Neil Crickmore

How Bacillus thuringiensis has evolved specific toxins to colonize the insect world

Bacillus thuringiensis is a bacterium of great agronomic and scientific interest. Together the subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specificity. This is mainly determined by the arsenal of crystal proteins that the bacterium produces during sporulation. Here we describe the properties of these toxin proteins and the current knowledge of the basis for their specificity. Assessment of phylogenetic relationships of the three domains of the active toxin and experimental results indicate how sequence divergence in combination with domain swapping by homologous recombination might have caused this extensive range of specificities.

The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N

A 120 kDa glycoprotein in the larval midgut membrane of the Iepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) δ‐endotoxin, has been purified by a combination of protoxin affinity Chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin In the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N‐terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3‐fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.

Bacillus thuringiensis: an impotent pathogen?

Bacillus thuringiensis (Bt) is an insecticidal bacterium that has successfully been used as a biopesticide for many years. It is usually referred to as a soil-dwelling organism, as a result of the prevalence of its spores in this environment, but one that can act as an opportunistic pathogen under appropriate conditions. Our understanding of the biology of this organism has been challenged further by the recent publication of two reports that claim that Bt requires the co-operation of commensal bacteria within the gut of a susceptible insect for its virulence. It is our opinion that Bt is not primarily a saprophyte and does not require the assistance of commensal bacteria but is a true pathogen in its own right and furthermore that its primary means of reproduction is in an insect cadaver.

Parallel Evolution of Bacillus thuringiensis Toxin Resistance in Lepidoptera

Despite the prominent and worldwide use of Bacillus thuringiensis (Bt) insecticidal toxins in agriculture, knowledge of the mechanism by which they kill pests remains incomplete. Here we report genetic mapping of a membrane transporter (ABCC2) to a locus controlling Bt Cry1Ac toxin resistance in two lepidopterans, implying that this protein plays a critical role in Bt function.

Parallel Evolution of Bt Toxin Resistance in Lepidoptera

Despite the prominent and worldwide use of Bacillus thuringiensis (Bt) insecticidal toxins in agriculture, knowledge of the mechanism by which they kill pests remains incomplete. Here we report genetic mapping of a membrane transporter (ABCC2) to a locus controlling Bt Cry1Ac toxin resistance in two lepidopterans, implying that this protein plays a critical role in Bt function.

Optimizing pyramided transgenic Bt crops for sustainable pest management.

Transgenic crop pyramids producing two or more Bacillus thuringiensis (Bt) toxins that kill the same insect pest have been widely used to delay evolution of pest resistance. To assess the potential of pyramids to achieve this goal, we analyze data from 38 studies that report effects of ten Bt toxins used in transgenic crops against 15 insect pests. We find that compared with optimal low levels of insect survival, survival on currently used pyramids is often higher for both susceptible insects and insects resistant to one of the toxins in the pyramid. Furthermore, we find that cross-resistance and antagonism between toxins used in pyramids are common, and that these problems are associated with the similarity of the amino acid sequences of domains II and III of the toxins, respectively. This analysis should assist in future pyramid design and the development of sustainable resistance management strategies.

Diversity of Bacillus thuringiensis Crystal Toxins and Mechanism of Action

Parasporal crystals produced by Bacillus thuringiensis (Bt) bacteria are the main virulence factors underlying Bt toxicity to insects. Parasporal crystals are composed primarily of Cry and Cyt proteins that act on the midgut of susceptible insects. Cry proteins are an important component of Bt biopesticides and are vital tools for insect control via expression in transgenic crop plants. Some members of the Cry group are more distantly related including ETX/MTX and binary type toxins. Cry toxin structure and action involves critical steps in toxin activation, binding to receptors such as cadherin and then aminopeptidase or alkaline phosphatase probably in a ‘sequential binding’ manner. Specific Cry toxin–receptor interactions are a focus of this review. Recently, the importance of midgut ATP-binding cassette proteins to Cry intoxication of insects has been demonstrated. Mechanistic details involved in ‘sequential binding’ and ‘pore formation’ models are examined. The Cyt toxin of Bt subspecies israelensis is an important and interesting component in Cry–midgut interactions in mosquitoes. For some Cry toxins, Cyt serves as a receptor for docking to midgut membrane. Recent engineering work has demonstrated that Cyt can be re-targeted generating novel toxins for insect control. Overall, we review the remarkable progress made in the past 20 years in discovering novel Cry toxins and in elucidating complex mechanisms of Cry and Cyt toxin action; subjects relevant to the long-term control of insects that damage crops and vector human disease.

Involvement of a possible chaperonin in the efficient expression of a cloned CryllA δ‐endotoxin gene in Bacillus thuringiensis

The Bacillus thuringiensis cryllAδ‐endotoxin gene is found as the third‐gene in a three‐gene operon, with a sporulation‐dependent promoter lying upstream of the first gene, orf1. We show here that the polypeptide product of the middle gene (orf2) is required for efficient expression of the toxin gene. In the absence of a functional ORF2 polypeptide the toxin does not form the crystalline inclusions characteristic of other known Bacillus thuringiensis toxins. We discuss the importance of this finding with respect to the possible role of chaperonins in the crystallization of these proteins.

Genetic, Biochemical, and Physiological Characterization of Spinosad Resistance in Plutella xylostella (Lepidoptera: Plutellidae)

Abstract Bioassays (at generation G2) with a newly collected field population (designated MN) of Plutella xylostella (L.) (Lepidoptera: Plutellidae) from Multan, Pakistan, indicated resistance to spinosad, indoxacarb, deltamethrin, abamectin, and acetamiprid. At G2 the field-derived population was divided into two subpopulations, one was selected (G2 to G11) with spinosad (Spino-SEL), whereas the second was left unselected (UNSEL). A significant reduction in the resistance ratio for each compound was observed in UNSEL at G12, indicating that the observed resistance to each insecticide was unstable. For Spino-SEL, bioassays at G12 found that selection with spinosad gave a resistance ratio of 283 compared with MN at G2. The resistance to indoxacarb and acetamiprid in the Spino-SEL population increased to 13- and 67-fold, respectively, compared with MN at G2. The toxicity of deltamethrin to Spino-SEL was similar to its toxicity to the MN population at G2. This suggests that spinosad selection maintained the otherwise unstable resistance to the compound. In contrast, resistance to abamectin decreased significantly from G2 to G12 in Spino-SEL. Logit regression analysis of F1 reciprocal crosses between Spino-SEL and the susceptible Lab-UK indicated that resistance to spinosad was inherited as an autosomal, incompletely recessive trait. The spinosad resistance allele significantly delays the developmental time, reduced pupal weight, number of eggs laid, and number of eggs hatched compared with Lab-UK. Further analysis suggests Spino-SEL exhibited a significantly lower intrinsic rate of population increase (rm) to all other populations tested.

Cloning and characterization of a novel Cry1A toxin from Bacillus thuringiensis with high toxicity to the Asian corn borer and other lepidopteran insects.

A novel cry1A was cloned from Bacillus thuringiensis strain BT8 and expressed in the B. thuringiensis acrystalliferous mutant HD73(-). The gene, designated cry1Ah1, encoded a protein with a molecular weight of 134 kDa. Reverse transcriptase-PCR and Western blotting showed that Cry1Ah was expressed in the host strain BT8. The toxin expressed in HD73(-) exhibited high toxicity against lepidopteran larvae of Ostrinia furnacalis, Helicoverpa armigera, Chilo suppressalis, and Plutella xylostella. The 50% lethal concentrations (LC(50)s) were 0.05, 1.48, 0.98 microg g(-1) and 1.52 microg mL(-1), respectively. The LC(50)s of Cry1Ah were significantly lower than that of Cry1Ac for H. armigera, C. suppressalis, and O. furnacalis, and lower than that of Cry1Ab and Cry1Ie for Ostrinia furnacalis. The high toxicity against a range of pest species makes this novel toxin a potential candidate for insect biocontrol.