Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis

University of Delhi, Old Delhi, NCT, India
FEMS Microbiology Letters (Impact Factor: 2.12). 03/2005; 243(2):467-72. DOI: 10.1016/j.femsle.2005.01.011
Source: PubMed


Twenty-four serovars of Bacillus thuringiensis (Bt) were screened by polymerase chain reaction to detect the presence of vegetative insecticidal protein gene (vip)-like sequences by using vip3Aa1-specific primers. vip-like gene sequences were identified in eight serovars. These genes were cloned and sequenced. The deduced amino acid sequence of the vip3Aa14 gene from Bacillus thuringiensis tolworthi showed considerable differences as compared to those of Vips reported so far. The vip3Aa14 gene from Bt tolwarthi was expressed in Escherichia coli using expression vector pET29a. The expressed Vip3Aa14 protein was found in cytosolic supernatant as well as pellet fraction, but the protein was more abundant in the cytosolic supernatant fraction. Both full-length and truncated (devoid of signal sequence) Vips were highly toxic to the larvae of Spodoptera litura and Plutella xylostella. Truncation of Vip3Aa14 protein at N-terminus did not affect its insecticidal activity.

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Available from: Siva K Panguluri, Dec 18, 2013
    • "The cry1, cry2, and cry9 genes are commonly found in isolates of B. thuringiensis toxic to Lepidoptera (Bravo et al 1998, Pinto & Fiuza 2003, Escudero et al 2007). Although vip genes are very toxic to many Lepidoptera (Yu et al 1997, Bhalla et al 2005, Fang et al 2007, Seifinejad et al 2008), they were not detected. Regarding the other toxins, it is likely that cry3, cry4, cry11, and cyt genes were not identified in the isolates tested because they were selected based on their activity against Lepidoptera: Cry3 proteins are Coleoptera specific and Cry4, Cry11, and Cyt proteins are Diptera specific (Höfte & Whiteley 1989, Aly 2007, van Frankenhuyzen & Nystrom 2015). "
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    ABSTRACT: Biopesticides based on Bacillus thuringiensis and genetically modified plants with genes from this bacterium have been used to control Plutella xylostella (L.) and Spodoptera frugiperda (J.E. Smith). However, the selection pressure imposed by these technologies may undermine the efficiency of this important alternative to synthetic insecticides. Toxins with different modes of action allow a satisfactory control of these insects. The purpose of this study was to characterize the protein and gene contents of 20 B. thuringiensis isolates from soil and insect samples collected in several areas of Northeast Brazil which are active against P. xylostella and S. frugiperda. Protein profiles were obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Polymerase chain reaction assays were used to determine toxin genes present within bacterial isolates. The protein profile of the majority of the isolates produced bands of approximately 130 kDa, suggesting the presence of Cry1, Cry8 and Cry9 proteins. The gene content of the isolates of B. thuringiensis investigated showed different gene profiles. Isolates LIIT-4306 and LIIT-4311 were the most actives against both species, with LC50 of 0.03 and 0.02× 108 spores/mL , respectively, for P. xylostella, and LC50 of 0.001× 108 spores/mL for S. frugiperda. These isolates carried the cry1, cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, cry1F, cry2, cry2A, cry8, and cry9C genes. The obtained gene profiles showed great potential for the control of P. xylostella and S. frugiperda, primarily because of the presence of several cry1A genes, which are found in isolates of B. thuringiensis active against these insects.
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    • "Vegetative insecticidal proteins (Vip) are produced by Bacillus thuringiensis (Bt) and secreted during the vegetative phase of growth. Vip1 and Vip2 proteins act as binary toxins and are toxic to coleopterans (Shi et al., 2004) and aphids (Sattar and Maiti, 2011), andVip3 proteins are toxic to lepidopterans (Estruch et al., 1996; Bhalla et al., 2005; Fang et al., 2007; Liu et al., 2007; Hernández-Rodríguez et al., 2009; Yu et al., 2010) (for a summary of toxicities, see van Frankenhuyzen and Nystrom, 2002 and Milne et al., 2008). Vip proteins are structurally different from B. thuringiensis Cry and Cyt d-endotoxins produced during the late growth phase, and share no sequence homology with them. "
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    ABSTRACT: Vip3 proteins have been described to be secreted by Bacillus thuringiensis during the vegetative growth phase and to display a broad insecticidal spectrum against lepidopteran larvae. Vip3Aa protoxin has been reported to be significantly more toxic to Spodoptera frugiperda than to Spodoptera exigua and differences in the midgut processing have been proposed to be responsible. In contrast, we have found that Vip3Ae is essentially equally toxic against these two species. Proteolysis experiments were performed to study the stability of Vip3A proteins to peptidase digestion and to see whether the differences found could explain differences in toxicity against these two Spodoptera species. It was found that activation of the protoxin form and degradation of the 62 kDa band took place at lower concentrations of trypsin when using Vip3Aa than when using Vip3Ae. The opposite effect was observed for chymotrypsin. Vip3Aa and Vip3Ae protoxins were effectively processed by midgut content extracts from the two Spodoptera species and the proteolytic activation did not produce a peptidase resistant core under these in vitro conditions. Digestion experiments performed with S. frugiperda chromatography-purified digestive serine peptidases showed that the degradation of the Vip3A toxins active core is mainly due to the action of cationic chymotrypsin-like peptidase. Although the digestion patterns of Vip3A proteins do not always correlate with toxicity, the peptidase stability of the 62 kDa core is in agreement with intraspecific differences of toxicity of the Vip3Aa protein.
    Full-text · Article · Jun 2014 · Journal of Insect Physiology
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    • "VIPs have excellent activity against black cutworms and armyworms (Yu et al., 1997) S. frugiperda (Barreto et al., 1999). S. litura and Plutella xylostella (Bhalla et al., 2005), Helithios zea, Trichoplusia ni, Ostrinia nubilalis (Fang et al., 2007; Sellami et al., 2011). "

    Preview · Article · Jun 2014 · AFRICAN JOURNAL OF BIOTECHNOLOGY
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