A novel method for biosynthesis of different polymorphs of TiO2 nanoparticles as a protector for Bacillus thuringiensis from Ultra Violet.

Bacillus thuringiensis (Bt) were used for biosynthesis of amorphous TiO2 converted to distinct polymorphs (anatase, rutile, mix) under different temperature conditions. Characterizations of TiO2 nanoparticles were performed by using X-ray diffraction spectroscopy (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and, energy-dispersive X-ray spectroscopy (EDX) analysis. Stability of five formulations under ultraviolet (UV) radiation with spore viability and mortality test on Ephestia kuehniella Zeller larvae were investigated. TiO2(mix) showed the highest viabilities of 79.76% after exposure to ultraviolet (UVA385 nm), while viabilities of non-protected spores under these conditions were 41.32%. The mortality of TiO2(mix), TiO2(anatase), TiO2(rutile), TiO2(amorphous) and free spore formulations on second-instar larvae of Ephestia kuehniella were 73.76%, 71.24%, 57.12%, 51.32%, and 50.32%, respectively on the 10th day of the experiment. The obtained results suggest that TiO2(amorphous) does not increase Bt resistance, but both phases of TiO2 nanoparticles synthesized (anatase and rutile) through the Bacillus thuringiensis and phase mixture can increase the persistence of Bt to the UV light. Furthermore, the combination of both crystalline phases of TiO2(mix) has the highest performance in improving the Bt resistance.

A combined treatment using ethylmethane sulfonate and ultraviolet light to compare amylase production by three Bacillus sp. isolates.

In this study, three Bacillus sp.-producing amylase enzymes were isolated from soil samples and identified using 16S rDNA sequence analysis. Amylase production and total protein productions were spectrophotometrically measured. The following media were tested to increase enzyme production: LB medium and molasses. Three Bacillus sp. were identified as follows: Bacillus subtilis subtilis, Bacillus thuringiensis, and Bacillus cereus. Amylase production levels were in the range of 10 U/mL, whereas total protein production levels were at 15 mg/mL. Higher amylase activity was found in the Bacillus subtilis isolate. Ethylmethane sulfonate (EMS) and ultraviolet (UV) mutagenesis in combination were applied to compare amylase production. Amylase activity was increased to around 58% in the treatment with 0.03 mL of EMS and UV when compared to the control group. A pilot scale bioreactor with a total working volume of 10 liters was used to produce amylase by B. subtilis subtilis. In conclusion, B. subtilis subtilis can be used to produce amylase enzyme for various industrial purposes, and, for the first time, the amylase activities of B. subtilis can be enhanced with EMS and UV treatment.

Purification, characterisation and biological activity of melanin from Streptomyces sp.

Melanins are enigmatic biological macromolecules that can be produced by plants, animals and microorganisms and are especially suited for production in fermentation broth, which is easily industrialised and requires few inputs. This research was conducted to purify, characterise and determine the biological activity of extracellular melanin extracted from Streptomyces sp. The extracellular melanin (M) was extracted from Streptomyces sp. with a yield of 1.46 g L-1. Ma, Mb, Mc and Md were fractionated from M using a Sephadex G-50 column. Ma was detected as the homogeneous component. After studying its ultraviolet-visible absorbance, infrared and electron paramagnetic resonance spectra, as well as scanning electron microscopy images, Ma was confirmed to be typical melanin. Based on its elemental analysis, Ma consisted of carbon, hydrogen, nitrogen, oxygen and sulfur, the percentages of which suggest that phaeomelanin was the main component. In addition, Ma showed significant anti-radiation and free radical scavenging activity. Thus, these results indicated that Streptomyces sp. could be a new source from which to obtain melanin. Ma might be a potential antioxidant and ultraviolet protector and could be used in cosmetics, pesticides, food and other industries.

Comparison and Mechanism of the UV-Resistant Mosquitocidal Bt Mutant LLP29-M19.

Bacillus thuringiensis (Bt) is one of the most widely used and studied biopesticides. However, it is vulnerable to the influence of ultraviolet (UV) radiation, causing shorter persistence under field conditions. To obtain a high-active and effective Bt new product, the main objective of this study is to obtain a highly UV-resistant Bt mutant from the mosquitocidal Bt LLP29 through UV exposure. After 19 rounds of UV exposure, a Bt mutant named LLP29-M19 was obtained, showing resistance to UV radiation for up to 67 min. The mosquitocidal fatality rate of LLP29-M19 was 95%, which was slightly higher than that of LLP29 (90%). Comparative characterization showed that there were no substantial differences in morphology between LLP29-M19 and the original strain, LLP29. However, some changes were detected in physiological and biochemical characteristic reactions, including fructose, glucose, and xylose metabolism. Furthermore, although both LLP29-M19 and LLP29 showed negative zeta potentials, the surface charge of LLP29 was -28.1 mV and that of LLP29-M19 was -42.8 mV. The size distribution of LLP29-M19 was also slightly larger than that of LLP29. Fourier transform infrared analysis indicated that amide functional groups might be involved in the resistance mechanism of LLP29-M19. Quantitative analysis using inductive coupled plasma emission spectrometry showed that some elements increased greatly in LLP29-M19, such as K. All of these results will be highly valuable for better understanding the mechanism of Bt resistance. Explanations regarding the resistance mechanism of this novel Bt mutant may lead to the development of new biopesticides with high mosquitocidal activity and persistence.

Improvement of Vip3Aa16 Toxin Production and Efficiency Through Nitrous Acid and UV Mutagenesis of Bacillus thuringiensis (Bacillales: Bacillaceae).

Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) strain BUPM95 was known by the efficiency of its vegetative insecticidal protein (Vip3Aa16) against different Lepidoptera such as Spodoptera littoralis (Lepidoptera: Noctuidae). To overcome the problem of the low quantities of Vip3 proteins secreted by B. thuringiensis strains in the culture supernatant, classical mutagenesis of vegetative cells of BUPM95 strain was operated using nitrous acid and UV rays. The survivors were screened on the basis of their hemolytic activity and classified in three groups: unaffected, overproducing, and hypo-producing mutants. Using different mutants improved in their hemolytic activity, the supernatants showed an improved toxicity toward S. littoralis larvae (83.33-100% of mortality) compared with the wild-type supernatant (76%). After Vip3 protein quantification in the different supernatants, bioassays against S. littoralis larvae demonstrated that mutants M62, M43, and M76 were improved in the efficiency of their toxin as demonstrated by the lower values of LC50 and LC90 compared with the wild-type Vip3Aa16 protein. However, M26 and M73 mutants were improved in the toxin quantities produced in the supernatant. The improvement of the production and the efficiency of B. thuringiensis Vip3 toxins should contribute to a significant reduction of the production costs of these very interesting B. thuringineis proteins and facilitate the use of these toxins in the pest control management.

Noble UV protective agent for Bacillus thuringiensis based on a combination of graphene oxide and olive oil.

The focus of this study is investigating the performance of graphene oxide (GO) in the protective effect of olive oil on Bacillus thuringiensis (Bt) after being exposed to UV radiations. Biological pesticides Bt subsp. Kurstaki is one of the most important biological control agents. We compared the protective effect of two UV protectant; GO and olive oil and also the combination of both, on the stability of the formulation of Bt after exposure to UV radiations. Spore viability was measured for protective effect and bioassay test was performed on the formulations of Bt. The combination of GO and olive oil revealed the highest viabilities of 50.62% after 96 h exposure to UV radiation, while viabilities of free spore, olive oil formulation and GO formulation were 32.54%, 37.19%,and 45.20%, respectively. The mortality of irradiated combination formulation on second-instar larvae Ephestia Kuehniella was 68.89%, while the same parameter for free spore, olive oil formulation and GO formulation were 40%, 46.66%,and 56%, respectively.

An in-depth characterization of the entomopathogenic strain Bacillus pumilus 15.1 reveals that it produces inclusion bodies similar to the parasporal crystals of Bacillus thuringiensis.

In the present work, the local isolate Bacillus pumilus 15.1 has been morphologically and biochemically characterized in order to gain a better understanding of this novel entomopathogenic strain active against Ceratitis capitata. This strain could represent an interesting biothechnological tool for the control of this pest. Here, we report on its nutrient preferences, extracellular enzyme production, motility mechanism, biofilm production, antibiotic suceptibility, natural resistance to chemical and physical insults, and morphology of the vegetative cells and spores. The pathogen was found to be ß-hemolytic and susceptible to penicillin, ampicillin, chloramphenicol, gentamicin, kanamycin, rifampicin, tetracycline, and streptomycin. We also report a series of biocide, thermal, and UV treatments that reduce the viability of B. pumilus 15.1 by several orders of magnitude. Heat and chemical treatments kill at least 99.9 % of vegetative cells, but spores were much more resistant. Bleach was the only chemical that was able to completely eliminate B. pumilus 15.1 spores. Compared to the B. subtilis 168 spores, B. pumilus 15.1 spores were between 2.67 and 350 times more resistant to UV radiation while the vegetative cells of B. pumilus 15.1 were almost up to 3 orders of magnitude more resistant than the model strain. We performed electron microscopy for morphological characterization, and we observed geometric structures resembling the parasporal crystal inclusions synthesized by Bacillus thuringiensis. Some of the results obtained here such as the parasporal inclusion bodies produced by B. pumilus 15.1 could potentially represent virulence factors of this novel and potentially interesting strain.

The impact of inducing germination of Bacillus anthracis and Bacillus thuringiensis spores on potential secondary decontamination strategies.

AIMS: Decontamination and remediation of a site contaminated by the accidental or intentional release of fully virulent Bacillus anthracis spores are difficult, costly and potentially damaging to the environment. Development of novel decontamination strategies that have minimal environmental impacts remains a high priority. Although ungerminated spores are amongst the most resilient organisms known, once exposed to germinants, the germinating spores, in some cases, become susceptible to antimicrobial environments. We evaluated the concept that once germinated, B. anthracis spores would be less hazardous and significantly easier to remediate than ungerminated dormant spores. METHODS AND RESULTS: Through in vitro germination and sensitivity assays, we demonstrated that upon germination, B. anthracis Ames spores and Bacillus thuringiensis Al Hakam spores (serving as a surrogate for B. anthracis) become susceptible to environmental stressors. The majority of these germinated B. anthracis and B. thuringiensis spores were nonviable after exposure to a defined minimal germination-inducing solution for prolonged periods of time. Additionally, we examined the impact of potential secondary disinfectant strategies including bleach, hydrogen peroxide, formaldehyde and artificial UV-A, UV-B and UV-C radiation, employed after a 60-min germination-induction step. Each secondary disinfectant employs a unique mechanism of killing; as a result, germination-induction strategies are better suited for some secondary disinfectants than others. CONCLUSIONS: These results provide evidence that the deployment of an optimal combination strategy of germination-induction/secondary disinfection may be a promising aspect of wide-area decontamination following a B. anthracis contamination event. SIGNIFICANCE AND IMPACT OF THE STUDY: By inducing spores to germinate, our data confirm that the resulting cells exhibit sensitivities that can be leveraged when paired with certain decontamination measures. This increased susceptibility could be exploited to devise more efficient and safe decontamination measures and may obviate the need for more stringent methods that are currently in place.

Efficacy of olive mill wastewater for protecting Bacillus thuringiensis formulation from UV radiations.

The effectiveness of 10 low-cost UV-absorbers in protecting Bacillus thuringiensis subsp. kurstaki BLB1 toxins against inactivation by UV-A and UV-B irradiation was evaluated in this study. Among them, two by-products, molasses and olive mill wastewater (OMW) were selected for further studies. They were tested at different concentrations of 0.05, 0.1, 0.15 and 0.2% using the para-aminobenzoic acid (PABA) as a common UV protectant. Interestingly, addition of PABA and OMW to BLB1 formulations was found to be most effective in protecting BLB1 spores at 90.8 and 76.4% respectively and in preserving delta-endotoxin concentration at a level of 81.7 and 72.2%, respectively when used at a concentration of 0.2%. The lowest preserved spores (46.3%) and delta-endotoxin level (12.4%) was found using molasses. In contrast, spore count and delta-endotoxin concentration were completely reduced after an exposure of unprotected Bt strain BLB1 to UV radiations up to 96h. SDS-PAGE analysis of protected and unprotected samples revealed that delta-endotoxin bands (130, 65-70kDa) were conserved until 96h of UV exposure in presence of PABA or OMW compared with their disappearance in presence of molasses after 72h of exposure and their dramatically decline from 8h of exposure in unprotected mixture. A complete loss of larvicidal toxicity against Ephestia kuehniella was found after 24h of exposure in absence of any UV-absorber. Addition of OMW or PABA offered the highest levels of insecticidal activity with 63.2 and 74.7% of residual toxicity, respectively. Whereas, molasses addition, as UV protectant retained only 26.3% of residual activity after 96h of exposure. Therefore, addition of OMW by-product to Bt formulation may be a suitable alternative to others synthetic chemical compounds. OMW may also provided added value, be environmentally friendly and less hazardous, when used at low concentration.

Online monitoring of Escherichia coli and Bacillus thuringiensis spore inactivation after advanced oxidation treatment.

Various studies have shown that advanced oxidation processes (AOPs) such as UV light in combination with hydrogen peroxide is an efficient process for the removal of a large variety of emerging contaminants including microorganisms. The mechanism of destruction in the presence of hydrogen peroxide (H2O2) is the enhanced formation of hydroxyl (·OH) radicals, which have a high oxidation potential. The goal of this study was to utilize in-line advanced oxidation to inactivate microbes, and document the inactivation via an in-line, real-time sensor. Escherichia coli cells and Bacillus thuringiensis spores were exposed to UV/H2O2 treatment in DI water, and the online sensor BioSentry(®) was evaluated for its potential to monitor inactivation in real-time. B. thuringiensis was selected as a non-pathogenic surrogate for B. anthracis, the causative agent of anthrax and a proven biological weapon. UV radiation and UV/H2O2 exposure resulted in a >6 log10 reduction of the viable culturable counts of E. coli vegetative cells, and a 3 log10 reduction of B. thuringiensis spores. Scanning electron microscopy of the treated samples revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the morphology of the B. thuringiensis spores. Following AOP exposure, the BioSentry sensor showed an increase in the categories of unknown, rod and spores counts, but overall, did not correspond well with viable count assays. Data from this study show that advanced oxidation processes effectively inactivate E. coli vegetative cells, but not B. thuringiensis spores, which were more resistant to AOP. Further, the BioSentry in-line sensor was not successful in documenting destruction of the microbial cells in real-time.

Bacillus thuringiensis as a surrogate for Bacillus anthracis in aerosol research.

Characterization of candidate surrogate spores prior to experimental use is critical to confirm that the surrogate characteristics are as closely similar as possible to those of the pathogenic agent of interest. This review compares the physical properties inherent to spores of Bacillus anthracis (Ba) and Bacillus thuringiensis (Bt) that impact their movement in air and interaction with surfaces, including size, shape, density, surface morphology, structure and hydrophobicity. Also evaluated is the impact of irradiation on the physical properties of both Bacillus species. Many physical features of Bt and Ba have been found to be similar and, while Bt is considered typically non-pathogenic, it is in the B. cereus group, as is Ba. When cultured and sporulated under similar conditions, both microorganisms share a similar cylindrical pellet shape, an aerodynamic diameter of approximately 1 µm (in the respirable size range), have an exosporium with a hairy nap, and have higher relative hydrophobicities than other Bacillus species. While spore size, morphology, and other physical properties can vary among strains of the same species, the variations can be due to growth/sporulation conditions and may, therefore, be controlled. Growth and sporulation conditions are likely among the most important factors that influence the representativeness of one species, or preparation, to another. All Bt spores may, therefore, not be representative of all Ba spores. Irradiated spores do not appear to be a good surrogate to predict the behavior of non-irradiated spores due to structural damage caused by the irradiation. While the use of Bt as a surrogate for Ba in aerosol testing appears to be well supported, this review does not attempt to narrow selection between Bt strains. Comparative studies should be performed to test the hypothesis that viable Ba and Bt spores will behave similarly when suspended in the air (as an aerosol) and to compare the known microscale characteristics versus the macroscale response.

The effect of gamma sterilization on the insecticidal toxicity of engineered and conventional Bacillus thuringiensis strains.

This study evaluates the effect of gamma radiation on the spore activity, toxicity, and crystal structures of two engineered Bacillus thuringiensis (Bt) strains, TnX and TnY, and the reference Bt strain HD-1. We attempted to identify dosages of cobalt-60 gamma radiation that would inactivate Bt spores but not affect its toxicity. In the radiation dosage range of 10-15 kilogray, no viable spore formation and no significant reduction of the efficiency of Bt against lepidopteran larvae were observed. However, further sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results show that the components of the protoxin are affected by gamma radiation and that some bands are absent after treatment compared with the controls; the change in the protoxin band pattern depends on the type of Bt strain. Furthermore, the spore crystal structure of three Bt strains was studied with scanning electron microscopy and transmission electron microscopy. The results show that there are no changes in the size or shape of the treated Bt spores and crystals compared with the controls. The use of gamma radiation is effective to inactivate the spores of engineered Bt strains while preserving stable Bt toxicity against the target insect larvae.

[Design of the composition of baculovirus agents].

The ability of 30 compounds to protect infection bacteria and baculoviruses from the damaging effect of ultraviolet (UV) irradiation was investigated. For this B. thuringiensis var. israelensis spores and gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus were mixed with different components and exposed to UV irradiation at 0.25 WI cm2 for 60 min. Then spore viability and viral pathogenicity were studied in third instar gypsy moth larvae. The composition comprising sodium alginate, albumin, and ascorbic acid ensured the most effective protection of the viruses and bacteria. These components were shown to provide protection from exposure to UV irradiation even at a low concentration. Their incorporation into biopesticides will assist in enhancing the efficiency of their use.

Characterization of a Mutant Bacillus thuringiensis d-endotoxin with enhanced stability and toxicity / Caracterización de una delta endotoxina mutante de Bacillus thuringiensis con estabilidad y toxicidad aumentadas

The centrally located a-helix 5 of Bacillus thuringiensis d-endotoxins is critical for insect toxicity through ion-channel formation. We analyzed the role of the highly conserved residue Histidine 168 (H168) using molecular biology, electrophysiology and biophysical techniques. Toxin H168R was ~3-fold more toxic than the wild type (wt) protein whereas H168Q was 3 times less toxic against Manduca sexta. Spectroscopic analysis revealed that the H168Q and H168R mutations did not produce gross structural alterations, and that H168R (Tm= 59 °C) was more stable than H168Q (Tm= 57.5 °C) or than the wt (Tm= 56 °C) toxins. These three toxins had similar binding affinities for larval midgut vesicles (Kcom) suggesting that the differences in toxicity did not result from changes in initial receptor binding. Dissociation binding assays and voltage clamping analysis suggest that the reduced toxicity of the H168Q toxin may result from reduced insertion and/or ion channel formation. In contrast, the H168R toxin had a greater inhibition of the short circuit current than the wt toxin and an increased rate of irreversible binding (kobs), consistent with its lower LC50 value. Molecular modeling analysis suggested that both the H168Q and H168R toxins could form additional hydrogen bonds that could account for their greater thermal stability. In addition to this, it is likely that H168R has an extra positive charge exposed to the surface which could increase its rate of insertion into susceptible membranes.

La a-Hélice 5 del domino I de las d-endotoxinas de Bacillus thuringiensis, es crítica para la toxicidad de las toxinas contra insectos al participar en la formación de canales iónicos. La participación en la función tóxica del residuo Histidina 168 (H168) –el cual es altamente conservado– fue estudiada mediante técnicas de biología molecular, electrofisiología y biofísica. La toxina mutante H168R fue ~ 3 veces más tóxica que la toxina silvestre (ts) en Manduca sexta, mientras que H168Q fue 3 veces menos tóxica. Los análisis espectroscópicos indicaron que las mutaciones no producen alteraciones estructurales significativas y que la toxina H168R (Tm= 59 °C) es más estable que las toxinas H168Q (Tm= 57.5 °C) y wt (Tm= 56 °C). Las tres toxinas exhibieron uniones de afinidad similares (Kcom) en vesículas de intestino de larvas de insecto, indicando que las diferencias en la toxicidad no se deben a cambios en la unión inicial al receptor. Los ensayos de unión/disociación y fijación de voltaje mostraron que la reducción de la toxicidad de la toxina H168Q se puede atribuir a una disminución en la inserción y/o en la formación de canales iónicos. De otro lado, H168R mostró una inhibición a la corriente de corto circuito mayor que la ts y un aumento en unión irreversible (kobs), lo cual es consistente con un menor valor de CL50. La modelación molecular sugiere que H168Q y H168R forman puentes de hidrógeno adicionales, lo que les confiere mayor estabilidad térmica. Adicionalmente, es probable que H168R tenga una carga positiva extra expuesta en la superficie, lo cual aumentaría su tasa de inserción en membranas susceptibles.

Bioterrorism: the effects of biological decontamination on the recovery of electronic evidence.

The investigation of a bioterrorism event will ultimately lead to the collection of vital data from electronic devices such as computers and mobile phones. This project sought to determine the use of gamma irradiation and formaldehyde gas as effective biological decontaminants, and the effect of these methods on the recovery of electronic evidence. Electronic items were contaminated with viable spores and then exposed to both decontaminants. Log values for each matrix were calculated with flash drives recording the highest value of 566 Gy for gamma irradiation and a maximum of 50 min exposure to formaldehyde saw the effective destruction of spores. The results indicate that recovery of data varied based on the decontaminant selected, formaldehyde gas giving the most promising results, with electronic data recovered after the required exposure time. Gamma irradiation proved damaging to electronic circuitry at levels required to render the items safe. The implications to computer intelligence and forensics will be discussed based on the outcomes of these findings.

Efficient screening and breeding of Bacillus thuringiensis subsp. kurstaki for high toxicity against Spodoptera exigua and Heliothis armigera.

Spodoptera exigua is one of the most renowned agricultural pest insects and relatively insensitive to Bacillus thuringiensis subsp. kurstaki strains which are widely used commercial products to control lepidopterans such as Heliothis armigera. In the current study, we have developed a new and efficient approach to screen and breed a B. thuringiensis subsp. kurstaki strain exhibiting high toxicity against S. exigua while retaining its high toxicity against H. armigera. UV and diethyl sulfate methods were used for mutagenesis, followed by an agar plug plate diffusion assay for preliminary screening of Zwittermicin A over-producing mutants, from which we obtained a mutant strain, designated here as B. thuringiensis subsp. kurstaki D1-23, with high toxicity against S. exigua. The toxicity of D1-23 against S. exigua and H. armigera was improved by 115.4 and 25.9%, respectively, compared to its parental commercial strain BMB005.

Photostabilization of Bacillus thuringiensis fermented wastewater and wastewater sludge based biopesticides using additives.

Photoprotection (against UV-A and UV-B radiations) of the active components of Bacillus thuringiensis var. kurstaki obtained from the fermentation of various culture media was investigated. The culture media comprised: starch industry wastewater; secondary wastewater sludge (non-hydrolyzed and hydrolyzed) and soya (used as a reference). Photoprotection was carried out by using various UV-protection additives, namely, para-aminobenzoic acid, lignosulfonic acid and molasses at different concentrations (0.1%, 0.15% and 0.2%, w/w). In the absence of UV-protection agents, secondary sludge demonstrated natural UV protection with half-lives ranging from 3.25 to 3.4 d. The half-life for soya and starch industry wastewater was 1.9 and 1.8 d, respectively. Para-amino-benzoic acid as a UV-protection agent at 0.20% (w/w) gave excellent UV-protection for soya and starch industry wastewater with half-lives being 5.9 and 7 d, respectively. Likewise, lignosulfonic acid at 0.20% (w/w) was an effective photostabilizer for hydrolyzed and non-hydrolyzed secondary sludge with half-lives of 7.25 and 8 d, respectively. Hence, when similar concentration of the UV-protection additives was used, photoprotection was higher for the alternative media than the conventional soya medium, validating the technical feasibility of using three additives.

Expression of mel gene improves the UV resistance of Bacillus thuringiensis.

AIMS: To improve ultraviolet (UV) resistance of Bacillus thuringiensis for increasing the duration of the Bt product applied in the field, a genetically engineered strain Bt TD841 that produced both melanin and Cry1A protein was constructed, and its UV resistance was evaluated in the laboratory. METHODS AND RESULTS: Melanin quantitative analysis revealed that the recombinant strain Bt TD841 could synthesize 0.15 mg melanin ml(-1) sporulated culture. Atomic force microscopy confirmed the production of diamond crystal and SDS-PAGE results showed the expression of the 130 kDa Cry1A protein. Bioassay results demonstrated that the LC(50) value of Bt TD841 was 3.69 microl ml(-1) against Helicoverpa armigera and the UV resistance of this recombinant was enhanced 9.7-fold compared to its parental strain Bt HC42 after 4-h UV irradiation. CONCLUSION: Expression of the mel gene can significantly increase UV resistance of B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on genetically engineered Bt strain with co-expression of melanin and the insecticidal crystal proteins gene, and the results may offer a practical solution for improving the photoprotection of Bt products in field application.

Screening of different adjuvants for wastewater/wastewater sludge-based Bacillus thuringiensis formulations.

Screening of different adjuvants, namely, suspending agents, phagostimulants, stickers, antimicrobial agents, and UV screens to develop aqueous biopesticidal suspensions of Bacillus thuringiensis (Bt) variety kurstaki HD-1 fermented broths, specifically, nonhydrolyzed sludge, hydrolyzed sludge, starch industry wastewater, and soya (commercial medium), were investigated. The selected suspending agents [20% (wt:vol)] included sorbitol, sodium monophosphate, and sodium metabisulfite with corresponding suspendibility of 74-92, 69-85, and 71-82%, respectively. Molasses [0.2% (wt:vol)] increased adherence by 84-90% for all fermented broths. The optimal phagostimulants [0.5% (wt:vol)], namely, soya and molasses, caused entomotoxicity increase of 3-13 and 7-13%, respectively. Sorbic and propionic acids showed high antimicrobial action [0.5% (wt:vol)], irrespective of fermentation medium. Sodium lignosulfonate, molasses, and Congo red, when used as UV screens [0.2% (wt:vol)], showed percent corresponding entomotoxicity losses of 3-5, 0.5-5 and 2-16, respectively. The Bt formulations, when exposed to UV radiation, showed higher half-lives (with and without UV screens) than the fermented broths or semisynthetic soya medium and commercial Bt formulation. UV screen-amended nonhydrolyzed, hydrolyzed, and starch industry wastewater formulations showed 1.3-1.5-fold higher half-lives than commercial Bt formulation. Thus, the recommended formulation comprises sorbitol, sodium monophosphate, sodium metabisulfite (suspending agents); molasses, soya flour (phagostimulants); molasses and skimmed milk powder (rainfasteners); sorbic and propionic acids (antimicrobial agents) and sodium lignosulfate; and molasses and Congo red (UV screens). These waste-based Bt formulations offer better UV resistance in comparison with commercial formulation.

Cloning and expression of mel gene from Bacillus thuringiensis in Escherichia coli.

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of L -tyrosine at elevated temperatures (42 degrees C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of L -tyrosine, via L -DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 alpha. A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 alpha conferred UV protection on the recipient strain.