Review. One Hundred Years of Bacillus thuringiensis Research and Development: Discovery to Transgenic Crops

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1 Journal of Insect Biotechnology and Sericology 70, 1-23 (2001) Review One Hundred Years of Bacillus thuringiensis Research and Development: Discovery to Transgenic Crops Takashi Yamamoto Maxygen, Inc., 515 Galveston Drive, Redwood City, California 94063, USA. (Received January 9, 2001; Accepted January 10, 2001) Bacillus thuringiensis (Bt), a bacterium known for its insecticide activity, was discovered in 1901 by a Japanese scientist who was studying a silkworm disease. The ailment, known as the "sotto" disease, is very virulent as it kills the insect almost instantaneously. He observed that death occurred before the bacterium multiplied and suggested that a toxin, not bacterial infection, was involved in the early stage of the pathogenicity. Modern biochemical studies, such as isolation of the protein responsible for insecticidal activity, began in the 1950's. During the same period, the first large scale, commercial production of sprayable Bt formulations in the U.S. took place in California. The industry flourished after a high potent strain, Bt kurstaki HD1, was reported in In 1981, the first gene for the crystalliferous insecticidal protein (crystal protein) was cloned from a variant strain of HD1 and sequenced. Since then, numerous genes have been cloned and characterized. These genes, called cry and cyt, have been classified into some 30 different groups by amino acid sequences of the proteins encoded by the genes. Extensive studies in biochemistry and molecular biology of the crystal proteins and their genes have been conducted. Receptor genes for the Bt crystal protein have been cloned, and regulation of the cry gene expression in Bt has been thoroughly studied. Technical and commercial success of transgenic crop plants expressing Bt insecticidal protein genes encourages further study on this scientifically and politically interesting subject matter. Key words: Bacillus thuringiensis, crystal protein, bioinsecticide, commercial development, transgenic crop, one hundred years INTRODUCTION Bacillus thuringiensis (Bt) is a spore-forming, rodshaped, gram-positive bacterium closely related to Bacillus cereus. A large number of Bt isolates have been found and grouped into subspecies, such as Bt thuringiensis, Bt kurstaki, Bt aizawai, etc., based on the classification scheme originally developed by Bonnefoi and de Barjac (1963). Bt is clearly distinguished from other bacilli by the production of intracellular crystals. During the sporulation process, Bt synthesizes massive amounts of one or more proteins which crystallize in a variety of shapes. Frequently, these crystal *Fax: Tel: maxygen.com proteins are highly toxic to specific insects. Besides the crystal protein, Bt is known to secrete additional insecticidal toxins. Heimpel (1967) designated Bt insecticidal toxins, including exotoxins and crystalline endotoxins, using Greek alphabets. Therefore, the crystalline toxin (or crystal protein as it is called in this article) is also known as "(s-endotoxin" by many researchers to distinguish it from other Bt toxins. Among all microbial insecticides, Bt has been the most successful commercial product. Because of its economic importance, intensive studies have been conducted. The gene coding for the crystal protein is called "cry" because of its crystal producing phenotype. The first cry gene, later designated as crylaa, was cloned about 20 years ago by Schnepf and whiteley (1981). Since then, numerous reports of cloning additional cry genes have been published. Bt has shown to produce a variety of crystal proteins that differ in insect specificities, even within one strain. More recently, selected Bt cry genes have been expressed in crop plants for insect control. Phenomenal technical and commercial successes have been obtained in corn and cotton with the crylab or crylac gene. In this article, a history of Bt research and development, especially research at the molecular level, is reviewed. A special attention is made to those who reported pioneer discoveries of agricultural importance. For recent progress, one should refer to an extensive review published by Schneff et al. (1998). More recently, a book (Charles et al., 2000), containing excellent reviews on entomopathogenic bacteria including Bt, has been published. DISCOVERY AND EARLY COMMERCIAL DEVELOPMENT In 1911, a German scientist, Ernst Berliner (1911),

2 2 Yamamoto isolated a rod-shaped bacterium from diseased Anagasta kuehniella found in Thuringia, Germany. He named the bacillus Bacillus thuringiensis Berliner. Ten years earlier in 1901, almost 100 years ago, a Japanese scientist, Sigetane Ishiwata, reported the isolation of a similar bacterium from diseased silkworm, Bombyx mori, larvae (Ishiwata, 1901) (Fig. 1). He was studying a silkworm disease, called "sotto-byo" or sotto disease, which kills the larvae almost instantaneously. Sotto means in Japanese sudden collapse. Therefore, he named the bacterium Bacillus lotto. In subsequent report (Ishiwata, 1905), he mentioned that death occurred before the bacterium multiplied and suggested that a toxin was involved in the pathogenicity of Bt. Since his reports were written in Japanese, the western world believed that Berliner was the first to discover Bt. During the following 10 to 15 years, several scientists reported repeatedly that the insecticidal activity was attributed to a toxin that was produced at the end of bacterial growth. In the 1950's, Edward Steinhaus at the University of California, Berkeley, promoted the use of Bt as an insect control agent (Steinhaus, 1951). He grew Bt on an agar-based medium. Steinhaus collected the spores and crystals and successfully demonstrated in the field that Bt efficiently controlled insect pests. Encouraged by this success, he persuaded a fermentation company, Pacific Yeast, to develop a Bt based insecticide product. Production began at Pacific Yeast's facility in Wasco, California. This is likely the first large-scale commercial production of a Bt insecticide formulation in the U.S. The business was then sold to Bioferm who developed a product called "ThuricideTM." Bioferm obtained, from the U.S. Food and Drug Administration, an exemption from residue tolerance for Bt products in agricultural use based on its safety for human, other animals, beneficial insects, etc. In Japan, Keio Aizawa at Kyushu University promoted the use of Bt as a sprayable insecticide. However, he met strong opposition from the silkworm industry who was concerned that Bt would spread the sotto disease. This concern was somewhat unsubstantiated as there were no reports of Bt epidemics in wild insect populations. Aizawa made extraordinary efforts to overcome the opposition by demonstrating the safety and environmental compatibility of Bt. On the other hand, a chemical company, Toa-Gosei, developed a formulation in which spores were "killed" in order to eliminate the concern with the sotto disease. Toa-Gosei obtained the first product registration and enjoyed early market dominance. This approach, however, hindered the development of ordinary formulations containing live spores that were sought by other companies. This episode is similar to the current controversy on transgenic food crops containing a Bt gene. More recently, a group of scientists are trying to raise issues, such as Bt killing non-target insect species and Bt's similarity to human pathogenic bacilli. There is sufficient documentation that Bt never has caused any substantial harm to humans even though a large amount of Bt formulations have been released every year into the environment (McClintock et al., 1995). Fig. 1 The first page of Ishiwata's report on the discovery of Bt (Ishiwata, 1901). Ishiwata wrote that H. Nomura at the Tokyo Agricultural Experimental Station isolated a similar Bacillus prior to Ishiwata's discovery. INSECTICIDAL ACTIVITY IS ATTRIBUTED TO THE CRYSTAL PROTEIN Bt has been often isolated as a pathogen from sick or dead insects. Insects killed by Bt can be found with bacterial cells and spores in their bodies suggesting that the death is caused by an infection. Even though a good number of reports had indicated that a toxin or toxins were responsible for insecticidal activity, some scientists thought that a spore count alone was a good indicator of the potency of commercial formulations.

3 One Hundred Years of Bacillus thuringiensis 3 This was based on the assumption that each spore contained one crystal with a certain amount of the insecticidal toxin. In fact, during a period in the 1960's, Bt commercial formulations were produced based on spore counts, and quantitative analysis of the crystal content in the products was often ignored. An unfortunate result of this widespread misunderstanding was Bt commercial formulations that contained only a small amount of toxins but a high spore count. Such products with a little toxin exhibited low insect control in the field. Production of modern formulations began after Dulmage (1970) found a high potency strain, Bt kurstaki HD 1. In the 1950's, two Canadian groups demonstrated that the crystal protein was mainly responsible for the insecticidal activity of Bt. The Canadian studies were published prior to the period when the industry thought that the spore was more important for the activity than the crystal protein. Hannay and Fitz- James (1955) reported a method to extract the crystal protein. Subsequently, Angus (1956) purified the crystal protein from the spore-crystal complex of a Bt sotto isolate and confirmed Ishiwata's observation that the insecticidal activity was due to the crystal protein. Bt CRYSTAL IS ALKALINE SOLUBLE Most protein purification techniques are for soluble proteins, and this is the case with the Bt crystals. NaOH was first used to dissolve the crystals. Hannay and Fitz-James (1955) solubilized the crystal in NaOH at ph Angus (1956) treated the crystal and spore complex with 0.05 M NaOH and showed that no insecticidal activity remained in the insoluble fraction (i.e., spores). These reports indicated that alkalinity higher than ph 12 was required for the complete dissociation of the Lepidoptera-specific, Cry 1-type protein. Nishitsutsuji-Uwo et al. (1977), however, found a sharp decline in toxicity when Bt aizawai crystals were exposed to an alkaline ph above ph 11. In order to avoid the activity loss at high ph, many researchers used chemicals that broke the disulfide bond. These chemicals included dithiothreitol (DTT) and 2-mercaptoethanol. Lecadet and Trefouel (1966) showed that the crystal was successfully dissolved in a solution containing a disulfide bond-reducing agent at a ph as low as ph 10. Huber et al. (1981) found that disulfide bonds of the crystal protein in the crystal were not accessible by the agent unless the crystal matrix was loosened in alkali. Once the crystal was solubilized, the crystal protein remained soluble at the neutral ph without a reducing agent. Bt CRYSTAL CONTAINS PROTEASES In the 1970's, there were numerous papers describing the use of SDS-PAGE to determine the molecular weights of the Lepidoptera-specific crystal proteins. The reported molecular weights were very contradictory. ranging from several thousands to well over 200,000. Most of the early reports, showing multiple proteins in the crystal, might be artifacts caused by proteases. Later, the gene cloning revealed that the protein, found in an ordinary bipyramidal Bt crystal that was often Lepidoptera-specific, had a molecular weight around 135,000. Zalunin et al. (1979) reported that the number of crystal proteins resolved by SDS- PAGE was reduced when the crystal was dissociated under a condition that the contaminating proteases were inactivated. MULTIPLE COMPONENTS OF Bt CRYSTALS The crystals of some Bt strains contain proteins other than the 135-kDa protein. Often, these strains are known to have additional specificities toward insect species other than those of Lepidoptera, for example, Bt kurstaki strains which produce Cry2-type proteins. Some of the multiple crystal components are distinguishable by immunoassay. Norris (1969) reported three antigens in the crystal of a Bt tolworthi strain. Krywienczyk et al. (1978) analyzed the crystals of many different Bt strains by immunodiffusion. She found the occurrence of two different crystal protein types even in one subspecies. She also found that the same types of proteins existed across different subspecies. These findings have been confirmed subsequently by molecular biologists who have shown that many Bt strains contain more than one crystal protein gene and that the genes are on plasmids which can be transferred by mating to different subspecies. Bt kurstaki HD 1 was isolated from Pectinophora gossypiella in Texas (Dulmage, 1970) and was shown to have a relatively wide spectrum against different insect species. Because of its spectrum, the strain has been extensively used in commercial products. Sharpe and Baker (1979) examined the HD 1 crystals by electron microscopy and found a foreign object partly embedded in the bipyramidal crystal of HD 1 (Fig. 2). On the other hand, Hall et al. (1977) reported a significant mosquito larvacidal activity in HD 1 and in some other Lepidoptra-specific Bt strains. Based on these reports, Yamamoto and McLaughlin (1981) isolated a 65-kDa protein from HD 1 crystals. The isolated protein was found to be toxic to mosquito larvae. This protein termed "P2" (now designated as Cry2A) was a minor

4 4 Yamamoto Fig. 2 Bt kurstaki HD1 crystals. The made of Cry1A proteins and the square Gene Sharpe, USDA, gave this picture and indicated more than one protein in bipyramidal crystal is crystal of Cry2A. Dr. to the author in 1979 the HD1 crystals. component of the HDI crystals and was immunologically distinct from the major component of the 135-kDa protein called "PI" (CrylA). They reported that Cry l A was toxic to only Lepidoptera larvae, but Cry2A was toxic to both Lepidoptera and Diptera larvae. Yamamoto and lizuka (1983) further characterized Cry2A and found that HDI synthesized Cry2A prior to the synthesis of Cry IA. Cry2A was crystallized in a cuboidal form, and CrylA was often crystallized partly wrapping around the cuboidal Cry2A crystal. Moreover, Yamamoto (1983) reported that some other Bt isolates that belong to subspecies thuringiensis, tolworthi, galleriae, and kenyae also produced Cry2A or a protein similar to Cry2A. Over a decade after the isolation of Cry2A, the gene coding for Cry2Aa were cloned and sequenced by Donovan et al. (1988a). The structure of Cry2Aa has been determined by Morse et al. (1998). Cryl-TYPE 135-kDa PROTEIN IS A PROTOXIN Over 40 years ago, Angus (1956) demonstrated that the crystal protein of Bt sotto did not kill a Bombyx mori larva when the protein was injected into the body cavity. When ingested orally, the same protein was highly toxic to the larva. He also showed that the crystal protein, which had been digested in the gut juice, killed the larva by injection. His report indicates undoubtedly that the crystal protein is a protoxin that has to be activated by proteases in the insect gut juice. Since then, numerous reports have attempted to characterize the activated toxin. In many cases, the size exclusion column chromatography was used to isolate the activated toxin. This chromatography separates molecules based on their sizes. For this reason, the molecular weight of the toxin was often estimated during separation from a few kda to over 100 kda. The size discrepancy may be due to aggregation of the activated toxin. The activated toxin, especially CrylA type, tends to aggregate under conditions normally used in column chromatography. SDS-PAGE eliminates the aggregation problem. Lilley et al. (1980) determined by SDS-PAGE the molecular weight of the trypsin-activated toxin as 70,000. The toxin was isolated from Bt tolworthi by column chromatography. At that time, there were several reports claiming that small fragments of the crystal protein, presumably resulting from protease digestion, were insecticidal. It was noteworthy that Lilley et al. (1980) confirmed that the small peptides were not toxic. Since then, an N-terminal part of the 135-kDa Lepidoptera-specific crystal protein, about one half of the molecule, has been found resistant to proteases, and this protease-resistant domain is fully responsible for insecticidal activity. A NEW CLASS OF CRYSTAL PROTEIN FOUND IN Bt israelensis In 1977, Goldberg and Margalit (1977) discovered a new subspecies of Bt that was highly pathogenic to Diptera, such as mosquito and blackfly larvae. The new isolate designated as Bt israelensis produces several differently shaped crystals. Tyrell et al. (1981) analyzed by SDS-PAGE the crystal complex that had been dissociated in an alkaline solution. This analysis revealed several proteins with molecular weights ranging from 10 to 120 kda including a major 27-kDa (otherwise referred as 28 or 26 kda) protein. Since then, molecular biology has established that Bt israelensis contains genes for four proteins, two proteins around 130 kda, one 72-kDa, and one 27-kDa protein. As early as 1983, Thomas and Ellar (1983) published a clear SDS-PAGE pattern showing these proteins. An unambiguous SDS-PAGE pattern like the one reported by them indicated a high integrity of the crystal preparation, which was presumably free from protease contamination.

5 One Hundred Years of Bacillus thuringiensis 5 Bt israelensis has a unique biological activity. Thomas and Ellar (1983) made an important discovery that an extract of Bt israelensis crystals was cytotoxic not only for cultured insect cells but also for mammalian cells including red blood cells. It was a surprise to many Bt researchers that an injection of the crystal extract killed suckling mice. This discovery of hemolytic activity was the first to show that Bt produced a crystal protein with specificity for animals other than insects. Consequently, Davidson and Yamamoto (1984) isolated a 25-kDa protein (processed from the 27-kDa protein) and showed that the protein was hemolytic. Armstrong et al. (1985) determined the N-terminal sequence of the 25-kDa protein. After the 27-kDa protein gene was cloned and sequenced, the N-terminal sequence of the 25-kDa protein was found to start at the 30th amino acid residues of the 27-kDa protein. Apparently, the 27-kDa protein was cleaved between the 29th and 30th amino acid residues during purification. Chestukhina et al. (1985) successfully isolated the intact 27-kDa protein and determined its N-terminal amino acid sequence. Chestukhina et al. (1985) isolated a 70-kDa protein from Bt israelensis crystals and sequenced its amino terminal sequence. This protein is now designated as the 72-kDa Cry llaa protein. Ibarra and Federici (1986) found with an electron microscope that a 65- kda protein of Bt israelensis, presumably the 72-kDa CryllAa protein, was crystallized into a bar-shaped inclusion body apart from the major crystal body. This protein was toxic to Aedes aegypti. COLEOPTERA-SPECIFIC CRYSTAL PROTEINS The first Bt strain specific for Coleoptera larvae was isolated by Krieg et al. (1983). The strain was classified as Bt tenebrionis but could not be distinguished from Bt morrisoni by serotyping. Bernhard (1986) showed that Bt tenebrionis crystal was easily dissolved in sodium bromide at a concentration higher than 3.3 M. When analyzed by SDS-PAGE, a 68-kDa protein, the major component, and a minor 50-kDa protein were detected. Krieg et al. (1987) referred to these proteins as the 74 and 68-kDa proteins. As reviewed in the molecular biology section, Bt tenebrionis contains only one gene called cry3aa which codes for a 73-kDa protein. The 73-kDa protein is converted by proteases contaminating the crystals into a 66-kDa protein. The proteases were found to cleave off the 57 aminoterminal amino acids (McPherson et al., 1988). Additional Cry3 proteins, Cry3B and Cry3C, have been reported (Rupar et al., 1991). They are discussed in the section about Coleoptera-specific genes. CRYSTAL PROTEIN TYPING BY IMMUNOASSAY AND HPLC Immunoassay and HPLC have been used to determine the types of crystal proteins. Krywienczyk et al. (1978) reported two types of the crystal proteins in Bt kurstaki isolates. Molecular biology studies have shown that these kurstaki strains contain three cryla genes which are expressed at different ratios. Hofte et al. (1988) surveyed 29 Bt strains with monoclonal antibodies and showed that some antibodies were specific to a part of the protein molecule that determined host specificity. Yamamoto (1983) utilized HPLC in determining the crystal protein types. In this method called peptide mapping, the crystal protein was purified, and the purified protein was digested with trypsin. HPLC separated these trypsin-digested fragments based on size, hydrophobicity and charge with remarkable reproducibility. Since the separation pattern was highly specific to the individual protein, the pattern was used as a fingerprint of each protein. Yamamoto et al. (1988) further applied this technique to show differences in the expression of two cryl A genes in Bt kurstaki HD 1 and HD263. Pang and Mathieson (1991) applied a different peptide mapping technique to quantify individual crystal proteins. They digested crystal proteins with cyanogen bromide and followed by SDS-PAGE analysis. Cyanogen bromide digested a protein at Met residues, and SDS-PAGE separated by size the fragments resulting from the digestion. Met residues occurred much less frequently than trypsin-recognition sites of Arg and Lys residues. Digestion with cyanogen bromide produced large size peptides which were more appropriate for SDS-PAGE than HPLC. Under this method, the two different proteins, CrylAa and CrylAc, found in Bt kurstaki crystals, produced distinctive bands on the SDS-PAGE gel and could be quantified. HPLC was also used to separate trypsin-resistant N-terminal cores (or activated toxins) of Cryl-type proteins at alkaline ph (Pusztai-Carey, 1994) or in a neutral ph buffer containing urea (Chestukhina et al., 1994). Under these conditions, the core proteins remained soluble, thus could be separated from each other in an ion-exchange column. These HPLC methods took advantage of the sequence heterogeneity that mainly existed in the core region. Using a technique similar to that of Chestukhina et al. (1994), the protein contents in commercial products were determined (Fig. 3).

6 6 Yamamoto In order to determine the bond type, the crystal protein was digested by a protease without reducing the disulfide bonds. The peptide fragments connected by the bond was isolated by electrophoresis from other free (i. e., not connected) fragments and characterized. Fig. 3 HPLC analysis of Bt products sold in Japan in Product A, a Bt kurstaki strain that lost the crylab gene; Product B, an aizawai strain; Product C, a Pseudomonas containing Bt crylac gene; Product D, a mixture of kurstaki and aizawai strains. This figure was reported by the author in 1996 at the annual meeting of the Society for Invertebrate Pathology in Cordoba, Spain. C-TERMINUS OF THE Cry1-TYPE PROTEIN IS DIGESTED STEPWISE DURING ACTIVATION Cryl-type crystal proteins, consisting of some 1,200 amino acid residues, are about 135 kda. As reviewed earlier, the 135-kDa protein is a protoxin which is converted to the true toxin by protease digestion. It has been shown that trypsin digests a part of the protoxin into small fragments and spares a 66-kDa N-terminal portion. Choma et al. (1990) followed the trypsin digestion of the CrylAc protein of Bt kurstaki HD73 and found that trypsin removed a 70-kDa carboxylterminal portion in seven consecutive events, cleaving a 10-kDa fragment at a time. When each l0-kda fragment was cleaved off from the crystal protein, the fragment was quickly digested into small fragments. Their finding suggested that removing a 10-kDa fragment exposed the next trypsin site to the enzyme. Choma et al. (1991) further reported that the trypsinactivated Cry 1 Ac toxin from HD73 was more structurally stable than the whole crystal protein. The crystal protein formed inter-molecule disulfide bonds between two Cys residues when crystallized in Bt cells. Bietlot et al. (1990) showed that disulfide bonds in the crystal were between the corresponding Cys residues of two identical proteins. This type of bonds is called symmetrical interchain disulfide bonds. STRUCTURES OF FOUR CRY PROTEINS HAVE BEEN DETERMINED BY X-RAY CRYSTALLOGRA- PHY The protein, in a crystal, diffracts X-ray to give precise three-dimensional coordinates of atoms that compose the protein molecule. Unfortunately, the crystal produced in a Bt cell is too small for this technique. By 1990, a crystal protein, Cry3Aa, had been artificially crystallized to a size big enough for X-ray crystallography. The Cry3Aa protein does not have any disulfide bonds and is easily dissolved in an alkaline solution or in 3 M sodium bromide. The protein was crystallized when it was dialyzed to remove alkali or sodium bromide (Garfield and Stout, 1988; Li et al., 1988). In 1991, Li et al. (1991) published the structure of the Cry3Aa protein, a Coleopteraspecific toxin. The structure of Cry3Aa revealed three structurally distinct, approximately equal-size domains. Starting from the N-terminus, Domain I contained a bundle of seven a-helices. This structure and other biochemical observations have indicated that domain I plays the major role in the membranedisrupting function of the toxin. Domain II comprised of three repeating sheets forming a triangular column. Domain III contained several antiparallel /3-chains forming a lectin-like structure. A substantial number of reports have shown that Domain II and III contain a receptor-binding site or sites (refer to the receptor section). The structure of Cry3Bb has been reported in a patent application (WO 99/31248) filed by Ecogen, Inc. But, the coordinates are not publicly available. Besides Cry3Aa and Cry3Bb, the structures of Cry 1 Aa (Grochulski et al., 1995) and Cry2Aa (Morse et al., 1998) have been determined. Primary sequence homology among Cry3, CrylAa and Cry2Aa is quite low, but X-ray crystallography has revealed a remarkable structural homology. Not only the basic threedomain structure but also almost all alpha and beta strands can be found commonly in these crystal protein structures (Fig. 4). Bt cry GENES ARE ON PLASMIDS Gonzalez and Carlton (1980) have found that Bt contains multiple plasmids as large as 75 MDa (110

7 One Hundred Years of Bacillus thuringiensis 7 CrylAa Cry2Aa Cry3Aa Fig. 4 Backbone structures of three Bt Cry proteins. The Cry2Aa structure was obtained from Drs. R. Morse and R. Stroud of the University of California, San Francisco and reproduced with their kind permission. CrylAa and Cry3Aa coordinates were from a public protein structure database. kb). They used a slot-lysis technique to prevent the degradation of large-size plasmids. In a subsequent paper, Gonzalez et al. (1981) isolated a series of plasmid-cured mutants from several different Bt strains including Bt kurstaki HD73 and Bt thuringiensis HD2. They demonstrated that the crystal production ceased when a large size plasmid, 75 MDa (110 kb) of HD2 or 50 MDa (75 kb) of HD73, was lost. Gonzalez et al. (1982) further reported that these plasmids were transmissible to Bacillus cereus. B. cereus produced the crystal after receiving one of the plasmids. Other Lepidoptera-specific strains were found to carry the crystal protein gene on one or more plasmids. For example, a Bt darmstadiensis strain was shown to have both crystal protein and heat-stable exotoxin genes on the 62-MDa plasmid (Ozawa and Iwahara, 1986). Ward and Ellar (1983) reported that mosquito-specific Bt israelensis carried all the crystal protein genes on the 72-MDa plasmid. All these assignments were made before the crystal protein gene had been cloned. A school of scientists believed that the isolation of the plasmid, which carried the crystal protein gene, was essential in cloning the gene. Today, the interest in isolating the plasmid before cloning is not as high. Progress in molecular biology techniques has made it practical to clone the gene from a complex DNA preparation. first gene coding for a crystal protein from Bt kurstaki. The strain used in this cloning had been isolated from Abbott's commercial Bt formulation, Dipe1TM, by Lee Bulla, Jr. and had been sent to Whiteley. The isolated culture was supposed to be the HD 1 strain. However, it was found later that the strain used in the cloning differed from the HD 1 strain available at the USDA Bt culture collection. The Bulla's strain, therefore, was named HD 1-Dipel (Kronstad et al., 1983). To clone the crystal protein gene, Schnepf and Whiteley (1981) fractionated Bt plasmids by size. The fraction containing the large plasmids was partly digested with Sau3AI, and the fragments were inserted into the BamHI site of an E, coli cloning vector, pbr322. An antibody raised against the HD 1-Dipel crystal protein was used to find the E, coli cells producing the crystal protein. The cloned cry gene in the recombinant plasmid, named pes 1, was expressed in E. coli and gave a positive reaction with the antibody. The gene product, a 133-kDa protein, was toxic to Manduca sexta. This cloning of the first crystal protein gene, now designated as CrylAa, is a very important breakthrough in the history of Bt research. Since the cry genes from different Bt isolates are often homologous to crylaa, the new genes, if homologous to crylaa, can be identified with a portion or the entire crylaa gene that is used as a probe. CLONING THE FIRST CRYSTAL PROTEIN GENE FROM Bt kurstaki In 1981, Schnepf and Whiteley (1981) reported the NOMENCLATURE OF Bt CRYSTAL PROTEIN GENES Since the cloning of the first crystal protein gene

8 8 Yamamoto from Bt kurstaki, numerous genes have been cloned from various Bt strains. In 1988, at the annual meeting of the Society for Invertebrate Pathology in San Diego, California, scientists agreed to designate the crystal protein gene as "cry." Several other names, such as icp (insecticidal crystal protein), were also proposed. The argument for selecting cry was that the gene phenotype was the formation of crystals and that some crystal proteins had no known insecticidal activities. After the meeting, Hofte and Whiteley (1989) published a review summarizing the cry genes which had been cloned and sequenced by the end of They classified the cry genes based on insect specificity. Their classification grouped the gene into four types: (i) cryl-type genes coding for proteins specific to only Lepidoptera species, (ii) cryll type specific to both Lepidoptera and Diptera species, (iii) crylll type specific to Coleoptera species and (iv) cryl V type specific to Diptera species. Some scientists disagreed with this classification scheme and pointed out that the scheme was based not only on the insect specificity but also on the similarity of nucleotide sequences. A good example of the mixed criteria is cryllb (now called cry2ab). Unlike crylla (cry2aa), the phenotype of cryllb, the CryIIB protein, is not active against mosquito larvae (Dankocsik et al., 1990; Widner and Whiteley, 1989). Nevertheless, the gene was classified as a dual-specificity cryll based on its high sequence homology to crylla. Regardless of the problem, the classification scheme was widely accepted by scientists working on Bt, and some additional genes, such as cryle and crylf, were added to the list. The 27-kDa protein gene of Bt israelensis was designated as cyta (now cytlaa). The gene was named after its cytotoxic activity that differed from other crystal proteins encoded by the cry genes. Within a few years after this nomenclature scheme was proposed, some confusion developed in the numbering of several newly cloned genes. A major issue was the failure to use the amino acid sequence when several different genes were classified into one group. For example, the crylll group contains short (truncated) and long (full protoxin) genes, such as cryllia (cry3aa) and cryllic (cry7aa). An additional problem was lack of a "clearinghouse" to establish the class for a new gene. Scientists who were interested in the classification scheme met in Paris in 1993 at the International Conference on Bacillus thuringiensis, and a nomenclature committee was formed. This committee published in 1998 a proposal for a revised Bt cry gene nomenclature (Crickmore et al., 1998). In the revision, the committee decided to use Arabic rather than Roman numerals and no parentheses. For example, cryla(a) was renamed as crylaa. More importantly, the new gene classification scheme is governed only by amino acid sequence homology. Also, a clearinghouse was set up at the Bacillus Genetics Stock Center (BGSC) at Ohio State University (Crickmore et al., 1998). The latest gene list and other related information can be seen at URL: Crickmore/Bt/index.html ADDITIONAL cryla-type GENES Bt sotto is a unique subspecies in respect to its plasmid content. Iizuka et al. (1981) studied the plasmid contents of a number of Bt strains and found that Bt sotto contained only one plasmid while all other subspecies they analyzed had multiple plasmids. Using the same Bt sotto strain, Shibano et al. (1985) cloned a crystal protein gene from the plasmid. This Bt sotto strain was obtained from Heimpel's collection at USDA Insect Pathology Laboratory in Beltsville, Maryland. Presumably, Heimpel acquired the sotto isolate from Angus who had examined its crystal protein. Sequencing of the cloned gene revealed a very close relation to the crylaa gene cloned by Schnepf and Whiteley (1981). Klier et al. (1982) cloned two cry genes from the berliner 1715 strain of Bt thuringiensis. The first gene was found in the bank of E. coli clones containing BamHI fragments derived from a complex of the chromosome and plasmids. The clone designated as pbt reacted with the sporulation-specific 265 mrna which was considered to be mrna of a cry gene. Since pbt15-88 did not hybridize with a plasmid preparation of the berliner 1715 strain, they suggested that the BamHI fragment in pbt came from the chromosome. The restriction map of the cry gene in pbtl5-88 shown by Klier et al. (1983) is very similar to the map of the crylaa gene cloned from Bt kurstaki HD 1-Dipel. Both kurstaki and berliner genes contain two PvuII sites separated by 2 kb. They cloned the second cry gene into a shuttle vector using a piece of pbt as a probe. The second gene, designated as pbt42-l, was in the 14-kb BamHI of the 42-MDa plasmid of the same Bt strain. This gene appears to be a crylab type. Subsequently, Klier et al. (1985) showed that the pbt42-1 plasmid contained transposable elements including Tn4430. The same Tn4430 arrangement was found with the crylab gene in the berliner 1715 strain by Menou et al. (1990). Wabiko et al. (1985) cloned in E. coli a 14.5-kb BamHI fragment of the 42-MDa plasmid of the berliner 1715 strain. The clone, designated as phw 13,

9 One Hundred Years of Bacillus thuringiensis 9 contained a cry gene, and the gene was subsequently sequenced (Wabiko et al., 1986). The sequence revealed significant differences from that of the crylaa gene of Bt kurstaki HD1-Dipel. Later, Hofte and Whiteley (1989) designated the seqeunce of the cry gene in phw13 as the holotype sequence of crylab, presumably because it was the first crylab gene that was sequenced. A new cry gene, almost identical to the crylab gene in phw 13, was cloned from the same berliner 1715 strain and sequenced by Hofte et al. (1986). This new crylab gene was on a 7.5-kb BamHI-PstI fragment as shown in the restriction map for the holotype crylab gene but with a few nucleotide differences between two crylab genes. The crylab gene is now known to be common in other subspecies including Bt kurstaki and Bt aizawai. The crylab gene codes for a 131-kDa protein. The molecular weight of CrylAb protein is smaller by 2 kda than that of the CrylAa protein due to a deletion towards the 5' (carboxyl) end. Kondo et al. (1987) pointed out that the deletion was possibly caused by a set of two direct repeat sequences found in the region. THREE cryla-type GENES IN Bt kurstaki A molecular biology technique, called Southern blotting, detects a specific nucleotide sequence in DNA samples. Using this technique, Kronstad et al. (1983) located cry genes in DNA preparations of several different Bt strains. The cloning of the crylaa gene from HDI-Dipel made it possible to conduct this study. They used an EcoRI fragment of the crylaa gene as a probe. The EcoRI fragment codes for a part of the N-terminal half of the 133-kDa CrylAa protein. A report by Kronstad et al. (1983) showed that Bt contained multiple copies of the cry gene on different plasmids. The Bt kurstaki HD 1 strain from USDA in Brownsville, Texas contained three genes with HindIII sites at different locations. They classified the 130-kDa crystal protein genes found in Bt kurstaki into three "4.5-kb, 5.3-kb and 6.6-kb HindIII classes" based on the sizes of the HindIII fragments. These genes correspond to the current crylaa, crylab and crylac genes, respectively. The gene cloned from HDI-Dipel was found to be the 4.5-kb HindIII class or crylaa. Subsequently, the 5.3-kb HindIII class or crylab was cloned from Bt kurstaki HD 1 by Thorne et al. (1986) and Geiser et al. (1986). The 6.6-kb HindIII class or crylac gene was cloned from Bt kurstaki HD73 by Whiteley et al. (1982) and Adang et al. (1985) and from Bt kurstaki HD244 by McLinden et al. (1985). A slightly different crylab gene was cloned also from HD 1 by Kondo et al. (1987). These reports indicate that, provided no sequence mistakes have been made, HD 1 contains at least two crylab type genes. POSSIBLE CHAPERONE PROTEINS IN THE cry2aa OPERON Donovan et al. (1988a) cloned the cry2aa gene and determined its nucleotide sequence. The gene was expressed in B. megaterium, and the Cry2Aa protein was active against both Lepidoptera and Diptera insects. Shortly after the cry2aa gene had been cloned, Widner and Whiteley (1989) cloned the same gene and an additional gene which was highly homologous to cry2aa. The additional gene, designated as crylib (cry2ab), was active only against Lepidoptera larvae. To clone these two cry2a genes, Widner and Whiteley (1989) used antiserum directed to the 65-kDa protein from the HD 1 strain. They found two clones, containing 5.0 and 9.0-kb HindIII fragments, produced a 65-kDa protein which reacted with the antiserum. The sequencing revealed that the 5.0-kb fragment contained cry2aa, and the 9.0-kb fragment had cry2ab. They further revealed that cry2aa was clustered with two open reading frames (orf) under one promoter like an operon. The cry2aa operon consisted of a promoter, orfl, orf2, the cry2aa coding region and a transcription terminator. The proteins encoded in orfl and orf2 were 20 kda and 29 kda, respectively. The ORF2 protein appeared to be expressed in Bt kurstaki HD 1 and had a 15-amino-acid sequence repeated 11 times. Possibly, the ORF2 protein is involved in crystallization but the functions of the ORF 1 and ORF2 proteins are not fully understood. Whether the ORF1 protein is synthesized in Bt is not known. The cry2ab appeared not to be expressed in Bt (Dankocsik et al., 1990; Widner and Whiteley, 1989). CLONING OF cry3aa FROM Bt tenebrionis AND OTHER SUBSPECIES Bt tenebrionis, isolated by Krieg et al. (1983), produces a flat-square crystal active against Coleoptera larvae. In 1987, three reports of cloning of the cry gene of this Bt isolate were published (Hofte et al., 1987; Jahn et al., 1987; Sekar et al., 1987). On this gene, there are two translation start sites for E. coli, one at the first colon for Met and the other at the 48th codon also for Met. The protein translated at the first codon is 73 kda and, the other started at the 48th colon is 68 kda. Since the same size proteins are also observed with the Bt tenebrionis crystal, it is highly likely that

10 10 Yamamoto Bt utilizes these translation start sites. A small part of these translated proteins appears to be processed by protease to smaller proteins. McPherson et al. (1988) have determined three processing sites, one of which is 58 Asp. Herrnstadt et al. (1986) isolated a Coleopteraspecific Bt strain from the soil in San Diego and claimed that the strain was a new subspecies, Bt sandiego. However, extensive comparisons between Bt tenebrionis and Bt sandiego including cloning and sequencing of these cry genes have not revealed any differences (Herrnstadt et al., 1987). Donovan et al. (1988b) isolated a Coleopteraspecific strain, EG2158, from a soybean grain dust sample. This strain differed from Bt tenebrionis as it produced a 30-kDa crystal protein in addition to a Cry3A protein. The 30-kDa protein, crystallized in a flat-bipyramidal shape, was not required for the Coleoptera-activity. The cry3a gene of EG2158 was cloned and sequenced but was found to be identical to the gene from Bt tenebrionis. A cry3 GENE SPECIFIC TO DIABROTICA Sick et al. (1990) published a one-page report showing the sequence of a cry3-type gene and designated the gene as cryllib based on the old nomenclature scheme. This gene is now called cry3ba. The gene was isolated from a Bt tolworthi strain and claimed to be specific for Coleoptera larvae. The sequence of the cry3ba gene differed significantly from that of cry3aa. The cry3ba gene codes for a 74-kDa protein which is slightly larger than the Cry3Aa protein. The cry3ba gene contains a possible translation start site at the 48th colon (Met) as shown with cry3aa. Another cry3b-type gene was independently discovered in Bt tolworthi EG2838 isolated from a grain dust sample by scientists at Ecogen (Rupar et al., 1991). They screened numerous Bt isolates with a cry3aa gene probe to find the EG2838 strain. They showed that the EG2838 strain produced 70- and 74-kDa proteins immunologically similar to the 70- and 73-kDa Cry3Aa proteins. Molecular weight differences between Cry3Aa and the EG2838 proteins and the insect specificity suggested that the gene in EG2838 was a cry3b type. With the gene in EG2838 as a probe, Rupar et al. (1991) discovered a new Bt strain (EG4961) producing a Cry3-type protein active against Diabrotica undecimpunctata. Since D. undecimpunctata, the corn rootworm, causes huge damage to corn, the discovery has a significant commercial value. The EG4961 strain was found to be Bt kumamotoensis, and its gene has been classified as cry3bb. The cry3 genes from Ecogen isolates are somewhat confusing. The cry3bb gene, M89794, was originally described as cryiiic in Ecogen's patents (USP , USP ). Later this gene was also called cryiiic(a) when a similar gene, cry3bb2 (U31633) described in USP and USP , was discovered in the EG5144 strain. U31633 was called cryiiic (b). M89794, now called cry3bbl, was then renumbered as cryiiib2 when it was published in a scientific journal (Donovan et al., 1992). According to USP , Cry3Bb 1 is 10 times more toxic to the southern corn rootworm than Cry3Bb2. This indicates that a small difference in the amino acid sequence makes a big difference in insecticidal activity. Recently, a patent application from Ecogen, WO 99/31248, showed that limited mutations on Cry3Bb 1 made the protein several times more potent to the corn rootworm. The application also revealed the tertiary structure of the protein. OTHER COLEOPTERA-SPECIFIC FAMILIES GENE Additional Coleoptera-specific genes, such as cry 7, cry8 and cry9 have been cloned and sequenced. Little information is available about cry7's in addition to their sequences. The cry7 genes were originally called cryiiic. As of this writing, there are three cry8 genes active against scarabs. A Japanese company, Kubota, has developed a commercial product with the Bt japoniensis buibui strain from which the cry8ca gene was cloned (Sato et al., 1994). The cry9-family genes are quite diversified. The cry9ca gene, originally called crylh, has a widespectrum activity against a number of Lepidoptera insects. This gene is used in Aventis' StarLinkTM transgenic corn. Since the Cry9Ca protein resists rapid degradation in simulated stomach fluid, it is considered to have a higher risk of causing allergenic reactions to human. Accordingly, StarLink obtained registration in the U.S. only for animal feeds. The crylab gene is widely used in the transgenic corn, and the CrylAb protein degrades quickly in simulated stomach fluid. Unfortunately, StarLink corn was detected in human foods (infamous Taco Bell recall) and caused public concern. According to Agrow (Oct. 27, 2000), Aventis has agreed with the U.S. EPA to cancel the registration of StarLink transgenic corn. The cry9aa is also Lepidoptera specific (Chestukhina et al., 1994), while others like cry9da have Coleoptera activity (Asano personal communication).

11 One Hundred Years of Bacillus thuringiensis 11 SEQUENCE HOMOLOGY AMONG CRYSTAL PROTEINS After several cryl -type genes had been cloned, it became clear that their sequences were quite homologous. Indeed, molecular biologists took advantage of this homology to clone additional cry genes. Most, if not all, 135-kDa crystal proteins have C- terminal halves that are remarkably similar to each other. It is widely believed that the C-terminal half plays an important role in the formation of crystals. Heterogeneity occurs in the N-terminal half and is considered to be a major contributing factor to host specificity. Cloning and sequencing of cry genes other than the cryl -type genes further revealed an extensive homology among those having different host specificities. Short cry genes, such as cry2 and cry3, are somewhat homologous to the corresponding part of long cry]- type genes. Hofte and Whiteley (1989) made an extensive comparison among the cry genes, which had been cloned by 1988, and found five highly conserved regions in the toxin (the N-terminal half). These conserved regions are presumed to be essential for the insecticidal activity, perhaps membrane-disrupting and protease-resistant functions of the toxin. Fig. 5 is a dendrogram of the crystal proteins showing the clustering relationship compiled from computer comparisons between any given two sequences. The dendrogram has revealed interesting relationships that suggest certain insect specificities. There is a cluster of Cry 1 B, Cry 1 K and Cry l I that is separated from other Cry l proteins. These proteins, except for Cry 1 Ka whose activity has not been widely tested, are known to have activity against Coleoptera insects. Another example is. an interesting observation in domain homology as reported by Crickmore (2000). As shown in the dendrogram, CrylAc is closely clustered with other CrylA-type proteins. The homology is mainly from their domains I and II. Domain III of CrylAc is quite unique not only among those in the cluster but also among the crystal proteins that have been reported. Only CrylBd domain III shows substantial homology with that of CrylAc. A similar situation has been found with CrylCa domain III. On the other hand, domain III of CrylAa is widely conserved among other proteins such as CrylAb, CrylFa and Cry1Ja. SIGMA FACTORS REGULATE cry GENE EXPRESSION IN Bt Klier et al. (1973) analyzed RNA polymerase in Bt and noted a specific sigma factor during sporulation. Klier et al. (1983) demonstrated that the cry]ab gene of Bt thuringiensis berliner 1715 required a sporulation-specific RNA polymerase and determined the promoter sequence of the cry gene. After the crylaa gene was cloned, Wong et al. (1983) found two transcription start sites on the gene which were recognized by independent sigma factors at different periods of bacterial growth. These sigma factors were isolated from the same Bt strain by Brown and Whiteley (1988, 1990) as 35- and 28-kDa proteins. The cloning and sequencing of these factors revealed that the 35-kDa and 28-kDa sigma factors were highly homologous to two B. subtilis GE and GK, respectively. These B. subtilis sigma factors are known to be specific for sporulation. Adams et al. (1991) successfully demonstrated in & or GE-deficient B. subtilis mutants that the 35-kDa and 28-kDa Bt sigma factors could replace corresponding sigma factors of B. subtilis. OTHER FACTORS THAT REGULATE cry GENE EXPRESSION The RNA polymerase terminates transcription when it encounters a terminator. Schnepf et al. (1985) defined the transcription stop site of crylaa by using S1 nuclease mapping. The translation of the crylaa gene was terminated at the second loop-and-stem structure. There have been only a few reports on the regulation of crystal protein synthesis in Bt during and after the translational stage. Perhaps, Bt does not need regulation at these periods, because the crystal protein synthesis is a sort of a runaway event at the end of Bt's life cycle. McLean et al. (1987) and Adams et al. (1989) cloned in E. coli a Bt israelensis gene coding for a 20-kDa protein. They demonstrated that a small amount of the 20-kDa protein was required for the efficient expression of the cyt]aa gene in E, coll. Their discovery was contrary to the conclusion made earlier that the CytlAa protein could not be produced in E. coli because of its toxicity to the host cells (Ward and Ellar, 1986). Subsequently, Visick and Whiteley (1991) reported that the 20-kDa protein protected the CytlAa protein from digestion by protease while it was being synthesized in E, coli. Possibly, the 20-kDa protein has dual functions. The 20-kDa protein may bind to the CytlAa protein to prevent this protein from degradation and to protect E, coli from the toxic effect of the Cyt 1 Aa protein.

12 12 Yamamoto Fig. 5 Dendrogram of Bt Cry and Cyt proteins. The figure was reproduced from that shown in the Bt Toxin Nomenclature web site with the kind permission from Dr. Neil Crickmore.

13 One Hundred Years of Bacillus thuringiensis 13 DISCOVERY OF TRANSPOSABLE ELEMENTS Multiple copies of the cry gene on different plasmids and on the chromosome imply that the cry gene is accompanied with transposable elements. The transposable element or transposon (Tn) codes for an enzyme called transposase which catalyzes the insertion of its own copy into a distant location of DNA. There are two classes of transposable elements. The simple element is called insertion sequence (IS) and is composed of a transposase gene or other proteincoding regions flanked by two short inverted repeat (IR) sequences. The functions of some of these proteins are not well understood but are implicated to act as agents that promote genetic exchange. The complex element is called the true transposon in which two insertion sequences enclose a gene or genes in addition to the genes needed for transposition. In this case, the whole structure, enclosed by the insertion sequences, moves as a block to a new location. The enclosed genes include an antibiotic-resistant gene and a toxin gene which bacterial cells requires in adaptation to a new environment. Insertion sequences have been found in Bt kurstaki HD73 (Kronstad and Whiteley, 1984; Menou et al., 1990), in Bt thuringiensis berliner 1715 (Mahillon et al., 1987; Mahillon et al., 1985) and in Bt israelensis (Bourgouin et al., 1988; Delecluse et al., 1989). In the HD73 strain, Kronstad and Whiteley (1984) found two types of insertion sequences termed IR2150 and IR1750, and Menou et al. (1990) designated them as IS232 and IS231, respectively. These insertion sequences flanking the crylac gene were on the 50- MDa (75-kb) plasmid of HD73. The arrangement suggests a complex transposon structure as defined above. Kronstad and Whiteley (1984) reported that these insertion sequences and their homologous sequences occurred commonly in other Bt strains. The insertion sequences, associated with crylab of the berliner 1715 strain, have been analyzed in detail. There are several variants of 1S231 and 1S232. 1S231A has been sequenced to confirm that it contains a 1.6-kb typical insertion sequence, consisting of 11-bp direct repeat and 20-bp inverted repeat sequences on both ends, and a 56-kDa transposase-like gene (Mahillon et al., 1985). Highly homologous IS231B and 1S231 C have also been cloned and sequenced (Mahillon et al., 1987). Menou et al. (1990) sequenced 2.2-kb IS232 and found two orf's coding for 50-kDa and 30-kDa proteins. The deduced sequence of the 50-kDa protein revealed structural characteristics of a DNA binding protein. Several different IS232's have been isolated from the berliner 1715 and HD73 strains. All IS232's possess a set of identical 37-bp terminal inverted repeat sequences, but some of them do not have a direct repeat. Both types of insertion sequences, and 15232, appear to be similar to the other insertion sequences found in gram-negative bacteria. An insertion sequence of Bt israelensis flanking the cry4aa gene has been designated as (Bourgouin et al., 1988) and sequenced by Delecluse et al. (1989). As in the case of 15231, contains a transposase gene flanked by 16-bp inverted repeats. Tn4430 IS A REPLICATIVE TRANSPOSON Another transposable element, a 3-MDa (4.5 kb) sequence of Bt thuringiensis berliner 1715, has been found as an insert in a Streptococcus faecalis plasmid when the plasmid is transferred to the berliner 1715 strain (Lereclus et al., 1983). Lereclus et al. (1984) revealed by Southern blotting that the 3-MDa transposon was on the 42=MDa plasmid of the berliner 1715 strain. In their report, Lereclus et al. (1984) called the sequence as Th sequence which contained inverted repeat sequences. Lereclus et al. (1986) cloned Th sequence in E. coli to confirm that it was a transposon and designated the sequence as Tn4430. Both ends of Tn4430 were sequenced and found that the inverted repeat of T n4430 was highly homologous to those of other transposons found in E, coli (Tn3) and B. subtilis (T n917). Mahillon et al. (1987) showed with the berliner 1715 strain that Tn4430 was positioned between two The complete sequence of Tn4430 has been determined by Mahillon and Lereclus (1988). The sequence shows two protein genes, a 32-kDa integrase and a 113-kDa transposase. The transposase inserts the transposable sequence by recognizing on its target DNA a specific sequence consisting of a few nucleotides. Tn4430 is considered to be a replicative transposon, because it contains an integrase gene. The replicative transposition duplicates a copy of the transposed sequence in the target DNA while keeping the original copy in the donor DNA. The simple transposition, on the other hand, leaves no copy of the original transposable sequence on the donor DNA. The replicative transposition scheme supports multiple copies of the cry genes normally found in Bt. An additional tranposon, Tn5401, has been reported by Baum (1994). Although this transposon has a gene organization similar to Tn4430, the sequence comparison indicates differences in origin.

14 14 Yamamoto DELINEATION OF THE PROTEASE-ACTIVATED CRYSTAL TOXIN Since Angus (1956) described the activation of Bt sotto crystal protein, others have studied the activation process of the 135 kda-type crystal proteins, especially Cryl-type proteins. The activation process removes about one half of the molecular weight of the crystal protein. In order to delineate the active toxin in the whole crystal protein, Nagamatsu et al. (1984) sequenced the N-terminal amino acids of the trypsin-activated toxin of Bt dendrolimus T84A 1. They isolated a trypsin-resistant domain of the crystal protein, which was presumably similar to, if not the same as, the toxin activated in vivo. The N-terminal amino acid sequence of the dendrolimus toxin was Ile-Glu-xxx-Gly-Tyr- Thr-. This sequence has been found at the site starting with the 29th amino acid residue of Cry1A-type crystal proteins. A similar study has been made with the CrylAb protein, isolated from E. coli, in which the crylab gene has been cloned from Bt thuringiensis berliner 1715 (Hofte et al., 1986). Trypsin has been found to cleave the crystal protein between 28 Arg and 29 Ile. In the cases of CrylD and Cry9Aa proteins from Bt galleriae 11-67, the toxins start at 28 Leu of CrylD and 24 Tyr of Cry9Aa (Chestukhina et al., 1988). Sequencing of C-terminal amino acids of the protease-activated toxin was performed by Bietlot et al. (1989) on the Cry 1 Ac protein isolated from Bt kurstaki HD73. In order to analyze the C-terminal amino acid sequence, all carboxyl groups of the trypsin-activated toxin was first blocked with methylamine. The toxin was then completely denatured and digested by pepsin. The digestion generated many endogeneous peptides and the N-terminal peptide with new carboxyl groups at cleavage points. However, the carboxyl group of the peptide from the original C- terminus of the toxin remained blocked. The C- terminal peptide was separated from others by electrophoresis, because it contained no carboxyl groups. The sequencing of the C-terminal peptide revealed Gln- Lys. The sequence corresponds to 623 Gln and 624 Lys residues of the Cry 1 Ac protein. Yamamoto et al. (1988) extensively sequenced the trypsin-activated CrylAa toxin. The CrylAa toxin was purified by ion-exchange column chromatography. The N-terminal amino acids were sequenced to confirm that it started with 29 Ile. The toxin was denatured in 8 M urea and further digested to completion with trypsin. Peptides, generated by the second trypsin digestion, were separated by HPLC and identified by mass spectrometry. The results showed that the trypsinactivated CrylAa toxin was terminated at either 618 Arg or 621 Lys. These two studies indicate that the trypsin-activated cryla-type toxins start with the N-terminal sequence of Ile-Glu-Thr-Gly-- and end with the C-terminal sequence of --Arg-Ala-Gln-Lys. DELINEATION OF THE ACTIVE TOXIN BY MOLECULAR BIOLOGY Further efforts to delineate the activated toxin have been done with two different molecular biology techniques. One technique utilizes a transposon and the other technique DNA-cleaving enzymes. When a transposon is inserted into a gene, the protein synthesis is disrupted at the site of insertion. The cry gene can be truncated with a proper restriction enzyme or with an exonuclease from the 3' end. The truncated genes are put into an expression system to produce shortened proteins, which are bioassayed to correlate the loss of activity to the size of the proteins. Schnepf and Whiteley (1985) examined, with Bt kurstaki HD1-Dipel, the region of the crylaa gene between two Hind III sites (the 563rd and 939th amino acid residues). Tn5, an E. coli transposon, was inserted into this region to produce truncated proteins. The 3' end of the region was removed at several restriction enzyme sites, such as Sau3AI, XmnI, BclI and EcoRI* (star activity). They demonstrated that the loss of insecticidal activity in vivo (i. e., whole insect bioassay) occurred between the 603rd and 645th amino acid residues of the protein. On the 5' (N-terminal) side, Schnepf and Whiteley (1985) found that the toxicity was destroyed between the 1st and 2nd XmnI sites, the 10th and 50th colons. A similar observation was made by Hofte et al. (1986) on the cryl Ab gene of the berliner 1715 strain. Deletions of the 3' side of the gene resulted in a loss of the activity between the 599th and 607th amino acid residues. Wabiko et al. (1986), with the same cryl Ab gene, observed an activity loss between the 607th and 612th amino acid residues. The region between residues appears to be very critical in maintaining insecticidal activity. HOST SPECIFICITY DOMAIN WAS DETERMINATED BY DOMAIN SWITCH The protease-activated Cryl-type toxins have molecular weights around 70,000. X-ray crystallography has revealed that the toxin is made of three structurally distinct domains. In order to find the domain or domains responsible for insect specificity, two major types of experiments have been conducted. One experiment, the domain switch, exchanges corresponding

15 One Hundred Years of Bacillus thuringiensis 15 domains of two cryl -type genes with different insect specificities. Ge et al. (1989; 1991) conducted an extensive domain switch between the crylaa and CrylAc genes. The crylaa gene is known to be highly specific to Bombyx mori and the crylac gene to Heliothis virescens. They found that the B. mori activity of the Cry 1 Aa protein was transferred to Cry 1 Ac when the residue domain of Cry 1 Aa replaced the corresponding domain of CrylAc. They also discovered that the residue domain of CrylAc was responsible for Trichoplusia ni activity and the residue domain of the same protein determined the H. virescens specificity. Schnepf et al. (1990) conducted a similar experiment on three cryla-type genes. They have concluded that the residue domain of CrylAc is important for Manduca sexta specificity. Raymond et al. (1990) swapped domains between the cryl A a and cryl Ab genes at the SstI site and found Manduca sexta specificity in the first 450 amino acid residues. The Cry2Aa protein is a dual toxin that is active against both Lepidoptera and Diptera larvae. The Cry2Ab protein homologous to Cry2Aa is not active against Diptera larvae. Widner and Whiteley (1990) applied the domain switch technique to establish the Diptera-specificity domain in the Cry2Aa protein. They showed that the Cry2Ab protein gained Diptera activity when the residue domain of Cry2Aa replaced the corresponding domain of Cry2Ab. HOMOLOGOUS RECOMBINATION A technique called homologous recombination has also been used to determine domain functions. This technique utilizes bacterial cells capable of recombining two homologous DNA sequences. Caramori et al. (1991) constructed in E. coli a plasmid containing a 5' (N) part of the crylaa gene and a 3' (C) part of the crylac gene. In the plasmid construction, the CrylAa gene, which terminated at the HindIII site (i.e., at the 564th colon), was connected to the EcoRI site (the 333rd colon) of the crylac gene with a tetracycline resistant gene. The plasmid was cloned in an E. coli strain capable of recombining DNA and was allowed to recombine within the plasmid. In recombinant plasmids, a 3' portion of the crylaa gene, the tetracycline gene, and a 5' portion of the CrylAc gene were deleted by looping out. Since about 200 colons having a homologous sequence overlapped between the crylaa and crylac genes in the original plasmid, a significant number of heterogeneous recombinant plasmids were observed. Proteins encoded in the recombinant plasmids were tested against four different insect species to determine the domain for Cry lac-type insecticidal spectrum. They found that a recombinant protein, with the cross over from CrylAa to CrylAc at the 347th amino acid residue, had an activity essentially the same as that of CrylAc. A similar study conducted with the cry] Ca gene revealed that the hybrid proteins containing CrylEa or CrylAb domain I and II and CrylCa domain III had substantially higher activity against Spodoptera exigua than any of the parent proteins (Bosch et al., 1994; de Maagd et al., 1996). Later de Maargd et al. (1999) reported that CrylCa domain III was involved in Spodoptera specificity. SLIGHT SEQUENCE DIFFERENCES CAUSE ACTIVITY VARIATION Moar et al. (1990) and Von Tersch et al. (1991) reported significant differences in the activity spectra among the same CrylA-type proteins from different Bt strains. Moar et al. (1990) showed that the activities of Cry 1 Ab from Bt kurstaki NRD 12 toward Spodoptera exigua and Trichoplusia ni were higher than those of CrylAb from the HDI strain. They also found that the T. ni activity of CrylAc from NRD12 or HD1 was higher than that of CrylAc from the HD73 strain. Von Tersch et al. (1991) cloned and sequenced a cry]ac gene from Bt kenyae HD588 and reported that the activity of the gene product, CrylAc, against Plutella xylostella and Ostrinia nubilalis was lower than that of CrylAc from Bt kurstaki HD263. The sequence of the HD588 CrylAc protein differs slightly from that of the original CrylAc protein of Bt kurstaki HD73. Dardenne et al. (1990) studied a variant of CrylAc whose sequence was almost identical to that of CrylAc from Bt kenyae. These CrylAc variants had mutations at the 440th and 442nd amino acid residues of the original HD73 CrylAc, and this suggested possible involvements of these residues in insecticidal activities. ACTIVITY SPECTRA OF THREE Cry1A-TYPE PROTEINS Three Cry 1 A proteins, Cry 1 Aa, Cry 1 Ab and CrylAc, have been found in most Bt kurstaki-based commercial strains currently being used. Among these CrylA proteins, CrylAc appears to be the most active protein against important agricultural pests, such as Pieris brassicae, Trichoplusia ni, and Heliothis species (Hofte and Whiteley, 1989). The CrylAc protein has especially high activity toward Heliothis virescens (Hofte and Whiteley, 1989) and perhaps other Heliothis species. In the case of Ostrinia nubilalis, CrylAb is

16 16 Yamamoto ten times more active than Cry 1 Ac. Among these Cry 1 A proteins, Cry 1 Ab acts best towards Spodoptera exigua (Moar et al., 1990). Aronson et al. (1991) reported a unique contribution of Cry 1 Ab to the overall activity of Bt aizawai HD133 crystal. They demonstrated, using plasmid curing technique to delete Cry 1 Ab from the crystal, that the crystal was less soluble and therefore less toxic. The Cry 1 Aa protein is very active against Bombyx mori. This is not a desired trait in commercial applications. On the other hand, Cry 1 Ac is essentially inactive against B. mori (Ge et al., 1989). CrylAa, however, is useful for some commercial applications. For example, Van Frankenhuyzen et al. (1992) reported that the activity of Cry 1 Aa was equally high as, if not better than, that of Cry 1 Ab against forest-defoliating Lepidoptera species. Numerous studies have been conducted to determine insecticidal activity of individual crystal proteins. In most cases, each report reveals assay results only against one or a few insect species. Since Bt has a very narrow insect specificity, assays are required on widely diversified insect species. In order to understand extensive insect specificity of any crystal protein, a large number of publications have to be reviewed. This had been a laborious task until a comprehensive database was put together by a Canadian group. The database is found at URL: res/bt HomePage/netintro.htm In-vitro ASSAY METHODS The insect specificity reports, which have been reviewed above, are all based on in-vivo bioassays with whole insects. Several in-vitro assay methods, including the cell-lysis and receptor-binding assays, have been utilized in the determination of crystal protein activity. A major purpose of using these methods is to elucidate the mode of action of the toxin at the cellular level and is not intended to replace the whole-insect assay. Nevertheless, the activity spectra of some crystal proteins, determined by the whole-insect assay, have been confirmed by cell-lysis assays. In the in-vitro assays, the crystal proteins first must be activated by protease. Trypsin has been a popular choice. Van Rie et al. (1990a) demonstrated that the activity spectra of CrylAa, CrylCa, and CrylEa toxins against Spodoptera littoralis, Manduca sexta, and Heliothis virescens corresponded to their binding affinities on the brush border membrane vesicles (BBMV) isolated from these insects. In contrast, Wolfersberger (1990) reported that the binding affinities of Cry 1 Ab and CrylAc to Lymantria dispar BBMV were not related to the whole insect activities of these proteins. CRYSTAL TOXIN BINDS TO THE RECEPTOR As early as 1984, Knowles et al. (1984) suggested the presence of a receptor for the crystal toxin. They found that a mixture of CrylA toxins, partly isolated from Bt kurstaki HD 1, behaved like a lectin, a protein known to bind to glycoproteins. They tested several monosaccharides and found that N-acetyl galactosamine (Gal- NAc) inhibited the toxicity of the HD 1 toxins towards Choristoneura fumiferana cells (CF1). Knowles and Ellar (1986) labelled the proteins on the CFl cell surface with radioactive isotopes and identified the toxin binding protein as a 146-kDa molecule. Hof mann et al. (1988a) later established a binding assay using the radio-labeled crystal toxin and Pieris brassicae BBMV. They demonstrated that a toxin preparation from Bt thuringiensis 4412 bound to BBMV of Pieris brassicae with a very high affinity. The binding site or molecule on BBMV was sensitive to protease or glcosidase treatment suggesting a glycoprotein. Using this technique, Hofmann et al. (1988b) further reported that the CrylAb toxin of Bt thuringiensis berliner 1715 binds to both Pieris brassicae and Manduca sexta BBMV but the toxin from Bt thuringiensis 4412 failed to bind Manduca sexta BBMV. Their binding study confirmed that differences occurred in activity spectra of these two proteins toward P. brassicae and M sexta. Both studies of Hofmann et al. (1988a, b) revealed that GalNAc did not affect the binding of these toxins. Subsequently, Knowles et al. (1991) showed that the binding inhibition of the CrylAc protein by Ga1NAc was dependent on the insect species from which BBMV had been isolated. Van Rie et al. (1989) introduced a theory of multiple binding sites on BBMV for CrylAa, CrylAb, and CrylAc toxins: two binding sites on BBMV from M. sexta, one site for all three CrylA toxins and the other site specific only to CrylAa. In Heliothis virescens, there were three sites with remarkably different concentrations. Site 1 bound to all CrylA's as in the case of M. sexta, site 2 to Cry 1 Ab and Cry 1 Ac, and site 3 to CrylAc only. The complexity of the binding sites, in particular those of H. virescens, has been confirmed, at least in part, by Knowles et al. (1991) and Garczynski et al. (1991) who have identified multiple binding or receptor proteins in Heliothis zea and H. virescens. This theory offers an explanation on the specificity of Cry 1 Ac. Cry 1 Ac has the highest activity against Heliothis species among the three CrylA's. The correlation between insect specificity and affinity of the receptor binding has been further endorsed with Spodoptera-specific CrylCa and CrylEa toxins (Van Rie et al., 1990a).

17 One Hundred Years of Bacillus thuringiensis 17 The protein identified as a receptor for CrylAc in Manduca sexta BBMV has been found to be aminopeptidase N (Knight et al., 1995; Sangadala et al., 1994). Ga1NAc is associated with the aminopeptidase N molecule that is attached to the midgut cell membrane through a glycosyl-phosphatidylinositol (GPI) anchor. Since then, numerous reports have indicated that other insect species have the same or similar molecule which acts as a receptor for CrylA-type toxins. On the other hand, Vadlamudi et al. (1995) have reported cloning of a 210-kDa cadherin-like protein from M sexta BBMV that binds to CrylA-type toxins. INSECTS RESISTANT TO Bt LOST THE SPECIFIC RECEPTORS In 1985, McGaughey (1985) demonstrated that insect resistance could develop against the Bt toxin. He showed that Plodia interpunctella became resistant to Bt when reared in several generations in the laboratory with a sublethal dose of a Bt commercial product, presumably Bt kurstaki HDI. When Van Rie et al. (1990b) examined the Bt-resistant P, interpunctella, he found that the receptor sites on BBMV for CrylAb were greatly reduced. The binding site for the CrylCa protein, however, remained in the resistant P. interpunctella. Ferre et al. (1991) further showed that a Btresistant colony found in Plutella xylostella had lost the binding sites for Cry 1 Ab but retained the sites for CrylBa and CrylCa. This indicates that CrylBa and CrylCa do not share the binding sites with CrylAb in these insects. P. xylostella was the first insect that developed, in the field, substantial resistance to Bt crystal proteins. Bt had been used heavily against this insect with a short life cycle. Tabashnik et al. (1994) found no binding of CrylAc to BBMV isolated from a resistant P. xylostella population. The resistance was lost when the Bt pressure was removed. Interestingly, BBMV from a revertant colony regained the binding to Cry 1 Ac. THREE MODELS FOR MEMBRANE INSERTION Von Tersh et al. (1994) have reported the insertion of domain I of a Cry toxin alone into phospholipid vesicles. Domain I is composed of seven alpha-helices similar to those of colicin A. The membrane insertion theory of colicin A suggests a similar mechanism for the Bt crystal toxins. Li et al. (1991), in their report of the Cry3Aa structure, suggested a model called the umbrella model. In their proposal, the hydrophobic hairpin between a-4 and a-5 helps these helices to be inserted into the membrane like an umbrella. Hodgman and Ellar (1990) have developed a model called the penknife model, which is a more direct adaptation of the colicin model. In this model, a-5 and a-6 penetrate like a penknife into the membrane. There are numerous reports that support or oppose these models. Alternatively, a school of scientists believe that the entire toxin molecule goes into the membrane. One major observation that supports this whole molecule theory is that the protection of the toxin from protease digestion only when the toxin is inserted into the membrane (Wolfersberger et al., 1987). ENVIRONMENTAL RELEASE OF GENETICALLY MODIFIED Bt Bt strains, used in commercial insecticides, often contain multiple crystal protein genes, because the combination of genes gives a wider host spectrum than that achievable with one gene. Efforts have been made to further broaden host specificity by inserting an additional gene or genes into Bt. Baum et al. (1990) at Ecogen and Gamel et al. (1992) at Sandoz constructed plasmid vectors utilizing the origin of replication from Bt. These vectors were relatively stable in Bt, and Ecogen developed a product based on this technology. On the other hand, Sandoz found that the vector was not stable enough in the host cell for commercial production. In order to circumvent the problems associated with the plasmid technology, Kalman et al. (1995) developed a method to insert a cry gene into the chromosomal DNA of a host Bt strain, a Bt kurstaki strain similar to HD 1. The inserted gene appeared to be very stable as no movement was observed in a conjugation type test. The Bt host continued to express indigent cryla-type genes at a level observed in the original strain with no inserted gene. In addition, the inserted gene expressed the protein as much as it did in the original Bt host. The results of the chromosomal integration encouraged Sandoz to conduct field trials in several locations in Mississippi and California. The recombinant strain, containing cryl Ca in its chromosome, showed substantially better crop protection against Spodoptera exigua than the non-recombinant, HD 1-type host strain. In a test done in California in 1994, the population of the recombinant strain in the soil was monitored for 264 days after its application. Yamamoto et al. (1998) reported that the population decreased exponentially at a rate of 20-day half-life. Although FUTURE FOR Bt Bt is the most commercially successful

18 18 Yamamoto microbial insecticide, it has not been used as much as a typical chemical insecticide. A problem with Bt is that the toxin must be ingested by insects. It has no activity by contact unlike certain chemical insecticides. Once sprayed in the field, the crystals are quickly washed off from the crop by rain or inactivated by sunlight. The efficacy lasts only for a day or two. On the other hand, the high host-specific activity of Bt crystal protein has distinct advantage. For a sensitive insect, the activity of a potent Bt toxin can be 10 times higher than that of a typical chemical insecticide. For these reasons, the Bt cry genes have been incorporated into crops, such as cotton (Perlak et al., 1990) and corn (Estruch et al., 1994). Within a few years, these Bt crops have demonstrated tremendous technical and commercial successes. According to Agrow (Sep. 1, 2000), over 11 million ha of Bt-corn were planted in the U.S. in Bt promotes less use of chemical insecticides. There is no doubt of saving beneficial insects, aquatic animals, birds and other higher animals from the intoxication caused by chemical insecticides. This is particularly true for cotton on which huge amounts of the chemical insecticide are sprayed at high frequencies. Planting of Bt transgenic crops, especially Bt-corn, has not increased in 2000 as expected because of political concerns. A controversial study done by Losey et al. (1999) claimed that the Bt corn was harmful to monarch butterfly larvae. The report has been widely cited by the press suggesting that the scientists have found Bt to kill non-target insects. There are two important aspects in this incident. First, the critics of the study have questioned the validity of the report. The study was done in a highly artificial laboratory condition, which was far from actual field conditions. No one has determined that monarch butterfly larvae are killed in mass in or near Bt-corn fields. A survey has shown that the monarch butterfly population has increased by 30% in 1999 while Bt-corn planting has increased by 40%. Another aspect is that this report has caused the public to believe that Bt can be harmful to other animals. The toxicity of CrylAb, the protein expressed in Bt corn, to the monarch butterfly is not surprising to those who have worked with Bt. The protein acts to kill Lepidoptera insects. ACKNOWLEDGMENTS The author thanks Dr. Yoshinori Tanada for critical reading of the manuscript. The author also thanks Dr. Toshihiko Iizuka for the opportunity of writing this review. REFERENCES Adams, L.F., Brown, K.L., and Whiteley, H.R. (1991) Molecular cloning and characterization of two genes encoding sigma factors that direct transcription from a Bacillus thuringiensis crystal protein gene promoter. J. Bacteriol. 173, Adams, L.F., Visick, J.E., and Whiteley, H.R. 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