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Recombinant Escherichia coli strains harboring pAG1, pAG2, pKBB100, and pKBB101 were cloned by using antiserum constructed against 130-kDa crystal protein antigen of Bacillus thuringiensis serovar japonensis strain Buibui. DNAs in the recombinant strains hybridized to the 26-base oligonucleotide probe corresponding to N-terminal amino acids of the 130-kDa crystal protein of strain Buibui. Cultures of the recombinant strains were toxic to larvae of the cupreous chafer, Anomala cuprea. Furthermore, the production of the 130-kDa polypeptide was demonstrated in the cells harboring pAG1 and pAG2 by immunoblot analysis with antiserum against the 130-kDa crystal protein. Southern hybridization analysis showed that the 130-kDa crystal protein gene is located on the chromosomal DNA of strain Buibui. On the other hand, DNA probes derived from cryIA(a) and cryIIIA genes did not hybridize to the DNA of strain Buibui.

Original ArticleSynergistic effects of chitinases A, B and C l, chitin binding protein and prodigiosin from Serratia marcescens on the insecticidal activity of delta-endotoxin, Cry 1C, of Bacillus thuringiensis against the common cutworm, Spodoptera litura, were investigated. Only prodigiosin showed a potent synergistic activity with Cry 1C on both the lethal and growth inhibitory activity. In the previous paper (Asano et al., 1999) a synergistic effect in the supernatants of S. marcescens culture on the insecticidal activity of Cry 1C was described. The supernatants from S marcescens and partially purified prodigiosin showed a similar synergistic activity on the insecticidal activity of Cry1C. The content of prodigiosin in the supernatants was estimated as 10% according to the absorbance at 467 nm where prodigiosin showed a peak and the content seemed to be enough to explain the synergistic activity of the supernatants on the activity of Cry 1C.

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A new isolate of Bacillus thuringiensis serovar japonensis strain Buibui, which was specific to scarab beetles (M. Ohba et al., Lett. Appl. Microbiol. 14:54, 1992), was shown to have a 130-kDa insecticidal crystal protein (ICP) (H. Hori et al., J. Appl. Bacteriol. 76:307, 1994). ClaI restriction enzyme fragments of total cell DNA of the isolate were cloned into E. coli (Sato et al., Curr. Microbiol. 28:15, 1994). Whole 3480-bp nucleotide sequence of the gene encoding 130-kDa ICP was determined, and the molecular weight of the ICP was estimated to be 130,424. The strongly conserved five blocks that occur in almost all ICP genes of B. thuringiensis were detected in the ORF with the same order and almost the same intervals as elsewhere. The amino acid sequence homologies of the whole ICP or N-terminus half portion to that of the CryIIIA, B, C, D, and CryV were about 35%.