Coexpression of chitinase and the cry11Aa1 toxin genes in Bacillus thuringiensis serovar israelensis.
At the spore stage, a cloned chitinase gene was coexpressed with the regulatory gene p19 and the toxin gene cry11Aa1 in the hosts Bacillus thuringiensis serovar israelensis strains 4Q2-72 and c4Q2-72. The chitinase gene was derived from a high-chitinase producer, Bacillus licheniformis TP-1. Two transcriptional fusion plasmids between the p19 or p19-cry11Aa1 genes and the promoterless chitinase gene were constructed. In transcription order, the p16-19CHI construct contained the p19 gene together with the chitinase gene only while the p16-1968CHI construct contained p19 together with the toxin gene cry11Aa1 and the chitinase gene. The inserted sequences were regulated by a spore-specific promoter located upstream of p19. The recombinant chitinase of all transformed B. thuringiensis serovar israelensis strains was initially synthesized at low level at about 9 h of growth when a portion of the cells started to sporulate. It increased thereafter and reached maximum levels of 5.5, 4.9, and 4.7 mU/ml at 48 h, for strain 4Q2-72 transformed with p16-19CHI and p16-1968CHI and strain c4Q2-72 transformed with p16-19CHI, respectively. This activity was approximately 2 times higher than the maximum activity (2.7 mU/ml) of the parental strain, B. licheniformis TP-1. Although crude chitinase alone from B. thuringiensis serovar israelensis c4Q2-72 (p16-19CHI) at 4.5 mU/ml caused 40% mortality in second instar Aedes aegypti larvae, transformants containing the chitinase alone or in combination with cry11Aa1 resulted in lower toxicity to A. aegypti larvae than the untransformed 4Q2-72 host. For example the LC(50) for the transformed 4Q2-72 harboring the chitinase gene only (p16-19CHI) was 5.6 x 10(4) +/- 0.7 x 10(4) cells, 40 times higher than that of the untransformed host at 1.4 x 10(3) +/- 0.19 x 10(3). The lower toxicity correlated with poor sporulation in the transformants (i.e., 35 times lower than that in the untransformed host). However, the transformed 4Q2-72 strain expressing both the chitinase and the cry11Aa1 toxin genes (p16-1968CHI) were only 4-fold less toxic (LC(50) = 5.6 x 10(3) +/- 1.99 x 10(3)) than the untransformed 4Q2-72 hosts even though their spore count was 300 times lower. Since coapplication of crude chitinase from the cloned gene in recombinant strain (c4Q2-72 harboring p16-19CHI) with cell suspensions of B. thuringiensis serovar israelensis 4Q2-72 and its transformants could enhance 3- to 50-fold larvicidal activity, improvement in sporulation ability of these genetically engineered strains and cocrystallization of chitinase with crystal toxins may increase their potential for future insect control.
Sirichotpakorn
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N
Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand. scwpb@mahidol.ac.th
Rongnoparut
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P
Choosang
K
K
Panbangred
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W
D000330:Aedes; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001427:Bacterial Toxins; D002688:Chitinases / Q000096:biosynthesis / Q000235:genetics*; D004731:Endotoxins / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D006460:Hemolysin Proteins; D007700:Kinetics; D007814:Larva
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D002688:Chitinases