Parasporal Inclusions Research Articles

The use of antigens derived from Bacillus thuringiensis bacteria for further differentiation

This study is devoted to studying Bacillus thuringiensis antigens and their insecticide activity as critical features in bacterial differentiation. Indeed, 190 samples were examined for flagellar antigenicity as well as the insecticidal activity exhibited. From a serological perspective, 122 isolates (64.2 %) were attributed to 8 H-serogroups, including 3 non-typeable and 65 unverified. The dominant serotype was H3abc (82 % frequency); H6 was less frequent (8.5 %). The other 6 serotypes accounted for a low frequency of occurrence (up to 1.5 %). Of the 190 isolates tested, 125 (65.8 %) formed bipyramidal, and 63 (33.2 %) represented spherical inclusions. All H3abc isolates contained bipyramidal inclusions. The same applied to H8ab and H7 isolates. Insecticide activity was noted in 70.1 % of the population. In general, 128 samples were toxic to both species (Bombyx mori, Aedes sp.). Another 3 samples were toxic only to B. mori, and 2 for Aedes sp. Among the samples exhibiting toxicity to both species, 97.6 % belonged to bipyramidal paraspore inclusions (H3abc). All H7 samples were toxic to two insect species. Monotoxic B. thuringiensis against Aedes sp. were found only among organisms producing spherical parasporal inclusions in the cell. Examples of such microorganisms include an isolate of the H4ab/43 serotype.

Prospects of Indigenous Bacillus subtilis Strain LS9.1 as a Potential Biocontrol Agent against Aedes aegypti Larvae

Biocontrol is one way that can be developed to overcome public health and agricultural problems. Biocontrol agents could be a microorganism, for example, from the Bacillus group. This study aims to determine the larvicidal toxicity of the Bacillus subtilis strain LS9.1 against Aedes aegypti larvae. The mode of action of the isolate caused larval death, which was detected with parasporal inclusions and secondary metabolites. Larvicidal toxicity with the bioassay method. Parasporal inclusion proteins were detected with the SDS-PAGE method. The Cry toxin coding gene with the PCR method and cell ultrastructure with transmission and scanning electron microscopy (TEM and SEM). The action of secondary metabolites was detected with hemolytic activity test. The isolate high toxicity to A. aegypti larvae. Parasporal inclusion proteins with molecular weights ranging from 72-7 kDa and the gene encoding a protein toxin in size 300 bp. The TEM and SEM results did not Cry-IV toxin. The hemolytic activity test showed a positive reaction correlated with biosurfactant production. Based on this study, the death of larvae is probably caused by secondary metabolites produced by Bacillus subtilis strain LS9.1. Thus, this isolate has the prospect of being an environmentally friendly biocontrol agent to disease vectors and insect pests.

Optimized high-yield preparation of alkaline-solubilizable crystalline inclusion of the Bacillus thuringiensis Cry4Aa δ-endotoxin expressed in Escherichia coli

The native Cry4Aa δ-endotoxin produced exclusively in Bacillus thuringiensis during sporulation as a ∼130-kDa inactive protoxin is confined within the parasporal crystalline inclusion that dissolves at alkaline pH in the midgut lumen of mosquito larvae. Here, the recombinant Cry4Aa toxin over-expressed in Escherichia coli at 30 °C as an alkaline-solubilizable inclusion was found inevitably lost during isolation from the cell lysate (pH ∼6.5) of which host cells were pre-suspended in distilled water (pH ∼5.5). When 100 mM KH2PO4 (pH 5.0) was used as host cell-suspending buffer, the cell lysate's pH became more acidic (pH 5.5), allowing the expressed protoxin to be entirely retained in the form of crystalline inclusion rather than a soluble form, and thus high-yield recovery of the partially purified inclusion was obtained. Upon dialysis of the alkaline-solubilized protoxin against the KH2PO4 buffer, the protoxin precipitate was efficiently recovered and still exhibited high toxicity to Aedes aegypti mosquito larvae. Additionally, the precipitated protoxin was completely resolubilized in 50 mM Na2CO3 buffer (pH 9.0) and proteolytically processed by trypsin to produce the 65-kDa activated toxin comprising ∼47- and ∼20-kDa fragments. In silico structural analysis suggested that His154, His388, His536 and His572 were involved in a dissolution of the Cry4Aa inclusion at pH 6.5, conceivably through interchain salt bridge breakage. Altogether, such an optimized protocol described herein was effective for the preparation of alkaline-solubilizable inclusions of the recombinant Cry4Aa toxin in large amounts (>25 mg per liter culture) that would pave the way for further structure-function relationship studies of different Cry toxins.

Genetic improvement of some microorganisms to increase the effect of bio-control on the potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)

BackgroundDuring the last five decades, chemical synthetic pesticides have been used extensively to control pests and protect crops. Use of synthetic pesticide has caused some unfortunate consequences like environmental pollution, pest resistance and toxicity to other non-target organisms. Due to the hazardous effects of their chemical residues to human and animal health, several studies have been carried out to determine effective alternative control methods. One of methods is the usage of entomopathogens such as bacteria, virus and fungi. Entomopathogenic bacteria have unique insecticidal properties mainly due to the production of larvicidal proteins that accumulate as parasporal crystalline inclusions within the cell. The bio-insecticidal bacterium Bacillus thuringiensis (Bt) has been widely used in agriculture for the control of pest insects which attack crops.ResultsThirteen genetically stable fusants strains were obtained as a result of protoplast fusion technique between a local Bacillus thuringiensis (Bt) and each of B. subtilis subsp. subtilis strain (Bs1), Bacillus licheniformis strain (Bl) or B. subtilis subsp. spizizeniie (Bs2). Thirteen fusants were obtained, including three fusants (group B) from Bt::Bs1 fusion, six fusants (group C) from Bt::Bl fusion and four fusants (group D) from Bt::Bs2 fusion. All fusants were chosen for bioassay treatments against the potato tuber moth (PTM) Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) which recorded a high mortality percentage of PTM ranged from 75 to 80% in F1 and F2 of first attempt B (Bt::Bs1). The accumulative larval mortality was notation the highest percentage reached to 90% in case of treatment by fusants F4 followed by F7, F8 and F9 which gave 75, 70 and 80% larval mortality, respectively, for second attempt C (Bt::Bl). The third attempt D (Bt::Bs2) fusants F10, F11, F13 achieved the highest mortality percentage up to 60, 60 and 70%, respectively. Expression of apoptosis-related encoding genes in PTM was determined in three fusants B (Bt::Bs1), C (Bt::Bl) and D (Bt::Bs2) and compared with A (Bt) and Control. The results showed a high expression of gene apoptosis to fusants D (Bt::Bs2) to Caspase-16 gene, Dronc and Dredd genes in tissues of the (PTM) treated with different biological pesticides.ConclusionsThe study used protoplast fusion technique between a local Bacillus thuringiensis (Bt) and B. subtilis subsp. subtilis strain (Bs1), B. licheniformis strain (Bl) and B. subtilis subsp. spizizeniie (Bs2). Thirteen fusants were chosen for bioassay treatments against PTM. Bacterial fusant, F1, F2, F4, F7, F9 and F13, achieved the highest mortality rates against PTM ranged 75–90% under laboratory conditions. The highest expression of gene apoptosis to fusants D (Bt::Bs2) to Caspase-16 gene, Dronc and Dredd genes was recorded in insect tissues treated with different bio-insecticides. As a result of the effected on the genes responsible for expression the vital processes (genes of apoptosis) in the insect as a result of treatment with bacteria, this led to deformities and death of PTM.

Potential biocontrol agent of indigenous Bacillus sp. EG6.4: Molecular identification, larvicidal toxicity, and mechanism of actions

Abstract. Salamun, Susetyo RD, Nafidiastri FA, Zain RA, Sari RP, Geraldi A, Fatimah, Ni’matuzahroh. 2022. Potential biocontrol agent of indigenous Bacillus sp. EG6.4: Molecular identification, larvicidal toxicity, and mechanism of actions Biodiversitas 23: 5431-5438. This research was carried out for molecular identification, as well as the determination and mechanism of action of larvicidal toxicity of Bacillus sp. EG6.4 isolated from breeding sites of Aedes aegypti from Gresik, East Java, Indonesia. Bacillus sp. EG6.4 was a Gram-positive endospore-forming bacteria. Molecular species identification using 16S rRNA gene sequencing showed that isolate had 97.89% similarity with Bacillus mojavensis. The isolate showed larvicidal toxicity against A. aegypti larvae. The lethal concentration 50% (LC50) values ??at 24 and 48hours exposure were 8.99±1.01 ×107 cells/mL and 8.43±1.01 ×107 cells/mL, respectively, while lethal time 50% (LT50) value was 11.9±1.1 hours. Production of chitinolytic enzymes or biosurfactants, chitinolytic and hemolytic assays were conducted to determine the larvicidal mechanism. As a result, Bacillus sp. EG6.4 showed hemolytic, but not chitinolytic activity, indicating its potency to produce biosurfactants. Transmission Electron Microscopy (TEM) result showed that isolate had oval-shaped endospores located subterminal with massive-shape parasporal inclusions. The detection of srfA-D gene showed that isolate produced surfactin biosynthesis thioesterase. Thus, Bacillus sp. EG6.4 produced biosurfactant that potentially to be developed as a biocontrol agent for disease vectors and plant pathogens.

Characterization and Evaluation of Indigenous Bacillus thuringiensis Isolate T352 against Fall Armyworm, Spodoptera frugiperda (J.E. Smith)

Bacillus thuringiensis (Bt) Berliner is a ubiquitous soil bacterium with commercial bio pesticidal value and widely used for effective control of various important agricultural insect pests. Invasion of Fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) into India caused potential yield loss in maize production, and also it threats the cultivation of other related crops. This study was aimed to characterize and evaluate the pathogenic activity of indigenous Bt isolate T352 against fall armyworm (FAW), S. frugiperda. The Bt isolate T352 was creamy white in colour and had irregular shaped flat colonies with undulated margin. Bipyramidal shape of parasporal inclusions was found in isolate T352. The isolate produced protein bands of ̴ 130 kDa and ̴ 65 kDa size in SDS PAGE analysis. PCR screening also confirmed the presence of cry1Ab, cry1Ac, cry2Aa, cry2Ab and vip3A genes. During probit analysis, isolate T352 exhibited the LC50 of 1.927 µg/ml as against 0.421µg/ml in positive standard strain HD- 1 based on mortality observed at 72 h after treatment in leaf disc bioassay with spore crystal mixtures.

Parasporin A13-2 of Bacillus thuringiensis Isolates from the Papaloapan Region (Mexico) Induce a Cytotoxic Effect by Late Apoptosis against Breast Cancer Cells

The protein A13-2 was obtained from Bacillus thuringiensis strains isolated from the Papaloapan watershed region (Oaxaca, Mexico). The cytotoxic activity of parasporal inclusions was studied against breast cancer cell line (MCF-7) and normal cell (human peripheral blood mononuclear cells). The MTT, the formation of reactive species, nitric oxide, free cell DNA, and the type of death cellular were assessed. The protein A13-2 shows the highest cytotoxic activity against MCF-7 (13% cell viability at 6 µg/mL), the extracellular DNA increases, and it shows no stress for reactive species or nitric oxide. Besides, the A13-2 parasporin shows no toxicity to peripheral blood mononuclear cells, and it does not generate changes in nitric oxide levels or free cell DNA. Due to that, the cytotoxic effect of A13-2 was specific for MCF-7, and it does not affect normal cells. According to microscopy and flow cytometry, A13-2 parasporin leads to the death of MCF-7 cells by late apoptosis together with necrosis and without allowing the triggering of the survival mechanisms. When analyzed together, our results show for the first time that the A13-2 protein isolated from Mexican strains of B. thuringiensis preferentially kills MCF- 7 (cancer cells) over HEK 293 and PBMC cell lines (normal cells), thus representing a promising alternative for the treatment of cancer breast.

Larvicidal toxicity and parasporal inclusion of native Bacillus thuringiensis BK5.2 against Aedes aegypti.

Native Bacillus thuringiensis BK5.2, isolated from soil of Baluran National Park, East Java, Indonesia, has been shown to be toxic against Aedes aegypti larvae. This study aims to determine the strength and the speed of the toxicity of B.thuringiensis BK5.2 against A.aegypti larvae in lethal concentration (LC) and lethal time (LT), as well as detection of toxin structure and parasporal inclusion. LC values were determined by the mortality of A.aegypti third instar larvae after 24 and 48h exposure to five various concentrations of B.thuringiensis BK5.2, while LT values were determined based on the mortality of A.aegypti third instar larvae due to exposure to LC90 concentration at 0; 0.5; 1; 2; 4; 8; 10; 20; 24; and 48h. Larvicidal toxicity was determined based on value of LC50 and LC90 (CFU/mL), as well as LT50 and LT90 (hours) analysed ​​with Probit analysis. Parasporal inclusion was detected using transmission electron microscope (TEM) and scanning electron microscope (SEM). Based on bioassay, LC50 and LC90 values ​​were 11.6×106 and 22.7×106CFU/mL, respectively, at 24h exposure, as well as 8.3×106 and 15.4×106CFU/mL, respectively, at 48h exposure, while the value of LT50 and LT90 were 19.0 and 26.6h, respectively. Morphological observation of the dead larvae showed there was damage on abdomen and thorax region. Detection by TEM and SEM showed there was cuboidal parasporal inclusion. Native B.thuringiensis BK5.2 has high toxicity against A.aegypti larvae and detected flatcuboidal toxin in parasporal inclusion.

Distinctive Molecular typing of 16S rRNA of Bacillus species isolated from farm settlement.

Distinctive Molecular typing of 16S rRNA of Bacillus species isolated from farm settlement.

Enhanced insecticidal activity of Bacillus thuringiensis using a late embryogenesis abundant peptide co-expression system

Bacillus thuringiensis (Bt) is a ubiquitous, gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing crystal protein, some of which are toxic against a wide range of insect orders like caterpillars, beetles, and flies, including mosquitoes. Regarding the biological control of insects, Bt is the mostly used microorganism worldwide and also alternatives to chemical insecticides for environmental conservation. Some strains of Bt are showing a promising activity against a wide variety of mosquito like Aedes, Culex, and Anopheles and so on with extremely damages in the larval midgut and ultimate death. Here, we introduced a late embryogenesis abundant (LEA) peptide co-expression system based on the expression vector pHT01 with a strong σA-dependent promoter to enhance the expression of insecticidal crystal proteins in native Bt. Two types of LEA peptide (LEA-II and LEA-K) were designed based on the sequence of group-3 LEA protein, which consists of a repetitive sequence of 11 amino acids. The LEA-II mediated co-expression system enhanced the production of crystal protein 3-fold after 12 h of induction of the peptide with 0.5 mM IPTG. Enhanced expression of crystal protein was confirmed by bioassay using 4th instar Aedes albopictus larvae. This unique approach has great potential to produce bio-pesticides by enhanced crystal protein expression not only for mosquitoes but also for other insects.

Exploration of insecticidal potential of Cry protein purified from Bacillus thuringiensis VIID1

Insect pests are a threat to agriculture as they cause a loss of 15–22% to economically important crops every year. Bacillus thuringiensis produces parasporal crystal inclusions that have insecticidal ‘Cry’ proteins which are toxic to insect larvae of the order Lepidoptera, Coleoptera and Diptera, etc. In the present study, 40 different soil samples from Amritsar and its surrounding areas were selected for isolation of B. thuringiensis. The rod shaped, gram-positive bacterial isolates were further analyzed for characteristic crystal formation using phase contrast and scanning electron microscopy. 6 Bacillus samples containing cry genes were identified using the universal primers for cry genes, of which one isolate exhibited a protein band of ~95 kDa. This protein was purified using a Sephadex G-75 column. The insecticidal assays conducted with purified Cry protein on insect larvae of lepidopteran and dipteran orders viz. Spodoptera litura, Galleria malonella, Bactrocera cucurbitae and Culex pipens revealed considerable detrimental effects. A significant increase in larval mortality was observed for the larvae of all insects in a concentration dependent manner when treated with Cry protein purified from B. thuringenisis VIID1. The purified Cry protein did not have any significant effect on honey bee larvae.

Aphicidal activity of Bacillus thuringiensis strain AH‐2 against cotton aphid (Aphis gossypii)

AbstractBacillus thuringiensis (Bt) is the most commercialized entomopathogenic species. However, the low efficacy against notorious sap‐sucking Hemipteran pests and the high costs of production, reduce the applicability of Bt biopesticides. In this context, B. thuringiensis AH‐2 (Bt‐2) isolated from soil was found to release parasporal inclusions and aphicidal activity. Bt‐2 showed optimal growth at pH 8 and 30°C in tryptic soy broth (TSB) and maintained normal growth when cultured in cost‐effective test media, pink brown (PB), supplemented with either glucose (PBG) or sucrose (PBS). To study aphicidal activity against cotton aphid (Aphis gossypii), Bt‐2 and a commercial B. thuringiensis subspecies kurstaki (Btk) were used. Using third nymph (3 N) instars, treatment with Bt‐2 caused 70.2, 78.1 and 88.4% mortality at 10, 25 and 50% culture concentrations, respectively. Similarly, Btk resulted in 79.9, 80.4 and 92.9% mortality at 10, 25 and 50%, respectively. When cultured in TSB, PBG and PBS media, Bt‐2 culture (50%) resulted in 85.9, 82.9 and 82.2% 3 N mortality, respectively. Equally, Btk caused a mortality of 79.9, 78.9 and 78.9% in TSB, PBG and PBS, respectively. Bt‐2 exerted a notably higher aphicidal efficacy at 24 h after treatment compared to Btk, especially at low concentration. Moreover, both Bt strains significantly suppressed adult fecundity compared to control. At 50% culture concentration, Bt‐2 and Btk suppressed adult fecundity by 98.3 and 91.1%, respectively. Our result demonstrates a strong aphicidal efficacy of Bt‐2, and its potential production using cost‐effective PB medium.

Promising active bioinsecticides produced by Bacillus thuringiensis strain BLB427

Bacillus thuringiensis is gaining worldwide importance in the control of pests in agriculture and public health. Characterization of new isolated strains with original, higher and broader spectrum of activity is an ever developing field. Parasporal inclusions from BLB427, a new B. thuringiensis strain isolated from Tunisian soil, contained 2 major Cry proteins having molecular weights of about 130-135 kDa and 65-70 kDa corresponding to cry1-type and cry2-type genes, respectively. These toxins exhibited high toxicities to Ephestia kuehniella and Spodoptera littoralis larvae, with LC50 values of about 81.91 µg/cm2 (+/- 15.30) and 79.70 µg/cm2 (+/- 36.73), respectively. BLB427 supernatant showed a promising activity against Lepidopteran pests due to the presence of vip3-type gene in this strain. The high toxicity of BLB427 supernatant compared to that of BUPM95, used as control, was due in part to the high copy number of vip3 gene of BLB427 compared to that of vip3Aa16 of BUPM95, as demonstrated by Real-time PCR. This gene was cloned, sequenced and the comparison with the most known vip3 genes in databases, demonstrated that vip3(427) gene and the corresponding protein showed differences that can influence the efficiency of the Vip3(427) toxin against Lepidoptera. After overexpression of Vip3(427) protein in Esherichia coli and its purification using His-Trap column, the toxin showed a promising toxicity against the lepidopteran pest S. littoralis with extremely damages in the larvae midgut traduced by the vacuolization of the apical cells, the damage of microvilli and the disruption of epithelial cells. The results described in the present study proved that B. thuringiensis BLB427 strain could be of a great interest for lepidopteran biocontrol by using its d-endotoxins and/or Vip3 toxins in bioinsecticides formulations.

Cytopathic effects of activated parasporal inclusion proteins produced from Iraqi isolates of Bacillus thuringiensis

Cytopathic effects of activated parasporal inclusion proteins produced from Iraqi isolates of Bacillus thuringiensis

Genome of Bacillus sp. strain QHF158 provides insights into its parasporal inclusions encoded by the S-layer gene.

Bacillus sp. strain QHF158, a Gram-positive, spore-forming and parasporal crystal-secreting bacterium, was isolated from soil of Limushan National Forest Park in China. Here we present the significant feature of parasporal inclusions of this organism, together with the draft genome sequence and annotation. Phylogenetic analysis suggested that strain QHF158 is possibly a novel species, most closely related to Bacillus mycoides. Genome annotation results revealed that strain QHF158 did not contain any typical Cry or Cyt toxin coding gene. Furthermore, the mass spectrometry analyses demonstrated that the parasporal crystalline inclusions were encoded by the orf_05273 gene, with 95% similarity to the S-layer protein (SLP) EA1 of B. mycoides, which indicated that the parasporal crystal from Bacillus sp. strain QHF158 was mainly formed by SLP, instead of the typical Cry or Cyt toxin proteins.

Enhancement of Purified Human Colon Cancer-Specific Parasporal Toxin from Bacillus thuringiensis-LDC-501.

Parasporal inclusion protein of Bacillus thuringiensis-LDC-501 (Bt-LDC-501) exhibits selective cytocidal action towards human colon cancer cells. The yield of this parasporal protein was minimum in the normal culture. In order to increase the yield of protein from Bt-LDC-501 various agro-based cost-efficient nutrient sources such as corn steep liquor (CSL), sesame oil cake extract (SOC), groundnut oil cake extract (GOC), neem oil cake extract (NOC), rice bran extract (RB), wheat bran extract (WB), red gram hull extract (RGH), green gram hull extract (GGH), black gram hull extract (BGH), Mysore gram hull extract (MGH), and maize flour waste extract (MFW) were screened. Statistical experimental designs such as Plackett-Burman design (PBD) and response surface methodology (RSM) were the tools employed for the optimization of medium. Groundnut cake extract (GOC) served as a potential carbon and nitrogen source, as it induced twofold higher production of parasporal protein. Among the optimized seven media components KH2PO4, K2HPO4, GOC, NaCl, MgSO4, MnSO4, and FeSO4, the concentrations of GOC, NaCl, and MgSO4 have significant effect on parasporin production as well as cytotoxicity against colon cancer cell line, HCT-116. Bt-LDC-501 was found to produce 0.88mg/ml of parasporal protein in optimized medium. In the un-optimized medium, the yield was 0.23mg/ml only. This indicated that there was 382% of increase in the production of Parasporal protein. Parasporin protein with the molecular weight of 27kDa has been purified with the purification fold of 27.1. It showed a LC50 value of 0.91 and 1.21µg/ml against colorectal cancer cell lines such as HCT-116 and HCT-15, respectively. Purified parasporin exhibited stable cytocidal activity between pH 4.0 and 9.0 at room temperature. The present study revealed that the quantity and quality of media composition were necessary for eliciting cytocidal activity against human colon cancer and the importance of alternate cost-effective production of clinically significant parasporin. Moreover, this is the first report regarding optimization of media components for parasporal protein production from Bt.

Crystal Protein of a Novel Bacillus thuringiensis Strain Inducing Cell Cycle Arrest and Apoptotic Cell Death in Human Leukemic Cells

Parasporal inclusions of a native non haemolytic Bacillus thuringiensis strain KAU 59 was screened for its cytotoxicity against human lymphocytic leukemic cell line jurkat and normal human lymphocytes. The cytotoxicity of proteinase activated and non activated solubilised parasporal inclusions against both cell lines was assessed by Cell Titer 96 Aqueous Non Radioactive Cell Proliferation Assay Kit using MTS. The 50 per cent effective concentration (EC50) values were deduced from log probit analysis at 48 h. Morphological changes associated with cytotoxicity were evaluated and molecular mechanisms of cell death were elucidated by TUNEL assay at 48 h post-inoculation. The fluorescence assisted cell sorting was done in the flow cytometer to assess the stage of cell cycle arrest. Relative quantification of caspase-3 expression in Jurkat cells treated with parasporal inclusion protein of KAU 59 was done by qRTPCR The results indicated that the protein was cytotoxic to jurkat cells at the same time non toxic to normal lymphocytes. Cytotoxicity was evident only after proteolytic activation. Apoptotic cell death was confirmed in the protein treated cells by TUNEL Assay and also up regulated caspase-3 gene expression (P < 0.001). S phase cell cycle arrest was confirmed by and fluorescence associated cell sorting.

Modification of Vip3Ab1 C-Terminus Confers Broadened Plant Protection from Lepidopteran Pests.

Vegetative insecticidal proteins (Vips) from Bacillus thuringiensis (Bt) are unique from crystal (Cry) proteins found in Bt parasporal inclusions as they are secreted during the bacterial vegetative growth phase and bind unique receptors to exert their insecticidal effects. We previously demonstrated that large modifications of the Vip3 C-terminus could redirect insecticidal spectrum but results in an unstable protein with no lethal activity. In the present work, we have generated a new Vip3 protein, Vip3Ab1-740, via modest modification of the Vip3Ab1 C-terminus. Vip3Ab1-740 is readily processed by midgut fluid enzymes and has lethal activity towards Spodoptera eridania, which is not observed with the Vip3Ab1 parent protein. Importantly, Vip3Ab1-740 does retain the lethal activity of Vip3Ab1 against other important lepidopteran pests. Furthermore, transgenic plants expressing Vip3Ab1-740 are protected against S. eridania, Spodoptera frugiperda, Helicoverpa zea, and Pseudoplusia includens. Thus, these studies demonstrate successful engineering of Vip3 proteins at the C-terminus to broaden insecticidal spectrum, which can be employed for functional expression in planta.

Solubility enhancement of Cry2Aa crystal through carboxy-terminal extension and synergism between the chimeric protein and Cry1Ac.

It was reported that the highly conserved C-terminal region of Bacillus thuringiensis Cry1A protoxins was very important for parasporal crystal formation and solubility feature in alkaline environment. In order to improve the solubilization efficiency of Cry2Aa crystal, the coding sequences of Cry2Aa protein and the C-terminal half of Cry1Ac were fused seamlessly through Red/ET homologous recombination and expressed in an acrystalliferous B. thuringiensis strain under the control of the cry1Ac promoter and terminator. Microscopic observation revealed that the recombinant strain containing the chimeric gene cry2Aa-1Ac produced distinct parasporal inclusion with semispherical to approximately cuboidal shape during sporulation. SDS-PAGE analysis showed that this strain expressed stable 130-kDa Cry2Aa-1Ac chimeric protein, which was confirmed to be the correctly expressed product by LC-MS/MS. The chimeric protein inclusion could be effectively dissolved at pH 10.5 and activated by trypsin like the parental Cry1Ac crystal. While, the parental Cry2Aa crystal exhibited very low solubility under this condition. Bioassays against third-instar larvae of Helicoverpa armigera proved that the chimeric protein was more toxic than Cry2Aa. Additionally, synergistic effect was clearly detected between the chimeric protein and Cry1Ac against H. armigera, while there was only additive effect for the combination of wild Cry2Aa and Cry1Ac. These results indicated that the developed chimeric protein might serve as a potent insecticidal toxin used in the field against lepidopteran pests.

LARVICIDAL ACTIVITY OF BACILLUS THURINGIENSIS ISOLATED FROM Bt COTTON RHIZOSPHERE SOIL AGAINST ANOPHELES MOSQUITO LARVAE (CULICIDAE)

Objective: The objective of the study was to isolate and identify Bacillus thuringiensis (Bt) from Bt cotton rhizosphere soil and analyze its larvicidal activity against Anopheles mosquito larvae.Methods: Soil samples were collected from Bt cotton field, and B. thuringiensis was isolated and characterized by biochemical and microscopical characterization. Crystal (Cry) proteins were extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and its larvicidal activity was checked by mortality analysis. Effective Bt isolates were identified by 16S rRNA sequencing.Results: A total of 24 isolates were characterized as Bacillus sp. by biochemical characterization. Further, parasporal inclusion and Cry proteins were extracted, and it was quantified by Bradford assay. The precipitated Cry proteins were analyzed by SDS-PAGE and the results have indicates Cry I protein at 100kDa, Cry3 protein at 44kDa, and cytolytic protein (Cyt) at 29kDa in most of the isolates. Larvicidal activity was checked by the 4th instar Anopheles mosquito larvae. Among the tested isolates, RBL10 and RBL20 have shown the highest percentage of larvicidal activity at 96 and 83%, respectively. Further, the two isolates RBL10 and RBL20 were identified as Bt by 16S rRNA sequencing.Conclusion: B. thuringiensis producing Cry and Cyt proteins posses potential larvicidal activity against Anopheles mosquito larvae which has biological and economic importance in mosquito control.