Search

The novel cry52Ca1 gene from an Indian Bacillus thuringiensis (Bt) isolate was cloned in an expression vector (pET301/CT-DEST, 6xHis). The gene expressed as a ∼77.2 kDa protein in E. coli BL21-CodonPlus (DE3)-RIPL cells upon induction with isopropyl-thio-galactoside (IPTG) for 18 h at 28 °C. Cry52Ca1 protein was toxic to Helicoverpa armigera (cotton bollworm) neonate larvae (LC50 36.66 µg per ml and MIC50 3.051 µg per ml) in diet-based laboratory assays. This gene has potential for deployment in insect-resistant transgenic crops.

Novel genes from Bacillus thuringiensis (Bt) are required for effective deployment in agriculture, human health, and forestry. In an improvement over conventional PCR-based screening, next generation sequencing (NGS) has been used for identification of new genes of potential interest from Bt strains, but cost becomes a constraint when several isolates are to be sequenced. We demonstrate the potential of a DNA pooling strategy known as pool deconvolution to identify commercially important toxin genes from 36 native Bt isolates. This strategy is divided into three steps: (a) DNA pooling, (b) short read sequence assembly followed by gene mining, and (c) host isolate identification. With this approach, we have identified insecticidal protein (ip) genes including nine three-domain (3D) cry genes, three cyt-type genes, three mtx genes (mosquitocidal toxin), and one bin and vip-type gene each. Three cry-type and three cyt-type genes were cloned, out of which, two cry-type genes, ip11 and ip13, were named as cry4Ca2 and cry52Ca1, respectively by the Bacillus thuringiensis nomenclature committee ( http://www.biols.susx.ac.uk/Home/Neil_Crickmore/BT/ ). Our results show that the pool deconvolution approach is well suited for high-throughput gene mining in bacteria.

Bacillus thuringiensis (Bt) strains produce Cry (crystal) and Cyt (cytolytic) proteins belonging to the group of bacterial toxins known as pore-forming toxins (PFTs), which interact with midgut cells of target insects to create pores, disruption of ion homeostasis and eventual death. PFTs have synergistic insecticidal activities and have been used as biopesticides against agriculturally important insects. Identification of new Cyt proteins is important because of their specific toxicity towards hemipteran pests, against which the Cry proteins are not effective. We have structurally characterized a cyt (cyt1007) gene from an Indian Bt isolate SK-1007. The presence of a "Bacillus thuringiensis toxin" domain and maximum identity of 36% with Cyt2Ca in the deduced amino acid sequence indicated Cyt1007 protein to be a new member of Cyt family. Three dimensional (3D) modeling (PMDB ID: PM0081490) revealed that it adopts a typical ferredoxin-like fold, and is composed of a single domain of α/β architecture, in which a single β sheet is surrounded by two α helical layers. The putative lipid binding site and probable mode of action of Cyt1007 protein were predicted through comparative analysis with other Cyt toxins and their distant homologs Evf (Erwinia virulence factor) and VVA2 (Volvatoxin A2). Heterologous expression of cyt1007 gene as a 25 kDa protein in Escherichia coli was achieved at high levels in both soluble and insoluble fractions. Affinity chromatography-based purification yielded 83.6% purified Cyt1007 protein, which can be used for downstream applications for the investigation of its toxicity. Graphical abstract Steps in the structural characterization and heterologous expression of a new cyt gene cloned from Bacillus thuringiensis.

As far as our understanding of the microbial world is considered, until the present only a small fraction of microbial communities from an ecosystem can be characterized, hence having enormous gene bioprospecting potential. Gene mining/prospecting is referring to the process of identifying gene(s) or their allele(s) involved in the economically important biological process of industrial importance. Industrial biotechnology and its vigorous growth over the decade increase the demand of novel genes with increased efficiency for industrial application. The metagenomic studies are one of the ways to explore the hidden potential of cultured and unculturable microbial communities of the ecosystem. Metagenome from the complex microbial community is a good bioresource for bioprospecting of industrial important biocatalyst. Despite having a remarkable contribution of metagenomics in gene prospecting from prokaryotes, it is still in its formative years. In the present chapter, we have tried to provide am efficacy of soil and marine metagenomic in gene mining/prospecting and describe various methods available to narrow down to the specific gene in the metagenome. Accomplishments made thus far, limitations, and future prospects of this new science are discussed.

The cry genes synthesized by Bacillus thuringiensis (Bt) during sporulation are a valuable source for the substitution of chemical pesticides. Exploration and evaluation of Bt toxins via novel gene isolation, DNA shuffling and in silico characterization are emerging as promising tools for the development of novel Bt toxins. In the current investigation, we isolated and structurally characterized a new cry gene from Indian Bt isolate, SK711. Furthermore, the theoretical three dimensional structure of a novel delta endotoxin belonging to the three domain Cry toxin family has been investigated through comparative homologous modelling. The refined model was evaluated by RAMPAGE, ERRAT and Verify 3D. Despite of its low sequence identity to the previously reported Cry protein structure, it not only resembles, but also shares five common conserved blocks of amino acid residues. This result is expected to be valuable in protein engineering and will serve as a starting point for the designing of experiment involving mutagenesis and bioassay aimed at the improvement of toxicity.

Bacterial Leaf Blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating disease and limiting factor for rice productivity worldwide. Since BLB pathogens are difficult to manage through chemical and other available cultural practices, development of host plant resistance is the most effective means of disease management. So far survey for resistance donor at molecular level in rice is scanty and the landraces of Gujarat state of India have received little attention. Therefore, a molecular screening was conducted to identify the presence of major BLB resistance genes Xa4, xa5 and Xa21 in landraces and local cultivars of Gujarat. In order to confirm the presence of BLB resistance genes, 25 genotypes were screened using PCR analysis. 10 genotypes (IR20, IR64, IR72, NWGR1095, NWGR2014, IET14726, GR7, GR102, Pankhali 203 and Ratna) were found to be Xa4 positive. In contrast, only IET18483 genotype was found to be Xa21 positive. Interestingly, not a single genotype was found to be positive for xa5 BLB resistance gene. The status of BLB resistance genes in landraces and cultivars of Gujarat will help design BLB-resistance breeding programs.

Transgenic hairy roots of Solanum lycopersicum were engineered to express a recombinant protein containing a fusion of rabies glycoprotein and ricin toxin B chain (rgp–rtxB) antigen under the control of constitutive CaMV35S promoter. Asialofetuin-mediated direct ELISA of transgenic hairy root extracts was performed using polyclonal anti-rabies antibodies (Ab1) and epitope-specific peptidal anti-RGP (Ab2) antibodies which confirmed the expression of functionally viable RGP–RTB fusion protein. Direct ELISA based on asialofetuin-binding activity was used to screen crude protein extracts from five transgenic hairy root lines. Expressions of RGP–RTB fusion protein in different tomato hairy root lines varied between 1.4 and 8 µg in per gram of tissue. Immunoblotting assay of RGP–RTB fusion protein from these lines showed a protein band on monomeric size of ~84 kDa after denaturation. Tomato hairy root line H03 showed highest level of RGP–RTB protein expression (1.14 %) and was used further in bench-top bioreactor for the optimization of scale-up process to produce large quantity of recombinant protein. Partially purified RGP–RTB fusion protein was able to induce the immune response in BALB/c mice after intra-mucosal immunization. In the present investigation, we have not only successfully scaled up the hairy root culture but also established the utility of this system to produce vaccine antigen which subsequently will reduce the total production cost for implementing rabies vaccination programs in developing nations. This study in a way aims to provide consolidated base for low-cost preparation of improved oral vaccine against rabies.