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

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... ogues 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. Abbreviations: BCET-RPLA, Bacillus cereus enterotoxin-reverse passive latex agglutination; BDE, Bacillus diarrhoeal enterotoxin; LC 50 , 50% lethal concentration; SOE, gene splicing by overlap extension. 3Present address: