Parasporal Protein Research Articles

Anticancer efficacy of biosynthesized silver nanoparticles loaded with recombinant truncated parasporin-2 protein

Bacterial toxins have received a great deal of attention in the development of cancer treatments. Parasporin-2 (PS2Aa1 or Mpp46Aa1) is a Bacillus thuringiensis parasporal protein that preferentially destroys human cancer cells while not harming normal cells, making it a promising anticancer treatment. With the efficient development and sustainable silver nanoparticles (AgNPs) synthesis technology, the biomedical use of AgNPs has expanded. This study presents the development of a novel nanotoxin composed of biosynthesized silver nanoparticles loaded with the N-terminal truncated PS2Aa1 toxin. MOEAgNPs were synthesized using a biological method, with Moringa oleifera leaf extract and maltose serving as reducing and capping agents. The phytochemicals present in M. oleifera leaf extract were identified by GC–MS analysis. MOEAgNPs were loaded with N-terminal truncated PS2Aa1 fused with maltose-binding protein (MBP-tPS2) to formulate PS2-MOEAgNPs. The PS2-MOEAgNPs were evaluated for size, stability, toxin loading efficacy, and cytotoxicity. PS2-MOEAgNPs demonstrated dose-dependent cytotoxicity against the T-cell leukemia MOLT-4 and Jurkat cell lines but had little effect on the Hs68 fibroblast or normal cell line. Altogether, the current study provides robust evidence that PS2-MOEAgNPs can efficiently inhibit the proliferation of T-cell leukemia cells, thereby suggesting their potential as an alternative to traditional anticancer treatments.

Parasporins of Bacillus thuringiensis Strain Exhibit Apoptosis-Mediated Selective Cytotoxicity to MDA-MB-231 Cells through Oxidative Stress

Historically, the most important source of both antibiotics and anticancer medications has been microorganisms. Bacillus thuringiensis (Bt) is one of the most prominent bacterial species used as a therapeutic agent targeting cancerous cells in recent worldwide investigations. This study was designed to isolate, molecularly identify, and discover novel Saudi Arabian Bt strains that selectively exhibit cytotoxic properties against MDA-MB-231, a human triple-negative breast cancer (TNBC) cell model. The bacterial strain under investigation was biochemically typed using API 20E and API CH50 and molecularly typed using 16S rDNA sequencing. Flow cytometry and immunoblotting were performed to elucidate the mechanism-of-action (MOA). Molecular typing confirmed the identity of the isolated non-hemolytic strain to be Bt and was named Bt HAU-145. Microscopic examination showed that the strain possessed a parasporal (PS) crystal protein with a spherical morphology. Data of cytotoxicity assay based on MTT revealed that Bt HAU-145 strain exhibited selective and potent cytotoxicity against MDA-MB-231, with a 50 percent inhibition (IC50) of 28 µg/ml. FACS analysis revealed that PS proteins induced both late and early apoptosis in a ROS-dependent manner. Immunoblotting assays showed increased expression of caspase-3 in response to PS treatment, paralleled by a reduction in Bcl-2 expression. This is the first study to investigate the MOA of PS proteins from the Saudi Arabian Bt strain, showing an induction of apoptosis through a ROS-dependent mechanism in TNBC cells. It is hoped that PS-based therapeutic strategies will be investigated at the preclinical scale in non-human primates prior to the clinical scale in randomized clinical trials.

Bacillus thuringiensis: a natural endophytic bacterium found in wild plants.

Despite the fact that Bacillus thuringiensis is the most widely used bacterium in biological pest control, its ecology has been notoriously neglected. Its role in nature is uncertain, and a defined habitat and niche are under discussion. In this report, wild-type strains were isolated from the inner plant tissues as natural endophytic bacteria in wild plants. Once a reliable superficial sterilization technique was standardized, leaf samples from 110 wildlife plant species within 52 families were processed to obtain their endophytic microflora, which were able to grow in artificial media. From 93 morphologically different isolates, 22 showed the typical sporangium morphology of B. thuringiensis (endospore and parasporal bodies). These isolates were identified and characterized by their 16S ribosomal RNA, hag gene, MLST, and cry gene sequences. Also, isolates were characterized by Bc-RepPCR and parasporal body protein content. All the isolates showed at least some of the typical B. thuringiensis features tested, but 10 showed information in all those features, which, in a rigorous selection, were taken as B. thuringiensis sensu stricto strains. Only three subspecies were identified: five kurstaki, four nigeriensis, and one thuringiensis. None showed toxicity against mosquito larvae or Caenorhabditis elegans, and only one showed significant toxicity against Manduca sexta larvae. The role of B. thuringiensis as a natural endophytic bacterium is discussed.

Characterization of a novel ETX/MTX2 domain-presenting parasporal crystal protein from Bacillus toyonensis HSY140 strain

Characterization of a novel ETX/MTX2 domain-presenting parasporal crystal protein from Bacillus toyonensis HSY140 strain

Isolation, molecular characterization and pathogenicity of native Bacillus thuringiensis, from Ethiopia, against the tomato leafminer, Tuta absoluta: Detection of a new high lethal phylogenetic group

Tuta absoluta (tomato leafminer) is one of the devastating agricultural pest that attack mainly tomatoes. The continuous use of chemical pesticides is not affordable and poses a collateral damage to human and environmental health. This requires integrated pest management to reduce chemical pesticides. B. thuringiensis is a cosmopolitan, antagonistic soil bacterium used to control agricultural pests. In this study, effective Bt strains were screened from different sample sources based on their lepidopteran specific cry genes and larvicidal efficacy against tomato leafminer, T. absoluta under laboratory conditions. Of the 182 bacterial isolates, 55 (30 %) of isolates harbored parasporal protein crystals. Out of these, 34 (62 %) isolates possess one or more lepidopteran specific cry genes: 20 % of isolates positive for cry2, 18.2 % for cry9, 3.6 % for cry1, 16.4 % for cry2 + cry9, 1.8 % for cry1 + cry9, and 1.8 % for cry1 + cry2 + cry9. However, 21 (38.2 %) isolates did not show any lepidopteran specific cry genes. Isolates positive for cry genes showed 36.7–75 % and 46.7–98.3 % mortality against second and third instar larvae of the T. absoluta at the concentration of 108 colony forming units (CFUs) ml−1. Cry1 and cry1 plus other cry gene positive isolates were relatively more pathogenic against T. absoluta. However, third instar larvae of the T. absoluta was more susceptible than second instar larvae. Two of the isolates, AAUF6 and AAUMF9 were effective and scored LT50 values of 2.3 and 2.7 days and LC50 values of 3.4 × 103 and 4.15 × 103 CFUs ml−1 against the third instar larvae, respectively. The phylogenetic studies showed some congruence of groups with cry gene profiles and lethality level of isolates and very interestingly, we have detected a putative new phylogenetic group of Bt from Ethiopia.

Quantitative Analysis of Parasporal Crystal Protein from Bacillus thuringiensis by Capillary Electrophoresis

The amount of parasporal crystal protein (δ-endotoxin) from the fermentation broth of the Bacillus thuringiensis is the best indication to assess the efficiency of the fermentation process or strain selection. Traditional methods for the assessment of insecticidal effect, such as bioassay or HPLC, were either time-consuming, inaccurate or inefficient. In this study, capillary electrophoresis (CE) was used for analyzing the amount of parasporal crystal protein after it was dissolved by adding a reducing agent, such as β-mercaptoethanol, to break the disulfide bonds. This soluble protein, δ-endotoxin, was then subjected to quantitative analysis by CE. The running buffer contained 300 mM boric acid, and pH was adjusted to 10.0 with 1 N NaOH. The dimensions of the capillary were 47 cm × 50 μm I.D. without coating. Lysozyme was used as internal standard for the quantitative assay of the δ-endotoxin. The migration time of the lysozyme peak was approximately 2 minutes earlier than that of the δ-endotoxin peak. The correlation between the concentration of δ-endotoxin and the ratio of the peak area of δ-endotoxin and the peak of lysozyme was calculated. The linear regression analysis showed that the correlation coefficient is equal to 0.9994, the slope is 0.4095 and the intercept is +0.0025. From this standard regression equation, the concentration of δ-endotoxin in fermentation broth or solution can be estimated easily by CE analysis.

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.

Genome sequencing of Bacillus thuringiensis isolate T414 toxic to pink bollworm (Pectinophora gossypiella Saunders) and its insecticidal genes

Bacillus thuringiensis is a spore-forming bacterium that is pathogenic towards a range of insect and nematode species and had been widely used as a biopesticide. In this study, we present the morphological, molecular and genetic characteristics of an indigenous Bt isolate T414 which displayed an effective toxicity against Pectinophora gossypiella. Scanning electron microscopy revealed the presence of bipyramidal, spherical and cubic shaped protein crystals in its spore-crystal suspension. SDS-PAGE analysis of its spore-crystal mixture showed the presence of two major protein bands viz.130 and 65 kDa. Whole genome sequencing with MiSeq divulged that it contains a chromosome and many plasmids. The assembled genome finally contained 6493494bp. Automated annotation of this genome draft predicted 6877 coding sequences and 152 RNAs (rRNAs, tRNAs and ncRNAs). NCBI blast analysis showed that assembled genome was distributed in a chromosome and 15 different types of plasmids. Further analysis of draft sequence revealed it harbors parasporal crystal protein genes (cry1Aa, cry1Ab, cry1Ac, cry1IAa, cry2Aa, cry2Ab and cyt1), vegetative insecticidal protein gene (vip3Aa), all plasmid borne and various additional virulence factors such as chitinases, proteases, bacteriocins and hemolysins. From the analysis it is evident that all the Cry, Cyt or Vip toxins are plasmid borne and are present on two types of plasmids named as p414A and p414E in the present study. A cry2A type gene was cloned and sequenced. It was named as cry2Aa21 by Bt nomenclature committee.

Aislados nativos de Bacillus thuringiensis del sureste de México

&#x0D; &#x0D; &#x0D; Con el objetivo de encontrar aislados nativos de Bacillus thuringiensis, en el presente trabajo se realizaron aislamientos a partir de diferentes materiales obtenidos del sureste de México, en el periodo de marzo 2014 a julio 2015. Se seleccionaron colonias bacterianas con base a las características típicas del genero Bacillus y se identificaron como Bacillus thuringiensis, con base a la presencia de cristales parasporales, gen cry, gen hag, perfil de proteínas y actividad insecticida. Se obtuvo un aislado de muestras de suelo (ITCBT34) y dos de larvas de Chlosyne lacinia (ITCBT61, ITCBT62). Estos aislados presentaron amplificación de los genes cry y hag. El aislado ITCBT34 presentó cristales con morfología oval, mientras que los aislados ITCBT61 e ITCCBT62 presentaron cristales bipiramidales. Se observaron diferencias en el tamaño del fragmento producido por la amplificación del gen hag y en el perfil de proteínas de los diferentes aislados. La actividad insecticida se determinó mediante bioensayo burdo con una concentración de 10 μg cm-2. Los tres aislados causaron 100% de mortalidad de larvas de Manduca sexta a las 96 h de exposición. Los aislados ITCBT61 e ITCBT62 mostraron características morfológicas y tamaño del gen hag similares a la cepa de referencia B. thuringiensis svar. kurstaki HD1, el aislado ITCBT34 no presentó similitud, por lo que podría tratarse de una aislado no relacionado al serovar kurstaki HD1.&#x0D; &#x0D; &#x0D;

Poly-β-hydroxybutyrate Metabolism Is Unrelated to the Sporulation and Parasporal Crystal Protein Formation in Bacillus thuringiensis.

Poly-3-hydroxybutyrate (PHB) is a natural polymer synthesized by many bacteria as a carbon-energy storage material. It was accumulated maximally prior to the spore formation but was degraded during the process of sporulation in Bacillus thuringiensis. Intriguingly, B. thuringiensis also accumulates large amounts of insecticidal crystal proteins (ICPs) during sporulation, which requires considerable input of carbon and energy sources. How PHB accumulation affects sporulation and ICP formation remains unclear to date. Intuitively, one would imagine that accumulated PHB provides the energy required for ICP formation. Yet our current data indicate that this is not the case. First, growth curves of the deletion mutants of phaC (encoding the PHB synthase) and phaZ (encoding the PHB depolymerase) were found to be similar to the parent strain BMB171; no difference in growth rate could be observed. In addition we further constructed the cry1Ac10 ICP gene overexpression strains of BMB171 (BMB171-cry), as well as its phaC and phaZ deletion mutants ΔphaC-cry and ΔphaZ-cry to compare their spore and ICP production rates. Again, not much change of ICP production was observed among these strains either. In fact, PHB was still degraded in most ΔphaZ-cry cells as observed by transmission electron microscopy. Together these results indicated that there is no direct association between the PHB accumulation and the sporulation and ICP formation in B. thuringiensis. Some other enzymes for PHB degradation or other energy source may be responsible for the sporulation and/or ICP formation in B. thuringiensis.

Erratum to: Screening and characterization of a non-insecticidal Bacillus thuringiensis strain producing parasporal protein with selective toxicity against human colon cancer cell lines

Parasporins are a heterogenous group of Cry proteins produced by non-insecticidal Bacillus thuringiensis strains that specifically act on human cancer cells without affecting normal ones. This unique cytotoxic property has driven researchers to explore novel non-insecticidal B. thuringiensis strains possessing cancer cell-killing proteins. In this study, we isolated 65 non-clonal native isolates from 21 coastal soil samples and subsequently tested 28 strains which were non-haemolytic and non-insecticidal for their cytotoxicity against human colon cancer cell line HCT 116 and human cervical cancer cell line SiHa. Parasporal protein from a strain designated as B.t.LDC 501 showed significantly higher cytolytic activity on HCT 116 cells than on SiHa cells. The activated protein also exerted specific cell lethality against two other colon cancer cell lines, SW480 and SW620. However, it was notably non-toxic to normal cells, such as human peripheral blood leukocytes, human embryonic kidney cell line (HEK293) and human corneal epithelial cell line (HCEC) and showed only modest toxicity on a murine fibroblast cell line (NIH/3T3). The purified 20-kDa crystal protein obtained through gel filtration chromatography exhibited a markedly higher cytopathic effect than the unpurified protein. Liquid chromatography–tandem mass spectrometry analysis of the 20-kDa fragment revealed it to be an uncharacterized protein containing a tumor necrosis factor-like domain. The non-apoptotic mode of cell death, extensive membrane permeability and aminopeptidase N-dependent cytotoxicity suggests the pore-forming nature of the protein. Further characterization of the protein and the receptor will facilitate its use as a potential therapeutic drug against cancer.

Screening and characterization of a non-insecticidal Bacillus thuringiensis strain producing parasporal protein with selective toxicity against human colon cancer cell lines

Screening and characterization of a non-insecticidal Bacillus thuringiensis strain producing parasporal protein with selective toxicity against human colon cancer cell lines

Early transcription of Bacillus thuringiensis cry genes in strains active on Lepidopteran species and the role of gene content on their expression

Six strains of Bacillus thuringiensis previously selected as highly toxic against Manduca sexta and Plutella xylostella were analyzed by PCR screening in order to identify the cry genes active on Lepidoptera. According to their gene content and insecticidal potency, these strains were cultured and aliquots taken at different pre- and post-sporulation times. Total RNA was extracted and used as template in RT-PCR analyses directed to identify mRNAs of the previously identified cry genes. Results showed transcription of genes cry1A, cry1E, cry1I, and cry2 even before the onset of sporulation. However, this early transcription did not lead to an appreciable parasporal protein synthesis until t5-t9, as deduced from SDS-PAGE profiles. As for cry1I gene, the corresponding protein was not detected, as expected, but cry1I mRNAs were present at least until t5. Interestingly, strains expressing four cry genes from the end of the log phase onwards exhibited kinetics characterized by a very long transition phase, whereas the strain expressing only one cry gene showed a very short transition phase. Strains expressing three genes showed an intermediate profile. These results indicate that the transcription of B. thuringiensis cry1 and cry2 genes in natural strains can start several hours before massive crystal synthesis occurs and that this translation is probably competing with transcriptional regulators required for the sporulation onset.

Protocol of SDS-PAGE for Proteomic Analysis of Bt Parasporal Crystals

Proteomic profiling is recognized as an effective way to directly understand the characteristics of parasporal protein, and SDS-PAGE technique can be used effectively to observe the parasporal proteins (proteomic profile) in Bt strain. However, the manipulation of SDS-PAGE electrophoresis is complex, while the experimental results are susceptible to experimental conditions as well as some experimental factors. This paper presents an experimental protocol and techniques of SDS-PAGE that can be used to obtain good results for genomic analysis of insecticidal crystal proteins formed by B.t. during sporulation. Case study confirmed that the results generated by this protocol are reliable, stable, and reproducible.

Bt S2160-1, a High Mosquitocidal Strain of &lt;i&gt;Bacillus thuringiensis&lt;/i&gt;

Bt strain S2160-1 was found to produce crystalline toxins with high insecticidal effects against mosquitoes ( Culex quinquefasciatus and Aedes albopictus ), which was characterized from the Bt isolate (Isolate No: 20070720S2160) from soil samples collected from Guangxi Dawangling Natural Reserves of Guangxi Province of China in 2007. Bt strain S2160-1 was stocked in the HITAR Bacillus Collections with Accession No 20070720S2160 and owned by HITAR. Scanning electron microscope observation showed that the spherical shape crystals of Bt S2160-1 with size at one fourth volume of spore to that of reference strain of Bti . SDS-PAGE analysis showed that the intact parasporal protein of Bt S2160-1 containing four major polypetide of about 140kDa, 130kDa, 75kDa and 30kDa was different from that of Bti . Furthermore, bioassays exhibited high toxicity to mosquito larva of Culex quinquefasciatus and Aedes albopictus . There are a great of differences in terms of the large plasmid patterns, parasporal proteins profiles and cry genes contents between Bt S2160-1 and Bti . The cloned genes of cry50Ba1 and cry54Ba1 expressed in BL21 (DE3) cells, but not success in Cry 30Ea1 and Cry 30Ga . Thus, it is believed that Bt S2160-1 could be applied into mosquito control as the potential alternative to prevent mosquito from developing the resistance to the toxins of Bti .

A Bacillus thuringiensis strain producing epizootics on Plodia interpunctella: A case study

After several disease outbreaks in laboratory cultures of pyralid moths in Tabriz University, Iran, during 2004 and 2005, a new Bacillus thuringiensis aizawai strain EF495116 (BTA) was isolated from a dead Plodia interpunctella larva. A complete characterization of the strain was performed, including serological identification, protein and plasmid pattern determination, a PCR-based identification of virulence-related genes, nucleotide sequence analysis of the 16S rDNA and gyrB genes (in order to find out relationships between the species with other virulent Bacillus pathogens), and biological activity assays. These studies revealed that BTA produced a major parasporal protein band of about 135kDa, bore seven out of the fourteen pyralid-active genes analyzed (cry1Aa, cry1Ab, cry1C, cry1D, cry1I, cry2A and cry9) and was toxic against P. interpunctella and P. xylostella larvae, with LC50 values of 7.13 and 3.1μg/mL, respectively. Although these features are common among other B. thuringiensis strains active on Lepidoptera, their role in epizootics is uncertain. However, sequence analysis of the 16S rDNA and gyrB genes revealed that BTA clustered with one of the few B. thuringiensis strains identified as a medical isolate. Interestingly, both strains, like many others reported to produce epizootics, belong to serovar aizawai. The implication of serovar or serovar-dependent genes in epizootics is discussed.

Mosquitocidal activity of two Bacillus bacterial endotoxins combined with plant oils and conventional insecticides

The full whole culture (FWC), containing parasporal protein toxins of Bacillus thuringiensis israelinsis (Bti) and Bacillus sphaericus 2362 (Bs), either singly or in combination with plant oils and commercial insecticides, was tested against larval and adult stages of Culex pipiens mosquitoes under controlled laboratory conditions. In terms of LC 50 values recorded after 24, 48, 72 and 96 h, the bacterial toxins showed high potency towards both larvae and adults of mosquitoes in a dose-dependent manner. Generally, the Bti toxin seemed to be more potent than the Bs toxin. For example, the Bti toxin showed a 24 h LC 50 of 8.2 ppm against mosquito larvae compared to 13.6 ppm for the Bs toxin. In the adult bioassay, the obtained 24 h LC 50 values were 0.064 and 0.085 mg/cm 2, respectively for the two bacterial toxins. The bacterial toxins mixed with plant oils or insecticides at equitoxic doses (e.g., LC 25 values) mostly showed potentiation effects, either against larvae or adults of the tested insect. Among a total of 14 paired mixtures, only the joint action estimated for the mixture of malathion + Bti or Bs was accounted as additively. Combining Bti or Bs endotoxins at LC 0 with different plant oils and insecticides at LC 50 concentration levels each, has resulted in considerable synergism against either larvae or adults. In the case of larval bioassays, the maximum synergistic factor (SF) obtained (ca. 2.0) was entitled to the mixture of Bti + spinosad. In the adult bioassays, the mixtures containing Curcuma longa or Melia azedarach oil extracts with Bti or Bs toxins achieved a SF accounted to 2.0. The results of the present study may be considered as an additional contribution to the area of joint toxicity of biocidal agents combining bacterial toxins, plant oils and traditional insecticides. The reached findings may encourage future research to elucidate its performance under practical field conditions.

New Vector pBUC3 can Express &lt;i&gt;Bt&lt;/i&gt; &lt;i&gt;Cry1Ac31&lt;/i&gt; during the Vegetative Phase in &lt;i&gt;Bacillus thuringiensis&lt;/i&gt;

The feature of Bacillus thuringiensis ( B. t .) is the ability to generate parasporal crystal protein during the spore-forming process. In order to realize the expression of the Bt Cry genes independent on spore formation, we attempted to design new vector that could express Bacillus thuringiensis insecticidal crystal protein in the vegetable growth phase in this research. First, we designed a pair of specific primers based on cry 3Aa promoter gene sequence to amplify cry3Aa promoter from B.t. subsp. Tenebrions ; then the DNA fragment with blunt-end was ligated to the cross-species vector pBU4 to build new pBUC3 vector which would be non-dependent sporulation vector expressing in the vegetative growth phase of Bacillus thuringiensis . In order to validate the vector whether express B.t cry gene in the vegetative phase, The B.t cry1Ac31 gene was ligated to the pBUC3 vector to be transformed into B.t non crystal mutant IPS strain by electronic transformation approach. Through the examinations by the PCR, microscopic observation and SDS-PAGE, the results showed that B.t cry1Ac31 was expressed in cell growth vegetable phase. Our study suggested that the feature of cry 3Aa promoter being non-dependent on spore formation might be useful expression element that could achieve the cry gene of Bacillus thuringiensis expressing in vegetative phase.

Bacillus thuringiensis: características gerais e fermentação

The insect control is carried out mostly by chemical products, whose cumulative effects cause serious losses to environmental and human health, highlighting rapid selection of resistant insects. Biological control by entomopathogenic bacteria is an efficient alternative, mainly due to high specificity, absence of resistance in the target insects and low environment residual effect. Bacillus thuringiensis is a Gram-positive spore-forming bacterium that produces a parasporal crystal protein toxic for many insect species. Despite the widespread use of B. thuringiensis based products in biological control of insects, there are little studies about its multiplication, sporulation and crystal production, as so many trade secrets information. This revision was aimed at gathering general information about B. thuringiensis, as well as its production in culture media.