Characterization of ISBth4, a functional new IS231 variant from Bacillus thuringiensis MEX312

A new insertion variant of IS231 family, designated ISBth4, was identified from Bacillus thuringiensis MEX312. ISBth4 is 2046bp in length and is delimited by two 17bp inverted repeats (IR) with one mismatch, flanked by two perfect 11bp direct repeats (DR). ISBth4 contains two open reading frames (ORFs), ORF1 and ORF2, which encode 72 and 473 amino acids, respectively. Multiple sequence alignments revealed that the potential transposase of ISBth4 contained five conserved domains N1, N2, N3, C1 and C2 that are similar to other IS231 elements; and the typical catalytic triad D(N2)-70-D(N3)-150-E(C1) and Y(2)R(3)E(6)K motifs as hallmarks of IS4 elements. Comparison of the amino acids of the potential ISBth4 transposase with those from other publicly available B. cereus group IS231 elements revealed a close similarity with ISBce7 (94% identity), ISBce5 (90%), IS231Y (89%) and ISBce8 (86%), and lower similarity to IS231N (49%), IS231M (48%) and ISBce12 (40%). Phylogenetic analysis of the evolutionary relationships between ISBth4 and the other IS231 elements showed that ISBth4 is more closely related to the IS231 sequences isolated from B. cereus strains than those from B. thuringiensis strains. In vivo transposition activity of ISBth4 was discovered in a mutant B18 from a MEX312 background, indicating that it is a functional insertion sequence in its B. thuringiensis natural host.