Bacillus Thuringiensis Strains Research Articles

The colonization of Bacillus thuringiensis strains in bryophytes

In our previous study, several Bacillus thuringiensis (Bt) strains were isolated from bryophyte samples, indicating that bryophytes could serve as Bt reservoirs in the wild. SFR13 is a wild strain isolated from the bryophyta Physcomitrium japonicum. In order to understand its ecological properties, green fluorescent protein (GFP)-labelled SFR13 (SFR13GFP) was generated to evaluate the colonization capability in bryophytes, using dynamic tracing and cell counting to observe the process and patterns of colonization. Our results showed that genetic stability, growth curve dynamics, and insecticidal crystal production were not affected by GFP expression in Bt. Fluorescence microscopy was used to track the dynamic distribution of SFR13GFP. Distribution patterns showed that SFR13GFP can establish stable and long-term colonization in leaves and stems by the 26th day after inoculation. A better understanding of how Bt colonizes plants in the wild will not only result in increased knowledge of plant-microbe interactions but will also lead to a more successful and reliable use of bacterial inoculants.

Complete Sequence and Organization of pFR260, the Bacillus thuringiensis INTA Fr7-4 Plasmid Harboring Insecticidal Genes

We report the complete sequence and analysis of pFR260, a novel megaplasmid of 260,595 bp from the Bacillus thuringiensis strain INTA Fr7-4 isolated in Argentina. It carries 7 insecticidal genes: 3 cry8 copies previously reported, 2 vip1, and 2 vip2. Also, it carries a gene encoding a putative atypical Cry protein. These genes are arranged in a region of approximately 105 kbp in size with characteristics of a pathogenicity island with a potential coleopteran-specific insecticide profile. DNA strand composition asymmetry, as determined by GC skew analysis, and the presence of a Rep protein involved in the initiation of replication suggest a bidirectional theta mechanism of replication. In addition, many genes involved in conjugation and a CRISPR-Cas system were detected. The pFR260 sequence was deposited in GenBank under accession number KX258624.

Isolation and characterization of native Bacillus thuringiensis strains from Saudi Arabia with enhanced larvicidal toxicity against the mosquito vector Anopheles gambiae (s.l.).

BackgroundWorldwide, mosquito vectors are transmitting several etiological agents of important human diseases, including malaria, causing millions of deaths every year. In Saudi Arabia, as elsewhere, vector-control is based mostly on chemical insecticides which may be toxic and cause environmental deprivation. Here, to support the development of bio-pesticide alternatives, a study was conducted to identify native Bacillus thuringiensis (Bt) isolates with improved toxicity against the malaria vector, Anopheles gambiae (s.l.).MethodsSixty-eight Bt isolates were obtained from 300 soil and other samples collected from 16 sites across Saudi Arabia. Bt identification was based on morphological characteristics of colonies, shape of parasporal crystals and biochemical profiles. After characterization of their mosquitocidal activity, larvicidal strains were described through 16S ribosomal DNA gene sequencing, cry, cyt and chi genes PCR-amplification profiles, and SDS-PAGE protein analyses.ResultsSpherical Bt crystals were predominant amongst the 68 isolates (34%), while irregular, bi-pyramidal and spore-attached crystals were found in 32, 13 and 21% of strains, respectively. LC50 and LC90 bioassays showed that 23/68 isolates were larvicidal, with distinct biochemical activity profiles compared to non-larvicidal Bt strains. Eight larvicidal strains showed larvicidal activity up to 3.4-fold higher (LC50 range: 3.90–7.40 μg/ml) than the reference Bti-H14 strain (LC50 = 13.33 μg/ml). Of these, 6 strains had cry and cyt gene profiles similar to Bti-H14 (cry4Aa, cry4Ba, cry10, cry11, cyt1Aa, cyt1Ab, cyt2Aa). The seventh strain (Bt63) displaying the highest larvicidal activity (LC50 = 3.90 μg/ml) missed the cry4Aa and cyt1Ab genes and had SDS-PAGE protein profiles and spore/crystal sizes distinct from Bti-H14. The eight strain (Bt55) with LC50 of 4.11μg/ml had cry and cyt gene profiles similar to Bti-H14 but gave a chi gene PCR product size of 2027bp. No strains harbouring cry2, cry17 + 27, cry24 + 40, cry25, cry29, cry30, or cyt2Ba were detected.ConclusionThis study represents the first report of several Saudi indigenous Bt strains with significantly higher larvicidal efficacy against An. gambiae than the reference Bti-H14 strain. The very high toxicity of the Bt63 strain, combined with distinct cry and cyt genes and SDS-PAGE-protein profiles makes it a promising candidate for future applications in mosquito bio-control.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1922-6) contains supplementary material, which is available to authorized users.

Comparative Efficacy of Bio-organic and Mineral Phosphate on the Growth, Yield and Economics of Wheat (Triticum aestivum L.) Grown by Different Methods

ABSTRACTThis study was conducted to assess the efficacy of bio-organic phosphate (BOP) and diammonium phosphate (DAP) fertilizers for improving the growth, yield, plant phosphorus (P) content and profitability of wheat cultivation under systems of wheat intensification (direct seeding and nursery transplantation) and conventional sowing (broadcasting). BOP was formulated by enriching the compost with rock phosphate and inoculated with plant growth-promoting rhizobacterial (PGPR) strain, Bacillus thuringiensis strain K5. Results revealed the supremacy of BOP over DAP, and the direct seeding method over the other sowing methods in almost all parameters studied. The application of BOP significantly (p ≤ 0.05) increased the plant height, root length, number of tillers per plant, wheat biomass, number of grains per spike, 1000-grain weight, grain yield, straw and grain P contents in direct seeded wheat in comparison to DAP fertilization. Furthermore, application of BOP and direct seeding of wheat generated more profit per hectare compared with the other treatments.

Glycan region of GPI anchored-protein is required for cytocidal oligomerization of an anticancer parasporin-2, Cry46Aa1 protein, from Bacillus thuringiensis strain A1547

Parasporin-2 (PS2), alternatively named Cry46Aa1, an anticancer protein derived from Bacillus thuringiensis strain A1547, causes specific cell damage via PS2 oligomerization in the cell membrane. Although PS2 requires glycosylphosphatidylinositol (GPI)-anchored proteins for its cytocidal action, their precise role is unknown. Here, we report that the glycan of GPI induces PS2 oligomerization, which causes cell death. Cytotoxicity, cell-binding and oligomerization of the toxin were not observed in GPI-anchored protein-deficient Chinese hamster ovary cells. Expression and protease-treatment analyses showed that the actions of the toxin were dependent on the glycan core, not the polypeptide moiety, of GPI-anchored proteins. However, surface expression of some GPI-anchored proteins is observed in PS2-insensitive cells. These data suggest that GPI-anchored proteins do not determine the target specificity, but instead function as a kind of coreceptor, in the cytocidal action of PS2.

Differential expression and characterization of cypermethrin-degrading potential proteins in Bacillus thuringiensis strain, SG4.

A cypermethrin-degrading bacterium (SG4) was isolated from the pesticide-contaminated soil in the agricultural field of the crop research centre of the University, and characterized as Bacillus thuringiensis strain, SG4. The bacterium degraded 78.9 % of cypermethrin (50 ppm) in 15 days when grown in a minimal medium. To understand the functional proteins of cypermethrin degradation in Bacillus thuringiensis strain SG4, a comparative proteomic analysis was performed in the presence/absence of cypermethrin after 5 days of incubation in minimal medium. More than 450 spots corresponding to different proteins were recorded by 2D electrophoresis. We report expression of 223 and 250 unique proteins under normal and induced conditions (cypermethrin stress), respectively. Identified proteins were categorized into different functional groups on the basis of their biological functions, viz., catabolic enzymes, translational and stress proteins, etc. Characterization of cypermethrin-specific proteins in a bacterial strain will help in biodegradation practices in situ.

Identification of a Bacillus thuringiensis Surface Layer Protein with Cytotoxic Activity against MDA-MB-231 Breast Cancer Cells.

In this work, we isolated a surface layer protein (SLP) from a Bacillus thuringiensis (Bt) strain to evaluate it cytotoxic effects against MDA-MB-231 human breast cancer cells. AP11 was selected from a group of Bt strains using SLP oligonucleotides developed from Bacillus conserved regions. The AP11 strain was grown in Luria Bertani medium until the late exponential phase; an 86 kDa protein was extracted using 5 M LiCl and identified by liquid chromatography- tandem mass spectrometry. It corresponded to a multispecies SLP highly similar to previously described SLPs in Bt. The MDA-MB-231 breast cancer cells LC50 was obtained using 0.25 μg/ml of the isolated SLP. HaCat non-cancerous cells presented 90% survival using the same protein concentration. Our data suggest that SLP cytotoxicity against MDA-MB-231 could be induced by an interaction with the CDH11 cell membrane receptor.

The response of soybean to nod factors and a bacteriocin

ABSTRACTMicrobe-to-plant signals can enhance the growth of a wide range of crops. The responses by soybean (Glycine max var. 91M01) to 2 signal molecules were investigated: Bradyrhizobium japonicum 532C lipo-chitooligosaccharide (Nod Bj V [C:18, MeFuc]) (LCO); and Bacillus thuringiensis strain NEB17 bacteriocin thuricin 17 (Th17). The objective was to assess and quantify the response by soybean, in terms of factors that contribute to yield, to the experimental signal molecules in germination experiments and field experiments. Soybean germination was stimulated by the experimental concentrations of Th17 under controlled 15°C and 22°C conditions, and 10−6 M LCO under 15°C. There were negative relationships between Th17 concentration and both the number of trifoliate leaves and the dry weight of nodules: lower concentrations resulted in plants with more leaves and nodules while higher concentrations resulted in plants with fewer leaves and nodules. The 10−8 M LCO treatment had a significant effect on the dry weight of nodules at the flowering stage of plant development (F4,21 = 6.06, p = 0.0019). Considering the harvest stage data from both field trials of 2011, the lower experimental concentrations of Th17 resulted in taller plants. The study of Th17 has the potential to expand our understanding of this relatively recent and unexpected finding; and to understand how best to apply this finding, to allow increased production of soybean. Collectively, these results indicate that Th17 has potential in this regard.

ChemInform Abstract: Diketopiperazines Derivatives Isolated from Bacillus thuringiensis and Bacillus endophyticus, Establishment of Their Configuration by X‐Ray and Their Synthesis.

Abstract Four known diketopiperazines have been isolated from Bacillus thuringiensis strain and two of them were isolated from Bacillus endophyticus also. Their structure was elucidated by a complete spectroscopy and the configuration of three of them was established by X-ray analysis. The dicetopiperazine cyclo-( l -Proline- l -Tyrosine), isolated from both strains, was polymorphic showing different physical properties. The synthesis has been successfully realized in two simple steps. Their biological activities were probed against some Gram-positive and Gram-negative bacteria and six fungi. The results indicated that these compounds had no antibacterial activity but they had antifungal activity.

Vip3Aa induces apoptosis in cultured Spodoptera frugiperda (Sf9) cells

The vegetative insecticidal proteins (Vip) secreted by many Bacillus thuringiensis strains during their vegetative growth stage are regarded as second generation insecticidal proteins, as they share no sequence or structural homology with known crystal insecticidal proteins (Cry) and have a broad insecticidal spectrum. Compared with insecticidal crystal proteins (ICPs), the insecticidal mechanisms of Vips have been little studied. Here we investigated the mechanism responsible for Vip3Aa toxicity in cultured insect cells. Using, flow cytometry analyzes, TUNEL staining and DNA fragmentation assays, we show that Vip3Aa can induce apoptosis in Spodoptera frugiperda (Sf9) cells and cause cells to arrest at the G2/M phase. We also show that Vip3Aa can disrupt mitochondrial membrane potential (ΔΨm), leading to the activation of Sf-caspase-1, suggesting that a mitochondrial mediated and caspase dependent pathway may be involved in Vip3Aa-induced apoptosis in Sf9 cells.

Selection and Characterization of Bacillus thuringiensis (Berliner) (Eubacteriales: Bacillaceae) Strains for Ecdytolopha aurantiana (Lima) (Lepidoptera: Tortricidae) Control.

The citrus fruit borer, Ecdytolopha aurantiana (Lima, 1927) (Lepidoptera: Tortricidae), is responsible for major losses to the citrus industry because it causes rot and drop of fruits. The current study aimed to select and characterize Bacillus thuringiensis (Berliner, 1911) strains toxic to E. aurantiana. For this purpose, 47 B. thuringiensis strains were evaluated in selective bioassays using first instar larvae of E. aurantiana. The lethal concentration (LC50) of the most toxic strains was estimated, and the strains were characterized by morphological, biochemical, and molecular methods. Of the 47 strains tested, 10 caused mortality above 85% and showed mean lethal concentrations between 1.05E+7 and 1.54E+8sporesmL-1. The lowest LC50 values were obtained for the HD-1 standard strain and the BR145, BR83, BR52, and BR09 strains. The protein profile showed the presence of Cry proteins of 60, 65, 70, 80, and 130kDa. The molecular characterization showed the presence of cry1, cry2, cry3, and cry11 genes. The morphological analysis identified three different crystalline inclusions: bipyramidal, round, and cuboidal. The cry1 and cry2 genes were the most frequent among the B. thuringiensis strains evaluated and encode Cry proteins toxic to insects of the order Lepidoptera, which agree with the toxicity results obtained by the selective bioassays against E. aurantiana. The results showed four different B. thuringiensis strains toxic to E. aurantiana at the same level as the HD-1 standard strain, and these strains have biotechnological potential for E. aurantiana control through the production of transgenic plants or the formulation of biopesticides.

Risks for public health related to the presence of Bacillus cereus and other Bacillus spp. including Bacillus thuringiensis in foodstuffs

The Bacillus cereus group, also known as B. cereus sensu lato, is a subdivision of the Bacillus genus that consists of eight formally recognised species: B. cereus sensu stricto, B. anthracis, B. thuringiensis, B. weihenstephanensis, B. mycoides, B. pseudomycoides, B. cytotoxicus and B. toyonensis. The current taxonomy of the B. cereus group and the status of separate species mainly rely on phenotypic characteristics. Bacillus thuringiensis strains display a similar repertoire of the potential virulence genes on the chromosome as B. cereus sensu stricto strains and it has been shown that these genes can also be actively expressed in B. thuringiensis strains. Bacillus cereus and B. thuringiensis strains are usually not discriminated in clinical diagnostics or food microbiology. Thus, the actual contribution of the two species to gastrointestinal and non-gastrointestinal diseases is currently unknown. Most cases of food-borne outbreaks caused by the B. cereus group have been associated with concentrations above 105 CFU/g. However, cases of both emetic and diarrhoeal illness have been reported involving lower levels of B. cereus. The levels of B. cereus that can be considered as a risk for consumers are also valid for B. thuringiensis. There is no evidence that B. thuringiensis has the genetic determinants for the emetic toxin cereulide. The Panel has recommended the application of whole genome sequencing to provide unambiguous identification of strains used as biopesticides and the detailed characterisation of outbreak strains allowing discrimination of B. thuringiensis from B. cereus. Data gaps include: dose–response and behavioural characteristics of B. cereus group strains and specifically of B. thuringiensis. Field studies after application of B. thuringiensis biopesticides are needed to enable the establishment of pre-harvest intervals.

A Method to Determine Lethal and Sublethal Concentrations of Recombinant Bioinsecticides Derived from Bacillus thuringiensis Against Larvae of Spodoptera litura

The increased awareness of environmental pollution have led to the growing interests of the application of biological agents to control insects such as lepidopteran family .Thus , this study aimed to produce genetically improved bioinsecticide agents to be used to control Spodoptera litura which would be useful in reducing environmental pollution ,preventing resistance to bioinsecticides and for safe food production .Four Bacillus thuringensis strains were used as recipients ' while ' Seratia marcescens was used as a doner strain in the conjugation to induce new recombinants in Bacillus thuringiensis over expressed chitinase.Four matings were conducted in this study between Serratia marcescence and the four Bacillus thuringiensis strains which having opposite genetic markers.Two efficient transconjugants based on chitin hydrolysis on agar medium were selected from each conjugation to be evaluated for toxicites against the larvae of cotton leafworm in relation to their parents .The efficient LC50 was appeared by Tr4 ( 1.93 x105 ppm ) followed by Tr6 and Tr7 ( 2x105 ppm). However , more than 995% toxicity index was recorded by Tr4,Tr6 and Tr7. Higher toxicity index observed by these transconjugants may be the related to the overexpression of chitinase genes.Therefore , the lethal concentration values obtained was necessary to kill 50% of larval population from Spodoptera litura in a certain period .The virulence of Bt strains showed that a lower concentration from Tr4 was required to kill 100% of larval population .It must be pointed out that higher toxicity index obtained by transconjugants reflected the interaction between crystal genes from Bt and chitinase genes from Seratia marcescens which enhanced the possibility of larval mortality as a genetic helpful tool in integrated pest management in cotton fields.

Replicates, Read Numbers, and Other Important Experimental Design Considerations for Microbial RNA-seq Identified Using Bacillus thuringiensis Datasets.

RNA-seq is being used increasingly for gene expression studies and it is revolutionizing the fields of genomics and transcriptomics. However, the field of RNA-seq analysis is still evolving. Therefore, we specifically designed this study to contain large numbers of reads and four biological replicates per condition so we could alter these parameters and assess their impact on differential expression results. Bacillus thuringiensis strains ATCC10792 and CT43 were grown in two Luria broth medium lots on four dates and transcriptomics data were generated using one lane of sequence output from an Illumina HiSeq2000 instrument for each of the 32 samples, which were then analyzed using DESeq2. Genome coverages across samples ranged from 87 to 465X with medium lots and culture dates identified as major variation sources. Significantly differentially expressed genes (5% FDR, two-fold change) were detected for cultures grown using different medium lots and between different dates. The highly differentially expressed iron acquisition and metabolism genes, were a likely consequence of differing amounts of iron in the two media lots. Indeed, in this study RNA-seq was a tool for predictive biology since we hypothesized and confirmed the two LB medium lots had different iron contents (~two-fold difference). This study shows that the noise in data can be controlled and minimized with appropriate experimental design and by having the appropriate number of replicates and reads for the system being studied. We outline parameters for an efficient and cost effective microbial transcriptomics study.

Diketopiperazines derivatives isolated from Bacillus thuringiensis and Bacillus endophyticus, establishment of their configuration by X-ray and their synthesis

Four known diketopiperazines have been isolated from Bacillus thuringiensis strain and two of them were isolated from Bacillus endophyticus also. Their structure was elucidated by a complete spectroscopy and the configuration of three of them was established by X-ray analysis. The dicetopiperazine cyclo-(l-Proline-l-Tyrosine), isolated from both strains, was polymorphic showing different physical properties. The synthesis has been successfully realized in two simple steps. Their biological activities were probed against some Gram-positive and Gram-negative bacteria and six fungi. The results indicated that these compounds had no antibacterial activity but they had antifungal activity.

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Screening of coastal soil samples from Ramnad district, Tamil Nadu, South India, revealed the presence of a novel non-haemolytic and non-insecticidal Bacillus thuringiensis strain that contained spherical inclusion body and exerted cytotoxic action on cultured human cancer cells. When protoplasmic extract of the B.t. LDC 501 isolate digest with proteinase K exerted significant toxicity on cell lines such as U937, HL-60 and primary leukemic cells. It also had mild influence on Jurkat (T cell lymphoma) but nontoxic to normal human leukocytes. Majority of the biochemical tests of the strain revealed similar characteristics like that of insecticidal strain B. thuringiensis (BGSC-4Q2) with slight variation. Moreover, antibiotic sensitivity assay discriminated the strain from B. anthracis due to its ability to resist ampicillin. Phylogenetic analysis revealed the distinctive nature of the strain as it forms a separate clade from other Bacillus strains.

Expression of cry1Ab gene from a novel Bacillus thuringiensis strain SY49-1 active on pest insects.

In this study, the cry1Ab gene of previously characterized and Lepidoptera-, Diptera-, and Coleoptera-active Bacillus thuringiensis SY49-1 strain was cloned, expressed and individually tested on Ephestia kuehniella (Lepidoptera: Pyralidae) and Plodia interpunctella (Lepidoptera: Pyralidae) larvae. pET-cry1Ab plasmids were constructed by ligating the cry1Ab into pET28a (+) expression vector. Constructed plasmids were transferred to an Escherichia coli BL21 (DE3) strain rendered competent with CaCl2. Isopropyl β-d-1-thiogalactopyranoside was used to induce the expression of cry1Ab in E. coli BL21(DE3), and consequently, ∼130 kDa of Cry1Ab was obtained. Bioassay results indicated that recombinant Cry1Ab at a dose of 1000 μg g−1 caused 40% and 64% mortality on P. interpunctella and E. kuehniella larvae, respectively. However, the mortality rates of Bt SY49-1 strains’ spore–crystal mixture at the same dose were observed to be 70% on P. interpunctella and 90% on E. kuehniella larvae. The results indicated that cry1Ab may be considered as a good candidate in transgenic crop production and as an alternative biocontrol agent in controlling stored product moths.

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.

Isolation and characterization of a new Bacillus thuringiensis strain with a promising toxicity against Lepidopteran pests

Insecticides derived from Bacillus thuringiensis are gaining worldwide importance as environmentally desirable alternatives to chemicals for the control of pests in public health and agriculture. Isolation and characterization of new strains with higher and broader spectrum of activity is an ever growing field. In the present work, a novel Tunisian B. thuringiensis isolate named BLB459 was characterized and electrophoresis assay showed that among a collection of 200 B. thuringiensis strains, the plasmid profile of BLB459 was distinctive. SmaI-PFGE typing confirmed the uniqueness of the DNA pattern of this strain, compared with BUPM95 and HD1 reference strains. PCR and sequencing assays revealed that BLB459 harbored three cry genes (cry30, cry40 and cry54) corresponding to the obtained molecular sizes in the protein pattern. Interestingly, PCR-RFLP assay demonstrated the originality of the BLB459 cry30-type gene compared to the other published cry30 genes. Insecticidal bioassays showed that BLB459 spore-crystal suspension was highly toxic to both Ephestia kuehniella and Spodoptera littoralis with LC50 values of about 64 (53–75) and 80 (69–91) μg of toxin cm−2, respectively, comparing with that of the commercial strain HD1 used as reference. Important histopathological effects of BLB459 δ-endotoxins on the two tested larvae midguts were detected, traduced by the vacuolization of the apical cells, the damage of microvilli, and the disruption of epithelial cells. These results proved that BLB459 strain could be of a great interest for lepidopteran biocontrol.

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