Cry4Ba toxin of Bacillus thuringiensis subsp. israelensis uses both domains II and III to bind to its receptor-Aedes aegypti alkaline phosphatase.

Receptor binding is one of the crucial steps to exhibit the insecticidal activity of Cry toxins. In addition, binding to receptors is a determining step for the specificity of toxins. In this work, receptor binding domain II was cloned from the full-length Cry4Ba toxin and heterologously expressed in Escherichia coli. The 21 kDa purified protein was characterized as Cry4Ba domain II using Western blotting and tandem mass spectrometry coupled to liquid chromatography. Circular dichroism revealed the correct folding of the isolated domain II fragment, similar to that found in the Cry4Ba protein. Binding analysis using an enzyme-linked immunosorbent assay revealed that the purified Cry4Ba-domain II had bound to the 54 kDa alkaline phosphatase cloned from Aedes aegypti (Aa-mALP) with a dissociation constant of approximately 116.27 ± 11.09 nM. The binding affinity of Cry4Ba-domain II to Aa-mALP was comparable to that of Cry4Ba domain III, suggesting that both domains II and III of the Cry4Ba contributed equally in binding to the Aa-mALP protein. Our findings should provide more valuable insight on the molecular mechanisms in the toxin-receptor interaction of the Cry4Ba toxin.

Aromatic Residues on the Side Surface of Cry4Ba-Domain II of Bacillus thuringiensis subsp. israelensis Function in Binding to Their Counterpart Residues on the Aedes aegypti Alkaline Phosphatase Receptor.

Receptor binding is a prerequisite process to exert the mosquitocidal activity of the Cry4Ba toxin of Bacillus thuringiensis subsp. israelensis. The beta-sheet prism (domain II) and beta-sheet sandwich (domain III) of the Cry4Ba toxin have been implicated in receptor binding, albeit the precise binding mechanisms of these remain unclear. In this work, alanine scanning was used to determine the contribution to receptor binding of some aromatic and hydrophobic residues on the surface of domains II and III that are predicted to be responsible for binding to the Aedes aegypti membrane-bound alkaline phosphatase (Aa-mALP) receptor. Larvicidal activity assays against A. aegypti larvae revealed that aromatic residues (Trp327 on the ß2 strand, Tyr347 on the ß3-ß4 loop, and Tyr359 on the ß4 strand) of domain II were important to the toxicity of the Cry4Ba toxin. Quantitative binding assays using enzyme-linked immunosorbent assay (ELISA) showed similar decreasing trends in binding to the Aa-mALP receptor and in toxicity of the Cry4Ba mutants Trp327Ala, Tyr347Ala, and Tyr359Ala, suggesting that a possible function of these surface-exposed aromatic residues is receptor binding. In addition, binding assays of the Cry4Ba toxin to the mutants of the binding residues Gly513, Ser490, and Phe497 of the Aa-mALP receptor supported the binding function of Trp327, Tyr347, and Tyr359 of the Cry4Ba toxin, respectively. Altogether, our results showed for the first time that aromatic residues on a side surface of the Cry4Ba domain II function in receptor binding. This finding provides greater insight into the possible molecular mechanisms of the Cry4Ba toxin.

Adding value to rice straw waste for high-level xylanase production using a new isolate of Bacillus altitudinis RS3025.

An investigation was carried out using rice straw as a low-cost substrate to study the optimization of xylanase production using a newly identified endospore-forming bacterium, Bacillus altitudinis RS3025. The highest xylanase activity was achieved using 2% rice straw (pretreated with 2% NaOH at 100 °C) at pH 7.0, 37 °C temperature, and with 72-h incubation time. Under the optimized conditions, xylanase activity reached 2518.51 U/mL, which was 11.56-fold higher than the activity under the initial conditions using untreated rice straw as substrate. Enzymatic hydrolysis of the rice straw using crude xylanase of B. altitudinis RS3025 demonstrated the hydrolyzation efficiency of the rice straw waste, especially alkaline rice straw. The highest level of released reducing sugars was 149.78 mg/g substrate. The study demonstrated the successful utilization of rice straw waste for high-level xylanase production using B. altitudinis RS3025 and reducing sugar production using low-cost crude enzyme, which has the advantages of reducing the processing cost and environmental concerns associated with rice straw waste management.

Proteomic Response of Aedes aegypti Larvae to Silver/Silver Chloride Nanoparticles Synthesized Using Bacillus thuringiensis subsp. israelensis Metabolites.

Silver/silver chloride nanoparticles (Ag/AgCl NPs) are an alternative approach to control the larvae of Aedes aegypti, a vector of mosquito-borne diseases. However, the molecular mechanisms of Ag/AgCl NPs to A. aegypti have not been reported. In this work, Ag/AgCl NPs were synthesized using supernatant, mixed toxins from Bacillus thuringiensis subsp. israelensis (Bti), and heterologously expressed Cry4Aa and Cry4Ba toxins. The images from scanning electron microscopy revealed that the Ag/AgCl NPs were spherical in shape with a size range of 25-100 nm. The larvicidal activity against A. aegypti larvae revealed that the Ag/AgCl NPs synthesized using the supernatant of Bti exhibited higher toxicity (LC50 = 0.133 µg/mL) than the Ag/AgCl NPs synthesized using insecticidal proteins (LC50 = 0.148-0.217 µg/mL). The proteomic response to Ag/AgCl NPs synthesized using the supernatant of Bti in A. aegypti larvae was compared to the ddH2O-treated control. Two-dimensional gel electrophoresis analysis revealed 110 differentially expressed proteins, of which 15 were selected for identification using mass spectrometry. Six upregulated proteins (myosin I heavy chain, heat shock protein 70, the F0F1-type ATP synthase beta subunit, methyltransferase, protein kinase, and condensin complex subunit 3) that responded to Ag/AgCl NP treatment in A. aegypti were reported for NP treatments in different organisms. These results suggested that possible mechanisms of action of Ag/AgCl NPs on A. aegypti larvae are: mitochondrial dysfunction, DNA and protein damage, inhibition of cell proliferation, and cell apoptosis. The findings from this work provide greater insight into the action of green synthesized Ag/AgCl NPs on the control of A. aegypti larvae.

High poly ε-caprolactone biodegradation activity by a new Acinetobacter seifertii isolate.

Poly(ε-caprolactone; PCL) is an attractive biodegradable polymer that has been increasingly used to solve environmental problems caused by plastic wastes. In the present study, 468 bacterial isolates were recovered from soil samples and screened for PCL degradation activity. Of the isolates, 37 (7.9%) showed PCL depolymerase activity on PCL agar medium, with the highest activity being by isolate S22 which was identified using 16S rRNA and rpoB gene sequencing as Acinetobacter seifertii. Scanning electron microscopy and Fourier transform infrared spectroscopy confirmed the degradation of PCL films after treatment with A. seifertii S22. The PCL depolymerase activity of A. seifertii S22 relied on the activity of esterase which occurred at an optimum temperature of 30-40 °C. The highest PCL depolymerase activity (35.5 ± 0.7 U/mL) was achieved after culturing A. seifertii S22 for 6 h in mineral salt medium (MSM) containing 0.1% Tween 20 and 0.02% ammonium sulfate as the carbon and nitrogen sources, respectively, which was approximately 20-fold higher than for cultivation in MSM supplemented with 0.1% PCL as sole carbon source. The results suggested that A. seifertii S22 or its enzymes could be used for PCL bioplastic degradation.

Bacillus thuringiensis Cry4Ba Insecticidal ToxinExploits Leu615 in Its C-Terminal Domain to Interact with a Target Receptor-Aedes aegypti Membrane-Bound Alkaline Phosphatase.

In addition to the receptor-binding domain (DII), the C-terminal domain (DIII) of three-domain Cry insecticidal δ-endotoxins from Bacillus thuringiensis has been implicated in target insect specificity, yet its precise mechanistic role remains unclear. Here, the 21 kDa high-purity isolated DIII fragment derived from the Cry4Ba mosquito-specific toxin was achieved via optimized preparative FPLC, allowing direct rendering analyses for binding characteristics toward its target receptor-Aedes aegypti membrane-bound alkaline phosphatase (Aa-mALP). Binding analysis via dotblotting revealed that the Cry4Ba-DIII truncate was capable of specific binding to nitrocellulose-bound Aa-mALP, with a binding signal comparable to its 65 kDa Cry4Ba-R203Q full-length toxin. Further determination of binding affinity via sandwich ELISA revealed that Cry4Ba-DIII exhibited a rather weak binding to Aa-mALP with a dissociation constant (Kd) of ≈1.1 × 10-7 M as compared with the full-length toxin. Intermolecular docking between the Cry4Ba-R203Q active toxin and Aa-mALP suggested that four Cry4Ba-DIII residues, i.e., Glu522, Asn552, Asn576, and Leu615, are potentially involved in such toxin-receptor interactions. Ala substitutions of each residue (E522A, N552A, N576A and L615A) revealed that only the L615A mutant displayed a drastic decrease in biotoxicity against A. aegypti larvae. Additional binding analysis revealed that the L615A-impaired toxin also exhibited a reduction in binding capability to the surface-immobilized Aa-mALP receptor, while two bio-inactive DII-mutant toxins, Y332A and F364A, which almost entirely lost their biotoxicity, apparently retained a higher degree of binding activity. Altogether, our data disclose a functional importance of the C-terminal domain of Cry4Ba for serving as a potential receptor-binding moiety in which DIII-Leu615 could conceivably be exploited for the binding to Aa-mALP, highlighting its contribution to toxin interactions with such a target receptor in mediating larval toxicity.

Evaluation of spent mushroom substrate after cultivation of Pleurotus ostreatus as a new raw material for xylooligosaccharides production using crude xylanases from Aspergillus flavus KUB2.

Xylooligosaccharides (XOS), a novel functional food and feed ingredient, can be produced from lignocellulosic biomass. In this study, spent mushroom substrate (SMS) gathered after Pleurotus ostreatus cultivation was investigated for its potential as a new raw material for XOS production using crude xylanases produced in-house from Aspergillus flavus KUB2. Xylan was extracted from SMS using the alkaline extraction method. The highest true recovery of xylan (20.76%) and the relative recovery of xylan (83.73%) were obtained from SMS extracted with 4 M NaOH. Enzymatic hydrolysis of SMS-extracted xylan using crude fungal xylanases from A. flavus KUB2 produced a maximum total XOS in the range 1.37-1.48 mg/ml, which was mainly composed of XOS with a low degree of polymerization (xylobiose and xylotriose). XOS derived from SMS-extracted xylan positively influenced the growth of probiotic bacteria, suggesting the prebiotic nature of XOS. The results indicated that XOS with prebiotic properties can be produced from SMS xylan using crude xylanases without any purification, which offers economic potential for food and feed applications.

Lignocellulose hydrolytic enzymes production by Aspergillus flavus KUB2 using submerged fermentation of sugarcane bagasse waste.

Lignocellulosic wastes, rice straw, sugarcane bagasse, rice bran and sawdust, and pure commercial carboxymethyl cellulose (CMC) and xylan were used as substrates to cultivate cellulolytic fungus, Aspergillus flavus KUB2, in submerged fermentation at 30°C. Of all the substrates, sugarcane bagasse was a good source for the production of cellulolytic and also hemicellulolytic enzymes. The maximum activities of endoglucanase (CMCase), total cellulase (FPase) and xylanase using sugarcane bagasse as substrate were 8%, 75% and 165%, respectively, higher than those of the commercial substrates. The time course determination of enzyme production revealed that the highest CMCase (1.27 U/ml), FPase (0.72 U/ml) and xylanase (376.81 U/ml) activities were observed at 14 days of fermentation. Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) analyses confirmed the efficient structural alteration of sugarcane bagasse caused by enzymatic actions during A. flavus KUB2 cultivation. Based on the results of the hydrolytic enzyme activities, FTIR and SEM, A. flavus KUB2 is suggested as an efficient hydrolytic enzymes producer and an effective lignocellulose degrader, while sugarcane bagasse can be applied as a low-cost carbon source for the economical production of lignocellulose hydrolytic enzymes by A. flavus KUB2.

Molecular characterization of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Thailand.

A total of 511 local isolates of Bacillus thuringiensis from different geographical regions of Thailand were analyzed for the presence of the cry1A, cry1B, cry2A, cry9, and vip3A genes encoding for lepidopteran-specific toxins. PCR results revealed that 94.32% (482/511) of B. thuringiensis isolates harbored at least one of the detected genes, of which the cry1A, cry1B, cry2A, cry9, and vip3A genes were detected at frequencies of 90.61%, 89.63%, 76.32%, 40.70%, and 48.18%, respectively. Nineteen gene-combination profiles were discovered among 482 B. thuringiensis isolates, of which the most frequently detected profile contained the cry1A, cry1B, cry2A, and vip3A genes. Sixty-one isolates (12.66%), which harbored all of the detected insecticidal toxin genes, were further detected for the exochitinase (chi36) gene and chitinase activity. The results revealed that all 61 isolates contained the chi36 gene and exhibited chitinase activity. Insect bioassays showed that five isolates were highly toxic (more than 80% mortality) against second instar larvae of Spodoptera litura, of which the highest insect mortality (93%) was obtained from the B. thuringiensis isolates 225-15 and 417-1. Scanning electron microscopy revealed that the crystal morphologies of the five effective isolates were bipyramidal and cuboidal shapes. SDS-PAGE analysis of the spore-crystal mixture showed major bands of approximately 65 and 130 kDa. These five effective strains are alternative candidates for use as a microbial insecticide for the control of the S. litura pest.

The C-Terminal Domain of the Bacillus thuringiensis Cry4Ba Mosquito-Specific Toxin Serves as a Potential Membrane Anchor.

Although the C-terminal domain (DIII) of three-domain Cry insecticidal toxins from Bacillus thuringiensis has been implicated in various biological functions, its exact role still remains to be elucidated. Here, the 21-kDa isolated DIII fragment of the 65-kDa Cry4Ba mosquito-specific toxin was analyzed for its binding characteristics toward lipid-bilayer membranes. When the highly-purified Cry4Ba-DIII protein was structurally verified by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, it revealed the presence of a distinct ß-sheet structure, corresponding to its structure embodied in the Cry4Ba crystal structure. Binding analysis via surface plasmon resonance (SPR) spectroscopy revealed that the 21-kDa Cry4Ba-DIII truncate displayed tight binding to immobilized liposome membranes in a two-step manner, exhibiting a dissociation rate constant (kd) comparable to the 65-kDa full-length toxin. Also similar to the Cry4Ba full-length toxin, its isolated DIII truncate was able to anchor a part of its molecule into the immobilized membrane as the SPR signal was still detected after prolonged treatment with proteinase K. However, unlike the full-length active toxin, the DIII truncate was unable to induce membrane permeability of calcein-loaded liposomes or ion-channel formation in planar lipid bilayers. Together, our present data have disclosed a pivotal role of C-terminal DIII in serving as a membrane anchor rather than a pore-forming moiety of the Cry4Ba mosquito-active toxin, highlighting its potential mechanistic contribution to the interaction of the full-length toxin with lipid membranes in mediating toxicity.

Antagonistic Activity against Dirty Panicle Rice Fungal Pathogens and Plant Growth-Promoting Activity of Bacillus amyloliquefaciens BAS23.

Bacterial strain BAS23 was isolated from rice field soil and identified as Bacillus amyloliquefaciens. Based on dual culture method results, the bacterium BAS23 exhibited potent in vitro inhibitory activity on mycelial growth against a broad range of dirty panicle fungal pathogens of rice (Curvularia lunata, Fusarium semitectum and Helminthosporium oryzae). Cell-free culture of BAS23 displayed a significant effect on germ tube elongation and mycelial growth. The highest dry weight reduction (%) values of C. lunata, H. oryzae and F. semitectum were 92.7%, 75.7% and 68.9%, respectively. Analysis of electrospray ionization-mass spectrometry (ESI-MS) and 1H nuclear magnetic resonance (NMR) spectroscopy revealed that the lipopeptides were iturin A with a C14 side chain (C14 iturinic acid) and with a C15 side chain (C15 iturinic acid), which were produced by BAS23 when it was cultured in nutrient broth (NB) for 72 h at 30°C.BAS23, the efficient antagonistic bacterium, also possessed in vitromultipletraits for plant growth promotionand improved rice seedling growth. The results indicated that B.amyloliquefaciens BAS23 represents a useful option either for biocontrol or as a plant growth-promoting agent.

Mosquitocidal potential of silver nanoparticles synthesized using local isolates of Bacillus thuringiensis subsp. israelensis and their synergistic effect with a commercial strain of B. thuringiensis subsp. israelensis.

Control of larval stages of Aedes aegypti is considered an effective approach for preventing outbreaks of dengue fever. In this work, silver nanoparticles (Ag NPs) were synthesized using the supernatant and insecticidal proteins from local isolates of Bacillus thuringiensis subsp. israelensis (Bti). Mosquitocidal activity assays against A. aegypti larvae revealed that the highest toxicity was obtained from the Ag NPs synthesized using supernatant of Bti K55 and the inclusion proteins of Bti K46 with a lethal concentration 50 (LC50) of 0.001 and 0.008µg/mL, respectively. The synthesized nanoparticles were characterized using UV-vis absorption spectrophotometry, scanning electron microscopy (SEM), SEM coupled with energy dispersive X-ray spectroscopy, X-ray diffraction and Fourier-transform infrared spectroscopy. The synergistic studies revealed that the Ag NPs synthesized using supernatant of Bti K55 were synergized with commercial Bti cells with a synergistic factor (SF) of 3.3 and 10.0 for LC50 and LC90, respectively. In addition, the Ag NPs synthesized using inclusion proteins of Bti K46 were synergized with commercial Bti cells with a SF of 1.6 and 4.2 for LC50 and LC90, respectively. This study provided the first report of the synergistic effect between Bti and Ag NPs. Such a combination could represent an effective approach for the control of the dengue vector and possibly reducing the likelihood of increased insect resistance to chemical control.

Efficient polyhydroxybutyrate production from Bacillus juice substrate thuringiensis using sugarcane.

The present study focused on the screening and optimization of biopolymer polyhydroxybutyrate (PHB) production by Bacillus spp. using cost-effective substrates. Among 602 local Bacillus isolates, Bacillus thuringiensis B417-5 produced the highest amount of PHB (2.278 g/L, 60.07% of dry cell weight, DCW). 1H NMR and FTIR analyses of the extracted polymer revealed the characteristic peaks of PHB. The optimization results showed that the highest PHB accumulation (2.768 g/L, 72.08% of DCW) was achieved when culturing B. thuringiensis B417-5 in a nitrogen-deficient medium containing 1% total sugar from sugarcane juice and 0.5% yeast extract, with a pH of 7.0 and an incubation temperature of 37 °C for 48 h. B. thuringiensis B417-5 can thus be considered a good candidate for large-scale production of PHB. We are reporting for the first time that sugarcane juice is a promising carbon source for economical PHB production by B. thuringiensis.

Enhanced xylose fermentation and hydrolysate inhibitor tolerance of Scheffersomyces shehatae for efficient ethanol production from non-detoxified lignocellulosic hydrolysate.

Effective conversion of xylose into ethanol is important for lignocellulosic ethanol production. In the present study, UV-C mutagenesis was used to improve the efficiency of xylose fermentation. The mutated Scheffersomyces shehatae strain TTC79 fermented glucose as efficiently and xylose more efficiently, producing a higher ethanol concentration than the wild-type. A maximum ethanol concentration of 29.04 g/L was produced from 71.31 g/L xylose, which was 58.95 % higher than that of the wild-type. This mutant also displayed significantly improved hydrolysate inhibitors tolerance and increased ethanol production from non-detoxified lignocellulosic hydrolysates. The ethanol yield, productivity and theoretical yield by TTC79 from sugarcane bagasse hydrolysate were 0.46 g/g, 0.20 g/L/h and 90.61 %, respectively, while the corresponding values for the wild-type were 0.20 g/g, 0.04 g/L/h and 39.20 %, respectively. These results demonstrate that S. shehatae TTC79 is a useful non-recombinant strain, combining efficient xylose consumption and high inhibitor tolerance, with potential for application in ethanol production from lignocellulose hydrolysates.

Improvement of ethanol production by ethanol-tolerant Saccharomyces cerevisiae UVNR56.

Ethanol tolerance is one of the important characteristics of ethanol-producing yeast. This study focused on the improvement of ethanol tolerance of Saccharomyces cerevisiae NR1 for enhancing ethanol production by random UV-C mutagenesis. One ethanol-tolerant mutant, UVNR56, displayed a significantly improved ethanol tolerance in the presence of 15% (v/v) ethanol and showed a considerably higher viability during ethanol fermentation from sugarcane molasses and sugarcane molasses with initial ethanol supplementation. A maximum ethanol concentration produced from molasses medium at 37°C by UVNR56 was 10.3% (v/v), productivity of 1.7 g/l/h and a theoretical yield of 98.7%, while the corresponding values for the wild-type were 8.6% (v/v), 1.4 g/l/h and 83.3%, respectively. In addition, during molasses fermentation under initial supplementation of 5% (v/v) ethanol, the maximum ethanol concentration and productivity of UVNR56 was 25.7% and 42.9% higher than the wild-type, respectively.

Aedes aegypti membrane-bound alkaline phosphatase expressed in Escherichia coli retains high-affinity binding for Bacillus thuringiensis Cry4Ba toxin.

Glycosylphosphatidylinositol-linked alkaline phosphatase (GPI-ALP) from the epithelial membrane of the larval midgut of Aedes aegypti was previously identified as a functional receptor of the Bacillus thuringiensis Cry4Ba toxin. Here, heterologous expression in Escherichia coli of the cloned ALP, lacking the secretion signal and GPI attachment sequences, and assessment of its binding characteristics were further investigated. The 54-kDa His tag-fused ALP overexpressed as an inclusion body was soluble when phosphate buffer (pH 7.5) was supplemented with 8 M urea. After renaturation in a nickel-nitrilotriacetic acid (Ni-NTA) affinity column, the refolded ALP protein was able to retain its phosphatase activity. This refolded ALP also showed binding to the 65-kDa activated Cry4Ba toxin under nondenaturing (dot blot) conditions. Quantitative binding analysis using a quartz crystal microbalance revealed that the purified ALP immobilized on a gold electrode was bound by the Cry4Ba toxin in a stoichiometry of approximately 1:2 and with high affinity (dissociation constant [K(d)] of ∼14 nM) which is comparable to that calculated from kinetic parameters (dissociation rate constant [k(off)]/binding constant [k(on)]). Altogether, the data presented here of the E. coli-expressed ALP from A. aegypti retaining high-affinity toxin binding support our notion that glycosylation of this receptor is not required for binding to its counterpart toxin, Cry4Ba.

PCR-based method for the detection of cry genes in local isolates of Bacillus thuringiensis from Thailand.

A total of 134 isolates of Bacillus thuringiensis obtained from different geographical and ecological origins in Thailand were analyzed to determine the distribution and diversity of cry1, cry2 and cry9 genes encoding for Cry proteins toxic to lepidopteran insects. Strains containing cry1-type genes (109/134 or 81.3%) were found at the same frequency as strains harboring cry2 gene (108/134 or 80.6%) whereas only 50 strains contained cry9 gene (50/134 or 37.3%). Seventeen percent (23/134) of the B. thuringiensis isolates did not harbor any cry1, cry2 or cry9 genes. Among cry1 containing isolates, cry1A (49.3%), cry1B (50.0%), cry1G (48.5%), cry1I (49.3%), cry1J (35.1%) and cry1L (47.0%) were considered abundant. The cry2 gene was distributed with high frequency (>70%) in every region of the country. The study of cry gene combinations revealed 14 cry gene profiles.