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

Abstract

A new insertion sequence (IS) element, IS679 (2,704 bp in length), has been identified in plasmid pB171 of enteropathogenic Escherichia coli B171. IS679 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 (Tn679) with the kanamycin resistance gene flanked by an intact IS679 sequence and an IS679 fragment with only IRR (IR on the right) was constructed to clarify the transposition activity of IS679. A transposition assay done with a mating system showed that Tn679 could transpose at a high frequency to the F plasmid derivative used as the target. On transposition, Tn679 duplicated an 8-bp sequence at the target site. Tn679 derivatives with a deletion in each ORF of IS679 did not transpose, finding indicative that all three IS679 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 IS679 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 IS66 family, as do other elements, including nine not previously 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 corresponding 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 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.Insertion sequences (ISs) comprise a large group of bacterial transposable DNA elements. These elements vary in size from 0.7 to 3.5 kb and have imperfect terminal inverted repeat sequences (IRs) of 10 to 40 bp in length (for recent reviews, see references 16 and 20). IS elements generally encode transposase, which is required for transposition, and duplicate a sequence of several base pairs at the target site on transposition. Based on the homology of their transposase genes, IS elements are classified into a number of families (see references 16 and 20). Most IS elements have an open reading frame (ORF) which is thought to encode transposase. Some elements, such as IS1 and IS3, have two ORFs, from which the transposase is produced by translational frameshifting (9,28,29,30). Unless frameshifting occurs, a protein(s) is produced that acts as a transposition inhibitor (31).IS679, which is present in two copies in plasmid pB171 of enteropathogenic ...

Everyman's Guide to Bacterial Insertion Sequences

“…It also carries an insertion domain between the second D and the E of the DDE motif (e.g., IS679, ISPsy5, and ISMac8) (31) The role of the products of tnpA (651 bp) and tnpB (345 bp) is less clear. Mutation of each orf separately (by introduction of an in-frame deletion) reduced transposition by at least two orders of magnitude (196). The three frames are disposed in a pattern suggesting translational coupling: tnpB is in general in translational reading frame −1 compared with tnpA and in most cases the termination codon of tnpA and the initiation codon of tnpB overlap (ATGA).…”

“…The IS66 reference copy from a plasmid of the enteropathogenic Escherichia coli B171, IS679, (196) is defined by three orfs: tnpA, tnpB, and tnpC and relatively well conserved terminal IR of about 20-30 bp flanked by an 8 bp DR at their insertion sites. Orf tnpC is 1,572 bp and its predicted product includes a typical DDE motif.…”

Insertion Sequence Diversity in Archaea

“…They are characterized by a large size (Ͼ2.5 kb), carry multiple ORFs (generally three: TnpA, TnpB, and TnpC), and are flanked by very similar IRs of between 15 to 27 bp, with a conserved terminal 5Ј-GTA. Insertion results in an 8-bp DR (34). Simpler but closely related elements carrying TnpC but not TnpA or TnpB can be found in both Archaea and Bacteria.…”

“…The IS66 family comprises elements found mainly in Proteobacteria (34). They are characterized by a large size (Ͼ2.5 kb), carry multiple ORFs (generally three: TnpA, TnpB, and TnpC), and are flanked by very similar IRs of between 15 to 27 bp, with a conserved terminal 5Ј-GTA.…”

“…These are shorter than IS66 (1,529 to 1,839 bp) and are bordered by IRs of 12 to 30 bp, which are closely related and include the conserved IS66 family terminal 5Ј-GTA trinucleotide. Insertion generates an 8-bp DR (34). The Tpases align well with TnpC and carry a DDE catalytic motif (34).…”