Characterization of the transposase encoded by IS256, the prototype of a major family of bacterial insertion sequence elements

Abstract

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.

FIG. 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. 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).

FIG. 2.

Identification of the DNA-binding domain of IS256 transposase. (A) Secondary structure prediction of the 390-aa transposase protein of IS256 based on PSIPRED analysis (http://bioinf.cs.ucl.ac.uk/psipred/psiform.html). Gray boxes indicate α-helices. β-Sheets are illustrated as black boxes. The three acidic residues of the catalytic DDE triad and their positions (in brackets) are shown above the diagram. The transparent box indicates the position of the putative DNA-binding domain identified in panels B and C. (B) Heterologous expression and purification of overlapping IS256 transposase fragments of various lengths. Dark gray boxes mark the CBP tag. Lanes: 1, full-length wild-type transposase; 2, N-terminal transposase fragment covering aa 1 to 130; 3, internal transposase fragment comprising aa 100 to 230; 4, C-terminal transposase fragment (aa 200 to 390). Proteins were overexpressed in E. coli, and purified fragments 1 to 4 were analyzed by SDS-PAGE (bottom left) and Western blotting with an anti-FLAG antibody (bottom right). (C) EMSAs using 15.5 nM DNA of the right-end IS256 terminus DNA as a substrate with increasing amounts (i.e., 200, 500, and 1,000 nM) of purified transposase fragments 1 to 4, respectively. The experiments were performed with 50 μg of poly(dI-dC) ml⁻¹ as a nonspecific competitor, and the molar DNA/protein ratios were 1:13 (200 nM protein), 1:32 (500 nM protein), and 1:64 (1,000 nM protein), respectively.

FIG. 3.

Analysis of a putative HTH DNA-binding motif of the IS256 transposase. (A) Amino acid sequence alignment (25) of the putative DNA-binding region of IS256 (aa 100 to 130) and other bacterial members of the IS256 family. ★, α-helix; C, coiled region. Rectangles mark the predicted α-helices α3 and α4, respectively. Highly conserved and conserved residues are highlighted in black and gray, respectively. In the consensus sequence, “x,” “p,” and “h” indicate variable, polar, and hydrophobic amino acid residues, respectively. (B) Amino acid exchanges of highly conserved and conserved residues within the putative HTH motif of the CBP-Tnp protein. (C) EMSAs with 15.5 nM IS terminus (right)-specific DNA as substrate and increasing amounts of the altered CBP-Tnp proteins described in panels A and B. Binding assays were performed in the presence of 50 μg of poly(dI-dC) ml⁻¹ as a nonspecific competitor, and molar DNA/protein ratios ranged from 1:3 (50 nM protein) to 1:52 (800 nM protein), respectively.

FIG. 4.

Influence of the integrity of the IRs on IS256 transposase binding. (A) Nucleotide sequence of IS256 ends harboring the IRs (uppercase boldface letters) and positions of nucleotide deletions (−) introduced into the IS terminus (left) and IS terminus (right) DNA substrates, respectively. (B) EMSAs in the presence of 50 μg of poly(dI-dC) ml⁻¹ with 1.9 or 3.8 μM CBP-Tnp transposase and a 15.5 nM concentration of the mutated IS termini as DNA substrates, respectively. The molar DNA/protein ratios were 1:122 (1.9 μM CBP-Tnp) and 1:245 (3.8 μM CBP-Tnp), respectively.

FIG. 5.

Effect of terminal IS deletions on IS256 circle formation. (A) Electron micrograph of an IS256 circular DNA molecule (white arrow). The picture was taken from an E. coli plasmid preparation of vector pIL2 carrying a wild-type IS256 insertion. The black arrowhead marks a 9.3-kb pIL2 vector molecule. (B) Illustration of an IS256 circle with abutted IS ends separated by a short stretch of foreign DNA (gray box). Arrows indicate the direction of primers used for PCR detections of IS256 circles in panels C and D. The orientation of the transposase gene tnp is marked by an arrow. The chart on the right details the nucleotide sequence of the last four nucleotides at the IS256 ends and their deletions introduced into the IS256 copy on vector pIL2, respectively. (C) Agarose gel electrophoresis of IS256 circle-specific PCR products amplified by using primers 1 and 2 and plasmid preparations of pIL2 (as a positive control) and pIL2 IS256Δtnp (as a negative control), as well as pIL2-derived mutants, with deletions in the IS256 termini as templates, respectively. (D) Comparison of the circle amounts detected in pIL2, carrying an IS256 wild-type copy, and various IS termini mutants, respectively, as determined by qPCR.