[Cloning, expression and sequence analysis of chiA, chiB in Bacillus thuringiensis subsp. colmeri 15A3]

Two DNA fragments encoding chitinase A and B were amplified from total genomic DNA of Bacillus thuringiensis subsp. colmeri 15A3, and then ligated with pUCm-T cloning vector. The recombinant plasmids pUCm-chiA and pUCm-chiB were transformed into Escherichia coli XL-Blue respectively. Both chiA and chiB were successfully expressed in E. coli with their natural promoters independent of any chitin. Additionally, the expressed ChiA and ChiB could be secreted from E. coli cells. It was proved that two chitinases were constitutively expressed in strain 15A3. The nucleotide sequence of chiA (GenBank Accession Number: EF103273) with a length of 1426bp included an open reading fram(ORF) of 1083 bp encoding for a protein of 360 amino acids . The deduced amino acid sequence showed that the mature protein ChiA with a predicted molecular mass of 36kDa consisted of a single catalytic domain. The nucleotide sequence of chiB (GenBank Accession Number: EF103273) with a length of 2279 bp included an ORF of 2031bp encoding for a protein of 676 amino acid residues. The deduced amino acid sequence showed that the mature protein ChiB with a predicted molecular mass of 70.6kD was composed of three domains: catalytic domain, chitin-binding domain and fibronectin type III-like domain. The comparison of their upstream sequences informed that there were differences between putative promoters of chiA and chiB.