Structural and Functional Characterization of IS 679 and IS 66 -Family Elements

Abstract

ABSTRACT A new insertion sequence (IS) element, IS 679 (2,704 bp in length), has been identified in plasmid pB171 of enteropathogenic Escherichia coli B171. IS 679 has imperfect 25-bp terminal inverted repeats (IRs) and three open reading frames (ORFs) (here called tnpA, tnpB , and tnpC ). A plasmid carrying a composite transposon (Tn 679 ) with the kanamycin resistance gene flanked by an intact IS 679 sequence and an IS 679 fragment with only IRR (IR on the right) was constructed to clarify the transposition activity of IS 679 . A transposition assay done with a mating system showed that Tn 679 could transpose at a high frequency to the F plasmid derivative used as the target. On transposition, Tn 679 duplicated an 8-bp sequence at the target site. Tn 679 derivatives with a deletion in each ORF of IS 679 did not transpose, finding indicative that all three IS 679 ORFs are essential for transposition. The tnpA and tnpC products appear to have the amino acid sequence motif characteristic of most transposases. A homology search of the databases found that a total of 25 elements homologous to IS 679 are present in Agrobacterium, Escherichia, Rhizobium, Pseudomonas , and Vibrio spp., providing evidence that the elements are widespread in gram-negative bacteria. We found that these elements belong to the IS 66 family, as do other elements, including nine not previously reported. Almost all of the elements have IRs similar to those in IS 679 and, like IS 679 , most appear to have duplicated an 8-bp sequence at the target site on transposition. These elements have three ORFs corresponding to those in IS 679 , 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 tnpA . In contrast, tnpC , separated from tnpB by a space of ca. 20 bp, is located in any one of three frames relative to tnpB . No common structural features were found around the intergenic regions, indicating that the three ORFs are expressed by translational coupling but not by translational frameshifting.