Characterization of the Transposase Encoded by IS256, the Prototype of a Major Family of Bacterial Insertion Sequence Elements

IS256 is the founding member of the IS256 family of insertion sequence (IS) elements. These elements encode a poorly characterized transposase, which features a conserved DDE catalytic motif and produces circular IS intermediates. Here, we characterized the IS256 transposase as a DNA-binding protein and obtained insight into the subdomain organization and functional properties of this prototype enzyme of IS256 family transposases. Recombinant forms of the transposase were shown to bind specifically to inverted repeats present in the IS256 noncoding regions. A DNA-binding domain was identified in the N-terminal part of the transposase, and a mutagenesis study targeting conserved amino acid residues in this region revealed a putative helix-turn-helix structure as a key element involved in DNA binding. Furthermore, we obtained evidence to suggest that the terminal nucleotides of IS256 are critically involved in IS circularization. Although small deletions at both ends reduced the formation of IS circles, changes at the left-hand IS256 terminus proved to be significantly more detrimental to circle production. Taken together, the data lead us to suggest that the IS256 transposase-mediated circularization reaction preferentially starts with a sequence-specific first-strand cleavage at the left-hand IS terminus.IS256 is an insertion sequence widespread in the genomes of multiresistant enterococci and staphylococci (3). The element, which is 1,324 bp in size, consists of a single open reading frame encoding a transposase protein flanked by noncoding regions (NCRs) harboring imperfect inverted repeats (IRs) (see Fig. ​Fig.1A).1A). IS256 occurs in multiple free copies in its host genomes but is also known to form the ends of composite transposon Tn4001 conferring aminoglycoside resistance (29). In Staphylococcus epidermidis, IS256 has been identified as a typical marker of hospital-acquired multiresistant and biofilm-forming clones causing opportunistic infections in immunocompromised patients (11, 20-22, 26, 34). The element has been shown to trigger heterogeneous biofilm expression by reversible transposition into biofilm-associated genes and regulators (4, 5, 19, 49, 56). Also, IS256 has the capacity to influence antibiotic resistance, either by insertion into regulatory genes or by modulating antibiotic resistance gene expression through formation of strong hybrid promoters resulting from transposition into the neighborhood of antibiotic resistance genes (6, 18, 31, 32). Finally, multiple genomic IS256 copies may serve as crossover points for homologous recombination events and thereby play an important role in genome flexibility, adaptation, and evolution of staphylococcal and enterococcal genomes (29, 42, 55).Open in a separate windowFIG. 1.IS256 transposase binding to IS termini. (A) Genetic organization of IS256. The transposase gene (tnp) is flanked by NCRs that harbor imperfect IRs (IR_L and IR_R) at the ends of the element. The nucleotide sequence of the IRs is indicated by uppercase boldface letters, with nucleotide numbering referring to GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"M18086","term_id":"152946","term_text":"M18086"}}M18086. Insertion of IS256 into the S. epidermidis icaC gene on plasmid pIL2 (27) is shown, and black boxes mark the 8-bp target site duplications (TSDs) generated upon transposition of the element. Black bars at the top indicate localizations of DNA fragments used in the EMSAs presented in panels B to D. (B to D) EMSAs of purified IS256 transposase protein (CBP-Tnp) with various IS256-specific DNA fragments. A 15.5 nM concentration of an IS terminus (left)-carrying DNA fragment (B) or an IS terminus (right)-carrying DNA-fragment (C), as well as an interal IS256 fragment (D), were used with increasing amounts of protein. All experiments were performed in the presence of unspecific competitor [50 μg of poly(dI-dC) ml⁻¹]. Molar ratios between DNA and protein comprised a range of 1:3 (50 nM CBP-Tnp) to 1:52 (800 nM CBP-Tnp).Given its important biological role, it is surprising that very little is known about the molecular function of IS256 and its lifestyle. Empirical analyses of IS256 insertion sites in various bacterial genomes and loci did not reveal nucleotide sequence specificity for target site selection (3, 29, 56). Typically, IS256 generates 8- or 9-bp target site duplications (TSDs) upon transposition that are caused by staggered nicks of the target DNA and refill of the resulting gaps by the host repair system (43). In the course of phase variation events, IS256 TSDs can be completely removed, with the original host sequence being restored (56). Such precise IS256 excisions are caused by an illegitimate recombination event that requires fully intact TSDs but no functional IS256 transposase (14). IS256 transposition itself was found to involve the formation of double-stranded circular IS256 molecules in which the insertion sequence (IS) ends abut, bridged by a few base pairs of host DNA originating from the original insertion site (27, 39). IS256 circle formation is a strictly transposase-dependent process and IS circles are regarded as transposition intermediates which are likely to be relinearized during transposition. However, details of the transposition reaction, including circle formation, putative relinearization, target site selection, and insertion of the element are far from being understood at the molecular level. We experimentally addressed here, for the first time for a bacterial transposase of the IS256 family, the DNA-binding properties of this protein. We identified a DNA-binding domain in the N-terminal region of the protein. The domain contains a putative classical helix-turn-helix (HTH) motif that is demonstrated to be involved in sequence-specific interactions of the IS256 transposase with the IRs present in the NCRs of the element. Moreover, we suggest a role for the terminal nucleotides of the IS256 nucleotide sequence in first-strand cleavage and subsequent circularization of the element.