Characterization of a novel insertion sequence, ISBp1, in Burkholderia pseudomallei

Abstract

During screening for antigenic proteins in Burkholderia pseudomallei, a novel insertion sequence, ISBp1, was found by sequence similarity searches. ISBp1 contains two overlapping ORFs of 261 bp (orfA) and 852 bp (orfB), encoding 87 and 284 amino acid residues, respectively, and an imperfect inverted repeat. The putative protein encoded by orfA (OrfA) is similar to the OrfA in insertion sequences of the IS3 family in other bacteria, showing 49% and 76% amino acid identity and similarity, respectively, with the transposase encoded by ISD1 of Desulfovibrio vulgaris vulgaris. The putative protein encoded by orfB (OrfB) is similar to the OrfB in insertion sequences of the IS3 family in other bacteria, showing 43% and 62% amino acid identity and similarity, respectively, with the transposase encoded by IS1222 of Enterobacter agglomerans. Sequence analysis of OrfA showed the presence of an α-helix-turn-α-helix motif, as well as the putative leucine zipper at its 3′ end, for possible DNA binding to the terminal inverted repeats. Sequence analysis of OrfB showed the presence of a DDE motif of aspartic acid, aspartic acid, and glutamic acid, a highly conserved motif present in OrfB of other members of the IS3 family. Furthermore, several other conserved amino acid residues, including the arginine residue located seven amino acids downstream from the glutamic acid residue, were observed. PCR amplification of the ISBp1 gene showed a specific band in 65% of the 26 B. pseudomallei strains tested. Southern blot hybridization after XhoI or SacI digestion showed nine different patterns of hybridization. The number of copies of ISBp1 in those strains that possessed the insertion sequence ranged from three to 12. Using several insertion sequences and a combination of insertion-sequence-based and non-insertion-sequence-based methods such as ribotyping will probably increase the discriminatory power of molecular typing in B. pseudomallei.