Requirement of IS911 replication before integration defines a new bacterial transposition pathway

Movement of transposable elements is often accompanied by replication to ensure their proliferation. Replication is associated with both major classes of transposition mechanisms: cut-and-paste and cointegrate formation (paste-and-copy). Cut-and-paste transposition is often activated by replication of the transposon, while in cointegrate formation replication completes integration. We describe a novel transposition mechanism used by insertion sequence IS911, which we call copy-and-paste. IS911 transposes using a circular intermediate (circle), which then integrates into a target. We demonstrate that this is derived from a branched intermediate (figure-eight) in which both ends are joined by a single-strand bridge after a first-strand transfer. In vivo labelling experiments show that the process of circle formation is replicative. The results indicate that the replication pathway not only produces circles from figure-eight but also regenerates the transposon donor plasmid. To confirm the replicative mechanism, we have also used the Escherichia coli terminators (terC) which, when bound by the Tus protein, inhibit replication forks in a polarised manner. Finally, we demonstrate that the primase DnaG is essential, implicating a host-specific replication pathway.     

The role of tandem IS dimers in IS911 transposition

Using a combined in vivo and in vitro approach, we demonstrated that the transposition products generated by IS911 from a dimeric donor plasmid are different from those generated from a plasmid monomer. When carried by a monomeric plasmid donor, free IS911 transposon circles are generated by intra-IS recombination in which one IS end undergoes attack by the other. These represent transposition intermediates that undergo integration using the abutted left (IRL) and right (IRR) ends of the element, the active IRR-IRL junction, to generate simple insertions. In contrast, the two IS911 copies carried by a dimeric donor plasmid not only underwent intra-IS recombination to generate transposon circles but additionally participated in inter-IS recombination. This also creates an active IRR-IRL junction by generating a head-to-tail IS tandem dimer ([IS]2) in which one of the original plasmid backbone copies is eliminated in the formation of the junction. Both transposon circles and IS tandem dimers are generated from an intermediate in which two transposon ends are retained by a single strand joint to generate a figure 8 molecule. Inter-IS figure 8 molecules generated in vitro could be resolved into the [IS]2 form following introduction into a host strain by transformation. Resolution did not require IS911 transposase. The [IS]2 structure was stable in the absence of transposase but was highly unstable in its presence both in vivo and in vitro. Previous studies had demonstrated that the IRR-IRL junction promotes efficient intermolecular integration and intramolecular deletions both in vivo and in vitro. Integration of the [IS]2 derivative would result in a product that resembles a co-integrate structure. It is also shown here that the IRR-IRL junction of the [IS]2 form and derivative structures can specifically target one of the other ends in an intramolecular transposition reaction to generate transposon circles in vitro. These results not only demonstrate that IS911 (and presumably other members of the IS3 family) is capable of generating a range of transposition products, it also provides a mechanistic framework which explains the formation and activity of such structures previously observed for several other unrelated IS elements. This behaviour is probably characteristic of a large number of IS elements.     

Truncated forms of IS911 transposase downregulate transposition

IS911 naturally produces transposase (OrfAB) derivatives truncated at the C-terminal end (OrfAB-CTF) and devoid of the catalytic domain. A majority species, OrfAB*, was produced at higher levels at 42 degrees C than at 30 degrees C suggesting that it is at least partly responsible for the innate reduction in IS911 transposition activity at higher temperatures. An engineered equivalent of similar length, OrfAB[1-149], inhibited transposition activity in vivo or in vitro when produced along with full-length transposase. We isolated several point mutants showing higher activity than the wild-type IS911 at 42 degrees C. These fall into two regions of the transposase. One, located in the N-terminal segment of OrfAB, lies between or within two regions involved in protein multimerization. The other is located within the C-terminal catalytic domain. The N-terminal mutations resulted in reduced levels of OrfAB* while the C-terminal mutation alone appeared not to affect OrfAB* levels. Combination of N- and C-terminal mutations greatly reduced OrfAB* levels and transposition was concomitantly high even at 42 degrees C. The mechanism by which truncated transposase species are generated and how they intervene to reduce transposition activity is discussed. While transposition activity of these multiply mutated derivatives in vivo was resistant to temperature, the purified OrfAB derivatives retained an inherent temperature-sensitive phenotype in vitro. This clearly demonstrates that temperature sensitivity of IS911 transposition is a complex phenomenon with several mechanistic components. These results have important implications for the several other transposons and insertion sequences whose transposition has also been shown to be temperature-sensitive.     

Assembly of a strong promoter following IS911 circularization and the role of circles in transposition.

When supplied with high levels of the IS911-encoded transposase, IS911-based transposons can excise as circles in which the right and left terminal inverted repeats are abutted. Formation of the circle junction is shown here to create a promoter, p(junc), which is significantly stronger than the indigenous promoter, pIRL, and is also capable of driving expression of the IS911 transposition proteins. High transposase expression from the circular transposon may promote use of the circle as an integration substrate. The results demonstrate that IS911 circles are highly efficient substrates for insertion into a target molecule in vivo. Insertion leads to the disassembly of p(junc) and thus to a lower level of synthesis of the transposition proteins. The observation that normal levels of IS911 transposition proteins supplied by wild-type copies of IS911 are also capable of generating transposon circles, albeit at a low level, reinforces the idea that the transposon circles might form part of the natural transposition cycle of IS911. These observations form the elements of a feedback control mechanism and have been incorporated into a model describing one possible pathway of IS911 transposition.     

Efficient transposition of IS911 circles in vitro.

An in vitro system has been developed which supports efficient integration of transposon circles derived from the bacterial insertion sequence IS911. Using relatively pure preparations of IS911-encoded proteins it has been demonstrated that integration into a suitable target required both the transposase, OrfAB, a fusion protein produced by translational frameshifting between two consecutive open reading frames, orfA and orfB, and OrfA, a protein synthesized independently from the upstream orfA. Intermolecular reaction products were identified in which one or both transposon ends were used. The reaction also generated various intramolecular transposition products including adjacent deletions and inversions. The circle junction, composed of abutted left and right IS ends, retained efficient integration activity when carried on a linear donor molecule, demonstrating that supercoiling in the donor molecule is not necessary for the reaction. Both two-ended integration and a lower level of single-ended insertions were observed under these conditions. The frequency of these events depended on the spacing between the transposon ends. Two-ended insertion was most efficient with a natural spacing of 3 bp. These results demonstrate that transposon circles can act as intermediates in IS911 transposition and provide evidence for collaboration between the two major IS911-encoded proteins, OrfA and OrfAB.     

IS911 transposition is regulated by protein-protein interactions via a leucine zipper motif

Efficient intermolecular transposition of bacterial insertion sequence IS911 involves the activities of two element-encoded proteins: the transposase, OrfAB, and a regulatory factor, OrfA. OrfA shares the majority of its amino acid sequence with the N-terminal part of OrfAB. This includes a putative helix-turn-helix and three of four heptads of a leucine zipper motif. OrfA strongly stimulates OrfAB-mediated intermolecular transposition both in vivo and in vitro. The present results support the notion that this is accomplished by direct interaction between these two proteins via the leucine zipper. We used both a genetic approach, based on gene fusions with phage lambda repressor, and a physical approach, involving co-immunoprecipitation, to show that OrfA not only undergoes oligomerisation but is capable of engaging with OrfAB to form heteromultimers, and that the leucine zipper is necessary for both types of interaction. Furthermore, mutation of the leucine zipper in OrfA inactivated its regulatory function. Previous observations demonstrated that the integrity of the leucine zipper motif was also important for OrfAB binding to the IS911 terminal inverted repeats. Here, we show, in gel shift experiments, using a derivative of OrfAB deleted for the C-terminal catalytic domain, OrfAB[1-149], that the protein is capable of pairing two inverted repeats to generate a species resembling a "synaptic complex". Preincubation of OrfAB[1-149] with OrfA dramatically reduced formation of this complex and favored formation of an alternative complex devoid of OrfA. Together these results suggest that OrfA exerts its regulatory effect by interacting transiently with OrfAB via the leucine zipper and modifying OrfAB binding to the inverted repeats.     

A target specificity switch in IS911 transposition: the role of the OrfA protein

The role played by insertion sequence IS911 proteins, OrfA and OrfAB, in the choice of a target for insertion was studied. IS911 transposition occurs in several steps: synapsis of the two transposon ends (IRR and IRL); formation of a figure-of-eight intermediate where both ends are joined by a single-strand bridge; resolution into a circular form carrying an IRR-IRL junction; and insertion into a DNA target. In vivo, with OrfAB alone, an IS911-based transposon integrated with high probability next to an IS911 end located on the target plasmid. OrfA greatly reduced the proportion of these events. This was confirmed in vitro using a transposon with a preformed IRR-IRL junction to examine the final insertion step. Addition of OrfA resulted in a large increase in insertion frequency and greatly increased the proportion of non-targeted insertions. The intermolecular reaction leading to targeted insertion may resemble the intramolecular reaction involving figure-of-eight molecules, which leads to the formation of circles. OrfA could, therefore, be considered as a molecular switch modulating the site-specific recombination activity of OrfAB and facilitating dispersion of the insertion sequence (IS) to 'non-homologous' target sites.     

IS200/IS605 family single-strand transposition: mechanism of IS608 strand transfer

Transposase, TnpA, of the IS200/IS605 family member IS608, catalyses single-strand DNA transposition and is dimeric with hybrid catalytic sites composed of an HUH motif from one monomer and a catalytic Y127 present in an α-helix (αD) from the other (trans configuration). αD is attached to the main body by a flexible loop. Although the reactions leading to excision of a transposition intermediate are well characterized, little is known about the dynamic behaviour of the transpososome that drives this process. We provide evidence strongly supporting a strand transfer model involving rotation of both αD helices from the trans to the cis configuration (HUH and Y residues from the same monomer). Studies with TnpA heterodimers suggest that TnpA cleaves DNA in the trans configuration, and that the catalytic tyrosines linked to the 5′-phosphates exchange positions to allow rejoining of the cleaved strands (strand transfer) in the cis configuration. They further imply that, after excision of the transposon junction, TnpA should be reset to a trans configuration before the cleavage required for integration. Analysis also suggests that this mechanism is conserved among members of the IS200/IS605 family.     

Host processing of branched DNA intermediates is involved in targeted transposition of IS911

A simplified system using bacterial insertion sequence IS911 has been developed to investigate targeted insertion next to DNA sequences resembling IS ends. We show here that these IR-targeted events occur by an unusual mechanism. In the circular IS911 transposition intermediate the two IRs are abutted to form an IR/IR junction. IR-targeted insertion involves transfer of a single end of the junction to the target IR to generate a branched DNA structure. The single-end transfer (SET) intermediate, but not the final insertion product, can be detected in an in vitro reaction. SET intermediates must be processed by the bacterial host to obtain the final insertion products. Sequence analysis of these IR-targeted insertion products and of those obtained in vivo revealed high levels of DNA sequence conversion in which mutations from one IR were transferred to another. These sequence changes cannot be explained by the classic transposition pathway. A model is presented in which the four-way Holliday-like junction created by SET is processed by host-mediated branch migration, resolution, repair and replication. This pathway resembles those described for processing other branched DNA structures such as stalled replication forks.     

In vitro reconstitution of a single-stranded transposition mechanism of IS608

Bacterial insertion sequences (IS) play an important role in restructuring their host genomes. IS608, from Helicobacter pylori, belongs to a newly recognized and widespread IS group with a unique transposition mechanism. We have reconstituted the entire set of transposition cleavage and strand transfer reactions in vitro and find that, unlike any other known transposition system, they strictly require single-strand DNA. TnpA, the shortest identified transposase, uses a nucleophilic tyrosine for these reactions. It recognizes and cleaves only the IS608 "top strand." The results support a transposition model involving excision of a single-strand circle with abutted left (LE) and right (RE) IS ends. Insertion occurs site specifically 3' to conserved and essential TTAC tetranucleotide and appears to be driven by LE. This single-strand transposition mode has important implications not only for dispersion of IS608 but also for the other members of this very large IS family.     

Control of IS911 target selection: how OrfA may ensure IS dispersion

IS911 transposition involves a closed circular insertion sequence intermediate (IS-circle) and two IS-encoded proteins: the transposase OrfAB and OrfA which regulates IS911 insertion. OrfAB alone promotes insertion preferentially next to DNA sequences resembling IS911 ends while the addition of OrfA strongly stimulates insertion principally into DNA targets devoid of the IS911 end sequences. OrfAB shares its N-terminal region with OrfA. This includes a helix-turn-helix (HTH) motif and the first three of four heptads of a leucine zipper (LZ). OrfAB binds specifically to IS911 ends via its HTH whereas OrfA does not. We show here: that OrfA binds DNA non-specifically and that this requires the HTH; that OrfA LZ is required for its multimerization; and that both motifs are essential for OrfA activity. We propose that these OrfA properties are required to assemble a nucleoprotein complex committed to random IS911 insertion. This control of IS911 insertion activity by OrfA in this way would assure its dispersion.     

Transposition in Shigella dysenteriae: isolation and analysis of IS911, a new member of the IS3 group of insertion sequences.

Twenty-nine clear-plaque mutants of bacteriophage lambda were isolated from a Shigella dysenteriae lysogen. Three were associated with insertions in the cI gene: two were due to insertion of IS600, and the third resulted from insertion of a new element, IS911. IS911 is 1,250 base pairs (bp) long, carries 27-bp imperfect terminal inverted repeats, and generates 3-bp duplications of the target DNA on insertion. It was found in various copy numbers in all four species of Shigella tested and in Escherichia coli K-12 but not in E. coli W. Analysis of IS911-mediated cointegrate molecules indicated that the majority were generated without duplication of IS911. They appeared to result from direct insertion via one end of the element and the neighboring region of DNA, which resembles a terminal inverted repeat of IS911. Nucleotide sequence analysis revealed that IS911 carries two consecutive open reading frames which code for potential proteins showing similarities to those of the IS3 group of elements.     

Lack of hotspot targets: a constraint for IS30 transposition in Salmonella

IS30 is an insertion element common in E. coli strains but rare or absent in Salmonella. Transfer of the IS30-flanked transposon Tn2700 to Salmonella typhimurium was assayed using standard delivery procedures of bacterial genetics (conjugation and transduction). Tn2700 'hops' were rare and required transposase overproduction, suggesting the existence of host constraints for IS30 activity. Sequencing of three Tn2700 insertions in the genome of S. typhimurium revealed that the transposon had been inserted into sites with a low homology to the IS30 consensus target, suggesting that inefficient Tn2700 transposition to the Salmonella genome might be caused by a lack of hotspot targets. This view was confirmed by the introduction of an IS30 'hot target sequence', whose sole presence permitted Tn2700 transposition without transposase overproduction. Detection of IS30-induced DNA rearrangements in S. typhimurium provided further evidence that the element undergoes similar activities in E. coli and S. typhimurium. Thus, hotspot absence may be the main (if not the only) limitation for IS30 activity in the latter species. If these observations faithfully reproduce the scenario of natural populations, establishment of IS30 in the Salmonella genome may have been prevented by a lack of DNA sequences closely related to the unusually long (24 bp) IS30 consensus target.     

The terminal inverted repeats of IS911: requirements for synaptic complex assembly and activity

The bacterial insertion sequence IS911 transposes via a covalently closed circular intermediate. Circle formation involves transposase-mediated pairing of both insertion sequence ends. While full-length transposase, OrfAB, binds poorly in vitro to IS911 DNA fragments carrying a copy of the IS911 end, truncated protein derivatives carrying the first 135 (OrfAB[1-135]) or 149 (OrfAB[1-149]) amino acid residues bind efficiently. They generate a paired-end complex containing two such fragments which resembles that expected for the first synaptic complex. Shorter protein derivatives lacking a region involved in multimerisation do not form these complexes but modify the binding of OrfAB[1-135] and OrfAB[1-149]. DNaseI footprinting demonstrated that OrfAB[1-149] protects a sub-terminal (internal) region of the inverted repeats which includes two blocks of sequence (beta and gamma) conserved between the left (IRL) and right (IRR) ends. DNA binding assays in vitro and measurement of recombination activity in vivo of sequential deletion derivatives of the two inverted repeats suggested a model in which the N-terminal region of OrfAB binds the conserved boxes beta and gamma in a sequence-specific manner and anchors the two insertion sequence ends into a paired-end complex. The external region of the inverted repeat is proposed to contact the C-terminal transposase domain carrying the catalytic site.     

The left end of IS2: a compromise between transpositional activity and an essential promoter function that regulates the transposition pathway

Cut-and-paste (simple insertion) and replicative transposition pathways are the two classical paradigms by which transposable elements are mobilized. A novel variation of cut and paste, a two-step transposition cycle, has recently been proposed for insertion sequences of the IS3 family. In IS2 this variation involves the formation of a circular, putative transposition intermediate (the minicircle) in the first step. Two aspects of the minicircle may involve its proposed role in the second step (integration into the target). The first is the presence of a highly reactive junction formed by the two abutted ends of the element. The second is the assembly at the minicircle junction of a strong hybrid promoter which generates higher levels of transposase. In this report we show that IS2 possesses a highly reactive minicircle junction at which a strong promoter is assembled and that the promoter is needed for the efficient completion of the pathway. We show that the sequence diversions which characterize the imperfect inverted repeats or ends of this element have evolved specifically to permit the formation and optimal function of this promoter. While these sequence diversions eliminate catalytic activity of the left end (IRL) in the linear element, sufficient sequence information essential for catalysis is retained by the IRL in the context of the minicircle junction. These data confirm that the minicircle is an essential intermediate in the two-step transposition pathway of IS2.