IS1 is an insertion element first identified in Escherichia coli (for a review, see reference 28). IS1 is only 768 bp in length and has imperfect terminal inverted repeats (IR) of about 30 bp (19,30). IS1 transposes to a new site and generates a duplication of a target sequence of 9 bp (in most cases) or 8 bp (in some cases) (13,14,17,29). IS1 has two open reading frames (ORFs), insA and BЈ-insB, which are required for transposing itself (18,25,26). insB is in the Ϫ1 frame with respect to insA. The Ϫ1 frameshifting occurs during translation at the AAAAAAC (A 6 C) sequence in the overlapping region between insA and BЈ-insB. The resulting transframe protein, InsA-BЈ-B, is IS1 transposase (10, 37).IS1 generates the InsA protein from insA unless translational frameshifting occurs. The InsA protein, which has the ability to bind to terminal IR sequences (43, 52, 54), inhibits transposition of IS1 (24, 53). IS1 with a 1-bp insertion in the A 6 C sequence, causing the two frames insA and BЈ-insB to be in the same frame, produces transposase without frameshifting and can thus transpose at a very high frequency (37, 39). These facts suggest that the InsB segment in IS1 transposase forms a catalytic domain. Many elements with structural features similar to those seen in IS1 have been identified in various bacteria and their plasmids (for a review, see reference 28). Most of them are highly homologous to IS1 and to one another (more than 90% sequence identity), whereas a few, such as IS1(NuXi) from Shigella dysenteriae, have low homology, about 55% (31).Transposases encoded by IS3 family elements and retroviral integrases have been found to have three acidic amino acid residues, D, D, and E, constituting a motif called the D-D-E motif, at positions where a polypeptide segment 35 amino acids long is usually present between the second D and third E residues (11, 23). The D-D-E motif has also been found in transposases encoded by IS630, Tn7, Tn552, and others and in integrases encoded by retrotransposons (7,34). Transposases encoded by IS10 and phage Mu also have the D-D-E motif, but a longer polypeptide segment of 130 or 55 amino acids is present between the second D and the E (4, 5, 49). The polypeptide segment with the D-D-E motif forms a catalytic domain in which the three acidic amino acid residues have an essential role in capturing Mg 2ϩ and other divalent cations (4). Tertiary structures of the catalytic core domains of human immunodeficiency virus type 1 (HIV-1) integrase and phage Mu transposase (MuA) and full-length Tn5 transposase have been determined by X-ray crystallography (6, 9, 35). These proteins characteristically have three ␤ sheets in tandem in the segment with the D-D-E motif, such that the first D residue is present in the first ␤ sheet.The transposase encoded by IS1 has been thought not to belong to the family of proteins with the D-D-E motif but to the phage integrase (Int) family (44) because IS1 transposase has the H-R-Y motif, which is conserved in all the Int family proteins (1, 3). In this study,...

IS1, the smallest active transposable element in bacteria, encodes a transposase that promotes inter-and intramolecular transposition. Host-encoded factors, e.g., histone-like proteins HU and integration host factor (IHF), are involved in the transposition reactions of some bacterial transposable elements. Host factors involved in the IS1 transposition reaction, however, are not known. We show that a plasmid with an IS1 derivative that efficiently produces transposase did not generate miniplasmids, the products of intramolecular transposition, in mutants deficient in a nucleoid-associated DNA-binding protein, H-NS, but did generate them in mutants deficient in histone-like proteins HU, IHF, Fis, and StpA. Nor did IS1 transpose intermolecularly to the target plasmid in the H-NS-deficient mutant. The hns mutation did not affect transcription from the indigenous promoter of IS1 for the expression of the transposase gene. These findings show that transpositional recombination mediated by IS1 requires H-NS but does not require the HU, IHF, Fis, or StpA protein in vivo. Gel retardation assays of restriction fragments of IS1-carrying plasmid DNA showed that no sites were bound preferentially by H-NS within the IS1 sequence. The central domain of H-NS, which is involved in dimerization and/or oligomerization of the H-NS protein, was important for the intramolecular transposition of IS1, but the N-and C-terminal domains, which are involved in the repression of certain genes and DNA binding, respectively, were not. The SOS response induced by the IS1 transposase was absent in the H-NSdeficient mutant strain but was present in the wild-type strain. We discuss the possibility that H-NS promotes the formation of an active IS1 DNA-transposase complex in which the IS1 ends are cleaved to initiate transpositional recombination through interaction with IS1 transposase.IS1 is an insertion element present in chromosomes and plasmids of enteric bacteria (for a review, see reference 38). IS1 (768 bp long) carries imperfect terminal inverted repeats (IRL and IRR) that are about 30 bp long (17,40). IS1 mediates the formation of cointegrates between the IS1-carrying plasmid and a target plasmid in which two copies of IS1 are duplicated at the two junctions in direct orientation (10,39). This element encodes two open reading frames, insA and BЈ-insB (16,32,33). Transcription occurs from a promoter present in the left-terminal region (IRL) preceding insA (31). A translational frameshift occurs in the Ϫ1 direction at the AAAAAAC (A 6 C) sequence in the overlapping region between the two open reading frames, producing the InsA-BЈ-InsB transframe protein, IS1 transposase (7, 49). Unless frameshifting occurs, IS1 produces InsA protein from insA which can bind to the IRs (51, 74) and inhibits transposition (30,75). An IS1 mutant (IS1-31) with a single adenine insertion at the frameshifting site efficiently produces transposase and therefore can frequently transpose and mediate cointegration (49). The plasmid with this IS1 mutant generates min...

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 ...

Background: IS1, the smallest active transposable element in bacteria, encodes transposase. IS1 transposase promotes transposition as well as production of miniplasmids from a plasmid carrying IS1 by deletion of the region adjacent to IS1. The IS1 transposase also promotes production of IS1 circles consisting of the entire IS1 sequence and a sequence, 6±9 bp in length, as a spacer between terminal inverted repeats of IS1. The biological signi®cance of the generation of IS1 circles is not known.