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CURRENT MICROBIOLOGY Vol. 41 (2000), pp. 65– 69 DOI: 10.1007/s002840010093 Current Microbiology An International Journal © Springer-Verlag New York Inc. 2000 Cloning of a New Bacillus thuringiensis cry1I-Type Crystal Protein Gene Soo-Keun Choi,1 Byung-Sik Shin,1 Eun-Mee Kong,1 Hyune Mo Rho,3 Seung-Hwan Park2 1 Laboratory of Microbial and Bioprocess Engineering, Korea Research Institute of Bioscience & Biotechnology (KRIBB), P.O. Box 115, Yusong, Taejon 305-600, Korea 2 Genome Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), P.O. Box 115, Yusong, Taejon 305-600, Republic of Korea 3 Department of Molecular Biology and Research Center for Cell Differentiation, Seoul National University, Seoul, 151-742, Republic of Korea Received: 19 January 2000 / Accepted: 22 February 2000 Abstract. A new cry1I-type gene, cry1Id1, was cloned from a B. thuringiensis isolate, and its nucleotide sequence was determined. The deduced amino acid sequence of Cry1Id1 is 89.7%, 87.2%, and 83.4% identical to the Cry1Ia, Cry1Ib, and Cry1Ic proteins, respectively. The upstream sequence of the cry1Id1 structural gene was not functional as promoter in B. subtilis. The Cry1Id1 protein, purified from recombinant E. coli cells, had a toxicity comparable to that of Cry1Ia against Plutella xylostella, but it was significantly less active than Cry1Ia against Bombyx mori. Cry1Id1 was not active against the coleopteran insect, Agelastica coerulea. Commercial products of B. thuringiensis strains have been used as alternatives to chemical insecticides in the control of various insect pests. However, reports of the occurrence of insect resistance to Cry proteins call for the strategies of resistance management [11]. It has been reported that the insect resistance was due to the reduction of the binding affinity of the active toxin to the membrane receptor in insect midgut [18]. This fact has led many researchers to screen new cry genes because the alternating use of two biochemically unrelated toxins is expected to significantly reduce the rate of resistance development. Many of the genes encoding the crystal proteins have been cloned and sequenced. They have been classified cry1 to cry28 and cyt according to the degree of amino acid sequence homology [1]. In this study, we observed a B. thuringiensis isolate having a new cry1Itype gene utilizing DNA dot blot hybridization and PCR. We have cloned the cry1I-type gene cry1Id1 and determined its nucleotide sequence. The toxicities of Cry1Id1 purified from recombinant E. coli cells against the larvae of Plutella xylostella, Bombyx mori, and Agelastica coerulae were also described. Correspondence to: S.-H. Park; e-mail: [email protected] Materials and Methods Bacterial strains and plasmids. B. thuringiensis isolate BR30 was previously isolated from the phylloplane in Korea [12]. E. coli DH5␣ was used for DNA manipulation and toxin expression. Plasmids pGEM威-7Zf(⫹) for cloning and pKK223-3 for construction of expression vector were purchased from Promega Co. and Amersham Pharmacia Biotech, respectively. Plasmid pDG1728 was obtained from P. Stragier. Nucleic acid hybridization. The cry1I-specific primers, cry5A and cry5B, and a cry1I-specific probe were described previously [15]. Total DNA from B. thuringiensis cells was extracted by the method of Kalman et al. [8]. DNA dot blot and Southern hybridizations were performed according to the method of Shin et al. [15]. Colony hybridization was performed with DIG Nucleic acids detection kit (Boehringer Mannheim Biochemicals) as recommended by the manufacturer. Sequencing. Nested deletion mutants for sequencing were obtained by the unidirectional progressive deletion method with exonuclease III [7]. Sequencing by the Sanger dideoxy-mediate chain termination method was performed with a Dig Taq DNA sequencing kit (Boehringer Mannheim Biochemicals) as recommended by the manufacturer. Insect toxicity assays. The preparation of the inclusion bodies from recombinant E. coli cells and insect toxicity assays against larvae of diamondback moth (Plutella xylostella), silkworm (Bombyx mori), and alder leaf beetle (Agelastica coerulea) were followed as previously described by Shin et al. [15]. Bioassays were repeated three times. 66 Results and Discussion During screening for new cry genes from B. thuringiensis strains isolated from plant leaves in Korea by DNA dot blot hybridization and PCR, we found a B. thuringiensis isolate, BR30, suggested to contain a new cry1Itype gene. The total DNA of this isolate responded positively to a cry1I-specific probe by DNA dot blot hybridization, but did not generate cry1I gene fragments by PCR. Generally, the results of DNA dot blot analysis reflect a degree of homology throughout the region hybridized to the specific probe, whereas the results of PCR reflect only a homology at the primer binding site. Thus, this result indicated that B. thuringiensis isolate BR30 carried cry1I-type genes differing from known cry1I genes, at least in the binding sites of the primers cry5A and/or cry5B. From the strain BR30, we have cloned a new cry1I-type gene, cry1Id1. When total DNA of B. thuringiensis BR30, digested with various restriction enzymes, was analyzed by Southern hybridization with a cry1I-specific probe, a 4-kb EcoRI fragment was detected (data not shown). The 4-kb EcoRI fragment was cloned into the plasmid pGEM威-7Zf(⫹) by colony hybridization. Partial sequence analysis of the 4-kb EcoRI fragment revealed that it contained the N-terminus half of the cry1I-type gene. Further Southern hybridization with the same probe showed that a 2.3-kb HindIII fragment contains the Cterminal part of the gene, and it overlapped 1 kb with the 4-kb EcoRI fragment. The 2.3-kb HindIII fragment was also cloned into the plasmid pGEM威-7Zf(⫹), and the full cry1I-type gene was obtained by annealing two fragments by using SpeI site located in the overlapping region. The resulting plasmid was designated to pSK27. The nucleotide sequence of the cloned fragment contains an open reading frame, designated cry1Id1, encoding a 719-amino acid protein having a predicted molecular weight of 81.4 kDa (Fig. 1). Alignment of the deduced amino acid sequence of the Cry1Id1 with known crystal proteins revealed that the Cry1Id1 protein is 89.7% identical to the Cry1Ia, 87.2% to the Cry1Ib, and 83.4% to the Cry1Ic protein. The Cry1Id1 protein contains five conserved blocks and truncated block 6 identified by Schnepf et al. [13]. The potential ribosome binding site is located 7 base pairs upstream of the translation start site of the cry1Id1 gene, and the inverted repeat structure was found downstream of the cry1Id1 structural gene. The cry1I-type genes have been found to be located 0.5 kb downstream of the cry1 genes, and this cry1– cry1I linkage seems to be very common in B. thuringiensis strains [15]. But the cry1Id1 gene is not located closely downstream of the cry1 gene. The plasmid pSK27 contains the 2.9-kb upstream region from the cry1Id1 structural gene. CURRENT MICROBIOLOGY Vol. 41 (2000) Partial sequence analysis of the upstream region of cry1Id1 gene showed that a cry1-type gene is located in the 2.5-kb upstream of the cry1Id1 gene (Fig. 2). Although it has been discussed that cry1I-type genes are silent in B. thuringiensis [4, 15], a recent report showed that Cry1I-type protein (CGCryV) is expressed early in the stationary phase and the protein is secreted into the media [9]. For the identification of upstream promoter activities of the cry1Id1 gene, we constructed a cry1Id1⬘-lacZ transcriptional fusion using the plasmid pDG1728 [6] and measured -galactosidase activities in B. subtilis JH642 cultured at 37°C in DS medium [14]. The fused region of cry1Id1 was prepared by PCR with primers nr5P3 (5⬘-CGAATTCATGTAAGAGACCCCCTT-3⬘) and nr5P4 (5⬘-CCCAAGCTTACTAACAAACGGCTCTACAC-3⬘), and covered nucleotide ⫺542 to ⫹167 with respect to the translational start site of cry1Id1 (nucleotide position ⫹1) (Fig. 2). The PCR product was confirmed by sequencing and cloned into the EcoRI and HindIII sites of the plasmid pDG1728 to construct plasmid pSK35. The plasmid pSK35 was introduced into B. subtilis JH642. Time course analysis of lacZ gene expression showed that the enzyme activities were background levels, below 4 Miller units, at vegetative and through the sporulation stage (data not shown). This result suggests that the cry1Id1 gene is silent or very weakly expressed in B. subtilis. For the overexpression of the cry1Id1 gene in E. coli, we cloned the gene into the plasmid pKK223-3 to allow that the cry1Id1 gene was under the control of tac promoter. A 309-bp fragment containing the N-terminus of cry1Id1 gene was amplified by PCR with primers nr5a(5⬘-TCCCCCGGGATGAAATCAAAGAATCAAA A-3⬘) and nr5b (5⬘-CCTTTGGGCCAAAGCTCACC3⬘), and substituted for the N-terminus of cry1Id1 gene by using SmaI and NheI sites generating plasmid pSK28. The PCR-amplified fragment was confirmed by sequencing. The plasmid pSK28 was digested with SmaI and NsiI, and the DNA fragment containing cry1Id1 gene was cloned into the SmaI and PstI sites of plasmid pKK223-3 to construct plasmid pSK29. Recombinant E. coli DH5␣ cells harboring the plasmid pSK29 produced inclusion bodies without the addition of isopropyl--Dthiogalactopyranoside when they were grown overnight at 37°C in LB medium containing ampicillin (50 g ml⫺1). We prepared the inclusion bodies from recombinant E. coli cells and performed insect toxicity assays against larvae of the diamondback moth (Plutella xylostella), silkworm (Bombyx mori), and alder leaf beetle (Agelastica coerulea). According to the analyses of crystal structures of Cry1Aa and Cry3Aa toxins [5, 10], domain II is involved in recognition and binding to cell surface receptors. In particular, the mutations in se- S.-K. Choi et al.: Cloning of a New Bacillus thuringiensis cry1I-Type Gene 67 Fig. 1. Nucleotide sequence of the cry1Id1 gene (GenBank accession number AF047579) and deduced amino acid sequence of Cry1Id1 protein. The box indicates the ribosome binding site. The inverted repeats are marked with long arrows below the sequence. Conserved amino acid blocks for the known crystal proteins are in boldface. The Cry1Id1 amino acid sequence was aligned with those of Cry1Ia and Cry1Ib proteins; only the differences in the open reading frame are shown. 68 CURRENT MICROBIOLOGY Vol. 41 (2000) Fig. 2. Restriction map of cry1Id1 compared with other cry1Igenes. Black boxes indicate open reading frames of the genes. The numbers indicate the distance between cry1I genes and other cry1 genes located in the upstream region of the cry1I genes. The dashed box indicates the DNA region used for lacZ transcriptional fusion. Table 1. Toxicity of Cry1Ia3 and Cry1Id1 proteins produced by E. coli recombinant cells LC50a (g/ml) against Proteins Bombyx mori Plutella xylostella Agelastica coerulea Cry1Ia3 Cry1Id1 7.08 (4.11–10.77) 439.56 (214.28–709.12) 2.57 (0.88–5.32) 4.26 (2.44–7.47) ⬎1,900 ⬎960 a 95% confidence intervals are shown in parentheses. quences encoding loop regions of domain II had either a negative or positive effect on receptor binding and toxicity [13]. The analysis of the deduced amino acid sequence of the Cry1Id1 protein showed that domain II (residues 294 – 492) is 97% identical to that of the Cry1Ia3 protein. Moreover, the amino acid sequences corresponding to three loops (loop 1, residues 349 –355; loop 2, residues 409 – 419; loop 3, residues 480 – 483) proposed by Tabashnik et al. [16] in domain II of Cry1Id1 are perfectly matched with those of Cry1Ia3. This enabled us to predict that the insecticidal spectrum of the Cry1Id1 protein might be similar to that of the Cry1Ia3 protein. Bioassay results showed that the toxicity of the Cry1Id1 protein was comparably to that of Cry1Ia3 against P. xylostella. However, counter to expectation, Cry1Id1 was significantly less active than Cry1Ia3 against B. mori (Table 1). It has been reported that the domain III of Cry protein might also participate in receptor binding [2, 3]. The domain III (residues 501– 642) of Cry1Id1 shares 90.1% homology with that of Cry1Ia3. Therefore, a possible explanation of the bioassay result was that the difference in the toxicity between Cry1Id1 and Cry1Ia3 against B. mori might originate from domain III of each toxin. Cry1Id1 did not show toxicity against a coleopteran insect, Agelastica coerulea. Similarly, other groups reported that Cry1I-type proteins were active solely against lepidopteran insects [4, 9, 15], even though the Cry1I protein was discovered to possess a property of dual insecticidal activity against lepidopterans and coleopterans [17]. ACKNOWLEDGMENTS We are grateful to P. 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