Structure, Diversity, and Evolution of Protein Toxins

3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE 10.1146/annurev.genet.37.110801.143042

STRUCTURE,DIVERSITY, AND EVOLUTION OF PROTEIN TOXINS FROM SPORE-FORMING ENTOMOPATHOGENIC BACTERIA

Ruud A. de Maagd,1 Alejandra Bravo,2 Colin Berry,3 Neil Crickmore,4 and H. Ernest Schnepf 5 1Plant Research International B.V., 6700 AA Wageningen, Netherlands; email: [email protected]; 2Instituto de Biotecnolog´ıa, Universidad Nacional Autonoma´ de Mexico,´ Cuernavaca, Mexico; email: [email protected]; 3Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3US, United Kingdom; email: [email protected]; 4School of Life Sciences, University of Sussex, Brighton BN1 9QG, United Kingdom; email: [email protected]; 5Dow AgroSciences, San Diego, California 92121; email: [email protected]

Key Words insects, Bacillus thuringiensis, Bacillus sphaericus, crystal proteins, insecticidal proteins ■ Abstract Gram-positive spore-forming entomopathogenic bacteria can utilize a large variety of protein toxins to help them invade, infect, and finally kill their hosts, through their action on the insect midgut. These toxins belong to a number of homology groups containing a diversity of protein structures and modes of action. In many cases, the toxins consist of unique folds or novel combinations of domains having known protein folds. Some of the toxins display a similar structure and mode of action to certain toxins of mammalian pathogens, suggesting a common evolutionary origin. Most of these toxins are produced in large amounts during sporulation and have the remarkable feature that they are localized in parasporal crystals. Localization of multiple toxin-encoding genes on plasmids together with mobilizable elements enables bacteria to shuffle their armory of toxins. Recombination between toxin genes and sequence divergence has resulted in a wide range of host specificities.

CONTENTS INTRODUCTION: SPORE-FORMING ENTOMOPATHOGENIC BACTERIA ...... 410 THREE-DOMAIN CRY PROTEINS ...... 412 Structure and Function ...... 412 Host Factors Driving Diversiﬁcation ...... 414 Mechanisms of Evolution ...... 415 THE CYT GROUP OF TOXINS ...... 416 THE BINARY TOXIN OF B. SPHAERICUS AND RELATED TOXINS ...... 417 0066-4197/03/1215-0409$14.00 409 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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The BinA/BinB Binary Toxin ...... 417 The Cry34/Cry35 Binary Toxin ...... 417 THE PORE-FORMING MTX TOXINS OF B. SPHAERICUS AND RELATED TOXINS ...... 418 Mtx2 and Mtx3 ...... 418 Mtx2/3 Related Toxins from B. thuringiensis ...... 418 THE MTX1 AND VIP TOXINS ...... 419 Mtx1 ...... 419 Vip1 and Vip2 ...... 420 Vip3 ...... 421 OTHER TOXINS ...... 421 Cry22 ...... 421 Cry6 ...... 422 TOXIN LOCALIZATION AND CRYSTALLIZATION ...... 422 TOXIN GENE ORGANIZATION AND ITS RELEVANCE FOR TOXIN EVOLUTION ...... 424 CONCLUDING REMARKS ...... 425

INTRODUCTION: SPORE-FORMING ENTOMOPATHOGENIC BACTERIA

Gram-positive entomopathogenic bacteria have received considerable attention because of their utility for control of insect pests in agriculture and insect vectors of human disease. Since the discovery of the first such pathogen, Bacillus thuringiensis (Bt), a little over a century ago (7), a number of other entomopathogenic bacterial species have been identified. Sprays of sporulated B. thuringiensis have a long history of safe use for pest control in agriculture, and more recently, sprays of B. thuringiensis subsp. israelensis (Bti) and of Bacillus sphaericus (Bsp)have been used to control disease-carrying mosquitoes and blackflies (6). Since 1996, transgenic crop plants have been commercialized that express entomocidal Cry proteins from B. thuringiensis, rendering these plants resistant to several insect pests (31). The gram-positive entomopathogenic bacteria under review here form endo- spores under adverse conditions and, coinciding with sporulation, form parasporal crystalline inclusions containing one or more insecticidal proteins (Figure 1). These proteins are toxic to insects by ingestion, usually through their action on midgut epithelium cells, and display a vast array of specificities. With one exception, all these bacteria were originally considered different species of the genus Bacillus. Developments in molecular systematics more recently prompted the redistribution of these species into several different genera (99). By far the most intensely studied species is B. thuringiensis. Screening programs have identified thousands of different strains, all of which have a limited host range but together span a wide range of insect orders (Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Orthoptera, and Mallophaga) and even other organisms 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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such as nematodes, mites, and protozoa (44). Bt appears to be a common bacterium found in various ecological niches such as soil, plant surfaces, dust from stored- products, and insects. Crystal production obviously increases the host range of Bt by providing it with a means of access to the nutrients of the hemocoel in a sensitive insect, but the true ecological role of Bt is still a matter of debate. Epizootics of Bt are rarely found, and Bt spores persist for a long time and may even germinate in soil and on plants; hence, it may best be described as a facultative entomopathogen. At the genome level, B. thuringiensis is so closely related to Bacillus cereus, which occupies the same niches but does not produce crystals, that they may be considered to belong to the same species (53). Spores of both Bt and B. cereus may cause septicemia in insects by themselves, and both are also opportunistic human pathogens (2). B. cereus is also a common cause of food-poisoning. This species may also include the human and animal pathogen Bacillus anthracis (containing two unique plasmids encoding virulence functions), although there may be some genetic differences that determine the maintenance of toxin gene-carrying plasmids, which in turn determine whether a bacterium is a human or an insect pathogen. Bacillus sphaericus (Bsp) forms spherical spores together with crystals, which are toxic to dipteran larvae. Some less active strains, which do not produce crystals, produce soluble proteins with activity against dipterans (21). Penibacillus larvae is the causative agent of American foulbrood disease of larvae of honeybee (Apis mellifera). Penibacillus lentimorbus and Penibacillus popilliae are the cause of milky disease of scarabaeid larvae (particularly the Japanese beetle, Popillia japonica, for which P. popilliae has been used as control agent). The Penibacillus insect pathogens are facultative anaerobes, are difficult to grow outside insects, and probably only sporulate in insects, thus making them probable obligate pathogens with poorly understood virulence (113). Brevibacillus laterosporus is also toxic to Diptera, and the more active strains produce a parasporal crystal, which is involved in this activity (91). Finally, anaerobic strains of Clostridium bifermentans with mosquitocidal activity have been identified (5). The most significant factors in determining the wide range of insect, and even some non-insect, species invaded by these entomopathogens are the insecticidal proteins contained in the parasporal crystals (Figure 1). These encompass an in- triguing variety of structures and modes of action, some of which are limited to strains of a single species, whereas others are found in different forms in several species. By far the greatest variety is found in the crystals of Bt, which may contain one or several proteins of the Cry (for Crystal) or Cyt (for Cytotoxic) type. As screening programs proceeded and more new genes were found, it became clear that the crystal proteins could be divided into a number of distinct homology groups (26) (Figure 2). The largest group contains what we call here the 3-domain Cry proteins (Figure 2A). This group contains over 30 different basic types (primary ranks), and over 100 subtypes, all with unique specificities. Curiously, members of this group are also found in P. popilliae (Cry18) (131) and in C. bifermentans (Cry16 and Cry17) (5), although the role of the Cry proteins in pathogenesis for 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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these examples is unclear. Additional Cry proteins, with no obvious homology to the 3-domain Cry proteins, fall into homology groups that may also contain some of the toxins of other bacteria, suggesting a common evolutionary origin. Crystal proteins of B. sphaericus are binary (two-component) toxins called BinA and BinB, which show homology to the Bt Cry35 and Cry36 proteins (Figure 2B). Cry35 proteins are only active as a binary toxin together with the unrelated Cry34 (41). Cry37 and Cry23 form an alternative binary toxin, with Cry23 being part of a diverse homology group that includes crystal proteins from B. thuringiensis serovar thompsoni (Cry15) and serovar dakota, and two (noncrystal) Mtx toxins from B. sphaericus (Figure 2C). Unlike most of the toxins reviewed here, the Mtx proteins and the Vips (vegetative insecticidal proteins) in Bt and B. cereus are produced as soluble proteins during vegetative growth. This review gives an overview of the known insecticidal crystal proteins as well as vegetative toxins of the entomopathogenic spore-forming bacteria. We explore what is known about their structure and their similarity to other toxins, including those of human pathogens, to provide an insight into how toxins may have evolved from common ancestors or have exploited common themes and mechanisms to arrive at biological activity, thereby helping the producing organisms to invade new ecological niches. We also review what we know of the genomic organization of insecticidal toxin-encoding genes and how this may explain the evolution of a new variant and new combinations of toxins.

THREE-DOMAIN CRY PROTEINS

Structure and Function The 3-domain toxins include proteins that are toxic to the insect orders Lepidoptera, Diptera, Coleoptera, and Hymenoptera as well as to nematodes (Figure 2A). These Cry protoxins generally have two different lengths (approximately 130 or 70 kDa) (108). The C-terminal extension found in the long protoxins is dispensable for toxicity (108) and may be involved in crystal formation within the bacterium (see below). The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores in the apical microvillar membranes (108). The crystal inclusions ingested by susceptible larvae dissolve in the environment of the gut, and the solubilized inactive protoxins are cleaved at the long C terminus (when present) by midgut proteases yielding 60-kDa protease-resistant fragments. The alignment of the active toxin sequences revealed the presence of up to ﬁve conserved blocks (108). It was proposed that Cry toxins that possess these blocks would share similar structures (69). The structures of activated toxins (Cry1Aa, Cry3Aa, and Cry3Bb) (46, 50, 69) and a short protoxin (Cry2Aa) (83) were determined and conﬁrmed the remarkably similar 3-domain structure, despite the low sequence similarity (Figure 3). Domain I is a seven -helix bundle where central helix 5 is completely surrounded by six amphipathic outer helices. This domain 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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Figure 3 Three-dimensional structure of the three-domain Cry protein Cry1Aa (PDB code: 1ciy) and comparison of its three domains with similar domains from other proteins (see text). All proteins are indicated by their PDB code. All ﬁgures were made with MolScript (65).

has been implicated in pore formation (108). It shows structural similarities with other pore-forming bacterial toxins, such as hemolysin E (PDB code: 1qoy), colicin Ia and N (1cii, 1a87), and the membrane translocation domain of diphtheria toxin (1ddt). Domain II consists of three antiparallel -sheets sharing similar topology in a Greek key conformation, forming a -prism. This domain plays an important role in interaction with the receptor (58). It has a remarkable similarity to the topology of some carbohydrate-binding proteins: vitelline (1vmo, 75% overlap of equivalent residues as determined by CATH),the lectin jacalin (1jac, 64% overlap), and the lectin Mpa (1jot, 65% overlap). Finally, domain III is a -sandwich of two antiparallel -sheets in a jellyroll formation. It is involved in receptor binding and additionally has a role in pore function (108). Domain III’s structure resembles that 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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of carbohydrate-binding protein domains such as the cellulose-binding domain of 1,4--glucanase CenC (1ulo, 75% overlap), galactose oxidase (1gof, 79% overlap), sialidase (1eut, 75% overlap) (Figure 3), -glucuronidase (1bhg, 60% overlap), the carbohydrate-binding domain of xylanase U (1gmm) and -galactosidase (1bgl, 60% overlap). Structural similarities were also found between domain III of Cry toxins and domains of other bacterial toxins as assessed by Dali (54) that ranked the output according to Z-score, which indicates the strength of structural similarity (where Z < 2 are structurally dissimilar). These are the domain HCN of tetanus toxin, which is involved in polysialogangloside-receptor binding (1a8d, Z 6.2), the C-terminal phospholipid binding domain of -toxin of Clostridium perfringens= (1ca1, Z 3) (35), and the P20 domain involved in heptamer formation of Anthrax protective= antigen PA (1acc, Z 4.8). PA inserts into the membrane and translo- cates the enzymes of anthrax toxin= into the cytosol (98). Finally, there is similarity to domain 4 of the pore-forming toxin aerolysin (1pre, Z 2.1; see below), which is involved in maintenance and stability of the heptameric= toxin complex (68). Besides C-terminal activation, all members of this family are processed at the N terminus. The 3-dimensional structure of Cry2Aa protoxin revealed that the N-terminal region occludes a putative binding region in the toxin (83). A Cry1Ac mutant resistant to trypsin cleavage at the N terminus is affected in binding and pore formation in membranes of Manduca sexta (11). The activated toxin binds to speciﬁc sites on the microvillar membranes before inserting into the membrane and forming lytic pores, followed by cell lysis and insect death (108).

Host Factors Driving Diversification Differences in physiological conditions of the gut such as pH, as well as midgut proteases and toxin receptors, may have been important selective forces for the evolution of toxins. Solubilization of long protoxins depends on the high pH in lepidopteran and dipteran midguts, contrasting with the rather acidic coleopteran midgut (39). Interestingly, most of the Lepidoptera-active toxins have arginine as the predominant basic amino acid rather than lysine. The high pKa of arginine might be required for maintaining positive charge at the midgut pH (32, 50). The main digestive proteases of Lepidoptera and Diptera are serine proteases, whereas those of Coleoptera are cysteine and aspartic proteases (116). Activation is a complex process; in addition to the protoxin proteolysis at the N and C termini, intramolecular processing within domains I and II have been reported for several toxins (19, 71, 79). Cleavage within domain I has been correlated with toxin activation (19, 48, 79). On the other hand, the lack of a major gut protease could result in insect resistance to Cry toxins (90), and the fast degradation of some Cry toxins was associated with low sensitivity for those Cry toxins (63). Interaction of Cry toxins with specific high-affinity receptors on the gut epithelium is a key factor in specificity. In fact, insect resistance to Cry toxins is often correlated with alterations in receptor binding (45). In the case of lepidopteran insects, different glycosylated proteins [aminopeptidase-N (APN), a cadherin-like protein 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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and a glycoconjugate] have been identified as Cry toxin receptors (84, 108, 125). Interaction with cadherin is necessary for toxin activation inducing the cleavage of helix -1 and the formation of a prepore structure that is an important intermediate in pore formation (48). All Cry1A APN receptors are anchored to the membrane by a glycosyl-phosphatidylinositol (GPI)-anchor, and the in vitro cleavage of this anchor prevents Cry1Ac pore-formation activity (74). GPI-anchored proteins are selectively included in differentiated microdomains called lipid rafts. These rafts are involved in signal transduction, sorting and trafficking of plasma membrane proteins (112), and function as pathogen portals for different toxins (18, 102). For several pore-forming toxins, the association of their receptors with rafts is a crucial step in their oligomerization and membrane insertion (1, 18, 102). The APN-receptor is located in rafts, and the integrity of lipid rafts is essential for Cry1A pore-forming activity (132). The regions of Cry toxins involved in receptor interaction, which include both domain II as well as domain III, have been mapped by different methodologies (108). The protruding loops of domain II were identified as important regions involved in protein-protein contacts with the receptors (38, 47, 59). Domain III has also a role in receptor binding as demonstrated by construction of hybrid proteins between different Cry1 toxins (30, 34, 67). Domain III of Cry1Ac has lectin-like properties and binds N-acetylgalactosamine on APN (16, 30, 66). The functional relevance of domains II and III’s similarity to the topology of carbohydrate-binding proteins is not completely clear, but the fact that putative Cry receptors are glycosylated proteins suggests that carbohydrate recognition may play an important role in this interaction. Indeed, resistance in Caenorhabditis elegans to Cry5B and Cry14 toxins was associated to changes in the expression of glycosylation enzymes (49). Also, altered glycosylation in Heliothis virescens correlates with increased resistance to Cry1 toxins (62).

Mechanisms of Evolution Phylogenetic studies suggested that the members of this family evolved from a common origin and that the high diversity of these proteins might be generated by sequence divergence and by homologous recombination that leads to domain III and carboxy-terminal extension interchange among different toxins (10, 32). For example, it was suggested that the Cry9Aa toxin evolved independently from other Cry9 toxins (see Figure 2A) but obtained the same C-terminal extension, probably by a recombination (10, 32). There are multiple clear examples of domain III swapping, particularly among Cry1 toxins (32). Particularly interesting are the toxins with dual (lepidopteran and coleopteran) insect-order specificity, Cry1I and Cry1B toxins, with domains I and II that share high similarity with coleopteran- specific toxins, whereas their domains III are more closely related to those of lepidopteran-active toxins (32). Moreover, interchange of domain III sequences in the laboratory (9, 30, 33) confirmed the hypothesis that domain III shuffling can be a mechanism for generating new specificities in nature. 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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Phylogenetic studies of domain I sequences indicate a correlation between clus- tering patterns and overall insect-order speciﬁcity. Because domain I is involved in pore formation, these data suggest the importance of other factors affecting toxin insertion, such as membrane composition, pH, and proteases in the evolution of these toxins (10, 32). Coevolution of Cry toxins and insects has been hypothesized, and interaction of Cry toxins with their receptors may play an important role in selection of new varieties. In this regard, domain II distribution on the phylogenetic tree correlates with insect speciﬁcity, and phylogenetic relationships of loop regions of domain II correlate with receptor recognition (61, 115).

THE CYT GROUP OF TOXINS

The Cyt toxins are a subset of the Bt crystal toxins, so-named because they possess a general cytolytic activity in vitro, although they show dipteran speciﬁcity in vivo. The structure of one member of this group, Cyt2Aa, has been solved and reveals a single domain, three-layer alpha-beta protein with a unique fold (Figure 4). Currently, two mechanisms of action are proposed for these toxins: Multimers of the toxin form a structured pore within the membrane (100), or the Cyt toxins exert their effect through a less speciﬁc detergent action. These two models, which may not be mutually exclusive, have recently been reviewed by Butko (17). What is well established is that the Cyt toxins synergize the activity of other toxins against a number of target insects (106). Although the mechanism of this synergism remains unclear, recent data (M.C. Wirth, personal communication) suggest that the Cyt toxin may facilitate binding, and perhaps internalization of its synergistic partner. In this respect, although toxic in its own right, the Cyt toxins could be thought of as having the functionality of the B component of the large group of A-B toxins such as in anthrax and diphtheria. cyt1Ca (8), which was found in the recently sequenced plasmid pBtoxis of B. thuringiensis subsp. israelensis, encodes what seems to be a fusion between an

Figure 4 Three-dimensional structure of the Cytotoxic crystal protein Cyt2Aa (PDB code: 1cby). 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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N-terminal Cyt domain and a tandem beta-trefoil module containing C-terminal domain that is homologous to ricin, B. sphaericus Mtx1 (see below), and Pieris brassicae pierisin-b. Thus Cyt1Ca may represent a Cyt protein that has acquired an extra target binding domain, possibly through recombination.

THE BINARY TOXIN OF B. SPHAERICUS AND RELATED TOXINS The BinA/BinB Binary Toxin The binary toxin (Bin) is found in the crystals of all highly mosquitocidal strains of B. sphaericus (Bsp). The crystals are composed of two separate, but homologous proteins, BinA (42-kDa) and BinB (51-kDa), and are deposited within the exosporium at the onset of sporulation (13). Although not many natural variants are known and the differences in these occur in only 6 amino acids of each of the components, there are differences in insect specificity (56). Produced in Escherichia coli, both proteins were required for toxicity to mosquito larvae, and an equimolar ratio of the proteins gave the highest activity (12). However, BinA produced alone in a recombinant Bt strain is toxic by itself (87). Like the Bt Cry proteins, the Bsp binary toxins are solubilized and activated by proteolysis in the target insect’s midgut (14, 29). The toxin binds to specific receptors on the gut epithelium of target insects, and binding affinity is correlated with toxicity (88, 110). The BinB component of the complex is required for strong and regionalized binding (89). The receptor from Culex pipiens larvae, to which mainly BinB binds with high affinity (23), was purified and cloned. It was shown to be a 60-kDa GPI-anchored membrane -glucosidase (28, 111). Binding to the receptor is followed by various sorts of ultrastructural changes in the epithelial cells, suggesting membrane pore formation (22). In vitro assays with artificial membranes have shown that mainly BinA, and to a lesser extent BinB, cause membrane channel formation (109). Thus, although BinA and BinB are homologous and probably have a common ancestor, a divergence in function (channel formation for BinA and receptor binding for BinB) seems to have occurred during evolution of these proteins. However, BinA does seem to have a role in receptor binding in Anopheles gambiae (23). The structure of a fragment of the 51-kDa component corresponding to residues 29 through 421 has been determined and shows an ellipsoid form predominantly of -strands (24; J.P. Allen, personal communication).

The Cry34/Cry35 Binary Toxin The Cry34 and Cry35 toxin families were isolated from Bt strains active on western corn rootworm (Diabrotica virgifera virgifera). A protein from each family is required for the lethal effects of the toxin, and they are therefore referred to as binary insecticidal crystal proteins (41). The symptoms displayed by western corn 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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rootworms exposed to Cry34 and Cry35 are focused on the midgut epithelium (81). The Cry34 proteins are not related to other Bt crystal proteins; however, the Cry35 proteins are homologous to the binary toxin proteins from B. sphaericus described above (Figure 2B), with 26% to 29% sequence identity to the majority of the sequence as well as having several of the conserved sequence elements (41). The Cry35 proteins probably have a similar fold to the Bsp binary proteins. In addition to Cry35, the Cry36 toxin from Bt shares signiﬁcant homology with the components of the Bsp binary toxin. Although this toxin alone possesses weak coleopteran activity, it may be part of an as yet uncharacterized binary toxin.

THE PORE-FORMING MTX TOXINS OF B. SPHAERICUS AND RELATED TOXINS

Mtx2 and Mtx3 Mtx2 and Mtx3 are widely distributed in mosquitocidal Bsp strains (73, 120). The 36-kDa Mtx3 is the least studied of the toxins, and its amino acid sequence seems to be highly conserved. The 32-kDa Mtx2 protein exhibits more amino acid sequence variation; in particular, residue 224 modulates toxicity between Culex quinquefasciatus and Aedes aegypti (20). These toxins are closely related to each other and, more distantly, to a number of Bt crystal proteins (Figure 2C; see below). They also show homology to the cy- totoxin of Pseudomonas aeruginosa, the epsilon toxin of Clostridium perfringens, alpha-toxin of Clostridium septicum, and aerolysin of Aeromonas hydrophila. The last of these three toxins form heptameric beta-barrel lined pores (80). The sequence similarity of the Mtx and related Bt proteins to aerolysin includes the distal portion of a sequence (residues 180–307) that is involved in maintaining the heptameric complex (68) (Figure 5), suggesting a similar mode of action for the insecticidal homologues. Interestingly, aerolysin contains an additional lectin- like domain that facilitates binding of the pore-forming domain (55).

Mtx2/3 Related Toxins from B. thuringiensis Cry15Aa protein from B. thuringiensis serovar thompsoni has signiﬁcant homology to Mtx2 and Mtx3 (Figure 2C). It is toxic to the lepidopteran Manduca sexta, and is found in crystals with a second apparently unrelated protein of apparent molecular weight of 40 kDa (15). The 40-kDa protein has no activity by itself nor does it affect the activity of Cry15A toward M. sexta, but it greatly enhances the activity of Cry15A on codling moth, Cydia pomonella (101). More recently, proteins with about 37% sequence identity to Cry15A and the 40-kDa protein were reported from a strain of B. thuringiensis serovar dakota (64). Like the B. thuringiensis serovar thompsoni genes, the genes encoding these proteins, cry33Aa and cryNT32 respectively, are arranged in an apparent operon. A crystal protein (CryC53) from a B. thuringiensis serovar cameroun strain is also in the Mtx2/Mtx3 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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Figure 5 Three-dimensional structures of the Cry23/37 complex (left) (104) and of a monomer of proaerolysin (PDB code: 1pre), with the domain numbers indicated. Aerolysin residues residues 180–307 in domains 2 and 3, which have sequence similarity to the Mtx2/Mtx3-like toxins, are highlighted in black.

homology group, along with another uncharacterized protein (CryC35), which is homologous to the 40-kDa and CryNT32 proteins above. Two additional Mtx2/3- like proteins from Bt are Cry38, of unknown activity, whose gene is found in association with those encoding Cry34 and Cry35 (103), and Cry23A. The 29-kDa Cry23A protein functions effectively only with the 13–14-kDa Cry37 protein on certain Coleoptera, notably boll weevil (Anthonomus grandis) (37). The sequence of Cry37 currently shows no signiﬁcant homology to any protein in the public databases. The crystal structure of Cry23Aa in association with Cry37Aa has been determined (104) (Figure 5), and both proteins were shown to consist mainly of -strands. Cry23Aa is an elongated structure dominated by antiparallel -sheets that is capable of forming channels in planar lipid bilayers, and has a structure and shape reminiscent of domains 2–4 of the toxin proaerolysin in Aeromonas hy- drophilia (see Figure 5) (94). Cry37, which binds near one end of Cry23, adopts a C2 -sandwich fold, like that observed in the calcium phospholipid binding domain of human cytosolic phospholipase A2 (97; T.J. Rydel, personal communication). Cry37 and the 40-kDa homology group proteins may function to facilitate binding of the channel-forming toxins analogous to the lectin-like domain of aerolysin.

THE MTX1 AND VIP TOXINS Mtx1 Mtx1 is a 100-kDa protein thought to be the principal toxin active in many of the so-called low-toxicity Bsp strains and, like Mtx2 and Mtx3, is also widely 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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distributed among most, but not all, high-toxicity strains (72, 119). Produced during the vegetative stage of growth, Mtx1 contributes little to the toxicity of Bsp since an inverted repeat region upstream of the gene serves as a repressor binding site (C. Berry & M.J. Humphreys, unpublished results), which leads to low-level production of the protein. Furthermore, the toxin is unstable in B. sphaericus as it is the target for degradation by a host serine protease (128). However, when expressed in E. coli, a 97-kDa form of the protein (lacking the signal peptide) was stable and showed potent mosquitocidal activity (95, 119). The Mtx1 protein has a C-terminal region with internal repeat elements (118), like the Cyt1Ca protein mentioned earlier, that are characteristic of the ricin-like beta trefoil repeats thought to be involved in carbohydrate binding (52). The N- terminal region contains areas of homology with known bacterial toxins, such as the pertussis and cholera toxins, that act by ADP-ribosylation of target G proteins (119). These toxins are two subunit (AB) toxins with an enzymatically active A subunit and a receptor binding B moiety that facilitates entry of the A peptide. Mtx1 is produced as a single polypeptide chain (A/B) by Bsp but on exposure to gut proteases from either susceptible or nonsusceptible mosquitoes is cleaved into an N-terminal 27-kDa protein (A subunit) and a 70-kDa C-terminal protein (B subunit) (118). ADP-ribosyl transferase activity has been demonstrated for the N-terminal region (107, 117) but the actual target in mosquito cells is yet to be determined. A close noncovalent association of the 27-kDa and 70-kDa proteins following Mtx1 activation (107) may help to deliver the A subunit on B subunit binding.

Vip1 and Vip2 Screening of cultures from B. cereus and B. thuringiensis has led to the discovery of novel toxins that are secreted during vegetative growth by many different strains. Although some Bt Cry proteins are also produced during vegetative growth, these new toxins do not form crystals, have no homology to the known crystal proteins, and hence were not called Cry, but rather Vip toxins. The vip2A(a) and vip1A(a) genes of strain AB78 of B. cereus are located in one operon and encode proteins of 52 kDa and 100 kDa, respectively (126, 127). Both contain a typical N-terminal signal sequence for secretion, and Vip1A(a) is further N-terminally processed to an 80-kDa protein. Both are required together for activity against some, but not all, coleopteran larvae (Diabrotica spp.) that were tested, thus constituting a binary toxin. Sequence homology searches suggest that the Vip1/Vip2-complex is a typical binary toxin of the A B type, where Vip2 is the cytotoxic A-domain and (part of) Vip1 contains the receptor-binding+ B-domain, and possibly a translocation domain. Indeed, Vip2 shows homology to the enzymatic domain of the toxin (CdtA) of Clostridium difﬁcile and the iota-toxin domain Ia of C. perfringens, which both have ADP-ribosyltransferase activity. X-ray diffraction analysis has conﬁrmed that Vip2 consists of two structurally similar domains, where the C-terminal domain has the enzymatic activity and shows high structural similarity with the active 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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C-terminal domain of iota-toxin (51, 124). The N-terminal domain, which is enzymatically inactive, may have diverged to enable interaction with Vip1. Vip1 is homologous to the CdtB toxin component of C. difﬁcile, the Ib-component of iota toxin of C. perfringens, and the protective antigen (PA) of B. anthracis. Analogous to the mode of action of these toxins, Vip1 may be binding speciﬁc receptors and, upon binding, form a channel-forming oligomer that allows translocation of the enzyme domain. The cellular target of Vip2 activity is not yet known, but because of its high homology with the other toxins, is most likely a form of actin.

Vip3 The vip3A(a) gene of strain AB88 of B. thuringiensis encodes an 88.5-kDa protein active against several, but not all, lepidopteran larvae tested, without sequence homology to any known protein (42, 43). It does not contain a typical N-terminal signal sequence (see below), and is not processed during secretion. In larval midgut ﬂuid this protein is processed by proteases at several sites, leaving an active 33-kDa fragment (amino acids 200–455). Vip3A binds to midgut epithelium of susceptible larvae but not to that of an insensitive species, causing cell death in a process resem- bling apoptosis (130). This suggests that binding is important in determining insect speciﬁcity. A cDNA encoding a putative receptor for Vip3A(a) was isolated from Agrotis ipsilon. It encodes a putative membrane protein with repeated EGF (epider- mal growth factor) domains showing homology to glycoproteins called tenascins or hexabranchion, as well as containing homology to the so-called death domain (42). Vip3A production by germinating spores is an important factor in the combined toxicity of spores and relatively inactive Cry toxins against several insects (36).

OTHER TOXINS

Cry22 The Cry22 proteins were initially identiﬁed in Bt strains active on ants (order: Hymenoptera) (96). Subsequently, additional Cry22 proteins were characterized with activity on certain Coleoptera, namely corn rootworm (Cry22Ab) (77) and boll weevil (Cry22Ba as well as the Cry22A proteins) (57). The proteins have apparent molecular weights of 75 to 86 kDa. The Cry22A proteins have four imperfect repeats of about 80 amino acids between residues 261 and 575, and Cry22Ba lacks essentially the ﬁrst of the repeats, accounting for its shorter length (57). A BLAST analysis (4) indicates that these repeated sequences are similar to cadherin-like repeated sequence elements found in some bacterial chitinases separating catalytic and chitin binding domains, perhaps functioning as a spacer (82). Similar repeated elements are also found in surface anchored proteins found in other low G C gram-positive bacteria, such as the C protein alpha antigen of group B streptococci+ (70). Although no mode of action studies have been reported, an X-ray crystallographic analysis indicates the protein is an elongated, 6-domain 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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protein with a 242-residue N-terminal domain, four cadherin-like domains, and a 142-residue C terminus with a fold similar to domain III of the 3-domain Cry proteins, which suggests a binding function (105).

Cry6 The Cry6 proteins were initially found in Bt strains active on nematodes (85, 86). Cry6A is active on several nematode species (129) as well as on corn rootworm (123), although Wei et al. recently failed to show toxic action of Cry6B against nematodes. The 390-residue Cry6Ba aligns to residues 1–387 of the 475-residue Cry6Aa. Truncation studies showed that a gene fragment of amino acids 11– 382 of Cry6A retains most of the activity on Caenorhabditis elegans (129). Two sequences in GenBank are similar to Cry6. YokGand YnzF were identiﬁed from the Bacillus subtilis genome (accession: CAB14078 and CAB13632, respectively) but represent currently uncharacterized proteins. The YokG protein, which shares 34% sequence identity with Cry6A, had previously been identiﬁed as being encoded by the SPbetac2 prophage of B. subtilis.

TOXIN LOCALIZATION AND CRYSTALLIZATION

A remarkable feature of the insecticidal toxins discussed in this review is that the vast majority are localized within crystalline inclusion bodies. The rarity of similar crystalline structures and the diversity of sequence and structure of the insecticidal proteins suggest that they have convergently evolved to be able to form these crystals. Suggestions for the purpose of a crystal include the need to package a high dose of toxin in a form that can survive in the environment for at least a limited time post-sporulation. The size and insolubility at neutral pH of the crystal would prevent it being rapidly leached away in the soil, for example. Consideration of the structural similarities between the toxins and other known proteins gives no indi- cation of conserved domains that might be involved in directing crystal formation. To date the only region implicated in crystallization is the C-terminal extension to some of the 3-domain toxins. Expressions of C-terminally truncated toxins, engi- neered for production without the extension, do not form crystals (93). Compared to the other three domains, the C-terminal extension to the larger toxins is more conserved, suggesting either a crucial function or a more recent evolutionary addition. Interestingly, three of the toxin genes cloned from Bt (cry10Aa, cry39Aa, and cry40Aa) do not code for an extension but in each case an open reading frame encoding an extension homologue is found some 100 bp downstream. The region separating the two open reading frames shows no homology to any known transposon or insertion sequence, yet this remains a possibility as to how the toxin gene and its extension became separated. An alternative possibility is that individual toxin genes are acquiring the extension through a recombination or transposition event. Despite the evidence showing the importance of the C-terminal extension in 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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crystallization, many of the toxins lacking the extension, for example Cry3A and Cry11A, are clearly capable of forming crystals in vivo. The fact that expression of the toxin genes in a range of gram-positive and gram-negative bacteria and even in planta results in crystal formation argues against speciﬁc host crystallization factors. Notable exceptions to the innate ability of the toxins to crystallize are the Cry2A toxins. These toxins are found as the third gene in a three-gene operon in which the product of the second gene (Orf2) is required for the formation of Cry2A crystals (114). The Orf2 crystallization factor is a protein largely consisting of 11 tandemly repeated 15 or 16 amino acid motifs. The acidic nature of these repeats provides an interesting parallel with reports that some toxin crystals contain DNA that may play a role in the formation of the crystals (25). Either the DNA or Orf2 molecules may form a scaffold around which the crystal is built using strategic ionic interactions. Interestingly, examination of the coding region of a number of other Bt protoxins reveals the presence of similar tandem repeat units C-terminal of the active toxin (Figure 6). Whether the toxin-encoded repeats have a similar crystallization facilitating function as they do in Orf2 is not known. We have compared the sequences of the repeat units found associated with these toxins (Follow the Supplemental Material link from the Annual Reviews home page at http://www.annualreviews.org/supmat/supmat.asp). A notable feature is that they are relatively rich in asparagine and glutamine. The Q/N richness of these repeats is particularly interesting given the involvement of Q/N-rich proteins in prion formation in eukaryotes (78). Although prions have not been described in bacteria, there are potentially similar interactions to those involved in the formation of amyloid plaques. The C-terminal extension is clearly important for the crystallization of certain toxins but appears to have become redundant in others. In addition to the

Figure 6 Diagrammatic representation of repeat motifs associated with some entomocidal toxins. Unless speciﬁed otherwise the open boxes represent the toxin proteins, the internal black boxes representing the presence of conserved regions (108). Each gray oval represents the presence of a single repeat motif. 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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crystalline inclusion bodies the presence of toxins has also been recorded within the protective layers of the spore of B. thuringiensis, where they can affect its germination competence (40). This interaction is believed to be mediated by disulﬁde bonding between the cysteine-rich C-terminal extensions and the disulﬁde-rich spore coat proteins (40). This and the observation that the C-terminal extension bears some sequence similarity to the CotA spore coat protein from B. subtilis lend some weight to the suggestion that at least this portion of the toxin may have evolved from a spore protein. In contrast to the crystalline toxins, a number of the insecticidal toxins have been reported as being secreted from the host bacterium (Figure 1). Although two of these (Vip1 and Vip2) contain canonical signal sequences, others (Cry1I, Cry16A, Cry17A, Vip3A) do not. N-terminal sequencing of the secreted forms of the latter three toxins additionally reveals the lack of N-terminal processing. The Mtx proteins all contain putative signal sequences, but their location in- or outside the cell has not been established (An analysis of putative signal sequences is available online. Follow the Supplemental Material link from the Annual Reviews home page at http://www.annualreviews.org/supmat/supmat.asp). Although the secreted Cry proteins (Cry1I, cry16A, Cry17A) are obviously homologous to the crystalline 3-domain Cry proteins, these proteins may have become part of an alternative secretory system. Such systems have been described in other pathogenic gram-positive bacteria (92).

TOXIN GENE ORGANIZATION AND ITS RELEVANCE FOR TOXIN EVOLUTION

Several factors are relevant in consideration of the evolution of Bt toxins: 1. Recombination: The Cry proteins of Bt are clustered into groups of related sequences as reflected in the current toxin nomenclature (27). Clearly, closely related toxins share significant homologies at the nucleotide level that may permit homologous recombination to reassort toxin gene sequences. Even between more distantly related toxins, we may find blocks of homology (Figure 6) that may also permit rearrangement of toxin gene sequences. Ev- idence for this kind of recombination event was discussed in the previous sections. 2. Transposition: Most Bt toxin genes are located close to sequences that appear to be related to transposition (75). This provides an obvious means for mobi- lizing the toxin sequences between resident plasmids and between plasmids and the host chromosome. Such transposition will permit new toxin gene assortments to be assembled within individual Bt strains. The recently published sequence of the toxin-coding plasmid from Bti, pBtoxis (8), shows numerous transposition-related sequences throughout, indicating that this process is involved not only in the movement of toxins but also in the evolution of the plasmids on which they may be carried. 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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3. Plasmids: Most Bt strains bear their toxin genes on large plasmids and although there is no evidence that the toxin-coding plasmids are self- mobilizable, other resident plasmids cause such mobilization, e.g., pX016 from Bti (60). Plasmid transfer between other Bt strains has been shown both in vitro and in insect larvae (121, 122). The sequence of pBtoxis from Bti (8) reveals several features of interest. Six of the seven toxin genes borne by this plasmid are clustered within an approximately 30-kb region of the 129-kb sequence. However, there is no evidence to suggest that this region represents a “pathogenicity island” within the plasmid as many of the toxins seem to be flanked by transposon sequences that would suggest that they were acquired independently. In addition to full-length toxin genes, the plasmid also bears what appear to be vestigial fragments of toxin coding sequences. These may be evidence of toxin evolution and may represent fragments of genes left behind after recombination or transposition events (each partial coding sequence has a nearby transposon sequence). Although many factors may contribute to the ability of Bt to increase the diversity of its toxin genes, the utility of many of the possible variants may be limited. One such limitation has been demonstrated by Almond & Dean (3) who showed that many chimeric variants of the Cry1A family are sensitive to degradation by host bacterial proteases. The fact that the majority of toxin genes identified seem to encode active toxins may imply a strong selection pressure to maintain activity, although the mechanism of this selection is not clear. A further interesting phenomenon is the target specificity of many Bt strains. For instance, Bti carries seven toxin genes and, for the five toxins for which specificity has been determined, all have demonstrated activity against mosquitoes and none of the four Cry toxins is active against non-dipteran insects. Bt strains with such limited target ranges are common and may suggest an evolutionary pressure on Bt strains to specialize in killing certain target insects rather than becoming generalist killers of all classes of insect by encoding proteins active against, for example, Diptera, Lepidoptera and Coleoptera. Factors such as impeding insect resistance and enhancing activity caused by synergism of multiple toxins against a single target are perhaps involved in this selection process.

CONCLUDING REMARKS

Entomopathogenic bacilli have evolved a large armory of toxins, as well as other virulence factors not discussed in this review, to help them better invade their host species. This obviously can help to colonize new ecological niches, but there must be a trade-off or ﬁtness cost to obtaining the ability to produce crystal proteins, since noncrystal-producing B. cereus and crystal-producing B. thuringiensis co- exist in the same habitats. Some of that trade-off probably comes from investing large resources into producing a protein crystal, and the negative effect on spore germination has been mentioned. It is also surprising that so many resources go 3 Nov 2003 16:51 AR AR201-GE37-16.tex AR201-GE37-16.sgm LaTeX2e(2002/01/18) P1: GCE

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into producing a crystal when often speciﬁc Bt strains are found in habitats with no obvious hosts for infection around, which has prompted speculation about other roles for the crystal (76). On the other hand, the co-occurrence of different toxins effective on the same insect in one strain (see previous section) does argue for a host-driven selection of speciﬁc combinations of toxin genes. With several insecticidal toxin structures already determined and some novel ones about to be published, it becomes clearer that entomopathogenic bacteria use a variety structural themes and modes of action to attack the insect gut. Structural and sequence homology suggest an evolutionary link with a number of known mammalian-active toxins, whereas in other cases such as the 3-domain Cry proteins and the Cyt proteins, the structure is (so far) unique, or as for the former, may be considered a novel combination of pre-existing domains involved in pore formation or in ligand binding. Further sequence divergence and recombination (domain swapping) has then led to a virtual burst of diversity.

ACKNOWLEDGMENTS We thank several colleagues for making available unpublished results for this review. Many colleagues have contributed to this ﬁeld, but space limitations did not allow all their contributions to be included. For this we apologize and we encourage readers to check previous reviews referred to in this paper to trace back the history of this research ﬁeld. We thank Dr. Jules Beekwilder (PRI) for assistance with molecular graphics. RdM was supported by Program subsidy 347 of the Dutch Ministry of Agriculture, Nature Management and Fisheries.

The Annual Review of Genetics is online at http://genet.annualreviews.org

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Figure 1 Schematic overview of the entomopathogenic spore-forming bacteria and their protein toxins. Localization in crystals or outside the cell (secreted) is depicted as far as known from literature. Mtx proteins contain signal sequences for secretion, but their localization has not been experimentally determined. Similar colors for toxins identify members of the same homology group (yellow for 3-domain Cry proteins; red-orange for Mtx2/3-like proteins; green for Bin-like proteins). HI-RES-GE37-16.qxd 11/3/2003 10:07 PM Page 2

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Figure 2 Phylogenetic relationships between the entomocidal toxins. Four distinct homology groups have been identified within the family of toxins (26), three of which are shown here. The Cyt group is not shown. The branches are color coded according to insect order specificity of the toxin where known: red, Coleoptera specific; green, Lepidoptera specific; blue, Diptera specific; magenta, nematode specific; yellow, Hymenoptera specific. Solid and dashed double-headed arrows indicate associations of proteins in binary toxins or suspected binary toxins, respectively. Panel A: 3-domain Cry proteins. The C-terminal extension (if present) was not included for the phylogenetic analysis. Hence, proteins that have the same primary rank based mainly on sequence similarity between their C-terminal extensions may sometimes end up on different branches. Panel B: Binary and Bin-like toxins; Panel C: Mtx2, Mtx3 and Mtx2/3-related proteins.