A quadruple-enterotoxin-deficient mutant of Bacillus thuringiensis remains insecticidal

Abstract

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...

How the insect pathogen bacteria Bacillus thuringiensis and Xenorhabdus/Photorhabdus occupy their hosts

“…Interestingly, a plcR mutant is blocked at the gut epithelial and transcriptional gfp promoter fusions showed that PlcR-regulated factors are expressed in the insect gut (Nielsen-LeRoux, unpublished data). However, deletion of enterotoxin genes (Nhe, Hbl complexes and CytK) did not significantly alter the insecticidal properties of B. thuringiensis towards three lepidopteran insects [19]. Then, further studies are The B. thuringiensis-based insecticidal products are a mixture of spores and insecticidal toxins forming a crystal inclusion.…”

Bacillus thuringiensis Recombinant Insecticidal Protein Production

“…Since the zwittermycin biosynthetic gene cluster has been identified by Kevany et al (2009) and others, and methods for manipulating large DNAs in Bt are being developed ), contemplating alteration of its synthesis is possible. Genome alterations to introduce crystal protein genes were noted above, and related methods have been used to eliminate enterotoxin genes (Klimowicz et al 2010). More drastic genome reduction in B. subtilis (Morimoto et al 2008) led to elongation of the transition phase to sporulation and enhancement of recombinant enzyme synthesis, which appears to be the same period over which Cry3A and Vip3 proteins are produced (see above).…”

“…Two of the three are still in available, and the third, Raven®, was abandoned in 2005, possibly due to lack of a market. In an assessment of insecticidal activity of an enterotoxin deleted Bt kurstaki strain (Klimowicz et al 2010), crystal protein production of the deleted and parent strains was estimated at about 10 g/l in 7.5 l fermenters. Evans (2004) reviewed the production of toxicology materials for transgenic plant registrations.…”

An efficient gene deletion system for Bacillus thuringiensis

“…Gene disruption is usually the method of choice to manipulate biosynthetic genes of B. thuringiensis (Delecluse et al 1991;Poncet et al 1993;Salamitou et al 2000). Klimowicz et al (2010) have used sequential gene replacement to replace the wild-type copies of genes with copies that contained internal deletions.…”