Isolation and sequence of a 2-kbp miniplasmid from Bacillus thuringiensis var. kurstaki HD-3a3b: relationship with miniplasmids of other B. thuringiensis strains

Abstract

The miniplasmid profiles of 18 Bacillus thuringiensis strains belonging to 8 different serotypes were determined using an alkaline hydrolysis method for isolation of low molecular mass plasmids. Nearly all the strains contained covalently closed circular (CCC) DNA species ranging from 2 to 5 species per strain and from 1.5 to 10.5 kbp in size (values corresponding to CCC forms). A 2-kbp plasmid from B. thuringiensis var. kurstaki HD-3a3b futura strain was used in Southern hybridization experiments to analyse relationships among the low molecular mass plasmids of different B. thuringiensis strains. This 2-kbp miniplasmid was present in most strains which show toxicity against lepidoptera. It was not present in those strains toxic against diptera (B. thuringiensis var. israelensis) or coleoptera (B. thuringiensis var. tenebrionis). The 2-kbp miniplasmid from B. thuringiensis var. kurstaki HD-3a3b futura was cloned and fully sequenced. Sequence analysis of the 2058 bp of the miniplasmid revealed the presence of an ORF (630 bp, 210 amino acids in size) that is preceded by a consensus sequence of B. thuringiensis crystal protein gene transcription promoters. No significant homology was observed with known B. thuringiensis toxin nucleic acid sequences or with other known sequences.