Identification Of Novel Nematicidal Crystal Proteins And Host-pathogen Interactions In Bacillus Thuringiensis

Bacillus thuringiensis(Bt)is an entomopathogenic bacterium,characterized by production of parasporal crystals during sporulation.These parasporal crystals include crystal proteins(Cry)which show high toxicity to many agricultural pests.So Bt becomes the most successful used microorganism in microbial pesticides.In addition,genes encoding Cry proteins can be used as resources to produce transgenic crops for controlling agricultural pests.Currently,there are relatively more researches on controlling insects with Bt,while limited studies have been conducted to study the role of Bt toxins to control nematodes.Plant parasite nematode cause significant loss to crops therefore it needs to find novel Cry proteins having higher activity and wider toxicity against nematodes.Therefore,we focus on isolating novel strains and using genomic analysis to mine novel nematicidal genes.1)About 20%of Bt genomes were found having partial genes by analyzing 100 Bt genomes.Functional analysis of one of these partial genes(cry5B-LN)showed that it could form typical parasproal crystal when fused with C-terminal coding sequence of crystal protein gene cry5Ba,and the fusion Cry protein(N+C protein)showed nematicidal activity.The results suggested that the partial cry genes can be used as a potential source of functional Cry proteins.Cry proteins have typical N-terminal playing critical role in its activity and specificity while C-terminal plays important role in expression of crystal protein.Genome sequences of Bt strains showed some implements that there are many truncated genes which encode the N-terminal or C-terminal of crystal proteins.In first part of this study,we have screened 100 genomes of Bt to investigate occurrence of partial cry genes.Twenty genomes were found having partial genes either in the form of N-terminal or C-terminal in addition to 65 full cry genes.To further investigate the expression and function of partial cry gene,it was focused on a partial encoding sequence from the genome of nematicidal strain C15.This partial gene fragment encodes a protein covering 47%of nematicidal crystal protein Cry5Ba at its N-terminal and having 28%similarity,so it was named as cry5B-like N-terminal gene(cry5B-LN).Firstly,cry5B-LN was expressed with traditional expression system in Bt,which revealed that cry5B-LN was unable to express.However,when fused with C-terminal coding sequence of crystal protein gene cry5Ba,it was not only expressed in protein but also form parasporal crystal body.Electron micrograph revealed that it produces crystals with pyramidal shape.By bioassay experiments with nematode Caenorhabditis elegans,it showed that this N+C fusion Cry protein was significantly toxic against nematode.Also this N+C fusion protein positively affects the nematodes life span,growth and survival rate with determined LC50 23.7 ?g/ml value.By observing the pathology changes of fusion proteins treated nematodes with light microscopy,it showed damage in the intestine of nematodes,thereby confirming the site of action of the fusion protein.The phenotypic changes of the GFP-labeled nematode mutant strain(FT63)after treatment with the fusion protein were observed by fluorescence microscopy.It was found that the fusion protein could cause the destruction of the intestinal epithelial cell junction of the nematode,thereby confirming the mode of action of the fusion protein.These results imply that partial cry gene could be source of novel crystal protein toxins with higher toxicity.It is supposed that further understanding about partial genes not only explore the source of cry genes but also help to study the evolutionary strategy of Bt to produce the multiple domain toxins.2)A novel Cry protein,Cry5Fa,was identified by genomic analysis of Bt strain F14.The protein is similar to Cry5Ba,which has been reported to be active against C.elegans.However,Cry5Fa was found to be toxicity to C.elegans mutants which have resistance against Cry5Ba.It suggests that Cry5Fa is a novel crystal protein with broad-spectrum nematicidal activity.In this study,a potential novel Cry protein was found by sequence analysis of Bt strain F-14.The putative encoded protein with conserved domain having similarity with Cry5Ba,so it was named as Cry5Fa.Sequence analysis showed that C-terminal part of Cry5Fa was more similar with C-terminal of Cry5Ba in range of 70-80%,however the N-terminal showed similarity in range of 20-30%,while overall similarity was 58%with Cry5Ba.Cry5Fa was expressed in Bt strain BMB171,it was found to produce square shape crystals.By bioassay experiments with C.elegans and C.elegans bre mutant,Cry5Fa was found toxic to the bre-mutant(mutant strain resistant to Cry5Ba)with LC5031 ?g/ml,revealing it may have different pattern of activity as compared to Cry5Ba.3)Hydralysin,aerolysin like protein,was encoded by Bt strain YBT-1520.With toxicity assay,it was found that the hydralysin alone had low toxicity to nematodes,while it could significantly increase the nematicidal activity of Cry5Ba when combined with Cry5Ba.The results indicated that hydralysin in Bt can act as a virulence factor to synergistically enhances the toxicity of Cry proteins.Bt produces a number of virulence factors besides Cry proteins.These proteins are grouped on the basis of their sequence and specificity.In this study we have found a gene(750bp)encodes a 32.5 kDa protein.The sequence analysis showed that it has significant similarity with beta-pore forming toxins hydralysin,which belong to aerolysin like protein.The protein was expressed and purified by heterologous expression in Escherichia coli.The bioassay of C.elegans showed that the hydralysin protein alone had low toxicity to nematodes,while when combined with Cry5Ba it significantly enhanced Cry5Ba activity.This result shows that hydralysin in Bt can act as a virulence factor to synergistically enhance the activity of Cry proteins.