Characterization Of Bacillus Thuringiensis HZM2 And Its Degradation Efficiency Of Chlorpyrifos

Bacillus thuringiensis (Bt) is an ubiquitous gram-positive, spore-forming bacterium. It accounts for 95% of the world market of microbial pest control agents. Besides to many insect orders, Bt is also toxic to nematodes, mites, protozoa and pathogens directly or indirectly. All of the properties attribute to a lot of toxins of Bt, such as insecticidal crystal proteins (ICPs), vegetative insecticidal proteins (VIPs), chitinase, AiiA, InhA, and so on. Of all of the toxins, ICPs are the most important. The application of Bt is not only on agriculture and forestry, but also in medicine and even in industry. We isolated Bt strains from industrial pollution sources, indentified the biochemical characteristics and the toxicity to Plutella xylostella, Ditylenchus dipsaci and Bactrocera dorsalis. Several bioactive genes of the isolate HZM2 was found and a new cry1 -type gene was cloned and sequenced. In addition, the degradation efficiency of the isolate to organophosphorus pesticide chlorpyrifos was also conducted.Of 220 samples from industrial pollution sources, 6 Bt isolates (2.7%) were obtained by sodium sacetate-antibiotic-heat treatment method. All tested strains differed in their biochemical characteristics. Bioassay showed that HZM2 was toxic to P. xylostella and D. dipsaci. The LC50 was 1.91μL/mL and 10.93μL/mL, respectively.PCR-RFLP and PCR technology were used to analyse the bioactive genes. The results showed that HZM2 harboured cry1Aa, cry1Ba, cry1Ea, cry1Ia, cry2Ab, vip3A, inhA, aiiA and chitinase genes. A cry1Ea gene was amplified with 3516 bp in length, encoding a protein of 1171 residues with a calculated molecular weight of 133.18 kDa and a predicted pI value of 5.01. The nucleotide sequence of cry1Ea had been deposited in GenBank at the accession number EU244426 and the cry1Ea had been designated as cry1Ea8 by the Bt delta-Endotoxin Nomenclature Committee.Bioinformatics analyse indicated the amino acid sequences encoded by cry1Ea8 contained 24.68% of helix, 30.66% of sheet and 44.66% of loop in secondary structure. The cry1Ea8 protein might be globular, but it was not as compact as a domain. cry1Ea8 might had three domains, Domain I consists of 219 residues(Thr₃₅ to Leu₂₅₃), Domain II consists of 197 residues (Thr₂₅₈ to Thr₄₅₄), Domain III consists of 138 residues (Ile₄₆₄ to Asp₆₀₁). The N-teminal helical domain is involved in membrane insertion and pore formation, the second and third domains are involved in receptor binding.In this paper, we firstly identified the tolerance ability and degradation rate of Bt to organophosphorus pesticide. The tolerance of the strain HZM2 to chlorpyrifos was up to 1600 mg/L. Tested with gas chromatography(GC), the degradation rate of strain HZM2 to chlorpyrifos was 88.9% at 48 hours. These results suggested that the strain HZM2 was tolerance to chlorpyrifos and it was potential to be developed as a new product for cleanup of chlorpyrifos.