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were collected in an apple orchard located in Frelighs- burgh (QC, Canada). Each mite was homogenized in 3 ml of phosphate-buffered saline (Sambrook et al.

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Isolation and characterization of a novel Bacillus thuringiensis strain expressing a novel crystal protein with cytocidal activity against human cancer cells Y.-C. Jung1, E. Mizuki2, T. Akao2 and J.-C. Coˆte´1 1 Agriculture and Agri-Food Canada, Research Centre, Saint-Jean-sur-Richelieu, QC, Canada 2 Biotechnology and Food Research Institute, Fukuoka Industrial Technology Centre, Aikawa-Machi, Kurume, Fukuoka, Japan

Keywords Bacillus thuringiensis, Cry protein, Cry31Aa2, cytocidal activity, human cancer cells. Correspondence Jean-Charles Coˆte´, Research Centre, Agriculture and Agri-Food Canada, 430 Gouin Blvd, St-Jean-sur-Richelieu, QC, Canada J3B 3E6. E-mail: [email protected]

Present address Y-C. Jung, The Center for Functional Genomics, ENH Research Institute, Northwestern University, 1001 University Place, Evanston, IL 60201, USA. 2006/0928: received 30 June 2006, revised 15 September 2006 and accepted 3 October 2006 doi:10.1111/j.1365-2672.2006.03260.x

Abstract Aims: To characterize a novel, unusual, Bacillus thuringiensis strain, to clone its cry gene and determine the spectrum of action of the encoded Cry protein. Methods and Results: The B. thuringiensis strain, referred to as M15, was isolated from dead two-spotted spider mites (Tetranychus urticae Koch; Arthropoda: Arachnida: Tetranychidae). It is an autoagglutination-positive strain and is therefore non-serotypeable. A sporulated culture produces a roughly spherical parasporal inclusion body, the crystal, tightly coupled to the spore. Although the crystal appears to be composed of at least two major polypeptides of 86 and 79 kDa as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Southern hybridization indicates that the corresponding crystal protein gene is likely present in only one copy. The crystal protein gene was cloned and, based on nucleotide sequence homology with an orthologous cry31Aa1 gene, assigned the name cry31Aa2. Although initially isolated from spider mites, B. thuringiensis M15 is non-toxic to spider mites and it does not produce the wide spectrum b-exotoxin. Assays on mammalian cells, however, reveal that Cry31Aa2, when cleaved with trypsin, is cytocidal to some human cancer cells but not to normal human cells. No cytocidal activity was induced after protease treatment of Cry31Aa2 with either chymotrypsin or proteinase K. Trypsin, chymotrypsin and proteinase K cleavage sites were determined. Conclusions: The B. thuringiensis strain M15 exhibits specific cytocidal activities against some human cancer cells. Significance and Impact of the Study: This study raises questions as to the actual role of this bacterial strain and its crystal protein in the environment. It may be possible to further develop the Cry31Aa2 protein to target specific human cancer cells.

Introduction Bacillus thuringiensis is characterized at the species level by the production upon sporulation of a parasporal inclusion body, the crystal. The crystal is made up of proteins, the d-endotoxins, classified into two families, the Cry and Cyt proteins, encoded by respective cry and cyt genes (Schnepf et al. 1998; de Maagd et al. 2001). Whereas Cyt proteins exhibit broad spectrum of activity in vitro against invertebrate and vertebrate cells including mammalian erythrocytes (Thomas and Ellar 1983) and

are lethal in vivo against larvae of dipteran insects (Armstrong et al. 1985), some Cry proteins, when processed, exhibit specific insecticidal activities against lepidopteran, dipteran or coleopteran larvae (Ho¨fte and Whiteley 1989; Schnepf et al. 1998). Some B. thuringiensis strains have been developed as insecticides in sprayable formulations and some cry genes have been transformed into transgenic plants to confer protection against specific insect pests. The mode of action of Cry proteins has been partly deciphered in insect models (Gill et al. 1992; Knowles and Dow 1993). Upon ingestion by a susceptible target, the

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crystal is solubilized in the alkaline pH of the insect midgut, the Cry proteins are released and processed by specific midgut proteases to yield active protein toxins. These bind to specific receptors on the surface of midgut epithelial columnar cells. This leads to the formation of pores in the apical membrane which causes osmotic balance disturbance, cell lysis and, ultimately, death. The development of B. thuringiensis as a microbial insecticide has driven the establishment of several screening programmes for novel B. thuringiensis strains expressing novel insecticidal and pesticidal activities. A decade ago, it was estimated that close to 50 000 B. thuringiensis strains were kept in various collections worldwide (Sanchis et al. 1996). Recently, it has been reported that some Cry proteins from strains with no known toxic activity exhibited cytolytic activities against various vertebrate cells including human cancer cells (Mizuki et al. 1999, 2000; Lee et al. 2000; Ito et al. 2004). These Cry proteins were named ‘parasporin’ (Mizuki et al. 2000). We report here the isolation and characterization of a novel B. thuringiensis strain expressing a novel crystal protein, Cry31Aa2, with unique activity against some human cancer cells. Materials and methods Bacterial strains and plasmids The Bacillus thuringiensis strain used in this study, referred to as M15, was isolated from dead two-spotted spider mites (Tetranychus urticae Koch; Arthropoda: Arachnida: Tetranychidae) as described elsewhere (Jung et al., 2001a, 2001b). Bacillus thuringiensis serovar kurstaki HD-1 and serovar israelensis HD-500 were obtained from the International Entomopathogenic Bacillus Centre, Institut Pasteur (Paris, France). Bacillus thuringiensis serovar higo BT205 was in our collection (Jung et al. 1998). Escherichia coli DH5a (Gibco BRL, Burlington, ON, Canada) was used as a bacterial host for the cloning vectors pUC18 (Amersham Pharmacia Biotech, Montreal, QC, Canada) and pBluescript II KS(+) (Stratagene, La Jolla, CA, USA). The E. coli–B. thuringiensis shuttle vector pHPS9 (Haima et al. 1990) was purchased from American Type Culture Collection (Manassas, VA, USA). The acrystalliferous B. thuringiensis serovar kurstaki HD-1 Cry) B strain (Stahly et al. 1978) was obtained from the Bacillus Genetic Stock Center, The Ohio State University (Columbus, OH, USA). The B. thuringiensis M15 strain was deposited on 1 February 2001 in the International Depository Authority (Winnipeg, MB, Canada), under accession number IDAC010201-5, in accordance with the ‘Budapest Treaty 66

on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure’. Isolation of the novel B. thuringiensis strain Dead two-spotted spider mites, parasitic on apple leaves, were collected in an apple orchard located in Frelighsburgh (QC, Canada). Each mite was homogenized in 3 ml of phosphate-buffered saline (Sambrook et al. 1989). The homogenate was incubated for 16 h at room temperature and heat shocked at 78C for 15 min. The homogenate was plated on 2YT agar (Sambrook et al. 1989), and incubated for 18 h at 30C. All bacterial colonies with a morphology similar to B. thuringiensis were streaked on T3 agar plates (Travers et al. 1987) and incubated at 30C for 48 h. Colonies were examined by phase-contrast microscopy (Carl Zeiss Canada Ltd, Toronto, ON, Canada) for the presence of spores and crystals. The 15th B. thuringiensis isolate was called strain M15. Biochemical profiles of the B. thuringiensis strains The B. thuringiensis strain M15 was biochemically characterized using the API 50CH and API 20E test strips following the manufacturer’s recommendations (bioMe´rieux, St-Laurent, QC, Canada). Bacillus thuringiensis serovar kurstaki HD-1, serovar israelensis HD-500 and serovar higo BT205 were used as controls. Analyses of parasporal inclusion proteins by SDS-PAGE The B. thuringiensis strain M15 was grown in T3 broth for 5 days at 30C on a rotary shaker to allow sporulation and crystal production. Spores and crystals were separated from each other in the tightly bound spore–crystal duplexes using an ultrasonic processor model VC130 (Sonics and Materials, Inc., Newtown, CT, USA) and purified by sucrose density-gradient centrifugation as described elsewhere (Thomas and Ellar 1983). For separation of proteins in the 20- to 200-kDa range, a 20-ll volume of the crystal suspension was added to three volumes of gel-loading buffer [2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, 0Æ05% bromophenol blue, 125 mmol l)1 Tris–HCl pH 6Æ8] in a 1Æ5-ml microfuge tube, incubated at 90C for 7 min and centrifuged for 2 min to pellet unsolubilized materials. A 30-ll volume of the supernatant was analysed by polyacrylamide gel electrophoresis (PAGE) in the presence of SDS. Electrophoresis was performed as described by Laemmli (1970) using 4% stacking and 10% resolving gels. Alternatively, for separation of proteins in the lower 1- to 25-kDa range, a tricine-SDS–PAGE was performed using Bio-Rad’s tricine sample buffer

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(Bio-Rad, Mississauga, ON, Canada), according to the manufacturer’s instructions, essentially as described by Scha¨gger and von Jagow (1987). Crystal protein N-terminal sequencing The purified crystals were added into 0Æ1 n NaOH– 3 mol l)1 HEPES solution and solubilized in 10 volumes of gel-loading buffer by incubating in boiling water for 5 min. The sample was run on 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The Cry protein bands stained with Coomassie brilliant blue R-250 (Bio-Rad) were excised and their N-terminal amino acid sequences were determined by Edman degradation liquid phase method using an Applied Biosystems Procise cLC-494 equipped with an on-line Applied Biosystems 140D Phenylthiohydantoin analyser (Applied Biosystems, Foster City, CA, USA) (Hewick et al. 1981). The phenylthiohydantoin amino acid derivatives were determined by comparison with standards analysed at the start of the sequence analysis.

A novel B. thuringiensis strain

(Gibco BRL), respectively, electrophoresed on a 0Æ7% agarose gel and transferred onto a Nytran nylon membrane (Schleicher and Schuell, Keene, NH, USA) by the method of Southern (1975). Southern blot hybridization using the DIG-labelled M15-M oligonucleotide was performed with the standard hybridization solution [5X SSC, 1% blocking reagent (Roche), 0Æ1% N-lauroylsarcosine, 0Æ02% SDS] for 13 h at 39C. After hybridization, the membrane was washed twice for 15 min each in 4X wash solution (4X SSC, 0Æ1% SDS) at 39C. Following the washes, detection of signals on the membrane was performed with the colour-substrate solution containing 4-nitroblue tetrazolium chloride (Roche) and 5-bromo-4chloro-3-indolyl-phosphate (Roche) following the manufacturer’s recommendations. Cloning and subcloning of the crystal protein gene Plasmid DNA purified from B. thuringiensis strain M15 was digested with HindIII and ligated with HindIII-diges(a)

Plasmid DNA preparation and Southern hybridization Bacillus thuringiensis strain M15 was grown in Luria-Bertani (LB) broth (bacto tryptone 10 g, bacto yeast extract 5 g, NaCl 5 g l)1) at 30C for 16 h on a rotary shaker. Plasmid DNA was isolated using the alkaline extraction method (Birnboim and Doly 1979) with the following modifications. Lysozyme [Sigma-Aldrich Canada Ltd (Sigma), Oakville, ON, Canada] was added at a concentration of 2 mg ml)1 and the cell suspension was incubated at 37C for 1 h. Recombinant plasmid DNAs from E. coli DH5a were isolated by the alkaline extraction method. The plasmid DNA for DNA sequencing was purified with Wizard Plus SV minipreps DNA purification system following the manufacturer’s recommendations (Promega, Nepean, ON, Canada). An 18-mer degenerate oligonucleotide, named M15-M, was designed based on the 86 and 79 kDa N-terminal amino acid sequences of the Cry proteins and used as a DNA probe for Southern hybridization. The M15-M oligonucleotide was labelled by the digoxigenin (DIG) oligonucleotide 3¢-end labelling kit containing DIG-11ddUTP following the manufacturer’s recommendations (Roche, Laval, QC, Canada). The labelled oligonucleotide was precipitated with 0Æ1 volume of 4 mol l)1 LiCl and 2Æ5 volumes of ice-cold ethanol, and transferred at )70C for 30 min. The reaction was centrifuged at 4C for 15 min. The washed pellet was resuspended in nucleasefree water, and stored at )20C until use. Plasmid DNA from B. thuringiensis strain M15 was digested with either HindIII, HindIII/EcoRI or EcoRI

(b)

Figure 1 Micrographs of Bacillus thuringiensis strain M15. (a) Phasecontrast micrograph of a lysed culture. (b) Transmission electron micrograph of a sporulated culture (magnification 25000·).

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Tests Fermentation of Glycerol D-arabinose L-arabinose Ribose D-xylose L-xylose D-galactose D-glucose D-fructose D-mannose L-sorbose Inositol D-mannitol D-sorbitol N-acetylglucosamine Arbutin Esculin Salicin D-cellobiose D-maltose Lactose Melibiose Sucrose Trehalose Starch Glycogen Gluconate Production of b-Galactosidase Arginine dihydrolase Ornithine decarboxylase Urease Tryptophane deaminase Gelatinase Oxidase Catalase H2 S Indole Acetoin Citrate utilization Nitrate reduction

Y.-C. Jung et al.

B. thuringiensis B. thuringiensis B. thuringiensis serovar kurstaki serovar israelensis serovar higo B. thuringiensis HD-1 HD-500 BT 205 M15

+ ) ) + ) ) ) + + ) ) ) ) ) + + + + + + ) ) ) + ) + +

+ ) ) + ) ) ) + + ) ) ) ) ) + + ± ) + + ) ) ) + + + +

+ ) ) + ) ) ) + + ) ) ) ) ) + + + + + + ) ) ) + + + ±

± ) ) + ) ) ) + + ) ) ) ) ) + ) ± + ) + ) ) ) + ) ) )

) + ) + ) + + + ) ) + + +

) + ) ) ) + + + ) ) + ) )

) + ) + ) + + + ) ) + ) )

) ) ) + ) + + + ) ) + ) )

Table 1 The biochemical profile of Bacillus thuringiensis M15 and selected control strains

+, ) and ± indicate positive, negative and weak reactions respectively.

ted shrimp alkaline phosphatase (Roche)-treated pBluescript II KS(+). After ligation, the recombinant DNA was transformed into E. coli DH5a. Preparation of E. coli DH5a competent cells and transformation were performed as described elsewhere (Sambrook et al. 1989). The transformants were grown on LB agar plates containing 100 lg ml)1 ampicillin (Sigma) and 40 lg ml)1 X– Gal (5¢-bromo-4-chloro-3-indolyl-b-d-galactopyranoside; Sigma) at 37C. White colonies were toothpicks trans68

ferred to 1 ml of fresh LB broth supplemented with 100 lg ml)1 ampicillin, and incubated overnight at 37C. The recombinant plasmid DNAs were isolated by the cracking procedure (Sambrook et al. 1989) and electrophoresed on a 0Æ7% agarose gel. Based on inserts estimated sizes, three recombinant plasmid DNAs were selected, digested with HindIII, electrophoresed on a 0Æ7% agarose gel, transferred onto a Nytran nylon membrane by the method of Southern (1975) and probed with the M15-M

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1

2

3

4

kDa 200·0

116·0 97·4

66·0

45·0

31·0

21·5

Figure 2 SDS–PAGE analysis of the crystal protein(s) of Bacillus thuringiensis strain M15. The B. thuringiensis strain M15 crystals purified by sucrose density-gradient centrifugation was subjected to a 10% SDS–PAGE electrophoresis (lane 4). Crude extracts of the fully lysed B. thuringiensis serovar kurstaki HD-1 were also subjected to electrophoresis on a 10% SDS–polyacrylamide gel (lane 3) as a control. High molecular (lane 1) and low molecular masses (lane 2) of standard protein markers in kDa are indicated on the left.

oligonucleotide. The recombinant selected by Southern hybridization was further digested using a series of restriction enzymes [EcoRI, BglII (Gibco BRL), DraI and SphI (Amersham Pharmacia Biotech)] for subcloning purposes and ligated to either pUC18 or pBluescript II KS(+).

Expression of the Cry protein in the B. thuringiensis Cry)B strain The 3Æ6-kb HindIII/SphI fragment containing the crystal protein gene was excised from the recombinant plasmid pYCH217 and cloned into the E. coli–B. thuringiensis shuttle vector pHPS9-doubly digested with HindIII/SphI. The B. thuringiensis Cry)B strain was transformed with the cloned B. thuringiensis M15 crystal protein gene by electroporation as described by Vehmaanpera¨ (1989) with the following modifications. Bacterial cells cultured in 200 ml of LB broth supplemented with 0Æ25 mol l)1 sucrose and 0Æ05 mol l)1 potassium phosphate, pH 7Æ0 (LBSP) to an optical density of 1Æ0 at 600 nm were centrifuged, washed three times with ice-cold SHMG buffer [250 mmol l)1 sucrose, 1 mmol l)1 HEPES, 1 mmol l)1 MgCl2, 10% (v/v) glycerol, pH 7Æ0], and resuspended in 1 ml of ice-cold SHMG buffer. The cell suspension was mixed with plasmid DNA at a final DNA concentration of 10 lg ml)1 in a 0Æ2-cm electroporation cuvette (BioRad), kept on ice for 30 min, and then electroporated using a Gene PulserTM model 1652076 (Bio-Rad) at 25lF, 2Æ5 kV and 400 ohms with a single pulse. After electroporation, 3 ml of LBSP supplemented with 10% (v/v) glycerol were immediately added into the cuvette and incubated at 37C for 2 h with shaking. The B. thuringiensis transformants were selected on nutrient agar plates containing 5 lg ml)1 of erythromycin (Sigma) and 5 lg ml)1 of chloramphenicol (Sigma) at 37C. The presence of crystals was examined by phase-contrast microscopy. The selected B. thuringiensis transformant was cultured in 250 ml of nutrient broth supplemented with 5 lg ml)1 of erythromycin and 5 lg ml)1 of chloramphenicol until cell autolysis was observed. The lysate was harvested and washed twice with 10 mmol l)1 EDTA (pH 8Æ0)–1 mol l)1 NaCl–1 mmol l)1 phenylmethylsulfonyl fluoride (Sigma). The crystal from the B. thuringiensis Cry)B transformant was purified by sucrose density-gradient centrifugation. The expressed Cry protein was analysed by SDS–PAGE. Electron microscopy

DNA sequencing DNA sequencing was performed with the near-infrared fluorescence automated DNA sequencer (LI-COR Model 4200; LI-COR, Inc., Lincoln, NE, USA). Nucleotide and protein sequences were analysed with the DNASIS program (Hitachi Software Engineering Co., Scarborough, ON, Canada). The 3313 nucleotide sequence presented here was deposited in GenBank under accession number AY081052.

The indigenous B. thuringiensis strain M15 was grown on T3 agar plate for 48 h at 30C. The B. thuringiensis Cry)B transformant was incubated on a nutrient agar plate supplemented with 5 lg ml)1 of erythromycin and 5 lg ml)1 of chloramphenicol for 48 h at 30C. The samples were mounted, fixed, ultra-thinly sliced (Beveridge et al. 1994) and electron micrographs were recorded at a nominal magnification of 25 000· and 20 000·, for the B. thuringiensis strain M15 and the B. thuringiensis Cry)B trans-

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(a)

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formant, respectively, at 80 kV on a JEOL model JEM1200EX transmission electron microscope (TEM) (Jeol, Tokyo, Japan).

5

kb

kb

23·13 9·42 6·56 4·36

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Detection of b-exotoxin

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2·32 2·03

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The presence of thermostable b-exotoxin in B. thuringiensis M15 was assessed using the house fly, Musca domestica L. (Diptera: Muscidae), as described elsewhere (WellmanDesbiens and Coˆte´ 2004). In summary, the supernatant of a sporulated culture of B. thuringiensis strain M15 was autoclaved, cooled, mixed with an artificial diet and presented as food source to house flies. Per cent mortality was recorded at different intervals. Activation of the crystal proteins and cleavage sites

(b)

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Figure 3 Southern blot analyses of Bacillus thuringiensis strain M15 plasmid DNA. Panel (a): three B. thuringiensis strain M15 plasmid DNA samples were digested with HindIII (lane 2), HindIII/EcoRI (lane 3), and EcoRI (lane 4), respectively, and separated by molecular weight on a 0Æ7% agarose gel. Molecular masses of lambda-DNA digested with HindIII (lane 1) and 1-kb DNA ladder (lane 5) are indicated on the left- and right-hand sides respectively. Panel (b): Southern hybridization of three B. thuringiensis strain M15 plasmid DNA samples digested with HindIII (lane 2), HindIII/EcoRI (lane 3), and EcoRI (lane 4) respectively. The digoxigenin-labelled 18-mer M15-M oligonucleotide hybridized with a 8-kb HindIII (lane 2), a 2Æ6-kb HindIII/EcoRI (lane 3), and a 2Æ6-kb EcoRI (lane 4) fragments. Molecular masses of each fragment detected by the M15-M probe are indicated on the right-hand side.

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The B. thuringiensis Cry)B transformant containing the B. thuringiensis M15 crystal protein gene was incubated in T3 broth at 30C for 4 days to allow the expression of toxin gene and crystal formation. Crystals were purified by sucrose density-gradient centrifugation. The crystal pellet was resuspended in 10 ml of solubilization buffer (50 mmol l)1 Na2CO3 (pH 10Æ0), 10 mmol l)1 dithiothreitol, and 1 mmol l)1 EDTA) and incubated at 37C for 2 h on a rotary shaker at 180 rev min)1. The mixture was centrifuged at 2900 g (Beckman TableTop Accuspin, Beckman Coulter, Fullerton, CA, USA) for 20 min at room temperature to remove unsolubilized materials, and the supernatant was transferred to a fresh tube. The pH was adjusted to 8Æ0 with Tris– HCl (pH 8Æ0). The protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as the standard and adjusted to 5 mg ml)1. To 70 ll of solubilized crystals, 1Æ5 ll of trypsin (from bovine pancreas; Sigma) at four different concentrations (10, 1, 0Æ1, and 0Æ01 mg ml)1) or 1Æ5 ll of a-chymotrypsin (from Tritirachium album; Sigma) at four different concentrations (10, 1, 0Æ1, and 0Æ01 mg ml1) or 2Æ5 ll of proteinase K (type II, from bovine pancreas; Sigma) at three different concentrations (100, 10 and 1 lg ml)1) were added, respectively, and incubated at 37C for 1 h. After protease treatment, phenylmethylsulfonyl fluoride was added to the solution to stop the proteolytic reaction and the mixture was run on an SDS–PAGE to assess the size of the cleaved fragments. N-terminal amino acid sequences of these fragments were determined as described previously. Mammalian cells and culture conditions The human cell lines UtSMC (originating from normal uterus smooth muscle) HeLa (from uterus–cervix–

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cancer), Sawano (from uterus cancer), TCS (from uterus– cervix–cancer), MOLT-4 (from leukaemic T cell), HL60 (from leukaemic T cell), Jurkat (from leukaemic T cell), MRC-5 (from normal embryonic lung fibroblast), A549 (from lung cancer), HC (from normal hepatocyte), HepG2 (from hepatocyte cancer), Caco-2 (from colon cancer), and animal cell lines Vero (from monkey normal kidney epithelial cell), COS-7 (from monkey SV-40-transformed kidney cell) and NIH3T3 (from mouse normal embryo fibroblast) were purchased from the RIKEN Cell Bank (Tsukuba, Ibaraki, Japan). Normal T cells were prepared from buffy coats obtained from the Fukuoka Red Cross Blood Center (Fukuoka, Japan) and were separated from lymphocytes and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 30 lg ml)1 kanamycin at 37C. HepG2, A549 and COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) FBS (ICN). HeLa, NIH3T3 and Vero cells were cultured in minimum essential medium (MEM) supplemented with 10% (v/v) FBS. Sawano cells were cultured in MEM supplemented with 15% (v/v) FBS. CACO-2 cells were cultured in MEM containing 10% (v/v) bovine serum (ICN) and 1% (v/v) non-essential amino acids. MRC-5 cells were cultured in HF-RITC80-7 supplemented with 10% (v/v) FBS. MOLT-4, HL60 and Jurkat cells were cultured in RPMI 1640 supplemented with 10% (v/v) FBS and 30lg ml)1 kanamycin. UtSMC and HC cells were cultured in SmGM and CS-C medium respectively. All cells were cultured at 37C in 5% CO2 in a humidified atmosphere. Assay of cytocidal activity The cytocidal activity was measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H tetrazolium bromide (MTT) method (Behl et al. 1992; Heiss et al. 1997), using a non-radioactive cell proliferation assay system (Promega). Briefly, each well of a microtest plate contained 90 ll of cell suspension containing 2 · 104 cells. After preincubation for 20 h at 37C, 10 ll of the trypsin-activated sample was added to the well at final concentrations 0Æ6 ng to 10 lg and the per cent cell survival was measured 24 h later. HindIII EcoRI

3·4 kb

DraI NdeI

SphI

DraI

DraI

BglII

Results Morphology and biochemical profile A Bacillus thuringiensis strain was isolated from dead twospotted spider mites and named M15. It was unusual in many respects. It was autoagglutination-positive, hence non-serotypeable strain. The crystal produced by a sporulated culture appeared roughly spherical when observed under phase-contrast microscopy and was tightly coupled to the spore even in lysed cultures (Fig. 1a). Further analysis under the TEM, however, revealed that the crystal had a two-dimensional polygonal shape (Fig. 1b). Bacillus thuringiensis strain M15 was characterized for its ability to ferment specific carbon sources, and for the production, utilization and reduction of specific compounds (Table 1). Although its biochemical profile shared several characteristics with the one of three other B. thuringiensis strains used as controls, serovar kurstaki HD-1, serovar israelensis HD-500 and serovar higo BT205, some differences were worth noting, namely poor fermentation of glycerol and inability to ferment arbutin, d-cellobiose, starch, glycogen and gluconate, and the absence of production of arginine dihydrolase. Although biochemical tests are not regarded as a key tool for the classification of B. thuringiensis strains (de Barjac and Frachon 1990), they are clearly useful for characterizing and distinguishing B. thuringiensis strains. SDS-PAGE analysis and crystal protein N-terminal sequence The B. thuringiensis strain M15 crystal was purified and its protein composition was analysed by SDS-PAGE (Fig. 2). At least two major bands of approx. 86 and 79 kDa in size were revealed. This was unusually given that most B. thuringiensis insecticidal crystal proteins have molecular masses in the range of 130–140 kDa for Cry1, Cry4A and Cry4B, and 65–80 kDa for Cry2A, Cry3A, Cry10A and Cry11A for examples (Aronson 1993; Baum and Malvar 1995). The N-terminal sequences were determined for the 86- and 79-kDa proteins. They both shared DraI

EcoRI

DraI XbaI

2·6 kb

PvuII

HhaI

EcoRI HindIII

1·4 kb

0·6 kb

Figure 4 The structural organization of the pYCH217 insert. A structural map of the 8-kb HindIII fragment was constructed. The open reading frame of a crystal protein gene, cry31Aa2, is indicated by a grey arrow. The hatched box indicates the region homologous to the digoxigeninlabelled 18-mer M15-M oligonucleotide probe. Selected restriction sites and subfragment sizes are indicated above and below the structural map respectively. ª 2007 The Authors Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 65–79

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Figure 5 Nucleotide sequence and deduced amino acid sequence of the cry31Aa2 gene. The cry31Aa2 gene is 2226 bp in length and codes for a polypeptide of 742 amino acids. The potential )35 and )10 boxes and a putative ribosome-binding site (RBS) are underlined. The stop codon is marked with asterisks. The sequence of the digoxigenin-labelled 18-mer oligonucleotide (M15-M) probe is indicated in bold capital letters. The terminal inverted repeats (IR) are underlined.

identical 20-amino acid residues. These were Met, Asp, Pro, Phe, Ser, Asn, Tyr, Ser, Glu, Gln, Lys, Tyr, Pro, Asp, Ser, Asn, Asn, Asn, Gln and Glu. This indicated that either both proteins were different while sharing identical N-terminal sequences or rather that the 86-kDa protein might have been processed at the C-terminus to yield the 79-kDa protein. 72

Southern hybridization An 18-mer oligonucleotide sequence, referred to as M15M, deduced from part of the N-terminal amino acid sequence (Glu, Gln, Lys, Tyr, Pro and Asp) of the 86- and 79-kDa proteins was synthesized with the following degenerate sequence: 5¢-GARCARAARTAYCCNGAY-3¢. This

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Figure 6 Comparison of the Cry31Aa2 and Cry31Aa1 deduced amino acid sequences. The capital letters and dotted lines under the amino acid sequence of Cry31Aa2 correspond to the differences and alignment gaps between the Cry31Aa2 and Cry31Aa1 proteins. Asterisks under the Cry31Aa2 sequence indicate identical amino acids in Cry31Aa1. The bold lines above the Cry31Aa2 sequence correspond to the five conserved amino acid blocks found in Cry proteins. Highly conserved residues in the five conserved amino acid blocks of the known Bacillus thuringiensis Cry proteins are underlined in bold under the Cry31Aa2 sequence; fl indicates trypsin cleavage sites in Cry31Aa2; l indicates the chymotrypsin and proteinase K cleavage site in Cry31Aa2.

was used as a probe in Southern hybridization to B. thuringiensis M15 plasmid DNA digested with various restriction enzymes and to clone the crystal protein gene. Hybridizations against plasmid DNA either digested with HindIII, doubly digested with EcoRI/HindIII, or digested with EcoRI, revealed single 8-kb HindIII, 2Æ6-kb EcoRI/HindIII or 2Æ6-kb EcoRI fragments [Fig. 3, panels (a) and (b)], respectively, indicating clearly that the cry gene was present in a single copy, in support to the C-terminal processing of the 86-kDa protein to yield the 79-kDa protein. Characterization of a new crystal protein gene, cry31Aa2 The 8-kb HindIII fragment which harbours the crystal protein gene was cloned and its nucleotide sequence was determined. A structural map is presented in Fig. 4. An open reading frame of 2226 bp in length that codes for a polypeptide of 742 amino acids with a predicted molecular mass of 83Æ12 kDa was found (Fig. 5). The start codon is not ATG but GTG. One potential promoter-like sequence in the 5¢-non-coding region (Lereclus et al. 1989; Baum and Malvar 1995) shows a 13-bp spacing between the putative )10 and )35 sequences located 138-bp upstream from

the start codon (GTG). The potential ribosome-binding site (RBS) (GAAAGGTGG) is located 7-bp upstream of the start codon (GTG) and is partially complementary to the 3¢-end (UCUUUCCUCC) of Bacillus subtilis 16S rRNA (McLaughlin et al. 1981; Moran et al. 1982). The calculated free energy of interaction (DG, 25C) between the B. subtilis 16S rRNA and the putative RBS is )14Æ8 kcal mol)1 (Tinoco et al. 1973). An inverted repeat that could form a stem-and-loop secondary structure with a calculated energy (DG, 25C) of )12Æ2 kcal mol)1 (Tinoco et al. 1973) is located 112-bp downstream from the stop codon (TAA) and may function as a transcription terminator. The 18-mer M15-M oligonucleotide sequence, 5¢-GARCARAARTAYCCNGAY-3¢, is homologous to a region located 24-bp downstream from the start codon (GTG), 5¢-GAACAAAAATACCAGAT-3¢. The amino acid sequence of the 83-kDa protein shares extensive (94%) amino acid sequence identities with Cry31Aa1 (Mizuki et al. 2000; Fig. 6) [also designated PS1Aa1 by the Parasporin Nomenclature Committee (Ohba et al. 2006)] except for the substitution of 25 amino acid residues scattered all over the sequence and the addition of 19 contiguous amino acids at positions

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Sma l BamH l Sal l Pst l Nhe l Nde l

EcoRI 8·64

Sphl 0·30

Bglll 1·11

cat86::lacZ Cm

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Er

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Hindlll 3·94

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Figure 7 Restriction map of the recombinant plasmid pYCP31A2 containing the cry31Aa2 gene. The 3Æ6-kb HindIII/SphI fragment containing the entire crystal protein gene, cry31Aa2, was cloned into the HindIII/SphI doubly digested Escherichia coli–Bacillus thuringiensis shuttle vector pHPS9 to yield recombinant plasmid pYCP31A2.

68–86 in the 83-kDa protein. The five conserved amino acid blocks of the 83-kDa protein were identical to those of Cry31Aa1 except for the substitution of an R (Arg) residue in Cry31Aa1 by a K (Lys) residue in the second conserved block of the 83-kDa protein. The 83-kDa protein was designated Cry31Aa2 by the Bacillus thuringiensis Nomenclature Committee (Crickmore et al. 1998, 2005). Recently, it was designated PS1Aa2 by the Parasporin Nomenclature Committee. )

The cry31Aa2 gene expression in B. thuringiensis Cry B strain The 3Æ6-kb HindIII/SphI fragment containing the entire crystal protein gene, cry31Aa2 (Fig. 4), was cloned into the HindIII/SphI doubly digested E. coli–B. thuringiensis shuttle vector pHPS9 to yield recombinant plasmid pYCP31A2 (Fig. 7). This construct was used to transform the acrystalliferous B. thuringiensis strain Cry)B. When observed under a phase-contrast microscope, the B. thuringiensis transformants expressing the cry31Aa2 gene contained, in addition to the spore, a roughly spherical parasporal inclusion, the crystal (data not shown). No inclusions were found in the B. thuringiensis transformant 74

Figure 8 Transmission electron micrograph of a sporulated, crystalproducing Bacillus thuringiensis Cry)B transformant expressing the cry31Aa2 gene (magnification 20000·).

45·0

31·0 21·5

Figure 9 SDS-PAGE analysis of the crystal protein of the crystal-producing Bacillus thuringiensis Cry)B transformant expressing the cry31Aa2 gene. The crystals were purified by sucrose density-gradient centrifugation and subjected to a 10% SDS-PAGE electrophoresis (lane 3). High molecular (lane 1) and low molecular masses (lane 2) of standard protein markers in kDa are indicated on the left-hand side.

harbouring the non-recombinant shuttle vector pHPS9 alone. Under the TEM, however, the crystal shows a nearly perfect hexagonal shape (Fig. 8). Both inclusions in the transformant, spore and crystal, are separated from each other, as opposed to what is found in B. thuringiensis strain M15 where they are tightly bound to each other. The crystal from the B. thuringiensis transformant was purified and analysed by SDS-PAGE. The crystal protein in the B. thuringiensis transformant is composed of a single major polypeptide of 83 kDa (Fig. 9). Detection of b-exotoxin To rule out the presence of the wide-spectrum b-exotoxin, assays were conducted on house flies. Assays were negative.

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A novel B. thuringiensis strain

Activation of the crystal proteins and cleavage sites

Dose–response analysis

Bacillus thuringiensis strain M15 crystals were alkali-solubilized, digested with three proteases, typsin, chymotrypsin and proteinase K, and the cleaved fragments were separated by electrophoresis (Fig. 10). Chymotrypsin and proteinase K each cleaves at least once generating a polypeptide with an estimated molecular mass of 70 kDa (Fig. 10). Trypsin, however, cleaves at least two times, generating polypeptides with estimated molecular masses of 70 and 55 kDa (Fig. 10). Additional electrophoresis ran longer showed the trypsin-generated 70-kDa band to be composed of at least two proteins with estimated molecular masses of 72 and 70 kDa respectively (data not shown). Electrophoresis under conditions aimed at the separation of lower molecular weight polypeptides, in the 1- to 25-kDa range, revealed an additional 15-kDa polypeptide (data not shown). Further digestion with an increased concentration of trypsin yielded only 55- and 15-kDa polypeptides (data not shown). The N-terminal sequences were determined for all fragments to identify the proteases cleavage sites. These are indicated in Fig. 6. Chymotrypsin and proteinase K cleave between residues Y (Tyr) and D (Asp) at amino acid positions 108–109 to yield a 70Æ4-kDa polypeptide (Fig. 6). Trypsin cleaves three times, between residues R (Arg) and D (Asp), R (Arg) and S (Ser), and R (Arg) and I (Ile), at amino acid positions 97–98, 112–113, and 250–251, to yield three polypeptides of 71Æ7, 69Æ9 and 54Æ6 kDa respectively (Fig. 6). The 15-kDa polypeptide detected by electrophoresis corresponds to the fragment between the second and third trypsin cleavage sites, a polypeptide of 15Æ3 kDa.

In the absence of protease digestion, alkali-solubilized Cry31Aa2 showed no cytocidal toxicity on any of the tested cells. Likewise, no cytocidal activity was induced after protease treatment of alkali-solubilized Cry31Aa2 with either chymotrypsin or proteinase K. Cytocidal activities were recorded only with alkali-solubilized trypsin-activated Cry31Aa2. Figure 11 shows the results of the dose–response study. Alkali-solubilized trypsin-activated Cry31Aa2 exhibited a similar dose-dependent activity on HeLa and TCS (cervix cancer cells), while not being toxic on Sawano (uterus cancer cells) and slightly toxic on UtSMC. Likewise, it exhibited a similar dose-dependent activity on HL-60 (leukaemic T cells) and Jurkat (leukaemic T cells), while not being toxic on MOLT-4 (leukaemic T cells) and normal T cells. It was highly toxic to Hep-G2 (hepatocyte cancer cells), but not to HC (normal hepatocytes). Finally, alkali-solubilized trypsin-activated Cry31Aa2 was neither toxic to A549 and MRC-5 (lung cancer cells and normal embryonic lung fibroblasts respectively), nor to Caco-2 (colon cancer cells), monkey (Vero and COS-7) and mouse (NIH3T3) cells tested. These results are summarized and compared with results obtained with Cry31Aa1 (Katayama et al. 2005) in Table 2. Both proteins are not cytocidal to Sawano, normal T cell, HC, A549, MRC5, CACO-2, both simian cell lines and the murine cell lines. The trypsin-activated Cry31Aa2 was, however, up to 500- and 150-fold more toxic than the activated Cry31Aa1 on Jurkat and HepG2 cells respectively. Discussion

1 2 3 4 5 6 kDa 200·0 116·0 97·4 66·0 45·0 31·0 21·5 -

Figure 10 SDS-PAGE analysis of the alkali-solubilized crystal cleaved with proteases. Alkali-solubilized crystals (lane 3), cleaved with trypsin (lane 4), chymotrypsin (lane 5) and proteinase K (lane 6). High (lane 1) and low molecular masses (lane 2) of standard protein markers in kDa are indicated on the left-hand side.

That an autoagglutination-positive, non-serotypeable B. thuringiensis strain expressing a crystal protein, Cry31Aa1, with proven cytocidal activity against human cancer cells was isolated from a soil sample in Hiroshima Prefecture, Japan (Mizuki et al. 2000), and that another autoagglutination-positive, non-serotypeable B. thuringiensis strain expressing a highly homologous crystal protein, Cry31Aa2, with proven cytocidal activity against human cancer cells was isolated from two-spotted spider mites in an apple orchard in Quebec, Canada (this work) is at the very least very puzzling and raises several questions. At the bacterial strain level, it would be interesting to compare both strains using different sets of characteristics and nucleotide sequences of rRNA and house-keeping genes to determine the level of phylogenetic relationship between both. Did they arise independently? What is their natural habitat? What is their role in their environment? Which role do their Cry proteins play in their natural

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Molt-4

100

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Cell survival (%)

HeLa

HL-60

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T-cell

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Figure 11 Cytocidal activity of the alkali-solubilized trypsin-activated Cry31Aa2 protein to cultured cells. To the cells (containing 2 · 104 cells) preincubated at 37C for 20 h, the toxin protein (final concentrations, 0Æ6 ng to 10 lg) were added, and the cells were further incubated at 37C for 24 h. Cell proliferation was assayed using MTT.

environment? These are questions of utmost importance from a microbial ecology standpoint. This is the first report of the isolation of a parasporin-producing B. thuringiensis strain outside Japan or Vietnam (Yasutake et al. 2006). It is worth pointing out that B. thuringiensis strain M15 is unable to ferment starch, a characteristic it shares with emetic Bacillus cereus strains (Ehling-Schulz et al. 2005). Whether this is true for other B. thuringiensis parasporin producers is unknown but worth addressing in future studies. Equally puzzling, Cry31Aa2 was, after Cry31Aa1, only the second member of the Cry31 class of Cry proteins reported. A third parasporin, PS1Aa3, which amino acid sequence is 100% identical to PS1Aa1 (Cry31Aa1) has been reported recently (GenBank accession number BAE79808). It is important to note that the amino acid sequence of Cry31Aa2, like Cry31Aa1, is very dissimilar 76

to that of the other classes of Cry proteins, with which they share very low (10 1Æ20 0Æ05 >10 0Æ02 >10 0Æ02 >10 >10 >10 >10 >10 >10 >10

0Æ12 – >10 >10 0Æ32 2Æ2 >10 >10 3Æ0 >10 >10 >10 >10 >10 >10 >10

Simian Murine

*The LC50 was calculated from the data of the dose–response curve of cytotoxicity against each cell line. Data from Katayama et al. (2005).

activity of Cry31Aa2 was only recorded following cleavage by trypsin. In Cry31Aa1, proteolytic treatment by proteinase K or trypsin generated four proteins. The cytocidal activity of Cry31Aa1 was recorded following cleavage by proteinase K and trypsin (Mizuki et al. 2000). Chymotrypsin treatment produced no cytotoxic proteins. Interestingly, the trypsin cleavage site between aminoacid residues 250–251 in Cry31Aa2, is absent in the corresponding region in Cry31Aa1, presumably due to the aminoacid substitution of M (in Cry31Aa1) to I (in Cry31Aa2) at residue 251 in Cry31Aa2. Of utmost importance, however, is that Cry31Aa2, when cleaved with trypsin, is up to 500and 150-fold more toxic than the activated Cry31Aa1 on Jurkat and HepG2 cells respectively. The requirement for protease activation of Cry31Aa2 (or Cry31Aa1) to reveal human cancer cell cytocidal activity is analogous to the requirement for protease activation of insecticidal Cry proteins. We are planning to follow-up, first with cell assays not only with alkalisolubilized trypsin-activated Cry31Aa2 but with specific trypsin-generated fragments to determine the toxic moiety, and secondly with in vitro assays on cultured human liver cancer tissue, especially in the light of the very high mortality rate of this type of cancer and a search for a cure. The spectrum of action of the Cry31Aa2 protein on human cells is also puzzling. When activated with trypsin, Cry31Aa2 is cytocidal to some human cancer cells from different tissues but not to normal cells from same tissues. This is exemplified by its toxicity on some leukaemic T cells and hepatocyte cancer cells and its absence of

toxicity on normal T cells and normal hepatocytes respectively. In insect models, Cry proteins exert their toxicity by binding to specific receptors. It is reasonable to speculate that a similar mechanism of ‘specific toxinspecific receptor’ might be at work here. As pointed out above, the amino acid sequence of the Cry31 proteins is very dissimilar to that of the other classes of Cry proteins. Dissimilarity in amino acid sequences would suggest dissimilarity in target cells. Cancer cells are usually less differentiated than the normal cells of the tissue where they arose. Whether this is due to cell dedifferentiation or rather to cancer arising in precursor cells, stem cells, might still be open to debate. Certainly, however, dedifferentiated cells or stem cells from different tissues may share common receptors capable of being recognized by similar binding proteins. Although the role of the B. thuringiensis crystal, and the Cry protein, has most often been associated with the killing of insects, its actual role may still be open to debate (Coˆte´ 2007). Certainly, the actual role of the B. thuringiensis crystal, and the Cry protein, is not to kill human cancer cells, yet in some cases they do, as shown here. It is reasonable to speculate that the number of binding protein-receptor duos, that is the number of key-lock duos, might be finite, and that there is redundancy in nature. Some receptor-binding proteins, such as a Cry protein here, might recognize similar receptors on different cells, including different human cancer cells as exemplified here with Cry31Aa2. Characterization of the putative receptors in sensitive human cancer cells and comparison with receptors from non-sensitive cells appear warranted. Additional experiments will include

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assay of caspase activity to determine whether or not apoptosis is involved in the mechanism of human cancer cells cytocidal activity. Acknowledgements We thank Suzanne Fre´chette for excellent technical assistance. Thanks are extended to Mona Boutros-Saleh and Pierre Lemoyne for assays on house flies for detection of b-exotoxin. References Armstrong, J.L., Rohrman, G.F. and Beaudreau, G.S. (1985) Delta-endotoxin of Bacillus thuringiensis subsp. israelensis. J Bacteriol 161, 37–46. Aronson, A. (1993) The two faces of Bacillus thuringiensis: insecticidal proteins and post-exponential survival. Mol Microbiol 7, 489–496. de Barjac, H. and Frachon, E. (1990) Classification of Bacillus thuringiensis strains. Entomophaga 35, 233–240. Baum, J.A. and Malvar, T. (1995) Regulation of insecticidal crystal protein production in Bacillus thuringiensis. Mol Microbiol 18, 1–12. Behl, C., Davis, J., Cole, G.M. and Schubert, D. (1992) Vitamin E protects nerve cells from amyloid b protein toxicity. Biochem Biophys Res Commun 186, 944–950. Beveridge, T.J., Popkin, T.J. and Cole, R.M. (1994) Electron microscopy. In Methods for General and Molecular Bacteriology ed. Gerhardt, P., Murray, R.G.E., Wood, W.A. and Krieg, N.R. pp. 42–71. Washington, DC: American Society for Microbiology. Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513–1523. Bradford, M.M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254. Coˆte´, J.-C. (2007) How early discoveries about Bacillus thuringiensis prejudiced subsequent research and use. In Biological Control: A Global Perspective ed. Vincent, C., Goettel, M. and Lazarovits, G. Wallingford, UK: CABI Publishing (in press). Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J. and Dean, D.H. (1998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62, 807–813. Crickmore, N., Zeigler, D.R., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J., Bravo, A. and Dean, D.H. (2005) Bacillus thuringiensis toxin nomenclature. Available at: http:// www.lifesci.sussex..ac.uk/Home/Neil_Crickmore/Bt/. Ehling-Schulz, M., Svensson, B., Guinebretiere, M.-H., Lindba¨ck, T., Andersson, M., Schulz, A., Fricker, M.,

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