Characterization of a cry2A Gene Cloned from an Isolate of Bacillus thuringiensis serovar sotto Jun Sasaki, 1, * Shinichiro Asano, 1 Naoki Hashimoto, 1 Bibiana W. Lay, 2 Sugyo Hastowo, 2 Hisanori Bando, 1 Toshihiko Iizuka 1 1 Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan 2 Faculty of Veterinary Medicine, Institut Pertanian Bogor, Indonesia Received: 17 July 1996 / Accepted: 5 December 1996 Abstract. A cry2A-type gene, designated as cry2(SKW), was cloned from Bacillus thuringiensis serovar sotto SKW01-10.2-06, and some unique features of the gene were revealed. The cry2(SKW) gene encoded a polypeptide of 635 residues with a predicted molecular mass of 71,137 Da. Cry2(SKW) had 95.4% identity with Cry2Aa in amino acid sequence and was two residues longer than Cry2Aa. Two open reading frames (ORFs), designated as orf1 and orf2, were present upstream of the cry2(SKW) and showed high homology with the corresponding ORFs in the cry2Aa operon. The Orf2 from SKW01-10.2-06 contained a region of repeated sequences. However, unlike Cry2Aa, Cry2(SKW) formed the cuboidal crystalline inclusions when the cry2(SKW) gene was expressed in an acrystalliferous B. thuringiensis strain in the absence of the upstream ORFs. Furthermore, Cry2(SKW) was less toxic to a lepidopteran species, Bombyx mori, than Cry2Aa in spite of high homology between the two proteins. Bacillus thuringiensis is a Gram-positive, spore-forming bacterium that produces insecticidal crystal proteins (ICPs) during sporulation. The ICPs are not only toxic to the larvae of lepidopteran, dipteran, and coleopteran insects, but also to nematodes, protozoan pathogens, animal-parasitic liver flukes (Trematodes), and mites [12]. To date, many ICP genes have been cloned and characterized, and the ICPs were divided into 17 groups on the basis of the amino acid sequence similarity [8, 14]. cry2A genes encode approximately 65-kDa proteins, which form cuboidal crystals [23, 27]. Three cry2A genes, cry2Aa [11, 23], cry2Ab [9, 23], and cry2Ac [25], have been reported. Cry2Aa is toxic to lepidopteran and dipteran species, while Cry2Ab and Cry2Ac are toxic only to lepidopteran species. cry2Aa and cry2Ac genes have a common characteristic in that these are the third genes in a three-gene operon. However, the crystalliza- tion of Cry2Aa protein requires the second gene (orf2) in the cry2Aa operon [6], while the two ORFs upstream of cry2Ac gene do not have the role in the formation of Cry2Ac inclusions [25]. On the other hand, cry2Ab gene is cryptic [7, 9, 23]. B. thuringiensis serovar sotto SKW01-10.2-06 was isolated from Indonesian soil and identified by H-antisera [18]. This isolate produced three types of crystal pro- teins—spherical, bipyramidal, and cuboidal [18]. The production of cuboidal proteins in SKW01-10.2-06 sug- gested that this isolate contained a cry2A gene. To date, it has been reported that strains of serovars kurstaki, tolworthi, kenyae, aizawai, and galleriae contain cry2A genes [2, 14], although no strain of serovar sotto with a cry2A gene has been reported. In this paper, we report that the cry2A gene, which was cloned from SKW01-10.2-06 of serovar sotto, has some characteristics different from those of the cry2Aa gene with respect to the corresponding amino acid sequences, the formation of crystalline inclusions, and the insecticidal activity against Bombyx mori. Materials and Methods B. thuringiensis. B. thuringiensis serovar sotto SKW01-10.2-06 was a soil isolate from Indonesia [18]. The soil sample was suspended in sterilized distilled water and shaken. The upper layer of the suspension was heat treated at 80°C for 5 min. Appropriate dilutions were plated on Nutrient Agar (Oxoid) and incubated at 28°C for 4 days. Colonies were * Present address: National Institute of Serological and Entomological Science, Tsukuba, Ibaraki 305, Japan. Correspondence to: T. Iizuka CURRENT MICROBIOLOGY Vol. 35 (1997), pp. 1–8 An International Journal R Springer-Verlag New York Inc. 1997