Improvement of Crystal Solubility and Increasing Toxicity against Caenorhabditis elegans by Asparagine Substitution in Block 3 of Bacillus thuringiensis Crystal Protein Cry5Ba

Abstract

The crystal proteins from Bacillus thuringiensis are widely used for their specific toxicity against insects and nematodes. The highly conserved sequence blocks play an important role in Cry protein stability and flexibility, the basis of toxicity. The block 3 in Cry5Ba subfamily has a shorter sequence (only 12 residues) and more asparagine residues than that of others which harbor about 48 residues but only one asparagine. Based on the theoretical structure model of Cry5Ba, all three asparagines in block 3 are closely located in the interface of putative three domains, implying their probable importance in structure and function. In this study, all three asparagines in Cry5Ba2 block 3 were individually substituted with alanine by site-directed mutagenesis. The wild-type and mutant proteins were overexpressed and crystallized in acrystalliferous B. thuringiensis strain BMB171. However, the crystals formed in one of the mutants, designated N586A, abnormally disappeared and dissolved into the culture supernatant once the sporulation cells lysed, whereas the Cry5Ba crystal and the other mutant crystals were stable. The mutant N586A crystal, isolated from sporulation cells by the ultrasonic process, was found to be easily dissolved at wide range of pH value (5.0 to 10.0). Moreover, the toxicity assays showed that the mutant N586A exhibited nearly 9-fold-higher activity against nematodes and damaged the host's intestine more efficiently than the native Cry5Ba2. These data support the presumption that the amide residue Asn586 at the interface of domains might adversely affect the protein flexibility, solubility and resultant toxicity of Cry5Ba.

Enhanced nematicidal potential of the chitinase pachi from Pseudomonas aeruginosa in association with Cry21Aa

“…Additionally, each toxin had a unique mechanism of killing hosts and damaging the host cell 11 . Many researchers believed that the ICP damaged the intestine of the host and that chitinases hydrolyzed the cuticle and egg shell 35 47 . Our results revealed that the chitinase showed high intestinal digestion activity, which would protect the ICP from proteolysis and help the ICP bind to its receptor.…”

Making 3D-Cry Toxin Mutants: Much More Than a Tool of Understanding Toxins Mechanism of Action

“…Single mutations in Domain III have also been described to be useful for toxicity enhancement. This was demonstrated in the nematocidal toxin Cry5Ba [101]. Investigating the role of the 3 asparagines present in block 3 of the toxin found that alanine substitution, by site directed mutagenesis, rendered a mutant (N586A) with a 9-fold increase in toxicity (GIC 50 from 42.11 to 4.75 ng/mL) toward Caenorhabditis elegans.…”

Specificity determinants for Cry insecticidal proteins: Insights from their mode of action

“…The crystals from the non-insecticidal Bt strains were active when pre-solubilized, supporting a critical role for crystal solubilization in determining specificity. Disparity in Bt crystal solubility has been attributed to differences in the disulphide bridges (Du et al, 1994) and blocks of amino acids highly conserved among similar Cry toxins (Wang et al, 2012) that contribute to the stability of the Bt crystal structure. In another example of the importance of crystal solubilization, activity of Cry1B toxin crystals against coleopteran larvae was only detected after in vitro solubilization (Bradley et al, 1995).…”