The basis of asymmetry in IS2 transposition

In the first step of IS2 transposition, the formation of an IS2 minicircle, the roles of the two IS ends differ. Terminal cleavage initiates exclusively at the right inverted repeat (IRR) - the donor end - whereas IRL is always the target. At the resulting minicircle junction, the two abutted ends are separated by a spacer of 1 or 2 basepairs. In this study, we have identified the determinants of donor and target function. The inability of IRL to act as a donor results largely from two sequence differences between IRL and IRR - an extra basepair between the conserved transposase binding sequences and the end of the element, and a change of the terminal dinucleotide from CA-3' to TA-3'. These two changes also impose a characteristic size on the minicircle junction spacer. The only sequences required for the efficient target function of IRL appear to be contained within the segment from position 11-42. Although IRR can function as a target, its shorter length and additional contacts with transposase (positions 1-7) result in minicircles with longer, and inappropriate, spacers. We propose a model for the synaptic complex in which the terminus of IRL makes different contacts with the transposase for the initial and final strand transfer steps. The sequence differences between IRR and IRL, and the behavioural characteristics of IRL that result from them, have probably been selected because they optimize expression of transposase from the minicircle junction promoter, Pjunc.     

Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS2 transposes by an unconventional pathway

The Escherichia coli insertion sequence, IS 2 , is a member of the IS 3 family of bacterial transposable elements. Its transposase is a fusion protein, OrfAB, made by a programmed −1 translational frameshift near to the end of orfA and just after the start of orfB . We have characterized two major products of IS 2 intramolecular transposition, which accumulate in cells that express the IS 2 OrfAB fusion protein at elevated levels. The more abundant product is a minicircle composed of the complete IS 2 with just a single basepair (occasionally 2 bp) separating the two IS ends. In all cases, this basepair is derived from the vector sequence immediately adjacent to the left IS 2 end (IRL). The second product is a figure-eight molecule that contains all the IS 2 and vector sequences present in the parental plasmid. One DNA strand contains the parental sequences unrearranged. The other contains a single-stranded version of the minicircle junction — the precise 3' end of IRR has been cleaved and joined to a target just outside the 5' end of IRL; the remaining vector sequences have a free 5' end, derived from cleavage at the 3' end of IRR, and a free 3' end, released upon cleavage of the target site adjacent to IRL. We propose that figure-eight molecules are the precursor to IS 2 minicircles and that the formation of these two products is the initial step in IS 2 intermolecular transposition. This proposed transposition pathway provides a means for a transposase that can cleave only one strand at each IS end to produce simple insertions and avoid forming co-integrates.     

Excision of IS492 Requires Flanking Target Sequences and Results in Circle Formation in Pseudoalteromonas atlantica

The gram-negative marine bacterium Pseudoalteromonas atlantica produces extracellular polysaccharide (EPS) that is important in biofilm formation by this bacterium. Insertion and precise excision of IS492 at a locus essential for extracellular polysaccharide production (eps) controls phase variation of EPS production in P. atlantica. Examination of IS492 transposition in P. atlantica by using a PCR-based assay revealed a circular form of IS492 that may be an intermediate in transposition or a terminal product of excision. The DNA sequence of the IS492 circle junction indicates that the ends of the element are juxtaposed with a 5-bp spacer sequence. This spacer sequence corresponds to the 5-bp duplication of the chromosomal target sequence found at all IS492 insertion sites on the P. atlantica chromosome that we identified by using inverse PCR. IS492 circle formation correlated with precise excision of IS492 from the P. atlantica eps target sequence when introduced into Escherichia coli on a plasmid. Deletion analyses of the flanking host sequences at the eps insertion site for IS492 demonstrated that the 5-bp duplicated target sequence is essential for precise excision of IS492 and circle formation in E. coli. Excision of IS492 in E. coli also depends on the level of expression of the putative transposase, MooV. A regulatory role for the circular form of IS492 is suggested by the creation of a new strong promoter for expression of mooV by the joining of the ends of the insertion sequence element at the circle junction.     

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.     

Transient promoter formation: a new feedback mechanism for regulation of IS911 transposition

IS911 transposition involves a free circular transposon intermediate where the terminal inverted repeat sequences are connected. Transposase synthesis is usually driven by a weak promoter, p(IRL), in the left end (IRL). Circle junction formation creates a strong promoter, p(junc), with a -35 sequence located in the right end and the -10 sequence in the left. p(junc) assembly would permit an increase in synthesis of transposase from the transposon circle, which would be expected to stimulate integration. Insertion results in p(junc) disassembly and a return to the low p(IRL)- driven transposase levels. We demonstrate that p(junc) plays an important role in regulating IS911 transposition. Inactivation of p(junc) strongly decreased IS911 transposition when transposase was produced in its natural configuration. This novel feedback mechanism permits transient and controlled activation of integration only in the presence of the correct (circular) intermediate. We have also investigated other members of the IS3 and other IS families. Several, but not all, IS3 family members possess p(junc) equivalents, underlining that the regulatory mechanisms adopted to fine-tune transposition may be different.     

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.     

Diversity of Tn4001 Transposition Products: the Flanking IS256 Elements Can Form Tandem Dimers and IS Circles

We show that both flanking IS256 elements carried by transposon Tn4001 are capable of generating head-to-tail tandem copies and free circular forms, implying that both are active. Our results suggest that the tandem structures arise from dimeric copies of the donor or vector plasmid present in the population by a mechanism in which an IS256 belonging to one Tn4001 copy attacks an IS256 end carried by the second Tn4001 copy. The resulting structures carry abutted left (inverted left repeat [IRL]) and right (inverted right repeat [IRR]) IS256 ends. Examination of the junction sequence suggested that it may form a relatively good promoter capable of driving transposase synthesis in Escherichia coli. This behavior resembles that of an increasing number of bacterial insertion sequences which generate integrative junctions as part of the transposition cycle. Sequence analysis of the IRL-IRR junctions demonstrated that attack of one end by the other is largely oriented (IRL attacks IRR). Our experiments also defined the functional tips of IS256 as the tips predicted from sequence alignments, confirming that the terminal 4 bp at each end are indeed different. The appearance of these multiple plasmid and transposon forms indicates that care should be exercised when Tn4001 is used in transposition mutagenesis. This is especially true when it is used with naturally transformable hosts, such as Streptococcus pneumoniae, in which reconstitution of the donor plasmid may select for higher-order multimers.     

Escherichia coli integration host factor binds specifically to the ends of the insertion sequence IS1 and to its major insertion hot-spot in pBR322

We report here that the ends of IS1 are bound and protected in vitro by the heterodimeric protein integration host factor (IHF). Under identical conditions, RNA polymerase binds to one of these ends (IRL) and protects a region that includes the sequences protected by IHF. Other potential sites within IS1, identified by their homology to the apparent consensus sequence, are not protected. Footprinting analysis of deletion derivatives of the ends demonstrates a correspondence between the ability of the end sequence to bind IHF and its ability to function as an end in transposition. Nonetheless, some transposition occurs in IHF- cells, indicating that IHF is not an essential component of the transposition apparatus. IHF also binds and protects four closely spaced regions within the major hot-spot for insertion of IS1 in the plasmid pBR322. This striking correlation of hot-spot and IHF-binding sites suggests a possible role for IHF in IS1 insertion specificity.     

Sequences essential for IS50 transposition. The first base-pair

Sequences near the ends of the insertion element IS50 are essential for its transposition, probably because they serve as sites upon which the IS50-encoded transposase protein acts. To determine if these essential sequences include the first base-pair at each end of IS50 we generated 5'C to 5'G transversions at these positions. Each mutation reduced the transposition frequency to 1% to 2% of wild-type. DNA sequence analyses showed that the mutant 5'G is preserved during transposition.     

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.     

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.     

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.     

Bias between the left and right inverted repeats during IS911 targeted insertion

IS911 is a bacterial insertion sequence composed of two consecutive overlapping open reading frames (ORFs [orfA and orfB]) encoding the transposase (OrfAB) as well as a regulatory protein (OrfA). These ORFs are bordered by terminal left and right inverted repeats (IRL and IRR, respectively) with several differences in nucleotide sequence. IS911 transposition is asymmetric: each end is cleaved on one strand to generate a free 3′-OH, which is then used as the nucleophile in attacking the opposite insertion sequence (IS) end to generate a free IS circle. This will be inserted into a new target site. We show here that the ends exhibit functional differences which, in vivo, may favor the use of one compared to the other during transposition. Electromobility shift assays showed that a truncated form of the transposase [OrfAB(1-149)] exhibits higher affinity for IRR than for IRL. While there was no detectable difference in IR activities during the early steps of transposition, IRR was more efficient during the final insertion steps. We show here that the differential activities between the two IRs correlate with the different affinities of OrfAB(1-149) for the IRs during assembly of the nucleoprotein complexes leading to transposition. We conclude that the two inverted repeats are not equivalent during IS911 transposition and that this asymmetry may intervene to determine the ordered assembly of the different protein-DNA complexes involved in the reaction.     

Transposon Tn10: genetic organization, regulation, and insertion specificity

Transposon Tn10 is a composite element in which two individual insertion sequence (IS)-like sequences cooperate to mediate transposition of the intervening material. The two flanking IS10 elements are not identical; IS10-right is responsible for functions required to promote transposition, and IS10-left is defective in transposition functions. We suggest that the two IS10 elements were originally identical in sequence and have subsequently diverged. IS10-right is compactly organized with structural gene(s), promoters, and sites important for transposition and (presumably) its regulation all closely linked and, in some cases, overlapping. IS10 has a single major coding region that almost certainly encodes an essential transposition function. A pair of opposing promoters flank the start of this coding region. One of these promoters is responsible for expression in vivo of transposon-encoded transposition functions. We propose that the second promoter is involved in modulation of Tn10 transposition. Genetic analysis suggests that transposon-encoded function(s) may be preferentially cis-acting. Insertion of Tn10 into particular preferred target sites is due primarily to the occurrence of a particular six-base pair target DNA sequence. The properties of this sequence suggest that symmetrically disposed subunits of a single protein may be responsible for both recognition and cleavage of target DNA during insertion.     

Single-stranded DNA intermediates in IS91 rolling-circle transposition

IS91 displays a number of characteristics unique among insertion sequence (IS) elements, suggesting that it transposes by a novel mechanism called rolling-circle (RC) transposition. We reported previously that IS91 transposase (TnpA) amino acid sequence shares a series of five conserved signatures with A proteins of RC replicating phages, including a pair of invariant tyrosines that catalyse two successive transesterification reactions during replication initiation and termination. To analyse their role in IS91 transposition, we constructed a series of TnpA derivatives in which the invariant Tyr-249 and/or Tyr-253 were mutated to either phenylalanine or serine. Mutation of either tyrosine resulted in complete loss of transposition activity in vivo. This result was taken as a first new line of evidence that TnpA is a functional analogue of phiX174 phage A protein. Secondly, RC replication plasmids and phages accumulate single-stranded DNA (ssDNA) intermediates as a result of uncoupled leading and lagging DNA strand synthesis. Using a plasmid carrying an IS91-derived IRLkan-IRR transposable cassette, in which the left (IRL)- and right (IRR)-terminal sequences of IS91 flank a kanamycin resistance gene (kan), we demonstrated the in vivo formation of two new DNA species after induction of transposase expression. The first was a circular ssDNA that contained the transposable cassette covalently joined at its exact termini, whereas the second was a double-stranded circle of the same element. When this experiment was repeated using the mutant transposases described above, the ssDNA and dsDNA intermediates could not be observed, indicating that the integrity of both Y249 and Y253 was essential for their appearance. The presence of ssDNA intermediate products is the first biochemical evidence for a RC mechanism of IS91 transposition.     

Intramolecular transposition of insertion sequence IS91 results in second-site simple insertions

A series of plasmids carrying an IRL-kan-IRR transposable cassette, in which IRL and IRR are the left- and right-terminal sequences of IS91, have been constructed. These cassettes could be complemented for transposition with similar efficiency when IS91 transposase was provided either in cis or in trans. A total of 87% of IS91 transposition products were simple insertions of the element, while the remaining 13% were plasmid fusions and co-integrates. When transposase expression was induced from an upstream lac promoter, transposition frequency increased approximately 100-fold. An open reading frame (ORF) present upstream of the transposase gene, ORF121, could be involved in target selection, as mutations affecting this ORF were altered in their insertion specificity. Intramolecular rearrangements were analysed by looking at transposition events disrupting a chloramphenicol resistance gene (cat ) located outside the transposable cassette. Plasmid instability resulting from insertion of an extra copy of IRL-kan-IRR within the cat gene was observed; transposition products contained a second copy of the cassette inserted either as a direct or as an inverted repeat. No deletion or inversion of the intervening DNA was observed. These results could be explained as a consequence of intramolecular transposition of IS91 according to a model of rolling-circle transposition.     

细菌插入序列的转座酶和转座机制

插入序列（insertion sequence, IS）是细菌中最简单的移动遗传因子,由两端的反向重复序列（inverted repeats, IR）和中间的转座酶 (transposase)编码序列组成。在细菌中,因为插入序列的转座酶催化活性中心氨基酸序列不同,所以将其转座酶分为DDE转座酶、DEDD转座酶、HUH转座酶和丝氨酸转座酶。在转座过程中,根据插入序列是否有复制,将插入序列的转座分为复制型转座（replicative -ansposition）和非复制型转座（non-replicative transposition）,而将形成夏皮罗中间体（Shapiro intermediate）的非复制型转座称为保守型转座（conservative transposition）。此外,插入序列通过不同的转座机制插入到基因编码区导致基因突变、缺失和倒置;或者插入到基因上游,通过自身启动子或与基因形成杂交启动子来影响插入序列下游基因的表达,从而帮助细菌抵抗复杂的环境变化。本文主要围绕细菌插入序列的特征、转座酶、转座机制和转座影响展开综述,以期为进一步研究插入序列的机制和插入序列在细菌中所起的作用提供参考。     

IS186 Insertion at a Hot Spot in the lon Promoter as a Basis for Lon Protease Deficiency of Escherichia coli B: Identification of a Consensus Target Sequence for IS186 Transposition

The radiation sensitivity of Escherichia coli B was first described more than 50 years ago, and the genetic locus responsible for the trait was subsequently identified as lon (encoding Lon protease). We now show that both E. coli B and the first reported E. coli K-12 lon mutant, AB1899, carry IS186 insertions in opposite orientations at a single site in the lon promoter region and that this site represents a natural hot spot for transposition of the insertion sequence (IS) element. Our analysis of deposited sequence data for a number of other IS186 insertion sites permitted the deductions that (i) the consensus target site sequence for IS186 transposition is 5'-(G)(> or =4)(N)(3-6)(C)(> or =4)-3', (ii) the associated host sequence duplication varies within the range of 6 to 12 bp and encompasses the N(3-6) sequence, and (iii) in a majority of instances, at least one end of the duplication is at the G-N (or N-C) junction. IS186-related sequences were absent in closely related bacterium Salmonella enterica serovar Typhimurium, indicating that this IS element is a recent acquisition in the evolutionary history of E. coli.