Coexpression of chitinase and the cry11Aa1 toxin genes in Bacillus thuringiensis serovar israelensis

At the spore stage, a cloned chitinase gene was co-expressed 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/mI 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 LC50 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 (LC50 = 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 p146-19CHI) 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. (C) 2001 Elsevier Science (USA).