RT Journal Article SR Electronic(1) A1 Klimowicz, Amy K. A1 Benson, Terry A. A1 Handelsman, JoYR 2010 T1 A quadruple-enterotoxin-deficient mutant of Bacillus thuringiensis remains insecticidal JF Microbiology, VO 156 IS 12 SP 3575 OP 3583 DO https://doi.org/10.1099/mic.0.039925-0 PB Microbiology Society, SN 1465-2080, AB 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, HBLa1 and HBLa2). 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 HBLa1 and HBLa2 were 96–97 % identical to each other and 76–84 % identical to those encoding HBL. The hbla2 operon was detected by PCR amplification only after hbla1 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., UL https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.039925-0