Is the Cry1Ab protein from Bacillus thuringiensis (Bt) taken up by plants from soils previously planted with Bt corn and by carrot from hydroponic culture?

The uptake of the insecticidal Cry1Ab protein from Bacillus thuringiensis (Bt) by various crops from soils on which Bt corn had previously grown was determined. In 2005, the Cry1Ab protein was detected by Western blot in tissues (leaves plus stems) of basil, carrot, kale, lettuce, okra, parsnip, radish, snap bean, and soybean but not in tissues of beet and spinach and was estimated by enzyme-linked immunosorbent assay (ELISA) to be 0.05 +/- 0.003 ng g(-1) of fresh plant tissue in basil, 0.02 +/- 0.014 ng g(-1) in okra, and 0.34 +/- 0.176 ng g(-1) in snap bean. However, the protein was not detected by ELISA in carrot, kale, lettuce, parsnip, radish, and soybean or in the soils by Western blot. In 2006, the Cry1Ab protein was detected by Western blot in tissues of basil, carrot, kale, radish, snap bean, and soybean from soils on which Bt corn had been grown the previous year and was estimated by ELISA to be 0.02 +/- 0.014 ng g(-1) of fresh plant tissue in basil, 0.19 +/- 0.060 ng g(-1) in carrot, 0.05 +/- 0.018 ng g(-1) in kale, 0.04 +/- 0.022 ng g(-1) in radish, 0.53 +/- 0.170 ng g(-1) in snap bean, and 0.15 +/- 0.071 ng g(-1) in soybean. The Cry1Ab protein was also detected by Western blot in tissues of basil, carrot, kale, radish, and snap bean but not of soybean grown in soil on which Bt corn had not been grown since 2002; the concentration was estimated by ELISA to be 0.03 +/- 0.021 ng g(-1) in basil, 0.02 +/- 0.008 ng g(-1) in carrot, 0.04 +/- 0.017 ng g(-1) in kale, 0.02 +/- 0.012 ng g(-1) in radish, 0.05 +/- 0.004 ng g(-1) in snap bean, and 0.09 +/- 0.015 ng g(-1) in soybean. The protein was detected by Western blot in 2006 in most soils on which Bt corn had or had not been grown since 2002. The Cry1Ab protein was detected by Western blot in leaves plus stems and in roots of carrot after 56 days of growth in sterile hydroponic culture to which purified Cry1Ab protein had been added and was estimated by ELISA to be 0.08 +/- 0.021 and 0.60 +/- 0.148 ng g(-1) of fresh leaves plus stems and roots, respectively. No Cry1Ab protein was detected in the tissues of carrot grown in hydroponic culture to which no Cry1Ab protein had been added. Because of the different results obtained with different commercial Western blot (i.e., from Envirologix and Agdia) and ELISA kits (i.e., from Envirologix, Agdia, and Abraxis), it is not clear whether the presence of the Cry1Ab protein in the tissues of some plants under field condition and in carrot in sterile hydroponic culture was the result of the uptake of the protein by the plants or of the accuracy and sensitivity of the different commercial kits used. More detailed studies with additional techniques are obviously needed to confirm the uptake of Cry proteins from soil by plants subsequently planted after a Bt crop.

Microbial populations and enzyme activities in soil in situ under transgenic corn expressing cry proteins from Bacillus thuringiensis.

Transgenic Bt crops produce insecticidal Cry proteins that are released to soil in plant residues, root exudates, and pollen and that may affect soil microorganisms. As a continuation of studies in the laboratory and a plant-growth room, a field study was conducted at the Rosemount Experiment Station of the University of Minnesota. Three Bt corn varieties that express the Cry1Ab protein, which is toxic to the European corn borer (Ostrinia nubilalis Hübner), and one Bt corn variety that expresses the Cry3Bb1 protein, which is toxic to the corn rootworm complex (Diabrotica spp.), and their near-isogenic non-Bt varieties were evaluated for their effects on microbial diversity by classical dilution plating and molecular (polymerase chain reaction-denaturing gradient gel electrophoresis) techniques and for the activities of some enzymes (arylsulfatases, acid and alkaline phosphatases, dehydrogenases, and proteases) involved in the degradation of plant biomass. After 4 consecutive years of corn cultivation (2003-2006), there were, in general, no consistent statistically significant differences in the numbers of different groups of microorganisms, the activities of the enzymes, and the pH between soils planted with Bt and non-Bt corn. Numbers and types of microorganisms and enzyme activities differed with season and with the varieties of corn, but these differences were not related to the presence of the Cry proteins in soil. The Cry1Ab protein of Bt corn (events Bt11 and MON810) was detected in most soils during the 4 yr, whereas the Cry3Bb1 protein was not detected in soils of Bt corn (event MON863) expressing the cry3Bb1 gene.

Release of the recombinant proteins, human serum albumin, beta-glucuronidase, glycoprotein B from human cytomegalovirus, and green fluorescent protein, in root exudates from transgenic tobacco and their effects on microbes and enzymatic activities in soil.

We determined the release in root exudates of human serum albumin (HSA), beta-glucuronidase (GUS), glycoprotein B (gB) from human cytomegalovirus, and green fluorescent protein (GFP) from genetically modified transgenic tobacco expressing the genes for these proteins in hydroponic culture and non-sterile soil. GUS, gB, and GFP were expressed in the plant but were not released in root exudates, whereas HSA was both expressed in the plant and released in root exudates, as shown by a 66.5-kDa band on SDS-PAGE and Western blot and confirmed by ELISA. Root exudates from GUS and gB plants showed no bands that could be attributed to these proteins on SDS-PAGE, and root exudates from GFP plants showed no fluorescence. The concentration of HSA in root exudates was estimated to be 0.021 ng ml(-1), whereas that in the plant biomass was estimated to be 0.087 ng ml(-1). The concentration of HSA in soil was estimated to be 0.049 ng g(-1). No significant differences in the number of microorganisms and the activity of selected enzymes were observed between rhizosphere soil of non-modified and HSA tobacco.

Antiviral effects on bacteriophages and rotavirus by cranberry juice.

Studies were undertaken to investigate the antiviral effects of comestible juices, especially cranberry juice, on non-related viral species. After exposure of bacteriophage T2 to a commercially available cranberry (Vaccinium macrocarpon) juice cocktail (CJ), virus infectivity titer was no longer detectible. After a 60-min exposure to orange (OJ) and grapefruit juices (GJ), phage infectivity was reduced to 25-35% of control, respectively. Similar data were observed for the bacteriophage T4. CJ inactivation of phage T4 was rapid, dose-dependent, and occurred at either 4 or 23 degrees C. Neither pH nor differences in sugar/carbohydrate levels among the juices may be ascribed to the recognized antiviral effects. Further studies were performed to identify the occurrence of antiviral activity by CJ to a mammalian enteric virus. The treatment of the simian rotavirus SA-11 with a 20% CJ suspension was sufficient to inhibit hemagglutination. Under scanning and transmission electron microscopy, CJ was observed to inhibit the adsorption of phage T4 to its bacterial host cells and prevented the replication of rotavirus in its monkey kidney (MA-104) host cells, respectively. The data suggest, for the first time, a non-specific antiviral effect towards unrelated viral species (viz., bacteriophages T2 and T4 and the simian rotavirus SA-11) by a commercially available cranberry fruit juice drink.

Larvicidal Cry proteins from Bacillus thuringiensis are released in root exudates of transgenic B. thuringiensis corn, potato, and rice but not of B. thuringiensis canola, cotton, and tobacco.

Larvicidal proteins encoded by cry genes from Bacillus thuringiensis were released in root exudates from transgenic B. thuringiensis corn, rice, and potato but not from B. thuringiensis canola, cotton, and tobacco. Nonsterile soil and sterile hydroponic solution in which B. thuringiensis corn, rice, or potato had been grown were immunologically positive for the presence of the Cry proteins; from B. thuringiensis corn and rice, the soil and solution were toxic to the larva of the tobacco hornworm (Manduca sexta), and from potato, to the larva of the Colorado potato beetle (Leptinotarsa decemlineata), representative lepidoptera and coleoptera, respectively. No toxin was detected immunologically or by larvicidal assay in soil or hydroponic solution in which B. thuringiensis canola, cotton, or tobacco, as well as all near-isogenic non-B. thuringiensis plant counterparts or no plants, had been grown. All plant species had the cauliflower mosaic virus (CaMV) 35S promoter, except rice, which had the ubiquitin promoter from maize. The reasons for the differences between species in the exudation from roots of the toxins are not known. The released toxins persisted in soil as the result of their binding on surface-active particles (e.g. clay minerals, humic substances), which reduced their biodegradation. The release of the toxins in root exudates could enhance the control of target insect pests, constitute a hazard to nontarget organisms, and/or increase the selection of toxin-resistant target insects.

Activity of free and clay-bound insecticidal proteins from Bacillus thuringiensis subsp. israelensis against the mosquito Culex pipiens.

Bacillus thuringiensis subsp. israelensis produces parasporal insecticidal crystal proteins (ICPs) that have larvicidal activity against some members of the order Diptera, such as blackflies and mosquitoes. Hydrolysis of the ICPs in the larval gut results in four major proteins with a molecular mass of 27, 65, 128, and 135 kDa. Toxicity is caused by synergistic interaction between the 25-kDa protein (proteolytic product of the 27-kDa protein) and one or more of the higher-molecular-mass proteins. Equilibrium adsorption of the proteins on the clay minerals montmorillonite and kaolinite, which are homoionic to various cations, was rapid (<30 min for maximal adsorption), increased with protein concentration and then reached a plateau (68 to 96% of the proteins was adsorbed), was significantly lower on kaolinite than on montmorillonite, and was not significantly affected by the valence of the cation to which the clays were homoionic. Binding of the toxins decreased as the pH was increased from 6 to 11, and there was 35 to 66% more binding in phosphate buffer at pH 6 than in distilled water at pH 6 or 7.2. Only 2 to 12% of the adsorbed proteins was desorbed by two washes with water; additional washings desorbed no more toxins, indicating that they were tightly bound. Formation of clay-toxin complexes did not alter the structure of the proteins, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the equilibrium supernatants and desorption washes and by dot blot enzyme-linked immunosorbent assay of the complexes, which was confirmed by enhanced chemiluminescence Western blot analysis. Free and clay-bound toxins resulted in 85 to 100% mortality of the mosquito Culex pipiens. Persistence of the bound toxins in nonsterile water after 45 days was significantly greater (mortality of 63% +/- 12.7%) than that of the free toxins (mortality of 25% +/- 12.5%).

Larvicidal toxins from Bacillus thuringiensis subspp. kurstaki, morrisoni (strain tenebrionis), and israelensis have no microbicidal or microbiostatic activity against selected bacteria, fungi, and algae in vitro.

The insecticidal toxins from Bacillus thuringiensis subspp. kurstaki (antilepidopteran), morrisoni strain tenebrionis (anticoleopteran), and israelensis (antidipteran) did not affect the growth of a variety of bacteria (8 gram-negative, 5 gram-positive, and a cyanobacterium), fungi (2 Zygomycetes, 1 Ascomycete, 2 Deuteromycetes, and 2 yeasts), and algae (primarily green and diatoms) in pure and mixed culture, as determined by dilution, disk-diffusion, and sporulation assays with purified free and clay-bound toxins. The insecticidal crystal proteins from B. thuringiensis subspp. kurstaki and israelensis had no antibiotic effect on various gram-positive bacteria.

Bt corn has a higher lignin content than non-Bt corn.

Bt corn has been genetically modified to express the Cry1Ab protein of Bacillus thuringiensis to kill lepidopteran pests. Fluorescence microscopy and staining with toluidine blue indicated a higher content of lignin in the vascular bundle sheaths and in the sclerenchyma cells surrounding the vascular bundle in all ten Bt corn hybrids, representing three different transformation events, studied than of their respective non-Bt isolines. Chemical analysis confirmed that the lignin content of all hybrids of Bt corn, whether grown in a plant growth room or in the field, was significantly higher (33-97% higher) than that of their respective non-Bt isolines. As lignin is a major structural component of plant cells, modifications in lignin content may have ecological implications.

Insecticidal toxin from Bacillus thuringiensis is released from roots of transgenic Bt corn in vitro and in situ.

The insecticidal toxin encoded by the cry1Ab gene from Bacillus thuringiensis was released in root exudates from transgenic Bt corn during 40 days of growth in soil amended to 0, 3, 6, 9, or 12% (v/v) with montmorillonite or kaolinite in a plant growth room and from plants grown to maturity in the field. The presence of the toxin in rhizosphere soil was determined by immunological and larvicidal assays. No toxin was detected in any soils from isogenic non-Bt corn or without plants. Persistence of the toxin was apparently the result of its binding on surface-active particles in the soils, which reduced the biodegradation of the toxin. The release of the toxin could enhance the control of insect pests or constitute a hazard to nontarget organisms, including the microbiota of soil, and increase the selection of toxin-resistant target insects.

Clay minerals protect bacteriophage PBS1 of Bacillus subtilis against inactivation and loss of transducing ability by UV radiation.

The effect of UV radiation on the survival of and transduction by phage PBS1 of Bacillus subtilis, free or adsorbed on the clay minerals montmorillonite (M) and kaolinite (K), was studied. After free or clay-associated phage (approximately 10(7) PFU.mL-1) was irradiated with UV light (254 nm) for 0, 1, 2, 5, 10, and 30 min and then allowed to infect B. subtilis FB300 (thiB4 metA29 argF4 Rfmr), the phage was titered, and Met+ transductants were enumerated on selective media. After 1 min of irradiation, the titer of free and clay-associated phage decreased significantly (approximately 1.6 times for free phage, and approximately 4.9 and 6.8 times for M and K, respectively), whereas the transduction frequency increased significantly (approximately 3 times for free phage and approximately 1.4 and 2.2 times for M and K, respectively). The titer and transduction frequency of clay-associated phage remain essentially constant between 1 and 10 min of irradiation, whereas the titer of free phage decreased by approximately 1 order of magnitude after 5 min of irradiation. When free phage was irradiated for 10 min, the titer and transduction frequency decreased by approximately 2 and 0.5 orders of magnitude, respectively, whereas 30 min of irradiation was necessary to obtain comparable decreases with clay-associated phage. These results indicated that phages are protected to some extent from UV radiation when adsorbed on clay minerals.

Interaction between bacteriophage PBS1 and clay minerals and transduction of Bacillus subtilis by clay-phage complexes.

Bacteriophage PBS1 of Bacillus subtilis was rapidly adsorbed on montmorillonite (M) and kaolinite (K), and adsorption was maximal after 30min on both clays. There was no correlation between adsorption and the cation exchange capacity of the clays. Studies with sodium metaphosphate (a polyanion that interacts with positively charged sites on clay) indicated that positively charged sites on K were primarily responsible for the adsorption of the phage, whereas other mechanisms appeared to be involved in adsorption of the phage on M. X-ray diffraction and electron microscopic analyses showed that the phage partially intercalated M. Survival of the phage was increased by adsorption on the clays, and adsorbed phage maintained its ability to transduce bacterial cells for at least 30 days (the longest time studied) after the preparation of the clay-phage complexes. Electron microscopic observations indicated that transduction by the clay-phage complexes was primarily the result of the phage detaching from the clays in the presence of host cells.

A simple method for enumerating bacteriophages in soil.

A plaque technique that uses antibiotic-resistant bacteria growing on antibiotic-containing agar for the assay lawn resulted in significantly better recovery of bacteriophages P1 of Escherichia coli and F116 of Pseudomonas aeruginosa from nonsterile soil than standard membrane filtration or centrifugation techniques. Adsorption of the phages on soil particles appeared to be involved in their recovery and survival in soil.

Monitoring the insecticidal toxins from Bacillus thuringiensis in soil with flow cytometry.

The accumulation and persistance in soil and other natural habitats of the insecticidal toxins from Bacillus thuringiensis may result in environmental hazards, such as toxicity to nontarget species and the selection of toxin-resistant target species. We describe the use of flow cytometry as a method for detecting and tracking the fate of these insecticidal toxins in soil that does not require their extraction and purification. The toxins from B. thuringiensis subspp. tenebrionis and kurstaki were bound on clay- or silt-sized particles separated from Kitchawan soil that was unamended (naturally contains predominantly kaolinite) or amended to 6% v/v with the clay minerals montmorillonite or kaolinite (as an internal control). The particle-toxin mixtures were suspended in 0.1 M phosphate buffer (pH 7) containing 3% nonfat milk powder to block nonspecific binding of antibody, resuspended in a solution of antibody to the toxin from B. thuringiensis subsp. tenebrionis, and then resuspended in a solution of anti-rabbit antibody conjugated with fluorescein isothiocyanate (FITC-Ab). Controls consisted of the particles alone and bound complexes of the particles with the toxin from B. thuringiensis subsp. kurstak. All particles that bound the toxin from B. thuringiensis subsp. tenebrionis showed a significant shift in the peak of fluorescence to the right on the x axis as compared with the nonspecific fluorescence from the control FITC-Ab complexes with particles in the absence of the toxin. There was also a slight shift in the peak to the right for some particles that bound the toxin from B. thuringiensis subsp. tenebrionis, as there is some cross-reactivity between the toxins from B. thuringiensis subspp. tenebrionis and kurstaki and the antibodies that they induce. This method is more sensitive and rapid than the dot-blot ELISA, and processing of many samples is easily accomplished.

Microbial Utilization of Free and Clay-Bound Insecticidal Toxins from Bacillus thuringiensis and Their Retention of Insecticidal Activity after Incubation with Microbes.

The insecticidal toxins produced by Bacillus thuringiensis subspp. kurstaki and tenebrionis were resistant when bound on clays, but not when free, to utilization by pure and mixed cultures of microbes as sources of carbon and carbon plus nitrogen, and their availability as a nitrogen source was reduced. The bound toxins retained insecticidal activity both before and after exposure to microbes or pronase. The insecticidal activity of the toxins persisted for 40 days (the longest time evaluated) in nonsterile soil continuously maintained at the -33-kPa water tension and room temperature, alternately air dried and rewetted to the -33-kPa water tension, or alternately frozen and thawed, although alternate drying and wetting reduced the activity.

Changes in the surface charge of bacteria caused by heavy metals do not affect survival.

Bacillus subtilis and Agrobacterium radiobacter remained viable when exposed to Ni (1 x 10(-4)M; ionic strength (mu) = 3 x 10(-4)) at pH values known to cause a change of the net negative charge of the cells to a net positive charge (charge reversal). The gross morphology, as determined by scanning electron microscopy, of these and other bacteria and of Saccharomyces cerevisiae was not altered in the presence of Ni, Cu, and Zn (1 x 10(-4) M; mu = 3 x 10(-4)), which caused a charge reversal at pH values between 6.0 and 9.0. Similar results were obtained in the presence of Na and Mg, which did not cause charge reversal at the same mu and pH values. These results confirmed that cells remain viable when their surface charge is changed in the presence of some heavy metals at high pH values.

Supplementation with selenium augments the functions of natural killer and lymphokine-activated killer cells.

This study examined the effects of dietary (2.0 ppm for 8 wk) and in vitro (1 x 10(-7)M) supplementation with selenium (Se, as sodium selenite) on the activity of spleen natural killer (NK) cells and plastic-adherent lymphokine-activated killer (A-LAK) cells from C57B1/6J male mice. Dietary supplementation with Se resulted in a significant increase in the lytic activity of activated NK cells, and cells from these highly lytic effector cell populations expressed significantly higher numbers of intermediate affinity interleukin-2 receptors (II-2R)/cell. In the presence of high concentrations of II-2 and 1 x 10(-7)M Se, resting populations of spleen NK cells developed into A-LAK cells that had a significantly enhanced ability to proliferate, as indicated by the significantly higher amounts of nuclear 3H-thymidine incorporation, and a significantly augmented cytolytic activity against both NK-sensitive and NK-resistant target cells. Se appears to enhance the lytic activity of activated NK cells and to augment the proliferation, expansion, and lytic activity of A-LAK cells in the presence of high concentrations of II-2 through its ability to enhance the expression of intermediate affinity II-2R on these cells.

Independent origin of mono-rifampin-resistant Mycobacterium tuberculosis in patients with AIDS.

Historically, infections caused by Mycobacterium tuberculosis have been treated simultaneously with isoniazid and rifampin. As a consequence of this combined therapy, strains resistant only to rifampin were rarely recovered. However, recently there has been an increasing number of reports describing HIV-positive patients infected with mono-rifampin-resistant M. tuberculosis strains. Organisms cultured from seven patients (including six with AIDS) with infections caused by mono-rifampin-resistant M. tuberculosis, and seen at one New York City hospital, were analyzed by molecular techniques to test the hypothesis that dissemination of a single clone had occurred. IS6110 DNA fingerprinting and automated DNA sequencing of a region of the RNA polymerase beta subunit structural gene (rpoB) containing mutations that confer rifampin resistance showed that all organisms independently acquired the mono-rifampin-resistant phenotype. Molecular analysis of mono-rifampin-resistant organisms cultured from 13 additional patients in New York City confirmed independent strain origin. The data rule out the possibility of person-to-person strain transmission among these patients, and they suggest that host factors such as poor compliance with antituberculosis medications or decreased absorption of rifampin have been a driving force in the origin of these strains.