pH-Dependent Changes in Structural Stabilities of Bt Cry1Ac Toxin and Contrasting Model Proteins following Adsorption on Montmorillonite.

The environmental fate of insecticidal Cry proteins, including time-dependent conservation of biological properties, results from their structural stability in soils. The complex cascade of reactions involved in biological action requires Cry proteins to be in solution. However, the pH-dependent changes in conformational stability and the adsorption-desorption mechanisms of Cry protein on soil minerals remain unclear. We used Derjaguin-Landau-Verwey-Overbeek (DLVO) calculation and differential scanning calorimetry to interpret the driving forces and structural stabilities of Cry1Ac and two contrasting model proteins adsorbed by montmorillonite. The structural stability of Cry1Ac is closer to that of the "hard" protein, α-chymotrypsin, than that of the "soft" bovine serum albumin (BSA). The pH-dependent adsorption of Cry1Ac and α-chymotrypsin could be explained by DLVO theory, whereas the BSA adsorption deviated from it. Patch-controlled electrostatic attraction, hydrophobic effects, and entropy changes following protein unfolding on a mineral surface could contribute to Cry1Ac adsorption. Cry1Ac, like chymotrypsin, was partly denatured on montmorillonite, and its structural stability decreased with an increase in pH. Moreover, small changes in the conformational heterogeneity of both Cry1Ac and chymotrypsin were observed following adsorption. Conversely, adsorbed BSA was completely denatured regardless of the solution pH. The moderate conformational rearrangement of adsorbed Cry1Ac may partially explain why the insecticidal activity of Bt toxin appears to be conserved in soils, albeit for a relatively short time period.

A dansyl-derivatized phytic acid analogue as a fluorescent substrate for phytases: experimental and computational approach.

A new myo-inositol pentakisphosphate was synthesized, which featured a dansyl group at position C-5. The fluorescent tag was removed from the inositol by a 6-atom spacer to prevent detrimental steric interactions in the catalytic site of phytases. The PEG linker was used in order to enhance hydrophilicity and biocompatibility of the new artificial substrate. Computational studies showed a favorable positioning in the catalytic site of phytases. Enzymatic assays demonstrated that the tethered myo-inositol was processed by two recombinant phytases Phy-A and Phy-C, classified respectively as acid and alkaline phytases, with similar rates of phosphate release compared to their natural substrate.

In vivo and in vitro 31P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals.

We used in vivo and in vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy to follow the change in transport, compartmentation and metabolism of phosphate in the ectomycorrhizal fungus Hebeloma cylindrosporum in response to root signals originating from host (Pinus pinaster) or non-host (Zea mays) plants. A device was developed for the in vivo studies allowing the circulation of a continuously oxygenated mineral solution in an NMR tube containing the mycelia. The in vitro studies were performed on fungal material after several consecutive treatment steps (freezing in liquid nitrogen; crushing with perchloric acid; elimination of perchloric acid; freeze-drying; dissolution in an appropriate liquid medium).

A Method for Radioactive Labelling of Hebeloma cylindrosporum to Study Plant-fungus Interactions.

In order to quantify P accumulation and P efflux in the ectomycorrhizal basidiomycete fungus Hebeloma cylindrosporum, we supplied 32P to mycelia previously grown in vitro in liquid medium. The culture had four main steps that are 1) growing the mycelium on complete medium with P, 2) transfer the mycelia into new culture solution with or without P, 3) adding a solution containing 32P and 4) rinsing the mycelia before incubation with or without plant. The main point is to rinse very carefully the mycelia after 32P supply in order to avoid overestimation of 32P efflux into the medium.

Establishing a Symbiotic Interface between Cultured Ectomycorrhizal Fungi and Plants to Follow Fungal Phosphate Metabolism.

In ectomycorrhizal plants, the fungal cells colonize the roots of their host plant to create new organs called ectomycorrhizae. In these new organs, the fungal cells colonize the walls of the cortical cells, bathing in the same apoplasm as the plant cells in a space named the 'Hartig net', where exchanges between the two partners take place. Finally, the efficiency of ectomycorrhizal fungi to improve the phosphorus nutrition of their host plants will depend on the regulation of phosphate transfer from the fungal cells to plant cells in the Hartig net through as yet unknown mechanisms. In order to investigate these mechanisms, we developed an in vitro experimental device mimicking the common apoplasm of the ectomycorrhizae (the Hartig net) to study the phosphorus metabolism in the ectomycorrhizal fungus Hebeloma cylindrosporum when the fungal cells are associated or not with the plant cells of the host plant Pinus pinaster. This device can be used to monitor 32Phosphate efflux from the fungus previously incubated with 32P-orthophosphate.

The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer, cytological staining and 31 P NMR spectroscopy study.

Ectomycorrhizal (ECM) association can improve plant phosphorus (P) nutrition. Polyphosphates (polyP) synthesized in distant fungal cells after P uptake may contribute to P supply from the fungus to the host plant if they are hydrolyzed to phosphate in ECM roots then transferred to the host plant when required. In this study, we addressed this hypothesis for the ECM fungus Hebeloma cylindrosporum grown in vitro and incubated without plant or with host (Pinus pinaster) and non-host (Zea mays) plants, using an experimental system simulating the symbiotic interface. We used 32 P labelling to quantify P accumulation and P efflux and in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and cytological staining to follow the fate of fungal polyP. Phosphate supply triggered a massive P accumulation as newly synthesized long-chain polyP in H. cylindrosporum if previously grown under P-deficient conditions. P efflux from H. cylindrosporum towards the roots was stimulated by both host and non-host plants. However, the host plant enhanced 32 P release compared with the non-host plant and specifically increased the proportion of short-chain polyP in the interacting mycelia. These results support the existence of specific host plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots.

Fate of insecticidal Bacillus thuringiensis Cry protein in soil: differences between purified toxin and biopesticide formulation.

BACKGROUND: Bacillus thuringiensis produces insecticidal proteins known as Cry, and its efficiency and absence of side effects make it the most widely used biopesticide. There is little information on the role of soils in the fate of Cry proteins from commercial biopesticide formulations, unlike toxins from genetically modified crops, which have been intensively studied in recent years. The persistence of Cry in soil was followed under field and laboratory conditions. RESULTS: Sunlight accelerated loss of detectable Cry under laboratory conditions, but little effect of shade was observed under field conditions. The half-life of biopesticide proteins in soil under natural conditions was about 1 week. Strong temperature effects were observed, but they differed for biopesticide and purified protein, indicating different limiting steps. CONCLUSION: For the biopesticide, the observed decline in detectable protein was due to biological factors, possibly including the germination of B. thuringiensis spores, and was favoured by higher temperature. In contrast, for purified proteins, the decline in detectable protein was slower at low temperature, probably because the conformational changes of the soil-adsorbed protein, which cause fixation and hence reduced extraction efficiency, are temperature dependent. © 2016 Society of Chemical Industry.

Efficiency of acid phosphatases secreted from the ectomycorrhizal fungus Hebeloma cylindrosporum to hydrolyse organic phosphorus in podzols.

Ectomycorrhizal fungi may improve the phosphate nutrition of their host plants by secreting, into the soil solution, acid phosphatases (AcPases) able to release orthophosphate (Pi) from soil organic phosphorus (Po). Using cation-exchange chromatography, we separated four fractions with AcPase activity secreted by the ectomycorrhizal fungus Hebeloma cylindrosporum grown in a pure culture under P-starved conditions. Each AcPase active fraction displayed strong ability in vitro to hydrolyse a wide range of phosphate monoesters, but none of them efficiently hydrolysed phytate. Their efficiency to release Pi from soil NaHCO(3)-extractable Po was studied in a sandy podzol used intact or autoclaved. Soils were collected in a 15-year-old Pinus pinaster stand, receiving regular fertilization or not. Autoclaving increased the NaHCO(3)-extractable Po concentrations by 55% in unfertilized and by 32-43% in fertilized soils. The efficiency of each AcPase fraction was affected significantly by the soil fertilization regime and the soil treatment (intact vs. autoclaved). The proportion of labile Po enzyme ranged from 0% to 11% and 14% to 48% after 1 h of incubation in bicarbonate solutions extracted from intact and autoclaved soils, respectively. This work suggests that AcPases secreted from H. cylindrosporum could be efficient in recycling Po pools from soil microorganisms that may be delivered by soil autoclaving.

Adsorption of alexa-labeled Bt toxin on mica, glass, and hydrophobized glass: study by normal scanning confocal fluorescence.

We studied the kinetics of adsorption of alexa-labeled Bt toxin Cry1Aa, in monomer and oligomer states, on muscovite mica, acid-treated hydrophilic glass, and hydrophobized glass, in the configuration of laminar flow of solution in a slit. Normal confocal fluorescence through the liquid volume allows the visualization of the concentration in solution over the time of adsorption, in addition to the signal due to the adsorbed molecules at the interface. The solution signal is used as calibration for estimation of interfacial concentration. We found low adsorption of the monomer compared to oligomers on the three types of surface. The kinetic adsorption barrier for oligomers increases in the order hydrophobized glass, muscovite mica, acid-treated hydrophilic glass. This suggests enhanced immobilization in soil if toxin is under oligomer state.

Dissimilar pH-dependent adsorption features of bovine serum albumin and alpha-chymotrypsin on mica probed by AFM.

We studied bovine serum albumin (BSA) and alpha-chymotrypsin adsorption onto mica surfaces over a large pH range by atomic force microscopy (AFM) measurements in liquid. Data analyses (height, roughness and roughness factor) brought new insights on the conformation of proteins in soil environments, with mica as a model of soil phyllosilicates and non-hydrophobic surfaces. Validation of AFM approach was performed on BSA, whose behavior was previously described by nuclear magnetic resonance and infra-red spectroscopic methods. Maximum adsorption was observed near the isoelectric point (IEP). A stronger interaction and a lower amount of adsorbed proteins were observed below the IEP, which contrasted with the progressive decrease of adsorption above the IEP. We then studied the adsorption of alpha-chymotrypsin, a proteolytic enzyme commonly found in soils. AFM pictures demonstrated a complete coverage of the mica surface at the IEP in contrast to the BSA case. Comparison of the AFM data with other indirect methods broadened the understanding of alpha-chymotrypsin adsorption process through the direct display of the protein adsorption patterns as a function of pH.

Fate of prions in soil: adsorption and extraction by electroelution of recombinant ovine prion protein from montmorillonite and natural soils.

Prions, the infectious agents thought to be responsible for transmissible spongiform encephalopathies, may contaminate soils and have been reported to persist there for years. We have studied the adsorption and desorption of a model recombinant prion protein on montmorillonite and natural soil samples in order to elucidate mechanisms of prion retention in soils. Clay minerals, such as montmorillonite, are known to be strong adsorbents for organic molecules, including proteins. Montmorillonite was found to have a large and selective adsorption capacity for both the normal and the aggregated prion protein. Adsorption occurred mainly via the N-terminal domain of the protein. Incubation with standard buffers and detergents did not desorb the full length protein from montmorillonite, emphasizing the largely irreversible trapping of prion protein by this soil constituent. An original electroelution method was developed to extract prion protein from both montmorillonite and natural soil samples, allowing quantification when coupled with rapid prion detection tests. This easy-to-perform method produced concentrated prion protein extracts and allowed detection of protein at levels as low as 0.2 ppb in natural soils.

Fate of prions in soil: adsorption kinetics of recombinant unglycosylated ovine prion protein onto mica in laminar flow conditions and subsequent desorption.

Prions can be disseminated in soils. Their interaction with soil minerals is a key factor for the assessment of risks associated with the transport of their infectivity. We did not examine here the infectivity itself but the adsorption kinetics of an ovine recombinant prion protein (ovine PrPrec), as a noninfectious model protein, on muscovite mica, a phyllosilicate with surface properties analogous to soil clays, in conditions of laminar flow through a channel. The protein was labeled with (125)I, and its adsorption examined between pH 4.0 and 9.0. At wall shear rate 100 s(-1), we found the process to be controlled mainly by transport at the beginning of the adsorption process. Additional experiments at 1000 s(-1) (pH 5 and 6) determined that the diffusion coefficient was in accordance with the hydrodynamic radius measured by size exclusion chromatography. The pseudo-plateau of the interfacial concentration with time was compatible with more than a monolayer and suggests the presence of aggregates. Desorption was not observed in the presence of buffer between pH 4 and 9 and sheep plasma, while the addition of alkaline detergent or 10(-1) M NaOH allowed an almost complete removal from the interface. The ensemble of results suggests that the largely irreversible adsorption of the ovine PrPrec onto mica is mainly due to electrostatic attraction between the protein and the highly negatively charged mica surface. Possible consequences for the mode of dissemination of prion proteins in soils are indicated.