Crystal structure of a Vip3B family insecticidal protein reveals a new fold and a unique tetrameric assembly.

Vegetatively expressed insecticidal proteins (VIPs) produced by Bacillus thuringiensis fall into several classes of which the third, VIP3, is known for their activity against several key Lepidopteran pests of commercial broad acre crops and because their mode of action does not overlap with that of crystalline insecticidal proteins. The details of the VIP3 structure and mode of action have remained obscure for the quarter century that has passed since their discovery. In the present article, we report the first crystal structure of a full-length VIP3 protein. Crystallization of this target required multiple rounds of construct optimization and screening-over 200 individual sequences were expressed and tested. This protein adopts a novel global fold that combines domains with hitherto unreported topology and containing elements seemingly borrowed from carbohydrate-binding domains, lectins, or from other insecticidal proteins.

Biogeochemical distribution of Pb and Zn forms in two calcareous soils affected by mycorrhizal symbiosis and alfalfa rhizosphere.

Using of arbuscular mycorrhizal fungi (AMF) has emerged as a new technique to alleviate the toxic metals stress through changing their chemical behavior. The present work was conducted as a factorial arrangement based on a completely randomized design to study the inoculation effects of Glomus intraradices, Glomus mosseae and Glomus etunicatum, on Pb and Zn fractions in the rhizosphere of alfalfa by using rhizobox technique in two agricultural soils with different Zn and Pb concentrations [with low (LH) and high (HH) concentration levels]. The results showed that AMF colonization promoted plant growth and lowered the shoot and root Pb and shoot Zn concentrations in the studied soils compared to uninoculated treatments. Mycorrhizal colonization significantly increased the Ca(NO3)2- extractable Zn and ORG-Zn (respectively 500 and 59.6% more than the uninoculated treatment) and decreased the OXI-Zn (20.32% less than the none inoculated treatment) in the HH soil. By contrast, mycorrhizae slightly increased the CARB, OXI and ORG-Zn forms in the LH soil compared to the uninoculation condition. In the AMF- treated HH soil, an increase was recorded in the Ca(NO3)2- extractable Pb, EXCH-Pb and CARB-Pb (respectively, 17.65, 3.09 and 14.22% compared to the none inoculated treatment) and a decrease in the OXI and ORG-Pb forms (respectively, 28.79 and 13.51% compared to the uninoculated treatment). A reverse status was observed for Pb changes in the LH soil. Depending on the contamination level, the mycorrhizal inoculation differentially affected the Pb and Zn fractions at different distances from the root surface. In the LH soil, at <5 mm distance (i.e. rhizospheric soil), the mycorrhizal inoculation decreased the CARB (about 17.99%) and OXI -Zn (about 29.63%) forms compared to bulk soil (i.e. > 5 mm distance) while ORG-Zn was increased up to 48.63%. However, Ca(NO3)2- extractable, CARB and ORG-Pb was increased in rhizosphere soil (respectively, 89.33, 3.84 and 6.14%) and OXI-Pb was decreased up to 10.36% compared to the bulk soil. In the HH soil, mycorrhizal inoculation increased the CARB and OXI-Zn (respectively, 1.76 and 5.71%) and OXI-Pb fractions (11.56%) compared to the <5 mm distances. Whereas, it reduced the Ca(NO3)2- extractable, EXCH, and ORG-Zn (Respectively, 52.70, 19.19 and 30.16%) and Ca(NO3)2- extractable, CARB and ORG-Pb (respectively, 47.18, 3.70 and 5.79%). These results revealed that depending on the soil contamination level and nature of the element, AMF colonization affects biogeochemical fractions of the metals and their accumulation in the plant tissues.

The quantity/intensity relation is affected by chemical and organic P fertilization in calcareous soils.

The use of organic fertilization increases the availability of phosphorus (P) in calcareous soils by affecting the colloidal properties of soils. Accordingly, it was hypothesized that chemical and organic fertilizers affect P availability in calcareous soils by influencing P sorption and buffering capacity. The objective was to investigate the quantity/intensity (Q/I) relation in calcareous soils as affected by chemical and organic P fertilization. Three different soil types with different Olsen-P values including Qazvin1 (very low P, VLP), Qazvin2 (low P, LP) and Dizan (medium P, MP) were fertilized with 50 mg P kg-1 soil using triple superphosphate (TSP), sheep manure (SM), and municipal solid waste compost (MSWC). The treated experimental soils were incubated for 90 days, and P sorption and buffering capacity indexes were determined using calcium chloride solutions in a range of 0-100 mg P L-1. A greenhouse experiment was conducted to determine wheat (Triticum aestivum L.) response to the experimental treatments. Wheat P content at tillering (60 days after planting) was determined. The SM and TSP treatments were the most efficient sources of P for plant use in the greenhouse, as they resulted in the highest wheat growth and P content. The incubation data were fitted to Langmuir, Freundlich, Temkin and surface sorption isotherm models. Langmuir model, as the best fitted one, indicated the highest P sorption (A) was resulted by the SM treatment for VLP and LP soils, compared to the other treatments. According to the model, the SM and MSWC treatments resulted in the least (0.04) and the highest (1.11) sorption energy (K) by the VLP soil, respectively. In the VLP soil the SM and MSWC treatments, and in the LP soil the MSWC treatment decreased P sorption, at the final concentration of P (100 mg L-1), compared to the control treatment. Organic fertilizers decreased buffering index, phosphorous buffering capacity, and K1 indexes in the VLP soil, compared to the control treatment. The corresponding reductions for SM were equal to 35.99, 2.7, 1.19 mL P g-1 and for MSWC were equal to 12.33, 36.2 and 1.19 mL P g-1. In the VLP and MP soils, (compared with control), the SM treatment decreased the rates of maximum buffering capacity at 0.38 and 0.52 mL P g-1, respectively. There were high and significant correlations among the soil P buffering indexes with soil and wheat P content. Fertilization affected soil P availability by affecting the Q/I relation and the buffering capacity indexes. It is possible to predict plant response to available P using the tested fitting models.

Disabled insecticidal proteins: A novel tool to understand differences in insect receptor utilization.

The development of insect resistance to pesticides via natural selection is an acknowledged agricultural issue. Likewise, resistance development in target insect populations is a significant challenge to the durability of crop traits conferring insect protection and has driven the need for novel insecticidal proteins (IPs) with alternative mechanism of action (MOA) mediated by different insect receptors. The combination or "stacking" of transgenes encoding different insecticidal proteins in a single crop plant can greatly delay the development of insect resistance, but requires sufficient knowledge of MOA to identify proteins with different receptor preferences. Accordingly, a rapid technique for differentiating the receptor binding preferences of insecticidal proteins is a critical need. This article introduces the Disabled Insecticidal Protein (DIP) method as applied to the well-known family of three-domain insecticidal proteins from Bacillus thuringiensis and related bacteria. These DIP's contain amino acid substitutions in domain 1 that render the proteins non-toxic but still capable of competing with active proteins in insect feeding assays, resulting in a suppression of the expected insecticidal activity. A set of insecticidal proteins with known differences in receptor binding (Cry1Ab3, Cry1Ac.107, Cry2Ab2, Cry1Ca, Cry1A.105, and Cry1A.1088) has been studied using the DIP method, yielding results that are consistent with previous MOA studies. When a native IP and an excess of DIP are co-administered to insects in a feeding assay, the outcome depends on the overlap between their MOAs: if receptors are shared, then the DIP saturates the receptors to which the native protein would ordinarily bind, and acts as an antidote whereas, if there is no shared receptor, the toxicity of the native insecticidal protein is not inhibited. These results suggest that the DIP methodology, employing standard insect feeding assays, is a robust and effective method for rapid MOA differentiation among insecticidal proteins.

Mobility of heavy metals in sandy soil after application of composts produced from maize straw, sewage sludge and biochar: Discussion of Gondek et al. (2018).

Gondek et al. (2018) investigate the mobility of heavy metals in sandy soil after application of composts produced from maize straw, sewage sludge and biochar. The authors made a little attention to role of the soil properties and soil condition on the metals chemical behavior. In different section of the paper, the authors reported that the content of the metals extracted with water or 1 M NH4NO3 was changed compared to the control treatment affected by the mentioned biosolids. Nowadays, different researches have shown that the trace metals nature have an important role in determining their fate in presence of different treatments. In addition to, organic compounds entered to the soil due to biosolids applications have a duplicate role in trace metals mobility. Therefore, it is needed this facts are considered to interpret the measurements in the original paper. Also, whenever effects of different materials are studied on the metals mobility, the metals status in the plant tissues presents valuable information to the scientists and readers for carefully interpreting the measurements, which it is ignored by the authors.

Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance.

The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.

Structure of the full-length insecticidal protein Cry1Ac reveals intriguing details of toxin packaging into in vivo formed crystals.

For almost half a century, the structure of the full-length Bacillus thuringiensis (Bt) insecticidal protein Cry1Ac has eluded researchers, since Bt-derived crystals were first characterized in 1965. Having finally solved this structure we report intriguing details of the lattice-based interactions between the toxic core of the protein and the protoxin domains. The structure provides concrete evidence for the function of the protoxin as an enhancer of native crystal packing and stability.

Criteria for performing extraction in the treatment of certain malocclusions.

This article identifies variables that should be assessed in patients with certain dental malocclusions that may require extraction. In addition, by presenting criteria for performing the more popular types of extraction, the article facilitates treatment decision-making. Pre- and post-treatment illustrations demonstrate the desired patient outcomes.

Dicamba monooxygenase: structural insights into a dynamic Rieske oxygenase that catalyzes an exocyclic monooxygenation.

Dicamba (2-methoxy-3,6-dichlorobenzoic acid) O-demethylase (DMO) is the terminal Rieske oxygenase of a three-component system that includes a ferredoxin and a reductase. It catalyzes the NADH-dependent oxidative demethylation of the broad leaf herbicide dicamba. DMO represents the first crystal structure of a Rieske non-heme iron oxygenase that performs an exocyclic monooxygenation, incorporating O(2) into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer (alpha(3)) in the crystallographic asymmetric unit with similar arrangement of neighboring inter-subunit Rieske domain and non-heme iron site enabling electron transport consistent with other structurally characterized Rieske oxygenases. While the Rieske domain is similar, differences are observed in the catalytic domain, which is smaller in sequence length than those described previously, yet possessing an active-site cavity of larger volume when compared to oxygenases with larger substrates. Consistent with the amphipathic substrate, the active site is designed to interact with both the carboxylate and aromatic ring with both key polar and hydrophobic interactions observed. DMO structures were solved with and without substrate (dicamba), product (3,6-dichlorosalicylic acid), and either cobalt or iron in the non-heme iron site. The substitution of cobalt for iron revealed an uncommon mode of non-heme iron binding trapped by the non-catalytic Co(2+), which, we postulate, may be transiently present in the native enzyme during the catalytic cycle. Thus, we present four DMO structures with resolutions ranging from 1.95 to 2.2 A, which, in sum, provide a snapshot of a dynamic enzyme where metal binding and substrate binding are coupled to observed structural changes in the non-heme iron and catalytic sites.

The Arabidopsis vitamin E pathway gene5-1 mutant reveals a critical role for phytol kinase in seed tocopherol biosynthesis.

We report the identification and characterization of a low tocopherol Arabidopsis thaliana mutant, vitamin E pathway gene5-1 (vte5-1), with seed tocopherol levels reduced to 20% of the wild type. Map-based identification of the responsible mutation identified a G-->A transition, resulting in the introduction of a stop codon in At5g04490, a previously unannotated gene, which we named VTE5. Complementation of the mutation with the wild-type transgene largely restored the wild-type tocopherol phenotype. A knockout mutation of the Synechocystis sp PCC 6803 VTE5 homolog slr1652 reduced Synechocystis tocopherol levels by 50% or more. Bioinformatic analysis of VTE5 and slr1652 indicated modest similarity to dolichol kinase. Analysis of extracts from Arabidopsis and Synechocystis mutants revealed increased accumulation of free phytol. Heterologous expression of these genes in Escherichia coli supplemented with free phytol and in vitro assays of recombinant protein produced phytylmonophosphate, suggesting that VTE5 and slr1652 encode phytol kinases. The phenotype of the vte5-1 mutant is consistent with the hypothesis that chlorophyll degradation-derived phytol serves as an important intermediate in seed tocopherol synthesis and forces reevaluation of the role of geranylgeranyl diphosphate reductase in tocopherol biosynthesis.

Metabolically engineered oilseed crops with enhanced seed tocopherol.

Tocochromanols (tocopherols and tocotrienols) are important lipid soluble antioxidants and are an essential part of the mammalian diet. Oilseeds are particularly rich in tocochromanols with an average concentration 10-fold higher than other plant tissues. Here we describe a systematic approach to identify rate-limiting reactions in the tocochromanol biosynthetic pathway, and the application of this knowledge to engineer tocochromanol biosynthesis in oilseed crops. Seed-specific expression of genes encoding limiting tocochromanol pathway enzymes in soybean increased total tocochromanols up to 15-fold from 320 ng/mg in WT seed to 4800 ng/mg in seed from the best performing event. Although WT soybean seed contain only traces of tocotrienols, these transgenic soybean accumulated up to 94% of their tocochromanols as tocotrienols. Upon crossing transgenic high tocochromanol soybean with transgenic high alpha-tocopherol soybean, the vitamin E activity in the best performing F2-seed was calculated to be 11-fold higher than the average WT soybean seed vitamin E activity.

The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad.

Patatin is a nonspecific lipid acyl hydrolase that accounts for approximately 40% of the total soluble protein in mature potato tubers, and it has potent insecticidal activity against the corn rootworm. We determined the X-ray crystal structure of a His-tagged variant of an isozyme of patatin, Pat17, to 2.2 A resolution, employing SeMet multiwavelength anomalous dispersion (MAD) phasing methods. The patatin crystal structure has three molecules in the asymmetric unit, an R-factor of 22.0%, and an R(free) of 27.2% (for 10% of the data not included in the refinement) and includes 498 water molecules. The structure notably revealed that patatin has a Ser-Asp catalytic dyad and an active site like that of human cytosolic phospholipase A(2) (cPLA(2)) [Dessen, A., et al. (1999) Cell 97, 349-360]. In addition, patatin has a folding topology related to that of the catalytic domain of cPLA(2) and unlike the canonical alpha/beta-hydrolase fold. The structure confirms our site-directed mutagenesis and bioactivity data that initially suggested patatin possessed a Ser-Asp catalytic dyad. Alanine-scanning mutagenesis revealed that Ser77 and Asp215 were critical for both esterase and bioactivity, consistent with prior work implicating a Ser residue [Strickland, J. H., et al. (1995) Plant Physiol. 109, 667-674] and a Ser-Asp dyad [Hirschberg, H. J. H. B., et al. (2001) Eur. J. Biochem. 268, 5037-5044] in patatin's catalytic activity. The crystal structure aids the understanding of other structure-function relationships in patatin. Patatin does not display interfacial activation, a hallmark feature of lipases, and this is likely due to the fact that it lacks a flexible lid that can shield the active site.

The Klebsiella pneumoniae cytochrome bd' terminal oxidase complex and its role in microaerobic nitrogen fixation.

Cytochrome bd' has been implicated in having an important role in microaerobic nitrogen fixation in the enteric bacterium Klebsiella pneumoniae, where it is expressed under all conditions that permit diazotrophy. In this paper the sequence of the genes encoding this terminal oxidase (cydAB) of Klebsiella pneumoniae and the characterization of a cyd mutant are reported. The deduced amino acid sequences support the proposal that His 19, His 186 and Met 393 provide three of the four axial ligands to the Fe of the three haems in the oxidase complex. The nitrogen-fixing ability of the mutant was severely impaired in the presence of low concentrations of oxygen compared with the wild-type bacterium. Only the wild-type organism was capable of microaerobic nitrogenase activity supported by fermentation products. It is proposed that formate dehydrogenase-O may be involved in supplying electrons to a respiratory chain terminated by the bd-type oxidase, which would remove inhibitory oxygen and supply ATP for nitrogenase activity.