Structural and functional characterization of IS679 and IS66-family elements

A new insertion sequence (IS) element, IS679 (2,704 bp in length), has been identified in plasmid pB171 of enteropathogenic Escherichia coli B171. IS6 79 has imperfect 25-bp terminal inverted repeats (IRs) and three open readi ng frames (ORFs) there called tnpA, tnpB, and tnpC). A plasmid carrying a c omposite transposon (Tn679) with the kanamycin resistance gene flanked by a n intact IS679 sequence and an IS679 fragment with only IRR (IR on the righ t) was constructed to clarify the transposition activity of IS679. A transp osition assay done with a mating system showed that Tn679 could transpose a t a high frequency to the F plasmid derivative used as the target. On trans position, Tn679 duplicated an 8-bp sequence at the target site. Tn679 deriv atives with a deletion in each ORF of IS679 did not transpose, finding indi cative that all three IS679 ORFs are essential for transposition. The tnpA and tnpC products appear to have the amino acid sequence motif characterist ic of most transposases. A homology search of the databases found that a to tal of 25 elements homologous to IS679 are present in Agrobacterium, Escher ichia, Rhizobium, Pseudomonas, and Vibrio spp., providing evidence that the elements are widespread in gram-negative bacteria. We found that these ele ments belong to the IS66 family, as do other elements, including nine not p reviously reported. Almost all of the elements have IRs similar to those in IS679 and, like IS679, most appear to have duplicated an 8-bp sequence at the target site on transposition. These elements have three ORFs correspond ing to those in IS679, but many have a mutation(s) in an ORF(s). In almost all of the elements, tnpB is located in the -1 frame relative to tnpA, such that the initiation codon of tnpB overlaps the TGA termination codon of tn pA. In contrast, tnpC, separated from tnpB by a space of ca. 20 bp, is loca ted in any one of three frames relative to tnpB. No common structural featu res were found around the intergenic regions, indicating that the three ORF s are expressed by translational coupling but not by translational frameshi fting.