IS50-mediated inverse transposition: specificity and precision

The IS50 elements, which are present as inverted repeats in the kanamycin-resistance transposon, Tn5, can move in unison carrying with them any interstitial DNA segment. In consequence, DNA molecules such as a lambda::Tn5 phage genome are composed of two overlapping transposons - the kan segment bracketed by IS50 elements (Tn5), and lambda bracketed by IS50 elements. During direct transposition, mediated by IS50 "O" (outside) ends, the kan gene is moved and the lambda vector is left behind. During inverse transposition, mediated by the "I" (inside) ends of the IS50 elements, the lambda vector segment is moved and the kan gene is left behind. Direct transposition is several orders of magnitude more frequent than inverse transposition (Isberg and Syvanen, 1981; Sasakawa and Berg, 1982). We assessed the specificity and precision of the rare events mediated by pairs of I ends by mapping and sequencing independent inverse transpositions from a lambda::Tn5 phage into the amp and tet genes of plasmid pBR322. Using restriction analyses, 32 and 40 distinct sites of insertion were found among 46 and 72 independent inverse transpositions into the amp and tet genes, respectively. Eleven sites were used in two or more insertion events, and the two sites in tet used most frequently corresponded to major hotspots for the insertion of the Tn5 (by direct transposition). The sequences of 22 sites of inverse transposition (including each of the sites used more than once) were determined, in eleven cases by analyzing both pBR322-IS50 junctions, and in eleven others by sequencing one junction. The sequence of the "I" end of IS50 was preserved and 9-bp target sequence duplications were present in every case analyzed. GC pairs were found at each end of the target sequence duplication in ten of the eleven sites used more than once, and also in seven of the other eleven sites. Our data indicate that transposition mediated by pairs of "I" ends is similar in its specificity and precision to the more frequent transposition mediated by IS50 "O" ends.     

Specificity of Tn9 insertion into the genome of bacteriophage lambda att80 depending on its preceding location

It was shown that the site of previous integration (the donor site) of Tn9 affects the specificity of its next integration into the target molecule--phage lambda att80 DNA. The transposon integration sites were mapped by restriction and heteroduplex analysis following Tn9 transposition from chromosomal sites of Escherichia coli K-12 differing in location and Tn9 stability. When transposed from chromosomal galT::IS1 gene, Tn9 inserted into the site with coordinates 44,5 +/- 2 kb of lambda att80; when transposed from chromosomal attTn9A site, the transposon inserted into the sites with coordinates 31 +/- 0,7 kb or 33,3 +/- 0,5 kb. In the course of transposition of Tn9 from chromosomal attTn9N site the transposon inserted into the lambda att80 site with coordinates 26,5 +/- 5 kb. In the latter case, the increase of Tn9 single-stranded loop and the appearance of two new HindIII cleavage sites were observed in heteroduplex experiments. The data were interpreted as indicating structural rearrangements of Tn9 or linked sequences in the course of transposition.     

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.     

A symmetrical six-base-pair target site sequence determines Tn10 insertion specificity

Transposon Tn10 inserts at many sites in the bacterial chromosome, but preferentially inserts at particular hotspots. We believe we have identified the target DNA signal responsible for this specificity. We have determined the DNA sequences of 11 Tn10 insertion sites and identified a particular 6 base pair (bp) symmetrical consensus sequence (GCTNAGC) common to those sites. The sequences at some sites differ from the consensus sequence but only in limited and well defined ways. The sequences at some sites differ from the consensus sequence than do sequences at other sites, and the consensus sequence and closely related sequences are generally absent from potential target regions where Tn10 is known not to insert. Other aspects of the target DNA can significantly influence the efficiency with which a particular target site sequence is used. The 6 bp consensus sequence is symmetrically located within the 9 bp target DNA sequence that is cleaved and duplicated during Tn10 insertion. This juxtaposition of recognition and cleavage sites plus the symmetry of the perfect consensus sequence suggest that the target DNA may be both recognized and cleaved by the symmetrically disposed subunits of a single protein, as suggested for type II restriction endonucleases. There is plausible homology between the consensus sequence and the very ends of Tn10, compatible with recognition of transposon ends and target DNA by the same protein. The sequences of actual insertion sites deviate from the perfect consensus sequence in a way which suggests that the 6 bp specificity determinant may be recognized through protein-DNA contacts along the major groove of the DNA double helix.     

Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes

DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene-operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing the transposition have to be introduced into the cell to be studied (microbial systems and applications are reviewed in ref. 1). However, all in vivo systems have a number of technical limitations. For instance, the transposase must be expressed in the target host, the transposon must be introduced into the host on a suicide vector, and the transposase usually is expressed in subsequent generations, resulting in potential genetic instability. A number of in vitro transposition systems (for Tn5, Tn7, Mu, Himar1, and Ty1) have been described, which bypass many limitations of in vivo systems. For this purpose, we have developed a technique for transposition that involves the formation in vitro of released Tn5 transposition complexes (TransposomesTM) followed by introduction of the complexes into the target cell of choice by electroporation. In this report, we show that this simple, robust technology can generate high-efficiency transposition in all tested bacterial species (Escherichia coli, Salmonella typhimurium, and Proteus vulgaris) We also isolated transposition events in the yeast Saccharomyces cerevisiae.     

Formation, characterization and partial purification of a Tn5 strand transfer complex

DNA transposition reactions typically involve a strand transfer step wherein the transposon ends are covalently joined by the transposase protein to a short target site. There is very little known about the transposase-DNA interactions that direct this process, and thus our overall understanding of the dynamics of DNA transposition reactions is limited. Tn5 presents an attractive system for defining such interactions because it has been possible to solve the structure of at least one Tn5 transposition intermediate: a transpososome formed with pre-cleaved ends. However, insertion specificity in the Tn5 system is low and this has hampered progress in generating target-containing transpososomes that are homogeneous in structure (i.e. where a single target site is engaged) and therefore suitable for biochemical and structural analysis. We have developed a system where the Tn5 transpososome integrates almost exclusively into a single target site within a short DNA fragment. The key to establishing this high degree of insertion specificity was to use a target DNA with tandem repeats of a previously characterized Tn5 insertion hotspot. The target DNA requirements to form this strand transfer complex are evaluated. In addition, we show that target DNAs missing single phosphate groups at specific positions are better substrates for strand transfer complex formation relative to the corresponding unmodified DNA fragments. Moreover, utilization of missing phosphate substrates can increase the degree of target site selection. A method for concentrating and partially purifying the Tn5 strand transfer complex is described.     

Factors responsible for target site selection in Tn10 transposition: a role for the DDE motif in target DNA capture.

Tn10, like several other transposons, exhibits a marked preference for integration into particular target sequences. Such sequences are referred to as integration hotspots and have been used to define a consensus target site in Tn10 transposition. We demonstrate that a Tn10 hotspot called HisG1, which was identified originally in vivo, also functions as an integration hotspot in vitro in a reaction where the HisG1 sequence is present on a short DNA oligomer. We use this in vitro system to define factors which are important for the capture of the HisG1 target site. We demonstrate that although divalent metal ions are not essential for HisG1 target capture, they greatly facilitate capture of a mutated HisG1 site. Analysis of catalytic transposase mutants further demonstrates that the DDE motif plays a critical role in ''divalent metal ion-dependent'' target capture. Analysis of two other classes of transposase mutants, Exc+ Int- (which carry out transposon excision but not integration) and ATS (altered target specificity), demonstrates that while a particular ATS transposase binds HisG1 mutants better than wild-type transposase, Exc+ Int- mutants are defective in HisG1 capture, further defining the properties of these classes of mutants. Possible mechanisms for the above observations are considered.     

A system of transposon mutagenesis for bacteriophage T4

We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24-deleted phage. The transposition occurred at a frequency of only 10(-7) per progeny phage, even though a dam- host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon-specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.     

Analysis of the zinc finger domain of TnpA, a DNA targeting protein encoded by mobilizable transposon Tn4555

The mobilizable transposon Tn4555, found in Bacteroides spp., is an important antibiotic resistance element encoding a broad spectrum beta-lactamase. Tn4555 is mobilized by conjugative transposons such as CTn341 which can transfer the transposon to a wide range of bacterial species where it integrates into preferred sites on the host chromosome. Selection of the preferred target sites is mediated by a DNA-binding protein TnpA which has a prominent zinc finger motif at the N-terminus of the protein. In this report the zinc finger motif was disrupted by site directed mutagenesis in which two cysteine residues were changed to serine residues. Elemental analysis indicated that the wild-type protein but not the mutated protein was able to coordinate zinc at a molar ration of 1/1. DNA binding electrophoretic mobility shift assays showed that the ability to bind the target site DNA was not significantly affected by the mutation but there was about a 50% decrease in the ability to bind single stranded DNA. Consistent with these results, electrophoretic mobility shift assays incorporating zinc chelators did not have a significant on affect the binding of DNA target. In vivo, the zinc finger mutation completely prevented transposition/integration as measured in a conjugation assay. This was in contrast to results in which a TnpA knockout was still able to insert into host genomes but there was no preferred target site selection. The phenotype of the zinc finger mutation was not effectively rescued by providing wild-type TnpA in trans. Taken together these results indicated that the zinc finger is not required for DNA binding activity of TnpA but that it does have an important role in transposition and it may mediate protein/protein interactions with integrase or other Tn4555 proteins to facilitate insertion into the preferred sites.     

Tn5 mutagenesis of the transforming genes of human adenovirus type 5

We have cloned the entire human adenovirus type 5 (Ad5) genome into the pBR322 plasmid in two segments: the BamHI-A fragment (21 kb) and the BamHI-B fragment (15 kb). We have also generated a series of clones with smaller Ad5 DNA inserts, all containing the left-end of the viral genome. One such clone, pXCl, containing the left 16% of the Ad5 DNA molecule, has been shown to transform rodent cells by DNA transfection. We have used the transposable element Tn5 as an insertion mutator to isolate pXCl mutants containing Tn 5 inserted at a large number of sites. By assaying transforming activity of selected pXC::Tn5 plasmids we have identified Ad5 sequences which are essential for DNA-mediated transformation. Our results with these mutants and with a plasmid pCDl, containing a deletion within the Ad5-transforming region, indicate that sequences present in both early region la and the N-terminal region of early region 1b are essential for DNA-mediated transformation.     

Tn7 transposition as a probe of cis interactions between widely separated (190 kilobases apart) DNA sites in the Escherichia coli chromosome.

We have used the bacterial transposon Tn7 to examine communication between widely separated DNA sites in the Escherichia coli chromosome. Using Tn7 target immunity, a regulatory feature of transposition which influences target selection, we have evaluated (i) how the presence of Tn7 sequences at one DNA site affects Tn7 insertion into another site in the same DNA molecule and (ii) the nucleotide distances over which the two sites are able to communicate. We demonstrate that Tn7 sequences at one chromosomal site act at a distance to inhibit insertion of Tn7 elsewhere in that DNA as far away as 190 kb, reflecting effective long-range cis interactions. We have found that while target immunity is effective over a substantial region of the chromosome, insertion of Tn7 into a more distant site 1.9 Mb away in the same DNA is not inhibited; this observation provides evidence that target immunity relies on DNA spacing. We also find that within the region of the chromosome affected by target immunity, the magnitude of the immune effect is greater at close DNA sites than DNA sites farther away, suggesting that target immunity is distance dependent. We also extend the characterization of the Tn7 end-sequences involved in transposition and target immunity and describe how Tn7 target immunity can be used as a tool for probing bacterial chromosome structure.     

The Bacteroides mobilizable transposon Tn4555 integrates by a site-specific recombination mechanism similar to that of the gram-positive bacterial element Tn916.

The Bacteroides mobilizable transposon Tn4555 is a 12.2-kb molecule that encodes resistance to cefoxitin. Conjugal transposition is hypothesized to occur via a circular intermediate and is stimulated by coresident tetracycline resistance elements and low levels of tetracycline. In this work, the ends of the transposon were identified and found to consist of 12-bp imperfect inverted repeats, with an extra base at one end. In the circular form, the ends were separated by a 6-bp "coupling sequence" which was associated with either the left or the right transposon terminus when the transposon was inserted into the chromosome. Tn4555 does not duplicate its target site upon insertion. Using a conjugation-based transposition assay, we showed that the coupling sequence originated from 6 bases of genomic DNA flanking either side of the transposon prior to excision. Tn4555 preferentially transposed into a 589-bp genomic locus containing a 207-bp direct repeat. Integration occurred before or after the repeated sequence, with one integration site between the two repeats. These observations are consistent with a transposition model based on site-specific recombination. In the bacteriophage lambda model for site-specific recombination, the bacteriophage recombines with the Escherichia coli chromosome via a 7-bp "crossover" region. We propose that the coupling sequence of Tn4555 is analogous in function to the crossover region of lambda but that unlike the situation in lambda, recombination occurs between regions of nonhomologous DNA. This ability to recombine into divergent target sites is also a feature of the gram-positive bacterial transposon Tn916.     

A comparison of intramolecular rearrangements promoted by transposons Tn5 and Tn10.

The bacterial transposon Tn10 has previously been shown to move to other genomic sites by a conservative mechanism, whereby the transposon is excised by double-strand breaks and inserted between a pair of staggered nicks at the target. Other transposons, like Tn3, have been shown to transpose by a replicative mechanism that involves symmetrical nicking of the element and formation of the 'Shapiro intermediate', which can mature into either a cointegrate or a simple insert. The situation with respect to Tn5 is unclear; it was originally reported to use a conservative mechanism, but other evidence suggests that the mechanism might be replicative. In this paper, rearrangements of adjacent DNA promoted by Tn10 and Tn5 have been compared using positive selection for galactose-resistance to detect such rearrangements. Tn10 promoted the formation of adjacent deletions (that started from an inside end of Tn10), deletion/inversions and simple IS10 insertions, but no cointegrates. This behaviour is fully consistent with a conservative mechanism. In contrast, Tn5 was found to promote formation of adjacent deletions (that started mainly from an outside end of Tn5), IS50 insertions (that were frequently accompanied by inversions of adjacent DNA) and cointegrates. These characteristics seem compatible with a replicative, rather than a conservative, mode of transposition. Clearly, Tn5 and Tn10 exhibit some significant differences in their transposition. These results, and results of some previous experiments, have been interpreted to mean that Tn5 could use a replicative mechanism for its transposition.     

Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones.

The 264 bp mini-transposon Tn5supF was constructed to sequence DNAs cloned in phage lambda without extensive shotgun subcloning or primer walking. Unique sequences near each transposon end serve as primer binding sites, and a supF gene is used to select transposition to lambda. We describe here PCR methods that facilitate Tn5supF-based sequencing. In a first pass, insertions are mapped relative to the ends of the cloned fragment using pairs of primers specific for vector DNA next to the cloning site and for a Tn5supF end. Most insertions not mapped in this step are near the center of the cloned fragment or in the vector arms, and are then mapped relative to the two innermost insertions by 'crossover' PCR. This involves amplification from primers on different DNA molecules, and generates hybrid DNA products whose lengths correspond to the distances between the two insertions. We routinely amplified more than 6 kb in direct PCR and 3 kb in crossover PCR; at the limit we amplified up to approximately 10 kb in direct PCR and approximately 6 kb in crossover PCR, but not reproducibly. Crossover PCR products were also obtained with insertions separated by only 200 bp, indicating that no rare sites are needed to switch templates. PCR products were purified by adsorption and then elution from glass slurry, and sequenced directly. Ladders of more than 400 bp were obtained from primer sites on each DNA strand; 2 kb was read from crossover PCR products, and showed that they were amplified with fidelity. In conclusion, direct and crossover PCR methods expedite transposon insertion mapping, and yield templates for accurate sequencing of both DNA strands.     

In vitro transposition of Tn552: a tool for DNA sequencing and mutagenesis.

We have explored the potential of the Tn 552 in vitro transposition reaction as a genetic tool. The reaction is simple (requiring a single protein component), robust and efficient, readily producing insertions into several percent of target DNA. Most importantly, Tn 552 insertions in vitro appear to be essentially random. Extensive analyses indicate that the transposon exhibits no significant regional or sequence specificity for target DNA and leaves no discernible 'cold' spots devoid of insertions. The utility of the in vitro reaction for DNA sequencing was demonstrated with a cosmid containing the Mycobacterium smegmatis recBCD gene cluster. The nucleotide sequence of the entire operon was determined using 71 independent Tn 552 insertions, which generated over 13.5 kb of unique sequence and simultaneously provided a comprehensive collection of insertion mutants. The relatively short ends of Tn 552 make construction of novel transposons a simple process and we describe several useful derivatives. The data presented suggest that Tn 552 transposition is a valuable addition to the arsenal of tools available for molecular biology and genomics.     

Transposition of Tn916 in the four replicons of the Butyrivibrio proteoclasticus B316(T) genome

The rumen bacterium Butyrivibrio proteoclasticus B316(T) has a 4.4-Mb genome composed of four replicons (approximately 3.55 Mb, 361, 302 and 186 kb). Mutagenesis of B316(T) was performed with the broad host-range conjugative transposon Tn916 to screen for functionally important characteristics. The insertion sites of 123 mutants containing a single copy of Tn916 were identified and corresponded to 53 different insertion points, of which 18 (34.0%), representing 39 mutants (31.7%), were in ORFs and 12 were where transposition occurred in both directions (top and bottom DNA strand). Up to eight mutants from several independent conjugation experiments were found to have the same integration site. Although transposition occurred in all four replicons, the number of specific insertion sites, transposition frequency and the average intertransposon distance between insertions varied between the four replicons. In silico analysis of the 53 insertion sites was used to model a target consensus sequence for Tn916 integration into B316(T) . A search of the B316(T) genome using the modelled target consensus sequence (up to two mismatches) identified 39 theoretical Tn916 insertion sites (19 coding, 20 noncoding), of which nine corresponded to Tn916 insertions identified in B316(T) mutants during our conjugation experiments.