Binary Toxins from Bacillus thuringiensis Active against the Western ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2004, p. 4889–4898 0099-2240/04/$08.00⫹0 DOI: 10.1128/AEM.70.8.4889–4898.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Vol. 70, No. 8

Binary Toxins from Bacillus thuringiensis Active against the Western Corn Rootworm, Diabrotica virgifera virgifera LeConte James A. Baum,1* Chi-Rei Chu,2† Mark Rupar,2‡ Gregory R. Brown,1 William P. Donovan,1 Joseph E. Huesing,1 Oliver Ilagan,1 Thomas M. Malvar,1 Michael Pleau,1 Matthew Walters,1 and Ty Vaughn1 Monsanto Company, Chesterfield, Missouri 63017-1732,1 and Ecogen Inc., Belmar, New Jersey 077192 Received 23 October 2003/Accepted 5 May 2004

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest. The gram-positive bacterium Bacillus thuringiensis is wellknown as a source of insecticidal proteins, most of which accumulate in crystalline inclusions during sporulation (26, 40, 43). These crystal proteins, or Cry proteins, are diverse and include distinct protein families as judged by amino acid sequence analysis and structural information derived from X-ray crystallography (for a review, see reference 40). Twenty-three years after the first reported cloning of a B. thuringiensis crystal protein gene (39), new proteins with varied activities against the larvae of insect pests, particularly species from the orders Lepidoptera, Coleoptera, and Diptera, continue to be identified. A comprehensive website provides ready access to Cry gene and protein sequences and assistance with nomenclature (http://www.biols.susx.ac.uk/home/Neil_Crickmore/Bt/index .html) (11). Historically, the use of the Cry proteins in agriculture for insect control has been restricted to spray-on bioinsecticide products composed of the spent fermentations of naturally occurring or transconjugant strains of B. thuringiensis as well as recombinant derivatives of B. thuringiensis and Pseudomonas fluorescens (5). Despite decades of use, these products remain a relatively minor component of the plant protection arsenal deployed by growers worldwide. In contrast, transgenic crops carrying B. thuringiensis cry genes and herbicide tolerance genes have enjoyed an unprecedented rate of adoption by farmers in the United States and elsewhere since their introduction in the mid-1990s. Worldwide acreage of transgenic

crops in 2002 was reported to be approximately 58.7 million hectares, or 145 million acres, representing a 12% increase over the year 2001 acreage (24). The larvae of the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, and related Diabrotica species are formidable pests of corn in the United States (17). Currently, there are two principal options for controlling rootworm feeding damage on corn: crop rotation, primarily with soybeans and alfalfa, and the application of granular insecticides for larval control (17, 27). The disadvantages of insecticide use include incomplete protection from rootworm feeding damage and adverse effects on the environment, human health, and beneficial organisms. Although generally effective in protecting first-year corn from rootworm damage, the crop rotation strategy has been circumvented, particularly in the eastern corn belt, by a behavioral adaptation: WCR adults lay eggs, which are then ready to hatch the following year, in nearby soybean fields (28). Likewise, an extended diapause enables eggs of the northern corn rootworm, Diabrotica barberi, to hatch 2 to 3 years after deposition, thereby reducing the effectiveness of crop rotation (25). An alternative approach to corn rootworm control is the cultivation of transgenic corn hybrids expressing insecticidal proteins derived from B. thuringiensis. A number of insecticidal proteins from B. thuringiensis have been reported to be toxic to rootworm larvae. These include several Cry3 (14, 15, 21) and Cry8 proteins (A. R. Abad, N. B. Duck, X. Feng, R. D. Flanagan, T. W. Kahn, and L. E. Sims, 2002, Genes encoding novel proteins with pesticidal activity against coleopterans, international patent application no. WO 02/34774 A2, World Intellectual Property Organization), Cry6Aa1 (33, 42), the binary toxins Cry34A-Cry35A (19), Cyt1Ba1 (34), Cyt2Ca1 (37), and the secreted proteins Vip1 and Vip2 (G. W. Warren, M. G. Koziel, M. A. Mullins, G. J. Nye, N. Desai, B. Carr, and N. K.

* Corresponding author. Mailing address: Monsanto Company, 700 Chesterfield Pkwy. West, Chesterfield, MO 63017-1732. Phone: (636) 737-7315. Fax: (636) 737-7015. E-mail: James.A.Baum@monsanto .com. † Present address: 226 Birchwood Dr., West Chester, PA 19380. ‡ Present address: Incyte Genomics, Stine-Haskell Research Center, Newark, DE 19714-0030. 4889

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APPL. ENVIRON. MICROBIOL. TABLE 1. Bacterial strains and plasmids used in this study

Strain

EG10650 EG10368 EG4550 EG5899 EG11521 EG11529 EG11582 EG11934 EG11935 EG11936 EG11937 EG9444 EG11648 EG4851 EG11658 EG12156 EG12158 SIC8004 SIC8006 SIC8008 SIC8096 a b

Organism

B. B. B. B. E. B. E. B. B. B. B. B. B. B. B. B. B. B. B. B. B.

thuringiensis thuringiensis thuringiensis thuringiensis coli thuringiensis coli thuringiensis thuringiensis thuringiensis thuringiensis thuringiensis thuringiensis thuringiensis thuringiensis thuringiensis thuringiensis thuringiensis thuringiensis thuringiensis thuringiensis

Plasmid

pEG1319 pEG1321 pEG1337 pEG1918 pEG1919 pEG1920 pEG1921 pEG1821 pEG1823 pEG2206 pEG2207 pIC17001 pIC17002 pIC17003 pIC17047

Relevant characteristic

Host Host Wild Wild

strain strain type type

Wild type Wild type ET76 knockout ET80 knockout ET84 knockout ET76 and ET84 knockout ET80 and ET84 knockout Deletion, ET84 5⬘ region

Cry protein produced

NDa ET39, ET39, ET39, ET39, ET75 ET74 ET39, ET39 ET71, ET71, ET76, ET76, ET80, ET76, ET76, ET80 ET76 None

ET74, ET74, ET74, ET74,

ET75 ET75 ET75 ET75

ET74 ET79 ET79 ET80, ET84 ET80, ET84 ET84 ET84 ET80

Reference or source

16 16 Rupar et al.b Rupar et al.b Rupar et al.b Rupar et al.b Rupar et al.b Rupar et al.b Rupar et al.b Rupar et al.b Rupar et al.b Rupar et al.b Rupar et al.b Rupar et al.b Rupar et al.b Rupar et al.b Rupar et al.b This study This study This study This study

ND, not determined. Rupar et al., 2000, international patent application no. WO 00/66742, World Intellectual Property Organization.

Kostichka, 1994, Novel pesticidal proteins and strains, international patent application no. WO 94/21795, World Intellectual Property Organization) that are produced during vegetative growth. Monsanto Company has obtained regulatory approval for the corn event designated MON863 (Environmental Protection Agency registration no. 524-528) that expresses a variant of the Cry3Bb1 protein active against southern and western corn rootworm larvae (20). Similarly, corn plants expressing the binary toxin Cry34Ab1-Cry35Ab1 have been shown to be protected from root damage caused by rootworm feeding (32). The long-term success of this transgenic approach to rootworm control will depend on appropriate insect resistance management strategies. An important component of a sustainable insect resistance management strategy is the discovery and deployment of insecticidal proteins with distinct modes of action. In this paper, we describe a new class of binary toxins that are active against corn rootworm larvae and could serve in this capacity. MATERIALS AND METHODS Bacterial strains and plasmids. Bacterial strains and plasmids used in this study are shown in Table 1. B. thuringiensis strain EG10368 and EG10650 are acrystalliferous host strains used for propagation on recombinant plasmids and for the expression of cloned cry genes. Strain EG10650 is a derivative of EG10368 that contains knockout mutations in both a neutral protease gene and an alkaline protease gene (16). Identification of B. thuringiensis strains active against rootworm. B. thuringiensis strains were isolated from crop dust samples obtained from various sources in the United States. The crop dust samples were treated and spread on agar plates (15) to isolate Bacillus-type colonies. Phase-contrast microscopy of sporulating colonies was used to identify Bacillus cereus-type sporangia producing parasporal crystalline inclusions characteristic of B. thuringiensis. Isolated B. thuringiensis strains were analyzed by a modified Eckhardt agarose gel electrophoresis procedure (22) to determine the array of resident plasmids found in each strain. Strains with apparently unique plasmid arrays were archived. To assess biological activity, B. thuringiensis strains were grown in C2 medium for 3 days at 28 to 30°C, after which the cultures were fully sporulated and lysed. The spores and crystalline inclusions (i.e., crystals) were pelleted by centrifugation,

resuspended in 0.005% Triton X-100, pelleted again by centrifugation, and resuspended in 0.005% Triton X-100 at 1/10 of the original culture volume. These 10⫻ spore-crystal concentrates were tested in bioassays against the southern corn rootworm (SCR [Diabrotica undecimpunctata howardi]). Molecular cloning and analyses. Genomic DNAs were prepared from vegetative B. thuringiensis cultures. Vegetative cells were resuspended in a lysis buffer containing 50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 4 mg of lysozyme/ml. The suspension was incubated at 37°C for 1 h. Following incubation, the suspension was extracted once with an equal volume of phenol, once with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), and once with an equal volume of chloroform-isoamyl alcohol (24:1). The DNA was precipitated from the aqueous phase by the addition of 1/10 volume of 3 M sodium acetate and then 2 volumes of 100% ethanol. The precipitated DNA was collected by centrifugation, washed with 70% ethanol, and dissolved in distilled water. Cry proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride membranes for N-terminal sequence analysis using standard Edman degradation procedures. DNA fragments were radioactively labeled by use of [␣-32P]dATP and a random primer labeling kit (Prime-a-Gene Labeling System; Promega Corporation, Madison, Wis.). Synthetic oligonucleotides were obtained from Integrated DNA Technologies, Inc. (Coralville, Iowa), and labeled with [␥-32P]ATP (3 ␮l of 3,000 Ci/mmol at 10 mCi/ml in a 20-␮l reaction volume) by using T4 polynucleotide kinase. Southern and colony blot analyses were performed essentially as described by Sambrook et al. (38). Blots probed with radiolabeled oligonucleotides were washed in 3⫻ SSC (1⫻ SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.1% SDS at 45°C, whereas blots probed with radiolabeled DNA fragments were washed in 0.1⫻ to 1⫻ SSC–0.1% SDS at 55°C prior to autoradiography with Kodak X-OMAT AR X-ray film (Eastman Kodak Company, Rochester, N.Y.). DNA sequencing was performed by using both manual and automated systems. Manual sequencing reactions were performed using the Sequenase Version 2.0 DNA sequencing kit (United States Biochemical/Amersham Life Science Inc., Cleveland, Ohio) following the manufacturer’s procedures and using 35S-dATP as the labeling isotope (DuPont NEN Research Products, Boston, Mass.). Denaturing gel electrophoresis of the reactions was performed with a 6% (wt/vol) acrylamide–42% (wt/vol) urea sequencing gel. The dried gel was exposed to Kodak X-OMAT AR X-ray film (Eastman Kodak) overnight at room temperature. For automated sequencing, DNA samples were sequenced by using the ABI PRISM DyeDeoxy sequencing chemistry kit (Applied Biosystems) according to the manufacturer’s suggested protocol. The completed-reaction mixtures were run on an ABI 377 automated DNA sequencer. DNA sequence chromatograms were analyzed by using Sequencher v3.0 DNA analysis software (Gene Codes Corp.). DNA and protein sequences were ana-

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B. THURINGIENSIS TOXINS AGAINST CORN ROOTWORM

lyzed by using the suite of programs provided in the Wisconsin Package version 10.0 or 10.2 (Accelrys Inc., San Diego, Calif.). Hidden Markov model (HMM) profiles were generated by using the program HMMer (18). Cloning and sequencing of crystal protein genes. Standard recombinant DNA procedures were used to clone and manipulate the cry genes from B. thuringiensis strains EG4851, EG5899, and EG9444 (38; M. Rupar, W. P. Donovan, C.-R. Chu, E. Pease, Y. Tan, A. Slaney, T. Malvar, and J. Baum, 2000, coleopteran toxic polypeptide compositions and insect resistant transgenic plants, international patent application no. WO 00/66742, World Intellectual Property Organization). An oligonucleotide, designated wd271 (5⬘-ATGTTAGATACAAATA AAGTATATGAAATTTCAAATCATGC-3⬘), was used as a hybridization probe for the 45-kDa protein genes of strains EG4550 and EG5899. The sequence of wd271 was designed by back translation of the N-terminal sequence obtained for the 45-kDa protein produced by strain EG4550. Southern blot analysis of genomic DNAs indicated that the wd271 oligonucleotide probe hybridizes to a 2.5-kb HindIII fragment from strain EG4550 and to an 8.4-kb HindIII fragment from strain EG5899 (data not shown). With wd271 as a hybridization probe, the 8.4-kb HindIII fragment from strain EG5899 was cloned into the pBluescript II SK(⫹) vector to yield the plasmid pEG1319 and the recombinant Escherichia coli strain EG11521 (Table 1). Subsequently, the 8.4-kb HindIII fragment was isolated from pEG1319 and subcloned into the unique HindIII site of the E. coli-B. thuringiensis shuttle vector pEG597 (3) to yield plasmid pEG1321. Plasmid pEG1321 was introduced into the acrystalliferous B. thuringiensis host strain EG10368 via electroporation (31) to generate the recombinant B. thuringiensis strain EG11529 (Table 1). To facilitate sequencing of the ET39, ET74, and ET75 cry genes, the 8.4-kb HindIII insert from plasmid pEG1319 was subcloned into the unique HindIII site of pUC18 to yield plasmid pEG1337. Sequencing of the cry genes harbored on plasmid pEG1337 was initiated by using the ET39-specific oligonucleotide wd271 as a sequencing primer. Subsequent sequencing primers were designed on the basis of sequencing data obtained from previous sequencing runs. Sequencing of the ET75 coding region was initiated by using a sequencing primer designed from the ET75 N-terminal sequence (5⬘-TCACAAAAATATATGAACAGC-3⬘). To identify related toxins with activity against corn rootworm larvae, B. thuringiensis strains producing crystal proteins of 40 to 50 kDa were identified by SDS-PAGE analysis of sporulated cultures. Genomic DNAs from such strains were then analyzed by Southern blotting using an ET39-specific hybridization probe. This probe consisted of an amplified DNA fragment generated by PCR from pEG1337 plasmid DNA using the primers mr13 (5⬘-TGACACAGCTAT GGAGC-3⬘) and mr24 (5⬘-ATGATTGCCGGAATAGAAGC-3⬘). Genomic DNAs prepared from these strains were used to prepare size-selected (6 to 10 kb) MboI-insert libraries in the E. coli-B. thuringiensis shuttle vector pHT315 (2). Probing these libraries with either the oligonucleotide wd271 (EG9444 library) or the amplified ET39 gene fragment (EG4851 library) yielded clones harboring the recombinant plasmids pEG1821 and pEG1823, respectively. Sequence analysis of the ET71 and ET79 genes was initiated by using pEG1821 as a template and the oligonucleotide wd271 as a sequencing primer. Sequence analysis of the ET76, ET80, and ET84 genes was initiated by using pEG1823 as a template and an oligonucleotide (mr18, 5⬘-GTACCAGAAGTAGGAGG-3⬘) designed from the ET39 gene sequence as a sequencing primer. Plasmids pEG1821 and pEG1823 were introduced into the acrystalliferous B. thuringiensis host strain EG10650 via electroporation (31) to generate the recombinant B. thuringiensis strains EG11648 and EG11658, respectively (Table 1). Preparation of crystal proteins for insect bioassay. To evaluate crystal protein production, recombinant and wild-type B. thuringiensis strains were typically grown in C2 sporulation medium (13) at 25 to 30°C for 3 to 4 days, at which point the cultures were fully sporulated and lysed. The sporulated and lysed cultures were harvested by centrifugation, washed once or twice in an equal volume of wash buffer (10 mM Tris-HCl [pH 7.5]–0.005% Triton X-100 or 0.005% Triton X-100 alone), and suspended at 1/10 of the original volume in the wash buffer. This 10⫻ spore-crystal concentrate was used directly in bioassays. Initial bioassays with ET39, ET74, and ET75 used protein crystals purified by sucrose gradient centrifugation. Sucrose step gradients (7.5 ml each of 79, 72, 68, and 55% sucrose in wash buffer) were prepared in 25- by 89-mm Ultra-Clear centrifuge tubes (Beckman Instruments, Inc., Palo Alto, Calif.). Spore-crystal suspensions (5 ml) were layered on top of the gradients. The gradients were centrifuged at 58,000 ⫻ g at 4°C in an L8-70 M ultracentrifuge (Beckman Instruments) overnight. The protein crystals of EG11529 separated into two distinct bands. One band, at the 68 to 72% interface, contained only the ET75 protein. The second band, at the 72 to 79% interface, contained both ET39 and ET74. The bands were recovered from the gradients by use of a pipette, and the protein crystals were concentrated by centrifugation. The protein crystals were washed twice in wash buffer. The ET39 and ET74 protein crystal preparation was subsequently

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run over a second gradient to ensure complete separation of ET75 from ET39 and ET74. Crystal proteins were quantified by SDS-PAGE and densitometry by using bovine serum albumin as a standard. Insect bioassay. SCR (D. undecimpunctata howardi) bioassay screenings were performed via surface inoculation of an artificial diet (30), but without formalin. Spore-crystal suspensions or gradient-purified protein crystals were prepared in a diluent (an aqueous 0.005% Triton X-100 solution) and applied to the surface of the diet. After the diluent had dried, first-instar larvae were placed on the diet and incubated at 28°C. Thirty-two larvae were tested per concentration. Mortality was scored after 7 days by using the diluent-only treatment as the untreated check. The WCR bioassay screen was run essentially in the same way as the SCR bioassay. Two different quantitative bioassays were run with WCR larvae. For the first WCR bioassay protocol, 200 ml of the WCR diet was prepared in a manner similar to that described by Pleau et al. (36). The following dilutions of the test samples were prepared by using water as a diluent: 300, 200, 133, 89, 59, 40, and 26 ppm of crystal protein. These were treated with 50 ␮g of rifamycin/ml overnight. The untreated control, sterile filtered water, was included. Twenty-five microliters of test sample was applied per well. Plates were allowed to dry before insect larvae were added. One WCR neonate larva was added per well with a fine paintbrush. Plates were sealed with polyester film (Mylar) and ventilated by use of an insect pin. Twenty-four larvae were tested per concentration. The bioassay plates were incubated at 27°C and 60% relative humidity in complete darkness for 5 to 7 days. The number of surviving larvae per concentration was recorded at the end of the experiment. The masses of the surviving larvae were recorded on a suitable microbalance (Cahn C-33). Data were analyzed by using JMP 4 statistical software (SAS Institute, Cary, N.C.). Levene’s test for homogeneity of variances was conducted on each data set, and where a significant lack of homogeneity was detected, masses were log transformed. General linear regressions were performed on the data sets to look for concentration-response effects. For the second WCR bioassay, crystal protein preparations were prepared in a diluent (10 mM Tris-HCl [pH 7.0], 0.1 mM EDTA, 0.005% Triton X-100, 50 ␮g of rifamycin/ml) and 20 ␮l of this preparation was applied to the surface of the WCR artificial diet (without formalin) dispensed in 200-␮l aliquots in 96-well plates. After the diluent had dried, one neonate WCR larva was added per well with a fine paintbrush, and then the plates were covered with a perforated Mylar seal. Twenty-four larvae were tested per concentration. The bioassay plates were incubated at 27°C and 60% relative humidity in complete darkness for 5 to 7 days. Mortality was scored, and the masses of the surviving larvae were recorded by using a microbalance. The Cry3Bb protein produced by B. thuringiensis strain EG11231 and active against corn rootworm (20) was used as a benchmark standard in the WCR bioassays. The Cry3Bb protein produced by B. thuringiensis strain EG11231 is a variant of Cry3Bb1 that contains the following amino acid substitutions: H231R, S311L, N313T, and E317K. Construction of cry expression plasmids. Standard recombinant DNA procedures (38) were used to subclone individual cry genes into expression vectors and to generate knockout mutations within the putative operon encoding the ET76, ET80, and ET84 crystal proteins (Table 1). The construction of plasmids pEG1918, pEG1919, pEG1920, and pEG1921 expressing the ET75, ET74, ET39 and ET74, and ET39 genes, respectively, is described in international patent application no. WO 00/66742. To construct the knockout mutations in the ET76, ET80, and ET84 genes, an ⬃3.2-kb BbuI-XbaI fragment from plasmid pEG1823 containing all three genes was inserted into the unique BbuI and BlnI sites of the B. thuringiensis-E. coli shuttle vector pEG854 (3) to yield plasmid pIC17000. To disrupt the ET84 gene, plasmid pIC17000 was cleaved with the restriction enzyme SpeI, the ends were blunt ended with T4 polymerase, and the blunt ends were ligated together to introduce a frameshift mutation at codon 86. This plasmid was designated pIC17001. To disrupt the ET80 gene, plasmid pIC17001 was cleaved with DraIII, the ends were blunt ended with T4 polymerase, and the blunt ends were ligated together to generate a single amino acid substitution and a deletion (H28R, deletion of codon 29) in ET80 that abolished ET80 crystal production in B. thuringiensis. The resulting plasmid, pIC17003, contains knockout mutations in both the ET80 and ET84 genes. To disrupt the ET76 gene, plasmid pIC17001 was cleaved with AgeI, the ends were blunt ended with T4 polymerase, and the blunt ends were ligated together to generate a frameshift mutation at codon 166. The resulting plasmid, pIC17002, contains knockout mutations in both the ET84 and ET76 genes. The same mutations were introduced into the ET76 and ET80 genes contained on plasmid pEG1823 to generate the recombinant plasmids pEG2206 and pEG2207, respectively. To generate plasmid pIC17047, plasmid pIC17000 was cleaved with SphI and SpeI, the ends were blunt ended with T4 polymerase, and the blunt ends were ligated together to generate a deletion derivative missing the ET84 upstream region. Nucleotide sequence accession numbers. The nucleotide sequences determined in this study were deposited in GenBank under the following accession

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APPL. ENVIRON. MICROBIOL.

FIG. 1. SDS-PAGE analysis of crystal proteins from various wild-type and recombinant strains of B. thuringiensis. Lanes: a, molecular mass standards (in kilodaltons); b, EG10650; c, EG4550; d, EG5899; e, EG11529; f, EG4851; g, EG11658; h, EG9444; i, EG11648; j, EG11936; k, EG11934; l, EG11935; m, EG11937.

numbers: AY036013 (ET75), AY036014 (ET39 and ET74), AY036015 (ET71 and ET79), and AY036016 (ET76, ET80, and ET84).

RESULTS Cloning and sequencing of the EG5899 crystal protein genes. Crystal proteins produced by two B. thuringiensis strains, designated EG4550 and EG5899, were determined to be toxic to SCR larvae in bioassay screens. Crystal proteins produced by strains EG4550 and EG5899 were resolved by SDS-PAGE, revealing prominent proteins not observed in a comparable sample prepared from the acrystalliferous strain EG10650 (Fig. 1A, lanes b through d). Strain EG4550 produced proteins with approximate molecular masses of 45 and 13 kDa, whereas strain EG5899 displayed proteins with approximate molecular masses of 45, 35, and 14 kDa as well as several proteins with molecular masses greater than 90 kDa. N-terminal sequence analysis of the 45-kDa protein from EG4550 yielded the sequence MLDTNKVYEISNHAN that was subsequently used to design an oligonucleotide probe for Southern blot analysis and molecular cloning. By this strategy, an 8.4-kb HindIII fragment from strain EG5899 was cloned into a shuttle vector capable of replication in both E. coli and B. thuringiensis (3). This plasmid, designated pEG1321 (Table 1), was introduced into the acrystalliferous B. thuringiensis strain EG10368 to test whether the 8.4-kb HindIII insert could direct the production of crystal proteins in B. thuringiensis. SDS-PAGE analysis of crystal proteins produced by the recombinant strain EG11529 containing pEG1321 revealed prominent proteins of approximately 45, 35, and 14 kDa (Fig. 1A, lane e), showing a level of crystal protein production apparently higher than that observed in the wild-type strain EG5899 (lane d).

DNA sequencing of the cloned EG5899 DNA, initiated by using an oligonucleotide primer complementary to the 5⬘ end of the ET39 gene, revealed two coding regions of the expected lengths for the ET39 and ET74 genes, separated by 120 bp. Mapping experiments indicate that the gene encoding ET75 is located well downstream of the ET39 gene. The N-terminal sequences obtained for the ET39 (LDTNKVYEISNHAN), ET74 (SARQVHIQINNKTRH), and ET75 (SILNLQDLSQKYMT AALNKI) proteins matched the 5⬘ ends of their corresponding open reading frames. Binary nature of the ET39-ET74 corn rootworm toxin. The spore-crystal suspension from EG11529 containing ET39, ET74, and E75 was tested in bioassays against SCR larvae and found to be toxic (data not shown). The protein crystals or inclusions produced by the recombinant strain EG11529 were subsequently purified from spores by sucrose gradient centrifugation. Inclusions containing the ET75 protein were observed to band at the 68 to 72% sucrose interface, while inclusions containing the ET39 and ET74 proteins were recovered at the 72 to 79% sucrose interface. Bioassays with these two protein preparations demonstrated that the corn rootworm activity of strain EG11529 is associated with the ET39 and ET74 proteins (data not shown). Subsequently, recombinant B. thuringiensis strains producing ET39, ET74, or both crystal proteins were constructed to test the activity of the individual proteins in bioassays (Fig. 1B; Table 1). In these assays, the crystal proteins were tested individually as a mixture produced by the recombinant strain EG11936 and as a 1:1 (wt/wt) physical mixture at total protein concentrations of 80 and 160 ␮g of protein per diet well. The results of those bioassays, summarized in Table 2, demonstrate that the individual crystal pro-


VOL. 70, 2004 TABLE 2. Binary nature of the ET74-ET39 toxin Strain(s)

EG11935 EG11935 EG11936 EG11936 EG11937 EG11937 EG11937 EG11937 EG11935 EG11935 EG11935 EG11935 a

⫹ ⫹ ⫹ ⫹

EG11937 EG11937 EG11937 EG11937

Crystal protein(s)

Test no.

Concn (␮g/well)

% WCR mortality

ET74 ET74 ET74⫹ET39 ET74⫹ET39 ET39 ET39 ET39 ET39 ET74⫹ET39 ET74⫹ET39 ET74⫹ET39 ET74⫹ET39

1 1 1 1 1 1 2 2 1 1 2 2

80 160 80 160 80 160 80 160 80a 160a 80a 160a

0 0 100 100 10.5 6.7 13.3 0 100 100 100 93.3

Crystal proteins were added in a 1:1 ratio (wt/wt).

teins possess little, if any, activity against WCR larvae whereas the recombinant mixture and the physical mixture of ET39 and ET74 cause significant mortality. These results are consistent with previous studies demonstrating that both the Cry34A and Cry35A proteins are required for full activity against Diabrotica species (19). Cloning and sequencing of related genes. SDS-PAGE analysis of crystal proteins produced by wild-type B. thuringiensis isolates identified two strains, designated EG4851 and EG9444, that produced proteins with apparent molecular masses similar to those of ET39 and ET74. Strain EG9444 produced proteins of approximately 44 and 13 kDa (Fig. 1A, lane h), while strain EG4851 produced proteins of approximately 42, 38, and 14 kDa as well as several prominent proteins larger than 90 kDa (Fig. 1A, lane f). Southern blot analysis of genomic DNAs confirmed the presence of ET39-related genes. The cloning of the EG4851 and EG9444 crystal protein genes

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is described in Materials and Methods. The recombinant plasmids containing the cloned EG9444 and EG4851 crystal protein genes were designated pEG1821 and pEG1823, respectively, and were introduced into an acrystalliferous B. thuringiensis host strain to generate the recombinant strains EG11648 and EG11658, respectively (Table 1). SDS-PAGE analysis of spore-crystal preparations demonstrated that EG11648 produces crystal proteins of approximately 44 and 13 kDa (Fig. 1A, lane i) and that EG11658 produces crystal proteins of approximately 42, 38, and 14 kDa (Fig. 1A, lane g). The 44- and 13-kDa proteins from EG9444 (lanes h and i) were designated ET71 and ET79, respectively, while the 44-, 38-, and 14-kDa proteins from EG4851 (lanes f and g) were designated ET76, ET84, and ET80, respectively (Table 1). Sequence analysis of the cloned EG4851 DNA on pEG1823 revealed a tight clustering of three open reading frames encoding, from the 5⬘ end to the 3⬘ end, ET84, ET80, and ET76. Similarly, sequence analysis of the cloned EG9444 DNA on pEG1821 revealed a tight clustering of two open reading frames encoding, from 5⬘ to 3⬘, ET79 and ET71. A schematic diagram of the organization of the cry genes cloned from B. thuringiensis strains EG4851, EG5899, and EG9444 is shown in Fig. 2. The ET39, ET74, and ET75 genes from EG5899 encode proteins of 44.2, 13.2, and 34.3 kDa, respectively. The ET71 and ET79 genes from EG9444 encode proteins of 43.8 and 13.6 kDa, respectively. The ET76, ET80, and ET84 genes from strain EG4851 encode proteins of 43.8, 14.8, and 37.5 kDa, respectively. Comparative sequence analysis. The deduced amino acid sequences for the eight crystal proteins were used for sequence database searches and sequence comparisons. FASTA (35) searches of the Allpeptides database with ET74, ET79, and ET80 revealed sequence similarity to the ⬃14-kDa Cry34A

FIG. 2. Schematic diagram of the apparent cry gene operons cloned from B. thuringiensis strains EG5899, EG9444, and EG4851. The restriction map for the EG4851 cry genes shows the restriction sites used to generate knockout mutations within the cry coding regions.

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TABLE 3. Pairwise sequence similarities for Cry34-type proteins Sequence identity (%) ET74

ET74 ET79 ET80 Cry34Ac1 Cry34Ab1 Cry34Aa1

79.2 50.8 80.0 76.7 100.0

ET79

ET80

Cry34Ac1

Cry34Ab1

Cry34Aa1

79.2

50.8 53.2

80.0 95.9 54.5

76.7 91.1 53.7 93.5

100.0 79.8 51.3 80.7 77.3

53.2 95.9 91.1 79.8

54.5 53.7 51.3

93.5 80.7

77.3

protein class, while searches with ET39, ET71, and ET76 revealed sequence similarity to the ⬃44-kDa Cry35A protein class (19). ClustalW (23) multiple sequence alignments were used to construct a pairwise sequence identity table for the Cry34- and Cry35-type proteins (Tables 3 and 4). This analysis demonstrates that (i) the ET74-ET39 pair is closely related to the Cry34Aa1-Cry35Aa1 binary toxin; (ii) the ET79-ET71 pair is closely related to the Cry34Ab1-Cry35Ab1 and Cry34Ac1Cry35Ac1 binary toxins; and (iii) ET80 and ET76 are highly divergent, displaying only 50 to 54% and 60 to 62% sequence identity, respectively, to previously described crystal proteins. Database searches with the ET75 and ET84 sequences provided less definitive results. A FASTA search of the nonredundant database with the ET75 sequence revealed significant sequence similarity to the Cry23Aa1 crystal protein of B. thuringiensis (expected number of false positives with scores at least as high as the hit [E-value] ⫽ 2 ⫻ 10⫺3). A subsequent pairwise alignment of ET75 and Cry23Aa1 (using the GAP program with the blosum62 scoring matrix) revealed a 27.4% sequence identity between the two proteins. FASTA and BLASTP (1) searches of the nonredundant database with the ET84 sequence failed to reveal any significant hits (however, see Discussion). Mutational analysis of the putative ET84-ET80-ET76 operon. The ET80 and ET76 genes are organized in an apparent three-gene operon, whereas the known cry34A- and cry35A-type genes are organized in apparent two-gene operons (19; this paper). The sequence of the presumptive operon encoding ET84, ET80, and ET76 is shown in Fig. 3 along with the deduced translation of the open reading frames. The ET80 and ET84 coding regions are separated by a 91-bp sequence (from the ET84 stop codon to the ET80 start codon) that is AT rich (83.5%), whereas the ET76 and ET80 coding regions are separated by a 92-bp sequence, also AT rich (83.7%). These intergenic regions do not share significant sequence similarity, nor do they contain sequences that resemble those characteristic of the ␴E- and ␴K-regulated sporulation stage promoters TABLE 4. Pairwise sequence similarities for Cry35-type proteins Sequence identity (%) ET39

ET39 ET71 ET76 Cry35Ac1 Cry35Ab1 Cry35Aa1

79.9 60.0 78.6 79.9 99.2

ET71

ET76

Cry35Ac1

Cry35Ab1

Cry35Aa1

79.9

60.0 61.1

78.6 93.5 61.6

79.9 99.2 61.9 93.2

99.2 80.4 60.3 79.1 80.4

61.1 93.5 99.2 80.4

61.6 61.9 60.3

93.2 79.1

80.4

of B. thuringiensis (for a review, see reference 4). Each open reading frame is preceded by a sequence that resembles the conserved Shine-Dalgarno sequence complementary to the 3⬘ end of the 16S rRNA (ET80, AAtGGAGGT; ET76, AAAGG tGtg; ET84, AAAGGAtGT [uppercase letters indicate a perfect match]). A series of knockout mutations were generated in the presumptive operon encoding the ET84, ET80, and ET76 crystal proteins to determine the role of each protein in cry gene expression and insecticidal activity. Unique restriction sites within the ET84 (SpeI), ET80 (DraIII), and ET76 (AgeI) genes (Fig. 2) were cleaved, and the termini were blunt ended with T4 polymerase and ligated together to generate knockout mutations at those sites. In addition, the region upstream of the SpeI site in the ET84 gene was removed, generating plasmid pIC17047, to test whether deletion of the ET84 gene promoter region abolished expression of the downstream ET80 and ET76 genes. Recombinant B. thuringiensis strains producing various combinations of the ET76, ET80, and ET84 knockout mutations (Table 1) were grown in C2 sporulation medium, and the washed 10⫻ concentrated spore-crystal suspensions were analyzed by SDS-PAGE to assess crystal protein production (Fig. 4). Each knockout mutation abolished production of the crystal protein encoded by the target gene but had no apparent impact on the production of other crystal proteins. For example, disruption of the ET84 gene at the SpeI site in strain SIC8004 abolished the production of ET84 protein but did not affect the accumulation of the ET80 and ET76 proteins (lane 5). In contrast, the recombinant strain SIC8096, containing a deletion of the ET84 region upstream of the SpeI site, failed to produce ET76 and ET80 (lane 8), providing evidence that this gene cluster comprises an operon. The crystal proteins in these samples were quantified by densitometry of the Coomassie blue-stained gels, with bovine serum albumin used as a standard, and were tested in bioassays against WCR larvae (Table 5). Crystal proteins from strains EG11658 (ET76, ET80, ET84) and SIC8004 (ET76, ET80) caused significant and statistically equivalent larval mass reduction, whereas crystal proteins from strains SIC8006 (ET80) and SIC8008 (ET76) had no effect on larval mass gain. Likewise, crystal proteins from strains EG12156 (ET80, ET84) and EG12158 (ET76, ET84) had no negative effect on larval mass gain (data not shown). As expected, a mock preparation from the crystal protein-negative host strain EG10650 was indistinguishable from the untreated control. These results demonstrate that disruption of either the ET80 gene or ET76 gene abolishes activity against the WCR whereas disruption of the ET84 gene has no significant impact on activity. The binary nature of the ET80-ET76 toxin was confirmed by using sucrose gradient-purified inclusions obtained from the recombinant strains SIC8006 and SIC8008, which produce ET80 and ET76, respectively (Fig. 4). When tested alone, neither ET76 nor ET80 exhibited activity towards WCR larvae (data not shown). Comparative bioassays against WCR. Crystal protein preparations from the recombinant B. thuringiensis strains SIC8004 (ET76 and ET80), EG11936 (ET39 and ET74), and EG11648 (ET71 and ET79) were tested in bioassays to compare the insecticidal activity of the novel ET76-ET80 binary toxin with that of the binary toxin homologs represented by ET39-ET74 and ET71-ET79. The ratio of the two proteins in each binary

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toxin preparation varied slightly (Fig. 5A); therefore, a 1:1 molar ratio of the Cry34- and Cry35-type proteins was assumed for the binary toxins, allowing us to normalize the amount of binary toxin submitted for bioassay against WCR larvae. The results of the bioassay analysis show a decreasing mean larval mass with increasing concentration of toxins, the untreated control showing a mean larval mass of 0.4 mg. The binary toxins ET80-ET76 and ET79-ET71 showed comparable activities (Fig. 5B). None of the binary toxins appeared to be more active than the Cry3Bb protein standard against WCR larvae.

4895

DISCUSSION We describe two new classes of crystal proteins from B. thuringiensis strain EG4851 that comprise a binary toxin active against WCR (D. virgifera virgifera LeConte). This binary toxin, designated ET80-ET76, is encoded by an apparent operon that encodes a third crystal protein of unknown function designated ET84. In addition to the ET80-ET76 binary toxin, we have identified two other binary toxins, ET79-ET71 and ET74ET39, that are closely related to previously described binary toxins from B. thuringiensis. Despite their divergent nature,

FIG. 3. Nucleotide sequence and translation of the apparent ET84-ET80-ET76 operon from B. thuringiensis strain EG4851.

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FIG. 4. SDS-PAGE analysis of knockout mutations within the ET84, ET80, and ET76 coding regions. Lanes: MW, molecular mass standards (in kilodaltons); 1, EG10650; 2, EG11658; 3, EG12156; 4, EG12158; 5, SIC8004; 6, SIC8006; 7, SIC8008; 8, SIC8096.

these binary toxins show comparable levels of activity against WCR larvae. The crystal proteins described in this paper have been assigned names according to the conventional B. thuringiensis toxin nomenclature (11). According to this nomenclature, ET80 and ET76 have been assigned the designations Cry34Ba1 and Cry35Ba1, respectively, and represent new secondary classes of crystal proteins. On the basis of its sequence similarity to Cry23Aa1 and related proteins (see below), ET75 was assigned the Cry designation Cry38Aa1 despite the lack of evidence for insecticidal activity. A summary of the Cry protein name designations is provided in Table 6. The roles of the individual Cry34- and Cry35-type proteins in mediating toxicity towards the corn rootworm have not been defined experimentally. The Cry35 proteins show sequence similarity to the 42- and 51-kDa crystal proteins (BinA and BinB, respectively) from Bacillus sphaericus, which comprise a binary toxin active against mosquitoes (6, 9, 19). The Bin proteins appear most effective as toxins when present in approximately equimolar concentrations. Binding to the target site (i.e., receptor) on the midgut epithelium is mediated by BinB, which appears to direct the subsequent binding of BinA (7, 10, 12). Ion channel studies with planar lipid bilayers (PLBs) and permeabilization assays with phospholipid vesicles indicate

TABLE 5. Effects of knockout mutations on WCR activity Strain

Untreated EG10650 EG11658 SIC8004 SIC8006 SIC8008

Protein produced (␮g/well)

ET76 ET80 ET84 ET76 ET80 ET80 ET76

(8.0) (3.2) (3.0) (8.0) (5.0) (5.0) (8.0)

Mean larval mass (mg)

SD

SEM

95% CIb

0.33 0.33 0.18a

0.06 0.06 0.07

0.02 0.03 0.04

0.30–0.37 0.26–0.40 0.09–0.26

0.10a

0.03

0.02

0.07–0.14

0.33 0.32

0.02 0.07

0.01 0.04

0.31–0.36 0.23–0.42

a Mean values significantly different from untreated (Dunnett’s method, alpha ⫽ 0.05). b 95% CI, 95% confidence interval.

that (i) BinA-BinB mixtures form ion channels in PLBs that resemble those of BinA alone, (ii) BinA is more efficient than BinB in promoting ion channel activity in PLBs, and (iii) BinB enhances the vesicle-permeabilizing activity of BinA (41). These results suggest that BinA is primarily responsible for the ion channel- and pore-forming activity of the binary toxin and is facilitated by BinB. By analogy, the Cry34- and Cry35- proteins of B. thuringiensis could exhibit a similar division of labor. For instance, the Cry34 proteins could be responsible for the initial membrane binding event, perhaps serving the same function as BinB, and providing specificity to the Cry35 protein interaction with insect target membranes. Recent observations that purified ET80 (Cry34Ba1) readily forms well-defined channels in PLBs while ET76 (Cry35Ba1) does not form such channels suggest an alternative mechanism in which ET80 is primarily responsible for the ion channel- and pore-forming activity of this binary toxin (V. Guzov, personal communication). On the basis of their close proximity and coordinate expression as parasporal crystal proteins, we presume that the genes encoding ET76, ET80, and ET84 comprise an operon. This conclusion is supported by the observation that deletion of the ET84 upstream region abolishes expression of the downstream ET76 and ET80 genes whereas mere disruption of the ET84 gene with a frameshift mutation has no impact on ET76 and ET80 expression. We have not mapped the promoter within this upstream region, nor have we identified the transcription terminator for the operon. It seems likely that the EG5899 and EG9444 binary toxin genes (Fig. 2) are also organized in operons. For instance, subcloning experiments with the ET39 gene from EG5899 demonstrated that the ET74-ET39 intergenic region is insufficient to direct the expression of ET39 in B. thuringiensis. Expression of the ET39 gene in B. thuringiensis was accomplished by placing the gene downstream of a cry2Ac promoter (Rupar et al., international patent application no. WO 00/66742). The ET75 and ET84 crystal proteins are not required for corn rootworm activity, nor are they required for the production of their associated binary toxin. A more detailed bioinfor-


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4897

FIG. 5. Bioassay evaluation of binary toxins against WCR larvae. (A) SDS-PAGE analysis of the binary toxins produced by the recombinant B. thuringiensis strains SIC8004 (lane 2, ET76 and ET80), EG11648 (lane 3, ET71 and ET79), and EG11936 (lane 4, ET39 and ET74). A purified Cry3Bb1 variant (lane 1) was used as a standard in the bioassays. (B) Chart plotting decreasing mean larval mass with increasing binary toxin concentration. The untreated control (UTC) yielded a mean larval mass of 0.4 mg.

matic analysis of these proteins reveals relationships with other crystal proteins. As noted above, a FASTA search of the protein databases with the ET75 (Cry38Aa1) sequence revealed significant sequence similarity to the Cry23Aa1 crystal protein of B. thuringiensis. FASTA searches with the Cry23Aa1 protein sequence in turn revealed significant sequence similarity to the B. thuringiensis crystal proteins Cry15Aa1 and Cry33Aa1. An HMM profile generated from a ClustalW multiple sequence alignment of Cry15Aa1, Cry23Aa1, Cry33Aa1, and ET75 (Cry38Aa1) and progressive refinements of that HMM profile based on search results were used to reiteratively search the protein databases and to demonstrate sequence similarity to the B. thuringiensis protein btc53 (EMBL accession no. X98616), the Mtx2 and Mtx3 proteins of B. sphaericus (GenBank accession no. U47301 and U42335) and the epsilon toxin of Clostridium perfringens (EMBL accession no. X60694 and related sequences). Although BlastP and FASTA searches of the protein databases with the ET84 sequence failed to identify a significant sequence similarity, a comparison of the ET84 protein sequence to the aforementioned HMM profile obTABLE 6. Cry protein designations Crystal protein

Cry designation

ET39 ET71 ET74 ET75 ET76 ET79 ET80 ET84

Cry35Aa2 Cry35Ab2 Cry34Aa2 Cry38Aa1 Cry35Ba1 Cry34Ac2 Cry34Ba1

Accession no.

Paired with:

AAK64561 AAK64563 AAK64560 AAK64559 AAK64566 AAK64562 AAK64565 AAK64564

Cry34Aa2 Cry34Ac2 Cry35Aa2 Cry34Ba1 Cry35Ab2 Cry35Ba1

B. sphaericus homologs

BinA, BinB BinA, BinB Mtx2, Mtx3 BinA, BinB

tained using HmmerPfam (18) yielded an E-value of 5.7 ⫻ 10⫺2, indicative of a significant sequence alignment. These analyses suggest that ET84, like ET75 (Cry38Aa1), belongs to a very diverse toxin family with representatives in several species of gram-positive bacteria. We note the interesting parallel between the Cry35- and Cry38-type proteins described here and the BinA/B and Mtx2/3 proteins, respectively, of B. sphaericus showing activity against diptera (Table 6). The Mtx2 and Mtx 3 proteins, like the BinA-BinB binary toxin, show activity against dipteran larvae (8, 9, 29) but have not been reported to exhibit anticoleopteran activity. Conversely, it is not known whether the crystal proteins described in this paper are active against larvae of dipteran pests. The binary toxin genes described in this paper and elsewhere (19, 32) may be useful in the development of second-generation transgenic corn hybrids protected from corn rootworm feeding damage. Since the Cry34 and Cry35 proteins are apparently unrelated to the Cry3 class of toxins against coleoptera and presumably have a distinct mode of action as a binary toxin, they could become an important component of a resistance management strategy against WCR. The binary toxins in our study, however, were not as active as the Cry3Bb protein standard used in the WCR bioassay, suggesting that the level of protein expression required in planta for effective rootworm protection may be higher than that required for the Cry3Bb protein produced by the corn event MON863. ACKNOWLEDGMENTS We thank Annette Slaney, YuPing Tan, and Elizabeth Pease for their technical assistance on this research project. REFERENCES 1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410.

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