Mining New Crystal Protein Genes from Bacillus thuringiensis on the Basis of Mixed Plasmid-Enriched Genome Sequencing and a Computational Pipeline

ABSTRACTWe have designed a high-throughput system for the identification of novel crystal protein genes (cry) fromBacillus thuringiensisstrains. The system was developed with two goals: (i) to acquire the mixed plasmid-enriched genomic sequence ofB. thuringiensisusing next-generation sequencing biotechnology, and (ii) to identifycrygenes with a computational pipeline (using BtToxin_scanner). In our pipeline method, we employed three different kinds of well-developed prediction methods, BLAST, hidden Markov model (HMM), and support vector machine (SVM), to predict the presence of Cry toxin genes. The pipeline proved to be fast (average speed, 1.02 Mb/min for proteins and open reading frames [ORFs] and 1.80 Mb/min for nucleotide sequences), sensitive (it detected 40% more protein toxin genes than a keyword extraction method using genomic sequences downloaded from GenBank), and highly specific. Twenty-one strains from our laboratory's collection were selected based on their plasmid pattern and/or crystal morphology. The plasmid-enriched genomic DNA was extracted from these strains and mixed for Illumina sequencing. The sequencing data werede novoassembled, and a total of 113 candidatecrysequences were identified using the computational pipeline. Twenty-seven candidate sequences were selected on the basis of their low level of sequence identity to knowncrygenes, and eight full-length genes were obtained with PCR. Finally, three newcry-type genes (primary ranks) and fivecryholotypes, which were designatedcry8Ac1,cry7Ha1,cry21Ca1,cry32Fa1, andcry21Da1by theB. thuringiensisToxin Nomenclature Committee, were identified. The system described here is both efficient and cost-effective and can greatly accelerate the discovery of novelcrygenes.

Cloning and Expression of Two Crystal Protein Genes, cry30Ba1 and cry44Aa1, Obtained from a Highly Mosquitocidal Strain, Bacillus thuringiensis subsp. entomocidus INA288

ABSTRACT Two novel crystal protein genes, cry30Ba and cry44Aa, were cloned from Bacillus thuringiensis subsp. entomocidus INA288 and expressed in an acrystalliferous strain. Cry44Aa crystals were highly toxic to second-instar Culex pipiens pallens (50% mortality concentration [LC50] = 6 ng/ml) and Aedes aegypti (LC50 = 12 ng/ml); however, Cry30Ba crystals were not toxic.

PCR-Based Identification of Bacillus thuringiensis Isolated from Soil Samples in Nigeria

Six isolates of Bacillus thuringiensis isolated from soil samples confirmed to be toxic to mosquito larvae were differentiated using a PCR-Based technique. Three of these isolates initially identified using a serological technique were further differentiated with the PCR amplification of the δ-endotoxin target sequences. Using the total DNA of isolates as tem plate, at least four isolates yielded amplicons one or all the crystal protein genes, cryl a, b, c, or II with sizes ranging from 238-1070 bp. None of these isolates yielded an amplicon for any of Cry IV A, B and D tested. Of the four isolates identified by PCR technique one isolate remained unidentified by serology.