Effect of entomocidal proteins from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> on ion permeability of apical membranes of <Emphasis Type="Italic">Tenebrio molitor</Emphasis> larvae gut epithelium

Effects of entomocidal Cry-type proteins, δ-endotoxins Cry3A and Cry11A produced by Bacillus thuringiensis, on ion permeability of the apical membranes of intestinal epithelium from Tenebrio molitor larvae midgut were studied. Using potential-sensitive dyes safranine O and oxonol VI and δpH indicator acridine orange, it was shown that placing brush border membrane vesicles (BBMV) (loaded with Mg²⁺ during their preparation) into a salt-free buffer medium resulted in spontaneous generation of transmembrane electric potential on the vesicular membrane (negative inside the vesicles) accompanied by acidification of the aqueous phase inside the vesicles. The generation of transmembrane ion gradients on the vesicular membrane was a result of an electrogenic efflux of Mg²⁺ from the vesicles as shown by abolishing of the membrane potential by such agents as MgSO₄ or CaCl₂ in centimolar concentrations, a highly lipophilic cation tetraphenylphosphonium, and some blockers of cell membrane Ca²⁺-channels in submillimolar concentrations. A passive generation of membrane potential on the vesicular membrane (but positive inside the vesicles) was also observed upon addition of centimolar concentrations of K₂SO₄. Addition of δ-endotoxins Cry3A and Cry11A to the vesicle suspension in a salt-free buffer medium or in the same medium supplemented with centimolar concentrations of K₂SO₄ exerted a pronounced hyperpolarization of the vesicular membrane. This hyperpolarization was sensitive to the same agents, which abolished the membrane potential generation in the absence of δ-endotoxin. It is concluded that Cry proteins induced in BBMV from T. molitor opening pores or ion channels, which were considerably more permeable for alkaline- and alkaline-earth metal cations than for the accompanying anions.     

Overexpression of <Emphasis Type="Italic">odc</Emphasis> (ornithine decarboxylase) in <Emphasis Type="Italic">Datura innoxia</Emphasis> enhances the yield of scopolamine

Scopolamine is widely used for its anticholinergic properties. Because of higher physiological activity and less side effects the world demand of scopolamine is estimated to be ten times greater than other anticholinergic agents, hyoscyamine and atropine. Since natural production is limited, alternatives are required to boost the production. We report the introduction of mouse odc gene of polyamine biosynthesis pathway which is also the primary pathway of tropane alkaloids in Datura innoxia. Polyamines, mainly putrescine, serve as the common metabolite for tropane alkaloids and nicotine. We have overexpressed odc gene to modulate the metabolic flux downstream and eventually achieved higher accumulation of scopolamine in transgenic plants. Among six independent transformed lines one line (O10) produced scopolamine (0.258 μg/g dry weight) almost six times higher than that produced by control plants (0.042 μg/g DW). To our knowledge, this is the first report of odc overexpression in D. innoxia leading to higher scopolamine yield.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the plant pathogenic fungus <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25°C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern b lot analy s is. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in R. necatrix and provide a simple and reliable method for genetic manipulation.     

pH tolerance of the biofouling invasive hydrozoan <Emphasis Type="Italic">Cordylophora caspia</Emphasis>

The hydrozoan Cordylophora caspia, with its origin in the Caspian and Black Seas, is considered an invasive biofouling species to power stations and water treatment plants in Brazil, causing economic and ecological impacts. Tolerance of the species to variation in pH was tested to evaluate its resistance to acidic and basic pH levels. Values of pH were tested over a range from 4.0 to 10.0. The colonies were exposed for 6 h to each treatment, using phosphoric acid and NaOH to set water pH to the desired acidic or basic levels, respectively. Colonies of C. caspia displayed 100% mortality only at pH 4 and 10, and showed high survival rates (>50%) within the pH range of 5.0–8.5. The species demonstrated tolerance to a wide range of pH levels, and the results will help in the development of techniques to reduce biofouling by this species. Treatments that keep the pH below 5 or above 8.5 for at least 6 h are suggested to control and eradicate C. caspia where fouling causes significant economic impacts.     

<Emphasis Type="Italic">In situ</Emphasis> carbon supplementation in large-scale cultivations of <Emphasis Type="Italic">Spirulina platensis</Emphasis> in open raceway pond

The objective of this study was to estimate the CO₂ absorptivity provided by an in situ carbon supply system using a photosynthetic culture of the cyanobacterium Spirulina platensis in an open raceway pond. The effects of initial total carbon concentrations (ranging from 0 to 0.1 mol/L), suspension depths (ranging from 5 to 20 cm) and pH values (ranging from 8.9 to 11.0) on the CO₂ absorptivity were studied. The results indicated that CO₂ absorptivity was positively correlated with pH value, negatively correlated with total carbon concentration, and only negligibly affected by the suspension depth. The optimum total carbon concentration range and pH range were 0.03 ∼ 0.09 mol/L and 9.7 ∼ 10.0, respectively. An average CO₂ absorptivity of 86.16% and average CO₂ utilization efficiency of 79.18% were achieved using this in situ carbon-supply system in large-scale cultivation of Spirulina platensis, with an initial total carbon concentration of 0.06 mol/L and pH value of 9.8. Our results demonstrated that this system could obtain a favorable CO₂ utilization efficiency in outdoor, large-scale cultivation of Spirulina platensis in open raceway ponds.     

An endophytic <Emphasis Type="Italic">Neurospora</Emphasis> sp. from <Emphasis Type="Italic">Nothapodytes foetida</Emphasis> producing camptothecin

The medicinal plant, Nothapodytes foetida, contains a number of important alkaloids like camptothecin (an anticancer drug molecule), but its concentration is less to meet the existing demand of this important molecule, in an effort for accessible availability of camptothecin. An endophyte (designated ZP5SE) was isolated from the seed of Nothapodytes foetida and was examined as a potential source of anticancer drug lead compound, i.e., camptothecin, when grown in Sabouraud liquid culture media under shake flask conditions. The presence of an anticancer compound (camptothecin) in this fungus was confirmed by chromatographic and spectroscopic methods in comparison with authentic camptothecin. Isolated endophyte (Neurospora crassa) producing camptothecin may become an easily accessible source for the production of a precursor anticancer drug molecule in the future at a large scale. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 2, pp. 225–231. The text was submitted by the authors in English.     

Isozymes of <Emphasis Type="Italic">α</Emphasis>-amylases from newly isolated <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> CKB19: Production from immobilized cells

The aim of this study was to produce two isozymes of α-amylase by immobilization of a newly isolated soil bacterium. The bacterium was identified as Bacillus thuringiensis CKB19 on the basis of its 16S rRNA profile. Enzyme production by free cells increased linearly with cell growth up to 34 h in starch containing enriched liquid media. The active bacterial cells were immobilized in Caalginate beads, and operational stability of the entrapped cell was optimized for amylase production. Enzyme production was optimal at an alginate concentration of 2 g% (w/v), calcium chloride concentration of 1 M, and with 300 beads (each bead contained 2 × 10⁷ cells)/250 mL flask. Amylase production by the immobilized cells was about 3 times higher than free cell fermentation after 34 h of incubation. It was observed that the immobilized bacterium secreted two different amylases (Am-I and Am-II) into the culture fluid. The molecular masses of Am-I and Am-II were 59.6 and 44.7 kd, respectively, and showed optimum activity at pH 5.0 and 9.0. Both amylases showed optimum activity at 40°C and were stable at the same temperature, with losses of only 10 and 20% (for Am I and Am II, respectively) of their original activities after 24 h of incubation. Further, both amylases were salt tolerant (up to 4 M NaCl) and hydrolyzed raw starchy foods into glucose. All these characteristics make this enzyme mixture suitable for use as a digestive aid and for the improvement of digestibility of animal feed ingredients.     

Accumulation of copper in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> transformants,constructed with the modified <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation technique

A modified Agrobacterium tumefaciens-mediated transformation method was established for the construction of mutants with improved copper tolerance and accumulation capability in Trichoderma reesei. One transformant, AT01, exhibited the highest copper accumulation capability. With copper at 0.7 mM, AT01 removed 13 mg copper/g biomass (removal rate of 96%), whereas the wild-type strain removed only 6 mg copper/g biomass (removal rate of 50%). Optimal conditions were a pH of 5.0 at 28°C. The pigment change of Trichoderma mycelia was a potential indicator of copper accumulation. Electron microscopy revealed that copper was mainly accumulated in cell vacuoles.     

Enhanced production and characterization of a β-glucosidase from <Emphasis Type="Italic">Bacillus halodurans</Emphasis> expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A putative β-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-β-D-1-thiogalactopyranoside, the enzyme expressed at ∼40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A ₆₀₀ 12 in auto-inducing M9NG medium, β-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the β-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45°C and 8.0, respectively, under the assay conditions. K _m and k _cat against p-nitrophenyl-β-D-glucopyranoside were 4 mM and 0.75 sec⁻¹, respectively. To our knowledge, this is the first report of high-level expression and characterization of a β-glucosidase from B. halodurans.     

Potential long-term storage of the predatory mite <Emphasis Type="Italic">Phytoseiulus</Emphasis><Emphasis Type="Italic">persimilis</Emphasis>

Increasing the ability to store mass-reared natural enemies during periods or seasons of low demand is a critical need of the biocontrol industry. We tested the hypothesis that chemicals can enhance long-term cold storage of a predatory mite Phytoseiulus persimilis Athias-Henriot. The research objective was to determine the effect of cryoprotectant and carbohydrate chemicals on in-storage survival of predators. In-storage survival at 8°C was greater for predators sprayed with glycerol (5%, v/v) or glucose (10% and 20%, v/v) than with water spray controls. After 74 days in the cryoprotectant experiment, predator survival declined to 11.5% in the 5% glycerol treatment and 7.8% in the water spray control. After 88 days in the carbohydrate experiment, predator survival declined to 22% in the 20% glucose treatment and 2% in the water spray control. Although many individuals expired within 50 days in both experiments, a few females survived more than 200 days. This research suggests that select cryoprotectants and carbohydrates have a limited capacity to facilitate long-term storage of P. persimilis.     

Spectroscopic and electrochemical characterization of cytochrome <Emphasis Type="Italic">c</Emphasis> encapsulated in a bio sol–gel matrix

Sol-gel technique represents a remarkably versatile method for protein encapsulation. To enhance sol-gel biocompatibility, systems envisaging the presence of calcium and phosphates in the sol-gel composition were recently prepared and investigated. Unfortunately, the low pH at which solutions were prepared (pH < 2.5) dramatically limited their application to proteins, because the acidic environment induces protein denaturation. In this paper we apply a new protocol based on the introduction of calcium nitrate to the inorganic phase, with formation of a binary bioactive system. In this case protein encapsulation results versatile and secure, being achieved at a pH close to neutrality (pH 6.0); also, the presence of calcium is expected to enhance system biocompatibility. To determine the properties of the salt-doped sol-gel and the influence exerted on entrapped biosystems, the structural and functional properties of embedded cytochrome c have been investigated. Data obtained indicate that the salt-doped sol-gel induces no significant change in the structure and the redox properties of the embedded protein; also, the matrix increases protein stability. Interestingly, the presence of calcium nitrate appears determinant for refolding of the acid-denatured protein. This is of interest in the perspective of future applications in biosensoristic area.     

Presence of <Emphasis Type="Italic">C. albidus</Emphasis>, <Emphasis Type="Italic">C. laurentii</Emphasis> and <Emphasis Type="Italic">C. uniguttulatus</Emphasis> in Crop and Droppings of Pigeon Lofts (<Emphasis Type="Italic">Columba livia</Emphasis>)

Columba livia is an important reservoir and carrier of Cryptococcus neoformans, Cryptococcus uniguttulatus, Cryptococcus laurentii and Cryptococcus albidus. Upper digestive tract of this species is also known as a habitat for Cryptococcus neoformans. Given the increasing clinical interest of this microorganism, 331 swabs from crop and 174 dropping samples from pigeon lofts in Grand Canary Island have been studied. The obtained results show an extensive presence samples 81 positive (24.47%) of Cryptococcus spp. in analysed crops: 32 (9.66%) for C. neoformans, 24 (7.2%) for C. uniguttulatus, 23 (6.9%) for C. albidus and 2 (0.6%) for C. laurentii. In the same way, Cryptococcus spp was also isolated in 82 (47.13%), dropping samples: C. neoformans in 59 (33.9%), C. uniguttulatus, in 9 (5.17%), C. laurentii in 8 (4.59%) and C. albidus in 6 (3.44%) of the investigated samples, respectively. The cryptococcosis produced by species of cryptococci other than C. neoformans has become more important during the last decade, supporting the study on the role of pigeon in the epidemiology of this disease.     

Statistical medium optimization and biodegradative capacity of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> toward <Emphasis Type="Italic">p</Emphasis>-nitrophenol

The effect of p-nitrophenol (PNP) concentration with or without glucose and yeast extract on the growth and biodegradative capacity of Ralstonia eutropha was examined. The chemical constituents of the culture medium were modeled using a response surface methodology. The experiments were performed according to the central composite design arrangement considering PNP, glucose and yeast extract as the selected variables whose influences on the degradation was evaluated (shaking in reciprocal mode, temperature of 30°C, pH 7 and test time of about 9 h). Quadratic polynomial regression equations were used to quantitatively explain variations between and within the models (responses: the biodegradation capacity and the biomass formation). The coefficient of determination was high (R _adjusted² = 0.9783), indicating the constructed polynomial model for PNP biodegradative capacity explains the variation between the regressors fairly well. A PNP removal efficiency of 74.5% occurred within 9 h (15 mg/L as the initial concentration of PNP with use of yeast extract at 0.5 g/L).     

<Emphasis Type="Italic">In vitro</Emphasis> biosynthesis of strictosidine using<Emphasis Type="Italic">lonicera japonica</Emphasis> leaf extracts and recombinant yeast

Strictosidine is a key intermediate in the biosynthesis of the terpenoid indole alkaloid (T1A) pathway. It results from a condensation reaction, catalyzed by strictosidine synthase (STR), between tryptamine and secologanin. We have now developed a useful method, based on enzyme-assisted synthesis, to produce strictosidine. Our procedure utilizes leaf extracts from Japanese honeysuckleLonicera japonica Thunb. as a secologanin source. In these experiments, an enzyme extract was prepared from transgenic yeastSaccharomyces cerevisiae that expresses theCatharanthus roseus STR (CrSTR) coding region. Strictosidine was then isolated with a 38% yield based on the initial amount of tryptamine in the enzymatic reaction.     

Overexpression of the Chitosanase Gene in <Emphasis Type="Italic">Fusarium solani</Emphasis> via <Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-Mediated Transformation

To overexpress the chitosanase gene (csn) in F. solani, a vector based on pCAMBIA 1300 was constructed. The csn gene, which is under control of the Aspergillus nidulans gpdA promoter and A. nidulans trpC terminator, was introduced back into the F. solani genome by Agrobacterium tumefaciens-mediated transformation, and the herbicide-resistance gene bar from Streptomyces hygroscopicus was used as the selection marker. Transformants which showed a significant increase in chitosanase production (~2.1-fold than control) were obtained. Southern blot analysis indicated that most transformants had a single-copy T-DNA integration.     

Anatomical observation of polyphenol changes in epidermal cells during the development of <Emphasis Type="Italic">Quercus acutissima</Emphasis>–<Emphasis Type="Italic">Scleroderma verrucosum</Emphasis> ectomycorrhizae

Quercus acutissima seedlings were cultivated in growth pouches and inoculated with Scleroderma verrucosum in order to assess the changes in polyphenol contents in epidermal cells during ECM development. Semithin sections stained with metachromatic Toluidine Blue O (TBO) were compared among non-inoculated lateral roots, early mantled lateral roots, and mycorrhizal roots with a mature mantle. Hyphae adhered closely or were embedded in mucilage-like materials on the epidermis. Epidermal cells and root hairs of the non-inoculated second-order lateral roots developing from the taproot harbored polyphenolic compounds that were stained by TBO. At non-inoculated stage, the average numbers of epidermal cells stained entirely (PC2), stained partially (PC1) or remaining unstained (PC0) were 16.5 ± 0.7, 0 ± 0, and 0 ± 0, respectively. At the early mantled stage, the numbers were 6.5 ± 1.6, 5.2 ± 1.4, and 4.2 ± 1.0, and at the mycorrhizal stage, it was 0 ± 0, 0 ± 0, and 32.8 ± 1.3 for PC2, PC1, and PC0, respectively. Total phenolic content in the root tips at each developmental stage declined with ECM development. The early mantled stage involved a dynamic process of polyphenol localization. However, some epidermal cells and endodermal cells of the proximal zone accumulated polyphenols. Eventually, polyphenolic compounds, which were found abundantly in the epidermal cells and root hairs of the non-inoculated lateral roots of the host, disappeared at the mycorrhization process with the symbiont.     

Cyanuric acid biodegradation by a mixed bacterial culture of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. in a packed bed biofilm reactor

Cyanuric acid (1,3,5-triazine-2,4,6-triol [OOOT]) is a common biodegradation byproduct of triazinic herbicides, frequently accumulated in soils or water when supplementary carbon sources are absent. A binary bacterial culture able to degrade OOOT was selected through a continuous selection process accomplished in a chemostat fed with a mineral salt (MS) medium containing cyanuric acid as the sole carbon and nitrogen source. By sequence comparison of their 16S rDNA amplicons, bacterial strains were identified as Agrobacterium tumefaciens, and Acinetobacter sp. When the binary culture immobilized in a packed bed reactor (PBR) was fed with MS medium containing OOOT (50 mg L⁻¹), its removal efficiencies were about 95%; when it was fed with OOOT plus glucose (120 mg L⁻¹) as a supplementary carbon source, its removal efficiencies were closer to 100%. From sessile cells, attached to PBR porous support, or free cells present in the outflowing medium, DNA was extracted and used for Random Amplification of Polymorphic DNA analysis. Electrophoretic patterns obtained were compared to those of pure bacterial strains, a clear predominance of A. tumefaciens in PBR was observed. Although in continuous suspended cell culture, a stable binary community could be maintained, the attachment capability of A. tumefaciens represented a selective advantage over Acinetobacter sp. in the biofilm reactor, favoring its predominance in the porous stone support.     

The influence of low pH on in vitro growth and biochemical parameters of <Emphasis Type="Italic">Plantago almogravensis</Emphasis> and <Emphasis Type="Italic">P. algarbiensis</Emphasis>

The effects of low medium pH (4.50, 5.00 and 5.75) on in vitro growth and on several biochemical parameters (lipid peroxidation, proline and carbohydrate content, antioxidant enzymes activities and total soluble protein) of Plantago almogravensis and P. algarbiensis micropropagated shoots were investigated. Overall, it was observed that medium pH did not affect in vitro proliferation and rooting. Interestingly, cultures of both species modify the initial pH value to the same final value. Results have shown that the lowest pH tested induced an increase in the level of lipid peroxidation in roots of both species and in shoots of P. algarbiensis, indicating plasma membrane damage. An accumulation of carbohydrates was observed in roots of P. almogravensis cultured in pH 4.50 and 5.00. It was observed a slight response of the enzymatic system to medium pH, particularly in P. almogravensis. Based on the results obtained we can conclude that Plantago species are apt to grow in vitro in medium with pH values much lower than the usually used in tissue culture, which is in agreement with the fact that both species colonize acid soils.