Importance of illegitimate recombination and transposition in IS30-associated excision events.

In the present study we report on the excision of IS30 elements and IS30-derived composite transposons. Frequent loss of IS30 was observed during dissolution of dimeric IS30 structures, containing IR-IR junctions, leading to resealed donor molecules. In contrast, unambiguous transpositional excision resulting in resealed remainder products could not be identified in the case of a monomeric element. The bias in the excision of monomeric and dimeric IS30 structures indicates a difference in the molecular mechanism of transposition of IS30 monomers and dimers. Sequence data on the rarely detected plasmids missing full IS or Tn copies rather suggest that all products were derived from illegitimate recombination. The reaction occurred between short homologies and was independent of the transposase activity. Similar IS30 excision events accompanied by multiple plasmid or genome rearrangements were detected in Pseudomonas putida and Rhizobium meliloti, yielding stable replicons that retained the selective marker gene of the transposon. We provide evidence that both transposition and illegitimate recombination can contribute to the stabilization of replicons through the elimination of IS elements, which emphasizes the evolutionary significance of these events.     

Insertion sequence-caused large-scale rearrangements in the genome of Escherichia coli

A majority of large-scale bacterial genome rearrangements involve mobile genetic elements such as insertion sequence (IS) elements. Here we report novel insertions and excisions of IS elements and recombination between homologous IS elements identified in a large collection of Escherichia coli mutation accumulation lines by analysis of whole genome shotgun sequencing data. Based on 857 identified events (758 IS insertions, 98 recombinations and 1 excision), we estimate that the rate of IS insertion is 3.5 × 10⁻⁴ insertions per genome per generation and the rate of IS homologous recombination is 4.5 × 10⁻⁵ recombinations per genome per generation. These events are mostly contributed by the IS elements IS1, IS2, IS5 and IS186. Spatial analysis of new insertions suggest that transposition is biased to proximal insertions, and the length spectrum of IS-caused deletions is largely explained by local hopping. For any of the ISs studied there is no region of the circular genome that is favored or disfavored for new insertions but there are notable hotspots for deletions. Some elements have preferences for non-coding sequence or for the beginning and end of coding regions, largely explained by target site motifs. Interestingly, transposition and deletion rates remain constant across the wild-type and 12 mutant E. coli lines, each deficient in a distinct DNA repair pathway. Finally, we characterized the target sites of four IS families, confirming previous results and characterizing a highly specific pattern at IS186 target-sites, 5′-GGGG(N6/N7)CCCC-3′. We also detected 48 long deletions not involving IS elements.     

Formation of the tandem repeat (IS30)2 and its role in IS30-mediated transpositional DNA rearrangements

Plasmids carrying two IS30 elements in the same orientation, as in the composite transposon Tn2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS30 elements. The resulting covalently closed replicon carries the two IS30 elements as tandem repeats separated by only 2 bp. This (IS30)₂ structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation. Among the descendants of transformants of recA ^– bacteria, replicated copies of the introduced (IS30)₂ structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identical ends of the IS30 elements involved, which results in the presence of just one intact IS30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS30 transposition, various transposition products of (IS30)₂ are also seen. Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS30 ends. In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration. A mutational study revealed the requirement in cis of one intact IS30 transposase gene and of both proximal ends of the two IS30 elements concerned not only for the formation of (IS30)₂, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions. A model is proposed, which postulates that (IS30)₂ intermediates play a key role in IS30 transposition pathways in which the formation of (IS30)₂ may be rate-limiting. Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS30)₂. Evolutionary implications of these findings are discussed.     

ISDra2 transposition in Deinococcus radiodurans is downregulated by TnpB

Transposable elements belonging to the recently identified IS200/IS605 family radically differ from classical insertion sequences in their transposition mechanism by strictly requiring single‐stranded DNA substrates. This IS family includes elements encoding only the transposase (TnpA), and others, like ISDra2 from Deinococcus radiodurans, which contain a second gene, tnpB, dispensable for transposition and of unknown function to date. Here, we show that TnpB has an inhibitory effect on the excision and insertion steps of ISDra2 transposition. This inhibitory action of TnpB was maintained when ISDra2 transposition was induced by γ‐irradiation of the host cells and required the integrity of its putative zinc finger motif. We also demonstrate the negative role of TnpB when ISDra2 transposition was monitored in a heterologous Escherichia coli host, indicating that TnpB‐mediated inhibition does not involve Deinococcus‐specific factors. TnpB therefore appears to play a regulatory role in ISDra2 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.     

Analysis of Tn5 inversion events inEscherichia coli plasmids

The ability of the bacterial transposon Tn5 to undergo sequence inversion in Rec⁺ Escherichia coli cells as a result of recombination between its duplicated IS50 elements was examined using specially designed plasmid constructs. Surprisingly, recombination events in the IS50 elements that led to crossover and therefore Tn5 inversion could be detected at a frequency of only 10^–5. This was approximately an order of magnitude lower than the frequency of IS50 recombination that led to conversion events (i.e. non-reciprocal recombination) without crossover, and at least two orders of magnitude lower than the frequency of intermolecular recombination between IS50 elements on two different plasmids. These rare conversion and inversion events in Tn5 appeared to be due to intramolecular recombination and not simply to multiple rounds of reciprocal crossing over, since the heterodimeric intermediates that would be generated during the latter process could be readily isolated but were shown to yield a completely different set of plasmid products upon resolution.     

High-Temperature-Induced Transposition of Insertion Elements in Burkholderia multivorans ATCC 17616

An efficient and quantitative method to analyze the transposition of various insertion sequence (IS) elements in Burkholderia multivorans ATCC 17616 was devised. pGEN500, a plasmid carrying a Bacillus subtilis-derived sacB gene, was introduced into ATCC 17616 cells, and 25% of their sucrose-resistant derivatives were found to carry various IS elements on pGEN500. A PCR-based experimental protocol, in which a mixture of several specific primer pairs was used, revealed that pGEN500 captured, in addition to five previously reported IS elements (IS401, IS402, IS406, IS407, and IS408), three novel IS elements, ISBmu1, ISBmu2, and ISBmu3. The global transposition frequency of these IS elements was enhanced more than sevenfold under a high-temperature condition (42°C) but not under oxidative stress or starvation conditions. To our knowledge, this is the first report demonstrating the elevated transposition activities of several IS elements at a high temperature. The efficient experimental protocol developed in this study will be useful in quantitatively and simultaneously investigating various IS elements, as well as in capturing novel functional mobile elements from a wide variety of bacteria.     

Genetic organization and transposition properties of IS 511

IS511 is an endogenous insertion sequence (IS) of the bacterium Caulobactercrescentus strain CB15 and it is the first Caulobacter IS to be characterized at the molecular level. We determined the 1266-bp nucleotide sequence of IS511 and investigated its genetic organization, relationship to other ISs, and transposition properties. IS511 belongs to a distinct branch of the IS3 family that includes ISRI, IS476, and IS1222, based on nucleotide sequence similarity. The nucleotide sequence of IS511 encodes open reading frames (orfs) designated here as orfA and orfB, and their relative organization and amino acid sequences of the predicted protein products are very similar to those of orfAs and orfBs of other IS3 family members. Nuclease S1 protection assays identified an IS511 RNA, and its 5′ end maps approximately 16 nucleotides upstream of orfA and about six nucleotides downstream of a sequence that is similar to the consensus sequence of C. crescentus housekeeping promoters. Evidence is presented that IS511 is capable of precise excision from the chromosome, and transposition from the chromosome to a plasmid. Transpositional insertions of IS511 occurred within sequences with a relatively high G + C content, and they were usually, but not always, flanked by a 4-bp direct repeat that matches a sequence at the site of insertion. We also determined the nucleotide sequence flanking the four endogenous IS511 elements that reside in the chromosome of C. crescentus. Our findings demonstrate that IS511 is a transposable IS that belongs to a branch of the IS3 family. Received: 18 August 1996 / Accepted: 17 September 1996     

Sub-terminal sequences modulating IS30 transposition in vivo and in vitro

Inverted repeats of insertion sequences (ISs) are indispensable for transposition. We demonstrate that sub-terminal sequences adjacent to the inverted repeats of IS30 are also required for optimal transposition activity. We have developed a cell-free recombination system and showed that the transposase catalyses formation of a figure-of-eight transposition intermediate, where a 2 bp long single strand bridge holds the inverted repeat sequences (IRs) together. This is the first demonstration of the figure-of-eight structure in a non-IS3 family element, suggesting that this mechanism is likely more widely adopted among IS families. We show that the absence of sub-terminal IS30 sequences negatively influences figure-of-eight production both in vivo and in vitro. These regions enhance IR-IR junction formation and IR-targeting events in vivo. Enhancer elements have been identified within 51 bp internal to IRL and 17 bp internal to IRR. In the right end, a decanucleotide, 5′-GAGATAATTG-3′, is responsible for wild-type activity, while in the left end, a complex assembly of repetitive elements is required. Functioning of the 10 bp element in the right end is position-dependent and the repetitive elements in the left end act cooperatively and may influence bendability of the end. In vitro kinetic experiments suggest that the sub-terminal enhancers may, at least partly, be transposase-dependent. Such enhancers may reflect a subtle regulatory mechanism for IS30 transposition.     

The IS4 family of insertion sequences: evidence for a conserved transposase motif

The eight IS 231 variants characterized so far (IS 231 A-F, V and W) display similar transposases with an overall 40% identity. Comparison with all the proka-ryotic transposable elements sequenced so far revealed that the IS231 transposases share two conserved regions with those of 35 other insertion sequences of wide origins. These insertion sequences, defining the IS4 family, have a common bipartite organization of their ends and are divided into two similarity groups. Interestingly, the transposase domains conserved within this family display similarities with the well known integrase domain shared by transposases of the IS3 and IS15 families, and integrases of retroelements. This domain is also found in IS30- related elements and Tn7 TnsB protein. Amino acid residues conserved throughout all these prokaryotic and eukaryotic mobile genetic elements define a major transposase/integrase motif, likely to play an important role in the transposition process.     

Isolation and Characterization of IS1416fromPseudomonas glumae,a New Member of the IS3Family

Isolation and characterization of four different insertion sequence (IS) elements fromPseudomonas glumaeMAFF 302744 through transposition into the entrapment vector pSHI1063 are described. One of the elements, IS1416,was further characterized. IS1416is 1322 bp long and carries 29-bp terminal inverted repeats flanked by a 3-bp direct duplication. IS1416contains three open reading frames (ORFs), which are designated ORFA1, ORFA2, and ORFB, on one strand. Both DNA sequence of IS1416and the deduced amino acid sequences of its ORFs strongly suggest that IS1416is a member of the IS3family, and is closely related to IS401fromPseudomonas cepaciaand IS51fromPseudomonas syringae.To our knowledge, IS1416is the first IS element isolated fromP. glumae.The gene organization and possible regulation of transposition functions of IS1416are also discussed.     

Formation of the tandem repeat (IS30)2 and its role in IS30-mediated transpositional DNA rearrangements

Plasmids carrying two IS30 elements in the same orientation, as in the composite transposon Tn2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS30 elements. The resulting covalently closed replicon carries the two IS30 elements as tandem repeats separated by only 2 bp. This (IS30)₂ structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation. Among the descendants of transformants of recA ^? bacteria, replicated copies of the introduced (IS30)₂ structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identical ends of the IS30 elements involved, which results in the presence of just one intact IS30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS30 transposition, various transposition products of (IS30)₂ are also seen. Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS30 ends. In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration. A mutational study revealed the requirement in cis of one intact IS30 transposase gene and of both proximal ends of the two IS30 elements concerned not only for the formation of (IS30)₂, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions. A model is proposed, which postulates that (IS30)₂ intermediates play a key role in IS30 transposition pathways in which the formation of (IS30)₂ may be rate-limiting. Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS30)₂. Evolutionary implications of these findings are discussed.     

Rates of transposition in Escherichia coli

The evolutionary role of transposable elements (TEs) is still highly controversial. Two key parameters, the transposition rate (u and w, for replicative and non-replicative transposition) and the excision rate (e) are fundamental to understanding their evolution and maintenance in populations. We have estimated u, w and e for six families of TEs (including eight members: IS1, IS2, IS3, IS4, IS5, IS30, IS150 and IS186) in Escherichia coli, using a mutation accumulation (MA) experiment. In this experiment, mutations accumulate essentially at the rate at which they appear, during a period of 80 500 (1610 generations × 50 lines) generations, and spontaneous transposition events can be detected. This differs from other experiments in which insertions accumulated under strong selective pressure or over a limited genomic target. We therefore provide new estimates for the spontaneous rates of transposition and excision in E. coli. We observed 25 transposition and three excision events in 50 MA lines, leading to overall rate estimates of u ∼ 1.15 × 10^–5, w ∼ 4 × 10⁻⁸ and e ∼ 1.08 × 10⁻⁶ (per element, per generation). Furthermore, extensive variation between elements was found, consistent with previous knowledge of the mechanisms and regulation of transposition for the different elements.     

Transposition of IS 117, the 2.5 kb Streptomyces coelicolor A3(2) 'minicircle': roles of open reading frames and origin of tandem insertions

IS 117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of 0RF1 of IS 117, presumed to encode a transposase, abolished transposition. Deletion or mutation of 0RF2 and 0RF3, which overlap each other on opposite strands of IS 117, caused a c. 20-fold reduction in integration frequency of the circular form of IS 117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS 117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. 0RF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild-type IS 117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS 117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS 117 derivatives yielded tandem chromosomal insertions, which arose by co-transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration.     

Identification and characterization of IS 1138, a transposable element from Mycoplasma pulmonis that belongs to the IS3 family

Insertion sequence (IS) elements are mobile genetic elements found in prokaryotes. We have identified a repetitive element from Mycoplasma pulmonis, a murine pathogen, that is similar to eubacterial IS elements. By subcloning a single strain of M. pulmonis, we isolated a variant clone in which the IS element had undergone an apparent transposition event. The nucleotide sequences of the element, designated IS 1138, and the target site into which it inserted were determined. IS1138 consists of 1288bp with 18bp perfect terminal inverted repeats. Sequence analysis of the target site before and after insertion of IS1138 identified a 3bp duplication of target DNA flanking the element. The predicted amino acids encoded by the major open reading frame of IS 1138 share significant similarity with the transposases of the IS3 family. Southern hybridization analysis indicates that repetitive sequences similar to IS 1138 are present in most, if not all, strains of M. pulmonis, but Is1138–like sequences were not detected in other mycoplasmal species.     

Evidence that Tn5565, which includes the enterotoxin gene in Clostridium perfringens, can have a circular form which may be a transposition intermediate

The Clostridium perfringens enterotoxin gene is on a transposon-like element, Tn5565, integrated in the chromosome in human food poisoning strains. The flanking IS elements, IS1470 A and B, are related to IS30. The IS element found in the transposon, IS1469, is related to IS200 and has been found upstream of cpe in all Type A strains. PCR and sequencing studies from cell extracts and plasmid isolations of C. perfringens indicate that Tn5565 can form a circular form with the tandem repeat (IS1470)₂, similar to the transposition intermediates described for a number of IS elements.     

Formation and transposition of the covalently closed IS30 circle: the relation between tandem dimers and monomeric circles

In the present study, we demonstrate that a circular IS30 element acts as an intermediate for simple insertion. Covalently closed IS and Tn circles constructed in vitro are suitable for integration into the host genome. Minicircle integration displays all the characteristics of transpositional fusion mediated by the (IS30 )2 dimer regarding target selection and target duplication. Evidence is provided for in vivo circularization of the element located either on plasmids or on the genome. It is shown that circle formation can occur through alternative pathways. One of them is excision of IS30 from a hot spot via joining the IRs. This reaction resembles the site-specific dimerization that leads to (IS30 )2 establishment. The other process is the dissolution of (IS30 )2 dimer, when the element is excised from an IR-IR joint. These pathways differ basically in the fate of the donor replicon: only dimer dissolution gives rise to resealed donor backbone. Analysis of minicircles and the rearranged donor replicons led us to propose a molecular model that can account for differences between the circle-generating processes. Our focus was to the dissolution of IR-IR joints located on the host genome, because these events promoted extensive genomic rearrangements and accompanied minicircle formation. The results present the possibility of host genome reorganization by IS30-like transposition.     

Absence of insertions among spontaneous mutants of Salmonella typhimurium

Summary While insertion sequences (IS) in Escherichia coli transpose frequently to generate spontaneous insertion mutants, such mutations are rare in Salmonella typhimurium: the only documented insertion mutation is a hisD mutation caused by the Salmonella-specific IS element IS200. To obtain more examples of IS200 insertion mutations and to seek additional types of IS elements in Salmonella, we selected and characterized 422 independent, spontaneous His^– mutants and some 2100 additional mutants that are not necessarily independent. None of the mutants showed the absolute polar effect characteristic of insertion mutations or the reversion properties characteristic of insertions (low spontaneous reversion frequency and no reversion induction by chemical mutagens). A few mutants, showing a high spontaneous reversion frequency, were screened physically. No insertion mutations were found. Thus insertion mutations appear to be rare in S. typhimurium, in strong contrast to E. coli and despite the possession in Salmonella of at least one type of insertion element (IS200). These results suggest that in Salmonella transposition of the endogenous elements has been controlled. The transposition ability of the elements may have been reduced or favored target sites removed from the host genome.     

Insertion Sequence-Driven Evolution of <Emphasis Type="Italic">Escherichia coli</Emphasis> in Chemostats

Insertion sequence (IS) elements are present in almost all bacterial genomes and are mobile enough to provide genomic tools to differentiate closely related isolates. They can be used to estimate genetic diversity and identify fitness-enhancing mutations during evolution experiments. Here, we determined the genomic distribution of eight IS elements in 120 genomes sampled from Escherichia coli populations that evolved in glucose- and phosphate-limited chemostats by comparison to the ancestral pattern. No significant differential transposition of the various IS types was detected across the environments. The phylogenies revealed substantial diversity amongst clones sampled from each chemostat, consistent with the phenotypic diversity within populations. Two IS-related changes were common to independent chemostats, suggesting parallel evolution. One of them corresponded to insertions of IS1 elements within rpoS encoding the master regulator of stress conditions. The other parallel event was an IS5-dependent deletion including mutY involved in DNA repair, thereby providing the molecular mechanism of generation of mutator clones in these evolving populations. These deletions occurred in different co-existing genotypes within single populations and were of various sizes. Moreover, differential locations of IS elements combined with their transpositional activity provided evolved clones with different phenotypic landscapes. Therefore, IS elements strongly influenced the evolutionary processes in continuous E. coli cultures by providing a way to modify both the global regulatory network and the mutation rates of evolving cells.     

Analysis of Tn5 inversion events inEscherichia coli plasmids

The ability of the bacterial transposon Tn5 to undergo sequence inversion in Rec⁺ Escherichia coli cells as a result of recombination between its duplicated IS50 elements was examined using specially designed plasmid constructs. Surprisingly, recombination events in the IS50 elements that led to crossover and therefore Tn5 inversion could be detected at a frequency of only 10^?5. This was approximately an order of magnitude lower than the frequency of IS50 recombination that led to conversion events (i.e. non-reciprocal recombination) without crossover, and at least two orders of magnitude lower than the frequency of intermolecular recombination between IS50 elements on two different plasmids. These rare conversion and inversion events in Tn5 appeared to be due to intramolecular recombination and not simply to multiple rounds of reciprocal crossing over, since the heterodimeric intermediates that would be generated during the latter process could be readily isolated but were shown to yield a completely different set of plasmid products upon resolution.