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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.

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.

The main purpose of this study was to investigate the effects of different silicon levels and sources on the efficiency of acquisition and utilization of silicon in seven wheat cultivars in a calcareous soil. The treatments consisted of silicon additions to the soil (control, 200, 400, and 1000 mg/kg as potassium silicate and 0, 50, and 100 mg/kg as nanoparticles) and seven wheat cultivars (Gonbad, Shiroudi, Shiraz, Mahdavi, Marvdasht, Bahar, and Parsi). The factorial experiment was carried out in three replications. The results showed that the application of silicon at different levels and from various sources, as well as wheat cultivars and their interactions with the silicon treatments, led to significant differences (p ≤ 0.01) in the root and shoot dry weights, the silicon concentration in the root and shoot, and the total silicon in the shoot. In addition, there was a significant relationship between the silicon level/source and wheat cultivars with all efficiency indices (at level of 1%). The results also show there is a significant (p ≤ 0.01) relationship between shoot silicon efficiency and silicon acquisition efficiency (0.72). Therefore, considering the role of silicon in stress alleviation, its application in wheat cultivars with higher acquisition efficiency can help the plant growth.

Production of oilseed crops requires balanced fertilization, while environmental problems of applied fertilizers must be considered. We evaluated seven winter canola cultivars (Brassica napus L.) f...

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.

Crops that acquire phosphorus more efficiently could reduce some of the environmental problems of fertilizers. We conducted two experiments to evaluate seven spring oilseed rape cultivars for their relative efficiency to use or acquire P under conditions of deficiency (4.6 mg Olsen P kg−1) and P application (80 mg P kg−1). Based on biomass, P content, P acquisition and use efficiency, cultivars were categorized as efficient responsive, non-efficient responsive, efficient non-responsive and non-efficient non-responsive. We also measured and compared root diameter, root surface area and total root length in two selected cultivars, and found that the Dalgan cultivar, which was efficient in P acquisition, was also an efficient accumulator of biomass under the P deficiency, and that P acquisition was related to higher Ca uptake. The responsible mechanism for P efficiency was suggested to be higher P uptake, which prompts the formation of larger root systems and improves plant access to soil P.

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.

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) 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

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.

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.

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.

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.

Most agricultural soils in Iran are usually low in organic matter (OM). Therefore, increasing OM in these soils is of great concern. Environmental pollution caused by chemical fertilizers has created an interest in the integrated use of organic wastes with inorganic fertilizers. The main purpose of this greenhouse study was to evaluate the impact of two organic wastes and phosphorus (P) on the growth, and elemental composition of spinach (Spinacia oleracea L.) and soil chemical properties. Treatments consisted of four levels of municipal waste compost, MWC (0, 1, 2, and 4%), five rates of poultry manure, PM (0, 1, 2, 3, and 4%), and three P levels (0, 25, and 50 mg kg−1 as KH2PO4). Application of P and MWC alone or in combination significantly increased the top dry weight of spinach. However, spinach growth was markedly increased up to 3% PM and suppressed with the higher rate, probably due to an excess of soluble salts in the soil. Moreover, the enhancing influence of P on spinach seedling growt...

The Langmuir, Freundlich, and Temkin isotherms were fitted to copper (Cu) adsorption data of 20 highly calcareous soils of Fars Province, ranging widely in relevant physical and chemical characteristics. One-gram samples of each soil were equilibrated for 24 h with 30 mL of 0.01 M CaCl2 containing 50 to 500 mg Cu L−1. The amount of Cu retained by soil solids was calculated based on the difference between the initial and equilibrium Cu concentrations. The Cu adsorption data showed a highly significant fit to the Freundlich, Temkin, and linear form of Langmuir adsorption isotherms. The fit to Langmuir and Temkin equations were, however, closer than that of Freundlich. Adsorption maxima (b) calculated from Langmuir isotherm was positively correlated with clay, cation exchange capacity (CEC), and calcium carbonate equivalent (CCE), whereas, Langmuir bonding energy coefficient (K) was related only to clay. Organic matter (OM), clay, and CEC were found to have a significant relationship with Freundlich coeffici...

The Azotobacter FeSII protein, also known as the Shethna protein, forms a protective complex with nitrogenase during periods when nitrogenase is exposed to oxygen. One possible mechanism for its action is an oxidation state-dependent conformational interaction with nitrogenase whereby the FeSII protein dissociates from the MoFe and Fe proteins of nitrogenase under reducing conditions. Herein we report the construction and characterization of five site-directed mutants of the FeSII protein (H12Q, H55Q, K14A, K15A, and the double mutant K14A/K15A) which were individually purified after being individually overexpressed in Escherichia coli. These mutant FeSII proteins maintain native-like assembly and orientation of the 2Fe-2S center on the basis of EPR and NMR spectroscopic characterization and their redox midpoint potentials, which are within 25 mV of that of the wild type protein. The abilities of the individual mutant proteins to protect nitrogenase were assessed by determining the remaining nitrogenase activities after adding each pure version back to extracts from an FeSII deletion strain, and then exposing the mixture to oxygen. In these assays, the H12Q mutant functioned as well as the wild type protein. However, mutation of His55, a few residues away from a cluster-liganding cysteine, results in much less efficient protection of nitrogenase. These results are consistent with pH titrations in both oxidation states, which show that His12 is insensitive to 2Fe-2S cluster oxidation state. His55's pK is weakly responsive to oxidation state, and the pK increase of 0. 16 pH unit upon 2Fe-2S cluster oxidation is indicative of ionization of another group between His55 and the 2Fe-2S cluster, which could modulate the FeSII protein's affinity for nitrogenase in a redox state-dependent manner. Both K14A and K15A mutant FeSII proteins partially lost their ability to protect nitrogenase, but the lysine double mutant lost almost all its protective ability. The nitrogenase component proteins in an Azotobacter strain bearing the double lysine mutation (in the chromosome) were degraded much more rapidly in vivo than those in the wild type strain under carbon substrate-limited conditions. These results indicate that the two lysines may have an important role in FeSII function, perhaps in the initial steps of recognizing the nitrogenase component proteins.

Summary: 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.

The Azotobacter vinelandii FeSII protein confers conformational protection to nitrogenase by binding to the MoFe and Fe proteins under periods of oxidative stress to create an inactive but O2-stabilized tripartite complex. In this work the FeSII protein has been overexpressed in Escherichia coli, and the recombinant protein has been purified to homogeneity, crystallized, and characterized in terms of its functional, spectroscopic, and redox properties. The recombinant protein is a homodimer and is expressed as a holoprotein with one [2Fe-2S]2+,+ cluster in each subunit. It is shown to be functional in reconstituting an O2-stable nitrogenase complex in vitro. Spectroscopic studies using the combination of UV-visible absorption, CD, and variable temperature MCD, EPR, and resonance Raman indicate that the [2Fe-2S]2+,+ cluster is coordinated exclusively by cysteine residues. The arrangement of coordinating cysteines in the primary sequence and the EPR properties of the [2Fe-2S]+ cluster (g = 2.04, 1.95, 1.88) are very similar to those of chloroplast ferredoxins. However, the variable-temperature MCD, resonance Raman, and redox properties (Em = -262 +/- 10 mV based on dye-mediated EPR redox titrations) are more characteristic of hydroxylase-type ferredoxins such as adrenodoxin. In contrast to chloroplast-type ferredoxins, the vibrational properties of the [2Fe-2S]2+,+ cluster in the FeSII protein indicate that none of the cysteinyl Fe-S-C-C dihedral angles are close to 180 degrees and that the cluster is not exposed to solvent. Preliminary X-ray diffraction analysis indicates that the protein crystallizes in an orthorhombic space group with unit cell dimensions a = 135 A, b = 135 A, and c = 38 A and that there are at least two dimers per asymmetric unit.

Summary Summary The FeSII protein of Azotobacter vinelandii has been proposed to mediate the ‘conformational protection’ of the molybdenum-dependent nitrogenase components against oxygen inactivation. We have cloned and characterized the structural gene for the FeSII protein (the fesII Iocus). Hybridization studies did not reveal the presence of fesII-like genes in a number of diverse species of well-studied nitrogen-fixing bacteria, with the exception of Azotobacter chroococcum. The fesll locus is transcriptionally expressed during both nitrogen fixing and non-nitrogen fixing conditions, although the level of its message is up-regulated by approximately 2.5-fold during nitrogen fixation. The promoter region was identified by primer extension analysis, and is similar to other σ70-type promoters. Mutants devoid of the FeSII protein were constructed. These mutants possessed growth characteristics on a variety of carbon substrates during non-diazotrophic as well as diazotrophic growth that were essentially indistinguishable from the wild-type strain. Nevertheless, the nitrogenase activity in cell-free extracts is significantly more sensitive to irreversible oxygen inactivation in the mutants as compared with the wild type. When treated with 250 mM NaCI (a condition known to dissociate FeSII from nitrogenase components), the wild-type and mutant extracts were equally hypersensitive to oxygen Inactivation. Upon energy starvation, conditions in which ‘respiratory protection’ is inoperable, the MoFe and Fe proteins of nitrogenase are degraded much more rapidly in vivo in the deletion mutants, compared to the wild type. Strains relying on either the vanadium or the ‘iron-only’ alternative nitrogenases exhibited similar growth rates irrespective of the presence of absence of the FeSII protein, and the in vitro inactivation of the vanadium nitrogenase components was not affected by the lack of the FeSII protein. All in all, these results are consistent with a model whereby ‘respiratory protection’ is the major physiological mechanism responsible for the protection of all three nitrogenases during energy supplemented growth. Upon energy starvation, however, ‘conformational protection’ mediated by the FeSII protein is capable of temporarily protecting the conventional molybdenum nitrogenase components from inactivation and subsequent degradation.

Bradyrhizobium japonicum , a symbiotic nitrogen-fixing bacterium, has a complex respiratory electron-transport chain, capable of functioning throughout a wide range of oxygen tensions. It does so by synthesizing a number of terminal oxidases, each appropriate for different environmental conditions. Several genes encoding terminal oxidases from B. japonicum have been cloned, but it is unknown what roles these individual oxidases play. In this paper, we describe the cloning and sequencing of the coxX gene encoding the large catalytic subunit for a fourth terminal oxidase from B. japonicum . The coxX gene encodes a 666-amino-acid (aa) protein ( M r 74527) that exhibits a high degree of homology to terminal oxidase proteins from a number of prokaryotic and eukaryotic species. This new oxidase exhibits greater homology to the Escherichia coli cytochrome o subunit I than any of the previously reported B. japonicum terminal oxidase genes.

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.

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.

Nickel is an essential component of all H2-uptake hydrogenases. A fragment of DNA that complements a H2-uptake-deficient but nickel-cured mutant strain (JHK7) of Bradyrhizobium japonicum was isolated and sequenced. This 4.5-kb DNA fragment contains four open reading frames designated as ORF1, hupN, hupO, and hupP, which encode polypeptides with predicted masses of 17, 40, 19, and 63.5 kDa, respectively. The last three open reading frames (hupNOP) are most likely organized as an operon with a putative sigma 54-type promoter. Based on its hydropathy profile, HupN is predicted to be a transmembrane protein. It has 56% identity to the previously described HoxN (high-affinity nickel transport protein) of Alcaligenes eutrophus. A subclone (pJF23) containing the hupNOP genes excluding ORF1 completely complemented (in trans) strain JHK7 for hydrogenase activity in low nickel conditions. pJF26 containing only a functional hupN complemented the hydrogenase activity of mutant strain JHK7 to 30-55% of the wild-type level. Mutant strain JHK70, with a chromosomal deletion in hupP but with an intact hupNO, showed greater activities than pJF26-complemented JHK7 but still had lower activities than the wild type at all nickel levels tested. pJF25, containing the entire hupO and hupP, but without hupN (a portion of hupN was deleted), did not complement hydrogenase activity of mutant strain JHK7. The results suggest that the products of the hupNOP operon are all involved in nickel incorporation/metabolism into the hydrogenase apoprotein. Based on (previous) nickel transport studies of strain JHK7, the hupNOP genes appear not to be involved in nickel transport by whole cells. The deleterious effects on hydrogenase expression are most pronounced by lack of the HupN product.

Many of the foundations of our current knowledge of nitrogen fixation have been based on studies of the Azotobacter species. These range from the early characterizations of the energy-utilizing nature of the nitrogenase reaction, to the demonstration of hydrogen evolution as an inherent by-product of the nitrogenase catalysis, and to the discovery and characterization of alternative nitrogenases. Now, with the recent solving of the crystal structure of the iron protein and molybdenum-iron protein of the nitrogenase from A. vinelandii, it is probably fair to say that the Azotobacter nitrogenase system is our most advanced model-system for nitrogenase structure and function.

The sensitivity of the membrane-bound hydrogenase of Bradyrhizobium japonicum to inactivation by proteases and membrane-impermeant protein modification reagents was compared under hydrogen versus oxygen. In membrane vesicles, the half-life of enzyme inactivation by trypsin of the H2-reduced enzyme was approximately 10 min, whereas O2-oxidized enzyme was much less sensitive to trypsin inactivation (half-life of over 90 min). Diazobenzene sulfonate (DABS) affected the enzyme activity in a manner similar to proteases. With DABS, the enzyme had a half-life of 2-3 min under H2 versus over 30 min under O2. Experiments in which the gas phase (containing either H2 or O2) available to the membranes was changed prior to the protease or chemical modification treatments indicated that it is the redox state of the enzyme at the time of the treatment which determines the sensitivity of the enzyme to inactivation. The redox-dependent differences in the behavior of the membrane-bound enzyme were attributed to changes in the accessibility of the small (33 kDa) subunit. The kinetics of enzyme inactivation by trypsin, under H2, correlated very well with the degradation of the intact 33-kDa subunit, whereas the large subunit (65 kDa) was rather resistant to proteolytic degradation. DABS treatment was found to decrease the reactivity of the small subunit to its antibody concomitant with enzyme inactivation under H2, but without such an effect on the O2-oxidized enzyme. In contrast to the results with the membrane-bound enzyme, purified dehydrogenase was found to be equally susceptible to inactivation by proteolysis or chemical modification irrespective of whether the treatments were performed under H2 or O2. These results indicate that, in the membrane, hydrogenase undergoes a redox-linked conformational change, whereby the small subunit of the enzyme becomes more accessible to external reagents when the enzyme is in its reduced form.

A technique involving the use of disposable cotton roll holders to isolate the lower arch is presented. The cotton roll holder serves two functions: maintenance of a dry field and enhanced visibility.

Upon analyzing the literature, it becomes apparent that the hard- and soft-tissue norms of blacks differ from those of whites. However, these established black norms are not complete when evaluating potential surgical cases. Therefore, the intent of this study was to establish black norms that will be valuable aids for diagnosis in such cases. Lateral cephalometric radiographs were taken of 50 white adults (25 male, 25 female) and 50 black North American adults (25 male, 25 female). All subjects met the following criteria: 18 to 50 years of age; a normal Class I skeletal and dental relationship without any severe anteroposterior, vertical, or transverse discrepancies; and a balanced profile. The tracings involved 24 measurements that were analyzed with a digitizer. Sexual differentiation was included within each measurement. Significant differences between white and black subjects were found in the following areas: hard tissue (SNA, ANB, mandibular length, and the Wits analysis); dental (anterior dental height [ADH], amount of tooth exposure at rest, upper incisor-palatal plane [UI-PP], and lower incisor-mandibular plane [LI-MP]); and soft tissue (nasolabial angle [NLA], upper lip length [ULL], lower lip length [LLL], throat length [TL], and the lip-chin-throat angle [LCTA]). From a practical point of view, this study can be used by orthodontists and oral surgeons to aid in the diagnosis of black patients contemplating orthognathic surgery.