level divergent lineages remaining unnamed so far, the results also highlight a high level (15%) of cryptic diversity within known species of both earthworms and collembolans. These results are supportive of recent local studies using a similar approach. Within an impeded taxonomic system for soil animals, DNA-assisted identification tools can facilitate and improve biodiversity exploration and description. DNA-barcoding campaigns are rapidly developing in soil animals and the community of soil biologists is urged to embrace these methods.

Towards a universal sampling protocol for soil biotas in the humid tropics

This paper reviews the methods for the inventory of below-ground biotas in the humid tropics, to document the (hypothesized) loss of soil biodiversity associated with deforestation and agricultural intensification at forest margins. The biotas were grouped into eight categories, each of which corresponded to a major functional group considered important or essential to soil function. An accurate inventory of soil organisms can assist in ecosystem management and help sustain agricultural production. The advantages and disadvantages of transect-based and grid-based sampling methods are discussed, illustrated by published protocols ranging from the original "TSBF transect", through versions developed for the alternatives to Slash-and-Burn Project (ASB) to the final schemes (with variants) adopted by the Conservation and Sustainable Management of Below-ground Biodiversity Project (CSM-BGBD). Consideration is given to the place and importance of replication in below-ground biological sampling and it is argued that the new sampling protocols are inclusive, i.e. designed to sample all eight biotic groups in the same field exercise; spatially scaled, i.e. provide biodiversity data at site, locality, landscape and regional levels, and link the data to land use and land cover; and statistically robust, as shown by a partial randomization of plot locations for sampling.

Why should we care about soil fauna?

The reasons why we care about soil fauna are related to their intrinsic, utilitarian and functional values. The intrinsic values embrace aesthetic or moral reasons for conserving below-ground biodiversity. Unfortunately, the protection of soil invertebrates has rarely been a criterion for avoiding changes in land use and management. Utilitarian, or direct use values, have been investigated more extensively for fungi, bacteria and marine invertebrates than for soil fauna. However, some traditional remedies, novel enzymes and pharmaceutical compounds have been derived from earthworms, termites and other groups, and gut symbionts may provide microbial strains with interesting properties for biotechnology. The functional importance of soil invertebrates in ecosystem processes has been a major focus of research in recent decades. It is suggested herein that it is rarely possible to identify the role of soil invertebrates as rate determinants of soil processes at plot and ecosystem scales of hectares and above because other biophysical controls override their effects. There are situations, however, where the activities of functional groups of soil animals, even of species, are synchronised in space or time by plant events, resource inputs, seasonality or other perturbations to the system, and their emergent effects are detectable as higher order controls.

Origin of needle fibre calcite (NFC) : a geochemical approach

ABSTRACTNeedle fibre calcite (NFC) is one of the most widespread habits of pedogenic calcite. It is a monocrystal of calcite, in the shape of a needle, with a diameter of one micron and a length between 4 and 103 times its width. NFC occurs in soils with different macroscopic and microscopic morphologies. Macroscopically, two main habits of NFC exist: "cotton ball'Mike clusters and as "powder" coating on pebbles inside the soil. It can also act as nucleation sites for precipitation of calcite cements of purely physicochemical origin (LCC - Late Calcitic Cement). Although many studies have been conducted on needle fibre calcite, its origin remains a subject of debate. The NFC having never been reproduced in the laboratory whatever the considered approach, the processes responsible for its precipitation as a so particular morphology remain unexplained. The shift between the length orientation of the needle crystal and the normal axis of calcite growth (c-axis) is also unresolved.Samples taken in two soils observed in quarries (Villiers and Savagnier) in the Swiss Jura Mountains are used to investigate the processes involved in the formation of these calcite crystals in pedogenic environments. Three groups of microscopic morphologies are distinguished: (i) simple needles (SN), (ii) simple needles with overgrowths (SNO), and (iii) simple needles with nanofibres (SNN), nanofibres being either organic or mineral in nature. These groups correspond to different stages in the formation and evolution of NFC.Comparison of carbon and oxygen isotope compositions of NFC with LCC, in relationship with the composition of the carbonate host rock (CHR), and the carbon isotope signature of dissolved inorganic carbon (DIC) from the soil waters, indicates that both NFC and LCC are precipitated in isotopic equilibrium with the soil solution. Similar Ô13C and Ô180 values of the elongated NFC and the rhombohedral calcite crystals of the LCC suggest that the precipitation of these different calcite habits is not due to changes in physicochemical conditions. The growth of NFC crystals inside an organic mould formed by the fungal hyphae could explain the formation of calcite ciystals in the shape of a needle in isotopic equilibrium with the local environment.Strontium and calcium isotope compositions of the three microscopic groups of NFC and LCC were also studied, in order to determine the origin of calcium (Ca2+) entrapped in the calcite lattice and to elucidate the processes responsible for the precipitation of NFC. The 87Sr/86Sr ratio of the SN is closer to the 87Sr/86Sr ratio of the carbonate host rock than the LCC, SNO, and SNN. This could be another clue for the formation of SN inside fungal hyphae, because fungi are known to dissolve the rocks to release and translocate through their hyphae nutrients necessary for their growth. SN and SNN are depleted in Sr and enriched in ^Ca compared to the LCC. In the context of Villiers quarry, where the two ciystal shapes precipitate at similar temperature (in a range of 0,6°C), such variations are the result of a slower precipitation rate of NFC, which is inconsistent with a purely physicochemically precipitated elongate crystal.Finally, NFC is enriched in major and trace elements (i.e. Fe, Zn, Cu, and Mo) compared to the LCC. This enrichment is ascribed to modification in precipitation processes between the NFC and LCC. Right now, it is not possible to preclude the influence of the particular shape of NFC or the biological influence. REEs are not fractionated in NFC, contrary to LCC. Thus, NFC does not precipitate from a percolation solution circulating downward the soil, which should lead to the fractionation of the REEs. As explained above, fungi, are able to dissolve and translocate nutrients. This kind of processes allows releasing elements in the soil solution without fractionation and could explain the particular chemical signature of NFC regarding the REEs.The geochemical approach to investigate the origin of NFC presented in this study does not allow providing direct clues to the fungal origin of NFC, but brings several new insights in the understanding of the formation of such a particular mineral, calcite needles, by precluding definitively their physicochemical origin.RESUMELa calcite en aiguilles (NFC) est l'une des formes les plus ubiquiste de calcite pédogénique. Il s'agit d'un monocrystal de calcite d'un diamètre d'un micron et d'une longueur 4 à 102 fois supérieure à sa largeur. A l'échelle macroscopique, la NFC a été observée sous deux principaux aspects : l'une « cotonneuse » et l'autre formant un mince croûte autour des graviers du sol. La NFC peut également servir de support à la nucléation de ciments secondaires précipités de manière purement physico-chimique (LCC). Malgré les nombreuses études menées sur la NFC, son origine demeure encore inconnue. A ce jour, aucune expérience en laboratoire n'a permis de créer de la calcite en aiguilles, et ce, quelque soit l'approche abordée. Par conséquent, les processus précis responsables de la précipitation calcite avec une telle morphologie restent inconnus. Le décalage existant entre l'axe d'allongement des aiguilles de calcite et l'axe normal de croissance de la calcite (axe c) reste inexpliqué.Des échantillons de NFC, LCC, roche mère, eau de pluie des différents horizons du sol ont été prélevés principalement au front de deux carrières de graviers, ainsi que dans un profil de sol adjacent à ces carrières, dans le but de mieux comprendre les processus à l'origine de la précipitation de calcite en forme d'aiguille. Trois micro-morphologies ont été distinguées: les aiguilles simples (SN), les aiguilles simples avec surcroissances calcitiques (SNO) et enfin les aiguilles simples avec nanofibres (SNN), celles-ci pouvant être de minérales ou organiques. Ces groupes, d'après nos résultats et les études antérieures pourraient correspondre à différentes étapes de formation de la calcite en aiguilles.Dans un premier temps, la comparaison des signatures isotopiques de la calcite en aiguilles, du LCC, de la roche mère et du carbone inorganique dissout dans la solution du sol (CID) indique que la NFC, tout comme le LCC, précipite en équilibre avec la solution du sol. Les valeurs similaires en Ô13C et δ180 de cristaux de calcite allongés (NFC) et rhombohédriques (LCC) formés dans un même milieu suggère que ces différences morphologiques ne peuvent pas être attribuées à des modifications purement physico-chimiques. La croissance de NFC à l'intérieur d'un moule organique comme les hyphes de champignons semble être la seule hypothèse plausible permettant d'expliquer la formation de monocrystaux allongés de calcite en équilibre avec leur environnement.La composition isotopique en strontium (Sr) et calcium (Ca) des LCC et des trois micro¬morphologies de la NFC ont été étudiées également, afin de déterminer l'origine du Ca2+ présent dans le réseau cristallin de la calcite en aiguilles, ainsi que les processus responsables de la formation de NFC. Les valeurs du rapport 87Sr/86$r de la forme SN sont les plus proches de celles de la roche mère par rapport aux formes SNN et SNO et du LCC. Ceci pourrait être un nouvel indice de l'implication des champignons dans la précipitation de calcite en aiguilles, puisqu'ils sont connus pour avoir la capacité de dissoudre les roches afin de libérer les nutriments nécessaires à leur croissance, ainsi que de les transloquer par leurs hyphes. De plus, les formes SN et SNN sont appauvries en Sr et enrichies en "Ca, comparativement au LCC. Dans le sol étudié, tous les carbonates de calcium précipitent à la même température, par conséquent, de telles variations sont dues à un taux de précipitation plus lent de SN et SNN, ce qui est contradictoire avec l'hypothèse physico-chimique. Pour finir, la NFC est enrichie en certains éléments majeurs et traces (i.e. Fe, Zn, Cu et Mo) par rapport au LCC. Ceci peut être attribué à différents processus de formation entre la NFC et le LCC. Pour le moment il est impossible d'exclure l'influence de la forme particulière de la NFC ou celle du champignon. Les terres rares (REEs) ne sont pas fractionnées dans la NFC, contrairement au LCC. Ceci peut être expliqué par le fait que la NFC précipite à partir d'une solution qui n'a pas percolé à travers le sol. Les champignons en dissolvant les roches mettent en solution éléments sans les fractionner. L'approche géochimique de l'étude de la calcite en aiguilles ne permets pas de produire des preuves directes sur sa potentielle origine fongique, mais permet de mieux comprendre comment un minéral aussi singulier que la NFC peut se former. D'autre pare cette étude permets d'exclure définitivement l'hypothèse physico-chimique de l'origine de la calcite en aiguilles

Elastic properties and secondary structure formation of single-stranded DNA at monovalent and divalent salt conditions

Single-stranded DNA (ssDNA) plays a major role in several biological processes. It is therefore of fundamental interest to understand how the elastic response and the formation of secondary structures are modulated by the interplay between base pairing and electrostatic interactions. Here we measure force-extension curves (FECs) of ssDNA molecules in optical tweezers set up over two orders of magnitude of monovalent and divalent salt conditions, and obtain its elastic parameters by fitting the FECs to semiflexible models of polymers. For both monovalent and divalent salts, we find that the electrostatic contribution to the persistence length is proportional to the Debye screening length, varying as the inverse of the square root of cation concentration. The intrinsic persistence length is equal to 0.7 nm for both types of salts, and the effectivity of divalent cations in screening electrostatic interactions appears to be 100-fold as compared with monovalent salt, in line with what has been recently reported for single-stranded RNA. Finally, we propose an analysis of the FECs using a model that accounts for the effective thickness of the filament at low salt condition and a simple phenomenological description that quantifies the formation of non-specific secondary structure at low forces.

Caspase-2 activation in the absence of PIDDosome formation.

PIDD (p53-induced protein with a death domain [DD]), together with the bipartite adapter protein RAIDD (receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a DD), is implicated in the activation of pro-caspase-2 in a high molecular weight complex called the PIDDosome during apoptosis induction after DNA damage. To investigate the role of PIDD in cell death initiation, we generated PIDD-deficient mice. Processing of caspase-2 is readily detected in the absence of PIDDosome formation in primary lymphocytes. Although caspase-2 processing is delayed in simian virus 40-immortalized pidd(-/-) mouse embryonic fibroblasts, it still depends on loss of mitochondrial integrity and effector caspase activation. Consistently, apoptosis occurs normally in all cell types analyzed, suggesting alternative biological roles for caspase-2 after DNA damage. Because loss of either PIDD or its adapter molecule RAIDD did not affect subcellular localization, nuclear translocation, or caspase-2 activation in high molecular weight complexes, we suggest that at least one alternative PIDDosome-independent mechanism of caspase-2 activation exists in mammals in response to DNA damage.