Characterization of a novel mosquitocidal strain of Bacillus thuringiensis serovar aizawai which harbors a rolling-circle replication plasmid, pBt1–3

Abstract

Bacillus thuringiensis 1–3, isolated from a Korean soil sample, was determined to belong to ssp. aizawai (H7) type by an H antiserum agglutination test, and produced bipyramidal-shaped crystal proteins with a molecular weight of 130kDa. PCR analysis with specific cry gene primers showed that B. thuringiensis 1–3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2A genes, differing from that of serovar of aizawai (reference strain) which contains cry1Aa, cry1Ab, cry1C and cry1D genes. In contrast to the reference strain, B. thuringiensis aizawai showed insecticidal activity against Plutella xylostella larvae, the B. thuringiensis 1–3 showed insecticidal activity against not only P. xylostella, but also Aedes aegypti, owing to its Cry2A crystal protein. In this study, we modified the plasmid capture system (PCS) through in vitro transposition to clone small cryptic plasmids from B. thuringiensis 1–3. Fifty-three clones were acquired, and their sizes were approximately 10kb. Based on the sequence analysis, they were classified into four groups, showing similarities with four known B. thuringiensis plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of the pGI3-like clones, pBt1–3, was fully sequenced, and its putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1–3 showed high similarity with pGI3, which is of the rolling-circle replication (RCR) group VI family.