Draft Genome Sequences of Two Bacillus thuringiensis Strains and Characterization of a Putative 41.9-kDa Insecticidal Toxin

Abstract

In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein (Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins (cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene (vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both strains, whose sequences exhibited 100% nucleotide identity. The predicted protein showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain (US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus, respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was expressed in Escherichia coli and tested for the first time against four lepidopteran species (Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the green-peach aphid Myzus persicae at doses as high as 4.8 µg/cm2 and 1.5 mg/mL, respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein’s target range or that the protein may not, in fact, be toxic. While the use of the polymerase chain reaction has allowed a significant increase in the number of Bt insecticidal genes characterized to date, novel NGS technologies promise a much faster, cheaper and efficient screening of Bt pesticidal proteins.

Complete Sequence and Organization of pFR260, the <b><i>Bacillus thuringiensis</i></b> INTA Fr7-4 Plasmid Harboring Insecticidal Genes

“…The present composition of the insecticidal genes could represent a dynamic state during the formation of new insecticidal genes within the great diversity in toxins observed in B. thuringiensis isolates. The fact that this remnant is located close to CDS with possible roles in transposition implies that transposition is the most probable mechanism to explain the insecticidal gene distribution in different strains [De Maagd et al, 2003], which is consistent with previous observations of B. thuringiensis insecticidal genes flanked by transposase sequences [Mahillon et al, 1994;Palma et al, 2014a]. Plasmid pBtoxis from B. thuringiensis serovar israelensis [Berry et al, 2002] also presents insecticidal gene remnants in correlation with a high number of transposition-related genes.…”

Insect Antifeedant Potential of Xanthohumol, Isoxanthohumol, and Their Derivatives

“…There is increasing application of microbial agents, so-called bioinsecticides, that contain live bacterial cultures, mostly of the genus Bacillus (Bacillus thuringiensis). 39 Another large group of biopreparations that inhibit plant pest development is based on viruses and nematodes. 40 Apart from interacting with insect chemoreceptors of taste, preparations of antifeeding activity may inhibit the fertility of insects, prolong their larval development, or inhibit the transmission of some viruses.…”

Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

“…In BinA, substitution of Trp222 in this feature, equivalent to Trp211 of Cry35Ab1, results in loss of activity although the protein is still able to permeabilize liposomes [63]. Ricin-B-like lectin repeats were also noted in the 41.9 kDa B. thuringiensis protein [51] although there are no QxW motifs in this protein.…”