CURRENT MICROBIOLOGY Vol. 45 (2002), pp. 299 –302 DOI: 10.1007/s00284-002-3755-0

Current Microbiology An International Journal © Springer-Verlag New York Inc. 2002

Isolation and Characterization of a Strain of Bacillus thuringiensis ssp. kurstaki Containing a New ␦-Endotoxin Gene M.S. Li,1 Y.H. Je,1 I.H. Lee,1 J.H. Chang,1 J.Y. Roh,1 H.S. Kim,1 H.W. Oh,2 K.S. Boo1 1 2

School of Agricultural Biotechnology, Seoul National University, 103 Seodoon-Dong, Suwon 441-744, Korea Korea Research Institute of Bioscience and Biotechnology, Taejeon 305-606, Korea

Received: 21 December 2001 / Accepted: 28 January 2002

Abstract. A strain of Bacillus thuringiensis that showed significantly high toxicity to Plutella xylostella and Spodoptera exigua was isolated from a Korean soil sample and characterized. The isolate, named B. thuringiensis K1, was determined to belong to ssp. kurstaki (H3a3b3c) type by an H antisera agglutination test and produced bipyramidal inclusions. Plasmid pattern of K1 was different from that of the reference strain, ssp. kurstaki HD-1, but the parasporal inclusion protein profile of K1 had two major bands that were similar in size to those of ssp. kurstaki HD-1. To verify the ␦-endotoxin gene types of K1, PCR analysis with specific cry gene primers was performed to show that K1 contained a new cry gene in addition to cry1Aa, cry1Ab, cry1Ac, cry1E and cry2 genes. PCR-amplified region of the new cry gene, cryX, showed 79% similarity to cry1Fa1 gene (GenBank Accession No. M63897). In an insect toxicity assay, K1 had higher toxicity against Plutella xylostella and S. exigua than ssp. kurstaki HD-1.

Bacillus thuringiensis is characterized by the production of parasporal crystals composed of proteins that have highly specific insecticidal activity against larvae of certain Lepidoptera, Coleoptera, or Diptera, and has been used for many years as a leading biorational pesticide [13, 18]. At present, B. thuringiensis ssp. kurstaki HD-1 is the most useful strain as an insecticide, because this strain exhibits powerful toxicities to various lepidopteran larvae. Historically, formulations of B. thuringiensis ssp. kurstaki HD-1, when applied at recommended field rates, are usually not efficacious against Spodoptera species [10]. Formulations of HD-1 typically contain Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A proteins, all of which have relatively low levels of toxicity to Spodoptera species [6]. Among several strains, B. thuringiensis ssp. aizawai, ssp. entomocidus, ssp. galleriae, and ssp. kenyae, which contain Cry1C, Cry1D, Cry1E, or Cry1F proteins, are the only significantly active strains against Spodoptera species [12, 16, 17].1 In this study, we isolated and characterized a strain of B. thuringiensis ssp. kurstaki, called K1, which had high toxicity against S. exigua and contained a new ␦-endotoxin gene. Correspondence to: K.S. Boo; email: [email protected]

Materials and Methods Bacterial strains and growth media. Several B. thuringiensis isolates were obtained from a soil sample in Korea, and a strain, temporarily named K1, was selected by primary bioassay [5]. B. thuringiensis strains used as references, ssp. kurstaki HD-1 (H3a3b3c) and ssp. aizawai (H7), were kindly provided by M. Ohba (Institute of Biological Control, Faculty of Agriculture, Kyushu University, Japan). For preparation of parasporal inclusions and purification of plasmid DNA, GYS and SPY media, respectively, were used [5]. Serology. H antisera to the previously reported strains of 33 serotypes (H1 to H27) of B. thuringiensis were prepared, and H antisera-antigens agglutination studies were performed with 96-well plates [5]. Plasmid DNA extraction. The plasmid DNAs of B. thuringiensis strains were isolated according to the manufacturer’s protocols of QIAGEN midi prep. kit (QIAGEN Co., Germany). The total plasmid DNA patterns of all B. thuringiensis strains were analyzed on agarose gel. Morphological observation and protein analysis. Parasporal inclusions were purified by the method of Thomas and Ellar [15], with a discontinuous 60 – 85% sucrose gradient. Crystal morphology of the isolates was examined by phase-contrast microscopy and scanning electron microscopy. For SDS-PAGE samples, cells were cultured in GYS medium at 30°C and harvested after autolysis. SDS-PAGE was performed on a 10% separating gel with a 3% stacking gel as described by Laemmli [9]. The gel was stained with 0.1% Coomassie brilliant blue (Sigma Co., St. Louis, MO, USA). Identification of cry-type genes by PCR. The 20 primers [cryIA(a),

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IA(b), IA(c), IB, IC, ID, IE, IF, IG, cryIIA, cryIIIA, IIIB, IIIC, IIID, cry IVA, IVB, IVC, IVD, cryV, and cytA] for the specific ␦-endotoxin genes used in the PCR analysis were synthesized as reported by Carozzi et al. [1], Gleave et al. [7], Ceron et al. [2, 3] and Kalman et al. [8]. The plasmid DNAs of B. thuringiensis strains, isolated according to the manufacturer’s protocols of QIAGEN midi prep. kit (QIAGEN Co., Germany), were used as templates. The reaction was conducted for 250 ng of sample DNA with 2.5 U of Taq DNA polymerase (Promega Co., USA), 200 nM each deoxynucleotide triphosphate, 100 pM each primer, and 3 mM MgCl2 in a final volume of 50 ␮l. Amplification was accomplished with the DNA Thermal Cycler (Perkin Elmer Cetus, USA) by using the Step-Cycle program set to denature at 94°C for 1 min, anneal at 55°C for 1 min, and extend at 72°C for 1 min, followed by a 4-s/cycle extension for a total of 35 cycles. Following amplification, the PCR products were ethanol-precipitated, centrifuged at 10,000g for 30 min, and rinsed with 70% ethanol. These DNAs were analyzed by 0.9% agarose gel electrophoresis. The purified PCR products were ligated to pGEM-T vector (Promega Co., USA) and analyzed by the dye termination method in ABI 377 automated sequencer (Applied Biosystems, USA) as specified by the manufacturer. Insect toxicity assays. Primary bioassay and quantitative bioassay were done as previously described [5]. In primary bioassay, Spodoptera exigua, Spodoptera litura, Plutella xylostella, and Culex pipiens were tested. In a quantitative bioassay, sporulated cultures of B. thuringiensis were serially diluted in 0.01% (vol/vol) Triton X-100, and 100-␮l aliquots of serial dilutions were applied to the surface of artificial diets for 24 larvae each of third instar P. xylostella and second instar S. exigua. Larval mortality was recorded in 3 days in the P. xylostella assay and in 5 days in the S. exigua assay. All tests were performed with spore-parasporal inclusion suspensions and were independently repeated three times. Statistical analysis of data was performed with probit analysis [11].

Fig. 1. Plasmid DNA pattern (A) and SDS-PAGE analysis of parasporal inclusions (B) of B. thuringiensis isolate K1. Lanes: H, B. thuringiensis ssp. kurstaki HD-1; K, K1. M indicates Lambda DNA digested with HindIII (A) and 10-kDa protein ladder (Gibco BRL, USA) (B).

Results A strain of B. thuringiensis, named K1, isolated from a Korean soil sample, was selected for further study on the basis of its insecticidal activity against Spodoptera species (data not shown). The isolate K1 belonged to B. thuringiensis ssp. kurstaki (H3a3b3c) by the H antisera agglutination test. The crystal morphology of K1, analyzed by phase-contrast microscopy and scanning electron microscopy, was found to be typically bipyramidal (data not shown). The plasmid pattern of K1 was different from that of the reference strain, ssp. kurstaki HD-1 (Fig. 1A). The parasporal inclusion proteins of K1 and ssp. kurstaki HD-1 were analyzed by SDS-PAGE. The isolate K1 had two major bands that were similar in size to those of ssp. kurstaki HD-1 (Fig. 1B). To identify crystal protein genes of B. thuringiensis strains, PCR analysis was performed with cry genespecific primers (Fig. 2). The reference strain, B. thuringiensis ssp. kurstaki HD-1, showed products of cry1Aa, cry1Ab, cry1Ac, and cry2A genes as reported before [13]. However, K1 amplified products of cry1Aa, cry1Ab, cry1Ac, cry1E, cry2A, and a new cry gene (Fig. 2). The insecticidal activity of K1 was tested against P.

Fig. 2. Detection of cry genes of B. thuringiensis K1 by PCR. Lanes: H, B. thuringiensis ssp. kurstaki HD-1; K, K1; C, control including cry1Aa, cry1Ab, cry1Ac, cry1E, cry1F, and cry2A specific PCR products. M indicates 100-bp DNA ladder (Promega Co., USA).

M.S. Li et al.: B. thuringiensis ssp. kurstaki Containing a New ␦-Endotoxin Gene

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Table 1. Toxicity of B. thuringiensis K1 against larvae of P. xylostella and S. exigua LC50 (⫻ 105 cfu 䡠 ml⫺1) Species

K1

KURa

AIZb

P. xylostella S. exigua

1.6 3.9

2.32 185.3

136.5 15.1

a b

KUR, B. thuringiensis ssp. kurstaki HD-1. AIZ, B. thuringiensis ssp. aizawai.

xylostella and S. exigua (Table 1). The insecticidal activity profile of K1 was different from that of B. thuringiensis ssp. kurstaki HD-1. The isolate K1 was significantly more active against S. exigua and more toxic against P. xylostella than ssp. kurstaki HD-1. The LC50 value of K1 to S. exigua was also lower than that of ssp. aizawai, which is highly active to S. exigua [12].

Discussion

Fig. 3. Comparison of nucleotide sequence between PCR products of cryX and cry1Fa1 (GenBank Accession No. M63897). The same nucleotide sequences are indicated by asterisk (*). The binding sites of forward and reverse primers are underlined.

The isolate K1 was isolated from a Korean soil sample and its biochemical and molecular biological characteristics were analyzed. K1 was selected for its significant toxicity against S. exigua through primary bioassay, but the serotype of K1 was determined as B. thuringiensis ssp. kurstaki. At present, the B. thuringiensis ssp. kurstaki strain shows broad activity to Lepidoptera species but has relatively low levels of toxicity to Spodoptera species [10]. From the comparison of protein profile between K1 and ssp. kurstaki HD-1, K1 showed a different plasmid composition. K1 does not contain the typical small plasmid isolated from ssp. kurstaki HD-1 (Fig. 1A). But the protein profile of K1 was similar to that of ssp. kurstaki HD-1 (Fig. 1B). It means that the proteins expressed by K1 may be similar in size to the Cry1 or Cry2 type proteins of ssp. kurstaki HD-1 [13]. The PCR analysis showed the different ␦-endotoxin gene profile of K1. K1 contained cry1Aa, cry1Ab, cry1Ac, cry2A, and cry1E-type genes and an additional cry1F-type gene (Fig. 2). The specificity of all PCR products was confirmed by restriction enzyme digestion patterns and DNA sequencing. Among six kinds of PCR products of K1, the existence of cry1Aa, cry1Ab, cry1Ac, cry1E, and cry2A genes was coincident with the previous reports [5, 13]. A PCR product of K1 with cry1F-specific primers was produced in the expected size of about 360 bp, but differed from cry1F genes in nucleotide sequence. From the comparisons of the PCR-amplified region of the putatively new cry gene, named cryX, with all cry genes in the GenBank, it showed the highest similarity to cry1Fa1 gene (GenBank Accession No.

M63897; Fig. 3). The cryX produced a 307-bp nucleotide that was shorter than cry1Fa1 by two bases and showed 79% similarity in DNA sequence. In addition, the cryX PCR-amplified region showed a similarity of 74.4% to cry1Ea1 (GenBank Accession No. X53985), 73.5% to cry1Ga1 (GenBank Accession No. Z22510), 73.4% to cry1Ha1(GenBank Accession No. Z22513), 73.1% to cry1Ca1(GenBank Accession No. X07518), and 70.8% to cry1Ac1(GenBank Accession No. M11068), respectively. The most related cry1Fa-type gene was isolated from B. thuringiensis ssp. aizawai, which showed higher toxicity to S. exigua than the cry1A-type gene [4] and the cryX gene may be responsible for the high toxicity against the Spodoptera species of K1. In an insect toxicity assay, B. thuringiensis ssp. kurstaki HD-1 and ssp. aizawai were used as controls (Table 1). The toxicity of K1 to P. xylostella was similar to that of ssp. kustaki HD-1, but higher than that of ssp. aizawai. The specific crystal genes working against P. xylostella are cry1Aa, cry1Ab, cry1Ac, cry1B, and cry1C [6]. As seen in PCR analysis, cry1Aa, 1Ab, and 1Ac genes were included by B. thuringiensis ssp. kurstaki HD-1 and K1; therefore, K1 might show as high toxicity to P. xylostella as ssp. kurstaki HD-1. In a toxicity assay against S. exigua, K1 was significantly more toxic than ssp. kurstaki HD-1 but similar to ssp. aizawai. Among several B. thuringiensis crystal protein genes, cry1C, cry1D, cry1E, and cry1F genes

302 showed strong activity to Spodoptera species [4, 8, 16, 17]. The high toxicity of K1 might be due to cry1E and cryX genes. This multiple gene content can make K1 overcome the low activity against Spodoptera species and the Cry1A and Cry1C type-resistance problem [10, 14]. B. thuringiensis biopesticide products composed of multiple endotoxins that interact with distinct membrane receptors may, therefore, be less likely to lead to resistant insect populations [4]. The cloning of the intact cryX gene and its characterization should proceed, and this will further diversify the endotoxin genes of B. thuringiensis. ACKNOWLEDGMENTS This research was supported by a grant (CG1416) from the Crop Functional Genomics Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of the Republic of Korea.

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