The plasmid pBMBt1 from Bacillus thuringiensis subsp. darmstadiensis (INTA Mo14-4) replicates by the rolling-circle mechanism and encodes a novel insecticidal crystal protein-like gene

This work describes a novel rolling-circle replicating (RCR) plasmid pBMBt1 from Bacillus thuringiensis subsp. darmstadiensis (INTA Mo14-4) encoding an insecticidal crystal protein-like gene. pBMBt1 (6700 bp) contains three ORFs and their putative transcription initiation sites and Shine-Dalgarno sequences were localized. ORF1 encodes a 34.6 kDa protein which showed identity with the protein CryC53 from B. thuringiensis subsp. cameroun (24.6%), the Cry15Aa insecticidal crystal protein from B. thuringiensis subsp. thompsoni (21.9%) and the Mtx3 protein from Bacillus sphaericus (27.8%). The ORF2 (52.3 kDa) showed a 74% identity with the Mob protein coded by pUIBI-1 from B. thuringiensis subsp. entomocidus and 64% identity with the Mob protein of pBMY1 from Bacillus mycoides; both Mob proteins belong to the pMV158 superfamily. To evaluate the Mob protein, the plasmid pHTMob14-4 was constructed. This plasmid shows transfer frequencies of 9.1 × 10−6 in B. thuringiensis subsp. israelensis (4Q7GmR). The ORF3 (23.6 kDa) gene product is homologous to the Rep protein from the plasmid pBMYdx of B. mycoides (37.6%). A putative double-strand origin with significant homology to that of B. thuringiensis plasmids, and an ssoA-type single-strand origin were also identified. Detection of single-stranded pBMBt1 DNA replicating intermediaries suggests that replication occurs via the rolling-circle mechanism.