TRENDS in Genetics Vol.17 No.4 April 2001 http://tig.trends.com 0168–9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(01)02237-5 193 Review Ruud A. de M aagd* Plant Research International, PO Box 16, 6700 AA Wageningen, The Netherlands. * e-mail: R.A.deMaagd@ plant.wag-ur.nl Alejandra Bravo Universidad Nacional Autónoma de México, Instituto de Biotecnología, Apdo. Postal 510-3, 62250 Cuernavaca, Morelos, México. Neil Crickmore School of Biological Sciences, University of Sussex, Brighton, UK BN1 9QG. Bacillus thuringiensis (Bt) is an endospore-forming bact er i um char act er i zed by t he pr esence of a pr ot ei n crystal within the cytoplasm of the sporulating cell (Fig. 1). The proteins within this crystal are toxic to insects, which explains the extensive use of Bt as a biological insecticide. The ecology of Bt remains unclear, this ubiquitous bacterium has been isolated from soil, stored grain, insect cadavers and the phylloplane (plant surfaces), and it is probably best described as an opportunist pathogen 1 . By synthesizing the crystal during sporulation, the bacterium can be thought of as providing for its future. A dead insect will provide sufficient nutrients to allow germination of the dormant spore. Despite the actual, or presumed, presence of various pathogenicity factors (summarized in Box 1), Bt does not have a significant history of mammalian pathogenicity, and research has concentrated on the insecticidal nature of the crystal proteins (Cry and Cyt proteins; also cal l ed δ-endotoxins). To date (January 2001), 89 different genes encoding crystal proteins have been cloned from Bt and two other species (a full list with further links can be found at http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/). In this article, we will focus on the Cry proteins only. A given strain will normally synthesize between one and five of these toxins packaged into a single, or multiple, crystals. Thousands of strains are kept in collections all over the world, and a multitude of gene combinations exists, although certain combinations of genes appear t o be mor e common t han ot her s. The genes encodi ng t he cr yst al pr ot ei ns ar e found (oft en clustered) on transmissible plasmids and flanking transposable elements, explaining how they can spread easily within the species 1 . Conjugation between different strains has been observed both in a soil environment and within insects 2 . Individual Cry toxins have a defined spectrum of insecticidal activity, usually restricted to a few species within one particular order of insects. To date, toxins for insect species in the orders Lepidoptera (butterflies and moths), Diptera (flies and mosqui t oes), Col eopt er a (beet l es and weevi l s) and Hymenoptera (wasps and bees) have been identified. A small minority of crystal toxins shows activity against non-insect species such as nematodes 3 . A few toxins have an activity spectrum that spans two or three insect orders– most notably Cry1Ba, which is active against larvae of moths, flies and beetles 4 . The combination of toxins within a given strain, therefore, defines the activity spectrum of that strain. Besides their long-term use as a biological insecticide in the form of sprays of spore–crystal mixtures, individual Cry toxins have been expressed in transgenic plants t o r ender cr ops r esi st ant t o i nsect pest s. Si nce 1996, t r ansgeni c mai ze, cot t on and pot at o car r yi ng a cr y gene have taken large crop market shares world wide, particularly in the US 5 . The structural diversity of Cry toxins Currently, the crystal toxins are classified on the basis of amino acid sequence homology, wher e each pr ot oxin acquired a name consisting of the mnemonic Cry (or Cyt) and four hierarchical ranks consisting of numbers, capit al let t er s, lower case let t er s and number s (e.g. Cry25Aa1), depending on its place in a phylogenetic t r ee. Thus, pr ot eins wit h less t hen 45% sequence identity differ in primary rank (Cry1, Cry2, etc.), and 78% and 95% identity constitute the borders for secondary and tertiary rank, respectively. This system replaces the old nomenclature using roman numerals 6 . Alignment of the Cry toxins reveals the presence of fi ve conser ved sequence bl ock s common t o a large majority of the proteins. Figure2a shows t he pr esence or absence of each of t hese bl ock s i n subgroups of the toxin family. Also apparent from this figure is the diversity in length between the different protoxins; in particular, one large group contains protoxins that are approximately twice as long as the majority of the rest. The C-terminal extension found in the longer protoxins is not part of the active toxin (it is digested by proteases in the 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 w ith domain swapping by homologous recombination might have caused this extensive range of specificities. How Bacillus thuringiensis has evolved specific toxins to colonize the insect world Ruud A. de Maagd, Alejandra Bravo and Neil Crickmore