Bacillus thuringiensis is the leading biopesticide used to control insect pests worldwide. Although they have a long record of safe use, under certain conditions commercial strains of B. thuringiensis have the ability to produce numerous putative enterotoxins that have been associated with food poisoning attributed to Bacillus cereus. Therefore, we designed a strategy to delete the genes encoding these toxins. B. thuringiensis strain VBTS 2477 contained genes encoding NHE, CytK-2 and three homologues of haemolysin BL (HBL, HBL a1 and HBL a2 ). This is the first report, to our knowledge, of a strain of B. cereus or B. thuringiensis containing three sets of hbl operons. The genes encoding HBL a1 and HBL a2 were 96-97 % identical to each other and 76-84 % identical to those encoding HBL. The hbl a2 operon was detected by PCR amplification only after hbl a1 was deleted. We used sequential gene replacement to replace the wild-type copies of the NHE and three HBL operons with copies that contained internal deletions that span the three genes in each operon. The insecticidal activity of the quadruple-enterotoxin-deficient mutant was similar to that of the wild-type strain against larvae of Trichoplusia ni, Spodoptera exigua and Plutella xylostella. This demonstrates that the genes for enterotoxins can be deleted, eliminating the possibility of enterotoxin production without compromising the insecticidal efficacy of a strain of B. thuringiensis.
INTRODUCTIONBacillus thuringiensis has been used globally for over 50 years as a bioinsecticide for the control of insect pests in agriculture, forestry and public health. B. thuringiensis produces one or more crystal proteins during sporulation that are toxic to insect larvae upon ingestion. The toxins exhibit a narrow host spectrum, with an individual toxin typically affecting a subset of species within an insect order (Lepidoptera, Diptera, Coleoptera). Numerous studies show B. thuringiensis to be harmless to humans and mammals (Fisher & Rosner, 1959; US Environmental Protection Agency, 1998) and it has become increasingly popular as a method of insect control that is more environmentally friendly than synthetic chemical insecticides.B. thuringiensis is a Gram-positive spore-forming bacterium that is a member of the Bacillus cereus group. Phylogenetic studies and whole-genome sequencing reveal that B. thuringiensis and B. cereus are members of the same species group (Carlson et al., 1994;Helgason et al., 2000;Hill et al., 2004;Rasko et al., 2005). Both B. thuringiensis and B. cereus are found in soils, in the guts of invertebrates and associated with plants (Smith & Couche, 1991; Margulis et al., 1998;Martin & Travers, 1989;Jensen et al., 2003;Jara et al., 2006;Bizzarri & Bishop, 2007), and the only distinguishing phenotype is the production in B. thuringiensis of plasmid-encoded crystal toxins upon sporulation. Some strains of B. cereus are known to be foodpoisoning agents and opportunistic pathogens (Kotiranta et al., 2000). Pathogenic strains of B. cereus are responsibl...
