S Thamthiankul | PubMed Health

PubMed2004

Improving the insecticidal activity of Bacillus thuringiensis subsp. aizawai against Spodoptera exigua by chromosomal expression of a chitinase gene.

A transcriptionally fused gene comprising the P19 gene from Bacillus thuringiensis subsp. israelensis fused with a chitinase gene (chiBlA) from B. licheniformis was integrated into the B. thuringiensis subsp. aizawai BTA1 genome by homologous recombination. The resulting B. thuringiensis subsp. aizawai strain (INT1) showed growth and sporulation comparable with that of the wild-type strain. INT1 produced four chitinases of different molecular masses (i.e., 66, 55, 39, 36 kDa). Three of these (66, 55, 36 kDa) were derived from the cloned chiBlA gene, whereas the 39-kDa chitinase originated from BTA1. Using surface contamination bioassays, the 50% lethal concentration of lyophilized whole culture broth of INT1 against Spodoptera exigua neonate larvae was 12.2 microg/cm2, compared with 30.8 microg/cm2 for BTA1. Bioassays using filtered culture supernatant of INT1 (110 microg/cm2) together with trypsin-activated purified Cry1C protein of B. thuringiensis (1,280 ng/cm2) showed 75.0% mortality, compared with 56.7% mortality for Cry1C combined with BTA1 at the same concentration. Using scanning electron microscopy, clear perforations were observed in S. exigua fifth instar peritrophic membranes incubated with either crude or purified chitinase, or isolated from fifth instar S. exigua fed purified chitinase since the first instar. These results show that chitinase can increase the activity of B. thuringiensis subsp. aizawai against S. exigua. This is the first documentation of expressing a chimeric chitinase gene on the chromosome of B. thuringiensis; and chromosomal integration might be used as a potential technique for strain improvement.

Animals↗

PubMed2001

Chitinase from Bacillus thuringiensis subsp. pakistani.

The chitinase gene (chiA71) from Bacillus thuringiensis subsp. pakistani consists of an open reading frame of 1,905 nucleotides encoding 635 amino acid residues with an estimated molecular mass of 71 kDa. Comparison of the deduced amino acid sequence of the mature enzyme to other microbial chitinases shows a putative catalytic domain and a region with conserved amino acids similar to that of the type III module of fibronectin and a chitin-binding domain. By activity detection of chitinase on SDS-PAGE after renaturation, the molecular mass of protein bands with chitinase activity were 66, 60, 47, and 32 kDa. The N-terminal amino acid sequence of each chitinase activity band was the same (Asp-Ser-Pro-Lys-Gln), suggesting that the 60-, 47-, and 32-kDa chitinases were derived from the 66-kDa chitinase by processing step(s) at the C-terminus. The enzyme was identified as an exochitinase, since it generated N-acetylglucosamine from early stage of colloidal chitin hydrolysis. The crude protein (2.3-18.4 mg/ml), containing chitinase at final activities of 8, 16, 32, and 64 mU/ml, was toxic to Aedes aegypti larvae and caused mortalities of 7.5, 15.0, 51.3, and 70.0% respectively, but the same amount of crude protein from a B. thuringiensis subsp. pakistani mutant lacking chitinase was not toxic.

Aedes↗