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Objective: To investigate the flexibility and mobility of the Bacillus thuringiensis toxin Cry1Aa., Methods: The graph theory-based program Constraint Network Analysis and normal mode-based program NMsim were used to analyze the global and local flexibility indices as well as the fluctuation of individual residues in detail., Results: The decrease in Cry1Aa network rigidity with the increase of temperature was evident. Two phase transition points in which the Cry1Aa structure lost rigidity during the thermal simulation were identified. Two rigid clusters were found in domains I and II. Weak spots were found in C-terminal domain III. Several flexible regions were found in all three domains; the largest residue fluctuation was present in the apical loop2 of domain II., Conclusion: Although several flexible regions could be found in all the three domains, the most flexible regions were in the apical loops of domain II., (Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

In order to assess possible enhancement of biopesticide activity, the fusion gene of crystal protein gene cry1Ac with the insect-specific neurotoxin ω-ACTX-Hv1a gene and egfp was expressed in Bacillus thuringiensis acrystalliferous strain Cry-B under the control of the native gene expression system. The fusion recombinant Cry-B(1Ac-ACTX-EGFP) generally produced two or three small crystal-like inclusion bodies in each cell and the GFP signal could be clearly observed. A 166 kDa full-length fusion protein was identified by immunoblot analysis. Virulence of the fusion inclusions was at least fivefold higher toward larvae of Spodoptera exigua. These results demonstrated that a foreign protein could be expressed and accumulate as parasporal inclusions in B. thuringiensis by C-terminal fusion with the native endotoxin while retaining partial insecticidal activity.

A unique residue W544 in the beta18-beta19 loop of the Bacillus thuringiensis Cry1Ac toxin has been implicated in its toxicity. In this study, the effects of mutations at this residue on protein stability during protease treatment, UV irradiation, and preservation were examined. Residue 544 of Cry1Ac was involved in maintaining structural stability, and substitution of a polar group at this position was unfavorable to protein stability. One mutant, W544F, produced larger crystals and was more stable. This mutant showed greater resistance to UV radiation than the wild type Cry1Ac but retained equal toxicity. This is the first report showing that residue 544 in the Cry1Ac domain III plays a significant role in toxin structural stability. Our W544F mutant is a significant development in terms of field applications of Cry1Ac toxin.

Previous studies revealed that chitinase could enhance the insecticidal activity of Bacillus thuringiensis and it has been used in combination with B. thuringiensis widely. However, the expression of B. thuringiensis chitinase is rather low and needs induction by chitin, which limits its field application. It would make sense to constitutively express the chitinase at a sufficiently high level to offer advantages in biological control of pests. In this study, a signal peptide-encoding sequence-deleted chitinase gene from B. thuringiensis strain 4.0718 under the control of dual overlapping promoters plus Shine-Dalgarno sequence and terminator sequence of cry1Ac3 gene was cloned into shuttle vector pHT315 and introduced into an acrystalliferous B. thuringiensis strain Cry(-)B. The recombinant plasmid was stably maintained over 240 generations in Cry(-)B. Chitinase was overexpressed within the sporangial mother cells in the form of spherical crystal-like inclusion bodies. The chitinase inclusions could be solubilized and exhibit chitinolytic activity in 30 mmol l(-1) Na(2)CO(3)-0.2% beta-mercaptoethanol buffer at a wide range of alkaline pH values, and what's more, the chitinase inclusions potentiated the insecticidal effect of Cry1Ac protoxin when used against larvae of Spodoptera exigua and Helicoverpa armigera.

Cry5Ba is a delta-endotoxin produced by Bacillus thuringiensis PS86A1 NRRL B-18900. It is active against nematodes and has great potential for nematode control. Here, we predict the first theoretical model of the three-dimensional (3D) structure of a Cry5Ba toxin by homology modeling on the structure of the Cry1Aa toxin, which is specific to Lepidopteran insects. Cry5Ba resembles the previously reported Cry1Aa toxin structure in that they share a common 3D structure with three domains, but there are some distinctions, with the main differences being located in the loops of domain I. Cry5Ba exhibits a changeable extending conformation structure, and this special structure may also be involved in pore-forming and specificity determination. A fuller understanding of the 3D structure will be helpful in the design of mutagenesis experiments aimed at improving toxicity, and lead to a deep understanding of the mechanism of action of nematicidal toxins.

A new fusion gene cry1Ac-tchiB was constructed to enhance the toxicity of crystal proteins, the cry1Ac gene of Bacillus thuringiensis strain 4.0718 was combined with a tchiB (deleted signal peptide and Enterokinase site sequence). In this process, the Enterokinase site sequence was inserted into the midst of these two genes. Then the combined fragment carrying the upstream promoter region and the downstream terminator region of cry1Ac gene were cloned into the shuttle vector pHT315. And after a series of enzyme digestions and subclonings two new expression vector pHUAccB6 and pHUAccB7 were obtained. The two vectors were transformed into B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant strain HAccB6 and HAccB7. The fusion gene was expressed and the expression product was detected by SDS-PAGE. The result indicated that the recombinant Cry1Ac-tchiB protein accumulated to the level of 61.38% of total bacterial proteins. Cry1Ac protein accumulated to the level of 42% of total bacterial proteins. Chitinase activities is 5.2 time more than that of the control strain. Under Atomic Force Microscopy and SEM of crystals protein, there were some bipy ramidal crystals with a size of 1.5 x 3.0 microm. Bioassay showed that the fusion crystals from recombinant strain HAccB6 and HAccB7 were high toxic against third-instar larvae of Helicourpa armigora with the LC50 (after 72h) value of 9.10 micro/mL and 11.34 micro/mL.The constructed fusion proteins had more toxicity than Cry1Ac crystal proteins. The study will enhances the toxicity of B. thuringiensis Cry toxins protein and makes a ground for constructing the fusion genes of B. thuringiensis cry gene and other foreign toxin genes and the recombinant strain with high toxicity.

The CrylA Crystal Protein from Bacillus thuringiensis is associated with DNA, but the role and sequences of these DNA molecules are unknown. CrylA bipyramidal crystals from B. thuringiensis strain 4.0718 was selectively dissolved and associated DNA was extracted from protoxin. The DNA was digested with Nde I to obtain 3 to 5 kb fragments and then the fragments were subcloned into pMD18-T vector, screening of recombinants were done by PCR-RFLP and sequencing. The ORF of cry1Ac gene was amplified by primers designed and then subcloned. The 3.5 kb BamH I and Sal I fragments of pMDX35 was inserted into the pET30a vector, giving 8.9 kb recombinant plasmid, pETX35. ETX35 strain were obtained by transformed pETX35 into B121 (DE3). A 141 kD fusion protein was superexpressed as inclusion bodies. Quantitative protein analysis indicated that the amount of 141 kD protein was above the level of 51.36% of total cellular protein. Plasmid pHTX42 constructed from shuttle vector pHT304 was transformed B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant HTX42. The recombinant protein was found with a molecular mass of 130 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scanning analysis indicated that the expressed protein accounted up to 79.28% of total cellular proteins and accumulated in the cells mounted up to 64.13% of cellular dry weight. Under Atomic Force Microscopy (AFM), typical bipyramidal crystals from HTX42 strain were found with a size of 1.2 microm x 2.0 microm. Bioassay showed that these inclusion bodies of ETX35 strain and crystals from HTX42 strain were highly toxic against the larvae of Plutella xylostella. On such a base, constructing insecticidal recombinant and analyzing the source, structure, and function of the 20 kb DNA can be further achieved.