Sequence requirements of Escherichia coli attTn7, a specific site of transposon Tn7 insertion.

Transposon Tn7 transposes at high frequency to a specific site, attTn7, in the Escherichia coli chromosome. We devised a quantitative assay for Tn7 transposition in which Tn7-end derivatives containing the cis-acting transposition sequences of Tn7 transpose from a bacteriophage lambda vector upon infection into cells containing the Tn7-encoded transposition proteins. We used this assay to identify a 68-base-pair DNA segment containing the sequences essential for attTn7 target activity. This segment is positioned asymmetrically with respect to the specific point of Tn7 insertion in attTn7 and lacks obvious homology to the sequences at the ends of Tn7 which participate directly in transposition. We also show that some sequences essential for attTn7 target activity are contained within the protein-coding sequence of a bacterial gene.     

Transposon Tn7. cis-Acting sequences in transposition and transposition immunity

We have identified and characterized the cis-acting sequences at the termini of the bacterial transposon Tn7 that are necessary for its transposition. Tn7 participates in two kinds of transposition event: high-frequency transposition to a specific target site (attTn7) and low-frequency transposition to apparently random target sites. Our analyses suggest that the same sequences at the Tn7 ends are required for both transposition events. These sequences differ in length and nucleotide structure: about 150 base-pairs at the left end (Tn7L) and about 70 base-pairs at the right end (Tn7R) are necessary for efficient transposition. We also show that the ends of Tn7 are functionally distinct: a miniTn7 element containing two Tn7R ends is active in transposition but an element containing two Tn7L ends is not. We also report that the presence of Tn7's cis-acting transposition sequences anywhere in a target replicon inhibits subsequent insertion of another copy of Tn7 into either an attTn7 target site or into random target sites. The inhibition to an attTn7 target site is most pronounced when the Tn7 ends are immediately adjacent to attTn7. We also show that the presence of Tn7R's cis-acting transposition sequences in a target replicon is necessary and sufficient to inhibit subsequent Tn7 insertion into the target replicon.     

Target DNA structure plays a critical role in Tn7 transposition

The bacterial transposon Tn7 utilizes four Tn7-encoded proteins, TnsA, TnsB, TnsC and TnsD, to make insertions at a specific site termed attTn7. This target is selected by the binding of TnsD to attTn7 in a sequence-specific manner, followed by the binding of TnsC and activation of the transposase. We show that TnsD binding to attTn7 induces a distortion at the 5' end of the binding site and TnsC contacts the region of attTn7 distorted by TnsD. Previous work has shown that a target site containing triplex DNA, instead of TnsD-attTn7, can recruit TnsABC and effect site- specific insertion of Tn7. We propose that the DNA distortion imposed by TnsD on attTn7, like the altered DNA structure via triplex formation, serves as a signal to recruit TnsC. We also show that TnsD primarily contacts the major groove of DNA, whereas TnsC is a minor groove binding protein. The footprint of the TnsC-TnsD-attTn7 nucleoprotein complex includes and extends beyond the Tn7 insertion site, where TnsC forms a platform to receive and activate the transposase to carry out recombination.     

Genetic analysis of attTn7, the transposon Tn7 attachment site in Escherichia coli, using a novel M13-based transduction assay

The large (14 kb; kb = 10(3) bases) bacterial transposon, Tn7 (encoding resistance to trimethoprim and streptomycin/spectinomycin), has unusual properties. Like other elements, Tn7 transposes with low efficiency and low target-site specificity, but Tn7 also transposes, with high frequency in a unique orientation, to a preferred "attachment" site, called attTn7, in the Escherichia coli chromosome and similarly into plasmids containing attTn7. We developed a novel bacteriophage M13-based assay system to measure the transposition frequency of Tn7 to M13mp phage vectors containing attTn7 on a cloned 1 kb fragment of chromosomal DNA. Phage harvested from a Tn7 donor strain were used to infect recipient bacteria with selection for trimethoprim resistance. Transposition frequency, expressed as the number of trimethoprim-resistant colonies per plaque-forming unit, was found to be approximately 10(-4) to M13mp::attTn7, in contrast to 10(-10) to M13mp recombinants with approximately 1 kb insertions of other, "generic brand", DNA. By deletion analysis of M13mp::attTn7, we show that attTn7 is contained within a 64 base-pair region; sequences adjacent to the actual insertion site and encoding the carboxy terminus of the glmS gene are required. This assay also provided evidence for transposition immunity conferred by the right end of Tn7.     

A Tn7-based broad-range bacterial cloning and expression system

For many bacteria, cloning and expression systems are either scarce or nonexistent. We constructed several mini-Tn7 vectors and evaluated their potential as broad-range cloning and expression systems. In bacteria with a single chromosome, including Pseudomonas aeruginosa, Pseudomonas putida and Yersinia pestis, and in the presence of a helper plasmid encoding the site-specific transposition pathway, site- and orientation-specific Tn7 insertions occurred at a single attTn7 site downstream of the glmS gene. Burkholderia thailandensis contains two chromosomes, each containing a glmS gene and an attTn7 site. The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginosa, establishing expression systems in P. aeruginosa and P. putida, and 'GFP-tagging' Y. pestis. This system will thus have widespread biomedical and environmental applications, especially in environments where plasmids and antibiotic selection are not feasible, namely in plant and animal models or biofilms.     

Transposon Tn7 protein TnsD binding to Escherichia coli attTn7 DNA and its eukaryotic orthologs

Transposon Tn7 inserts itself into the attTn7 target DNA sequence at the 3' end of the Escherichia coli glmS gene with high specificity and efficiency. This site in the E. coli genome displays amino acid conservation and nucleotide similarity with orthologous sequences in Archaebacteria and eukaryotes. On the basis of the high degree of nucleotide similarity, particularly with eukaryotes, we examined the interactions of a set of 20-bp duplex DNA sequences with the Tn7 protein TnsD. The protein was overexpressed in the IPTG-inducible vector pET14b-TnsD in E. coli BL21(DE3)-RIL-Codon-Plus, and purified by nickel chelation and ion exchange chromatography. Changes in the conformation of DNA duplexes upon interaction with TnsD were monitored by circular dichroism (CD) spectroscopy. TnsD binding to and dissociation from immobilized DNA duplexes were monitored by total internal reflectance (TIR). CD and TIR results were analyzed to calculate k(on), k(off), and K(D) values. The 20-bp DNA duplex corresponding to attTn7 at the 3' end of E. coli glmS displayed strong affinity for TnsD protein, with K(D) approximately 20 nM. Eukaryotic orthologs of attTn7 from yeast and mammalian GFPT1 displayed lower affinity, with K(D) approximately 500 nM. Mutant DNA sequences with a single central mismatch did not display any detectable interaction with TnsD. The physical studies validate our biological observation of Tn7 transposition into a plasmid containing the mammalian attTn7 ortholog sequence [Cleaver, S. H., and Wickstrom, E. (2000) Gene 254, 37-44], and suggest that 1-2 amino acid substitutions in TnsD might be sufficient to permit binding to mammalian orthologs that is as strong as wild-type TnsD binding to attTn7.     

mini-Tn7 insertion in bacteria with secondary, non-glmS-linked attTn7 sites: example Proteus mirabilis HI4320

We previously constructed a series of mini-Tn7 chromosome integration vectors that, when provided only with the site-specific transposition machinery, generally transpose to a naturally evolved, neutral attTn7 site that is located 25-bp downstream of the glmS gene. Here we provide a protocol for application of the mini-Tn7 system in Proteus mirabilis as an example of a bacterium with a secondary attTn7 site that is not linked to glmS but, in this case, located in the carAB operon. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into P. mirabilis by transformation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions; and last, optional Flp-mediated excision of the antibiotic-resistance selection marker present on the chromosomally integrated mini-Tn7 element. When transposon-containing cells are selected on rich medium, insertions occur at both attTn7 sites with equal efficiency and frequency. Because carA mutants are arginine and pyrimidine auxotrophs, single-site insertions at the glmS attTn7 sites can be obtained by selection on minimal medium. From start to verification of the insertion events, the whole procedure takes 5 d. This chromosome integration system in P. mirabilis provides an important tool for animal and biofilm studies based on this bacterium. Vectors are available for gene complementation and expression, gene fusion analyses and tagging with a green fluorescent protein (GFP)-encoding reporter gene.     

mini-Tn7 insertion in bacteria with multiple glmS-linked attTn7 sites: example Burkholderia mallei ATCC 23344

The mini-Tn7 vectors are universally applicable in gram-negative bacteria and thereby facilitate the manipulation of many organisms for which few genetic systems are available. These vectors, when provided with only the Tn7 site-specific transposition machinery, insert site and orientation specifically in the bacterial chromosome at an attTn7 site downstream of the essential glmS gene. A few bacteria, including Burkholderia spp., contain multiple glmS genes and therefore several attTn7 sites. Here we provide a protocol for application of the mini-Tn7 system in B. mallei as an example of bacteria with multiple glmS sites. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into B. mallei by conjugation, followed by selection of insertion-containing strains; and last, PCR verification of mini-Tn7 insertions. B. mallei possesses two glmS genes on chromosome 1 and Tn7 transposes to both sites, although transposition to attTn7-1 associated with glmS1 occurs in more than 90% of the clones examined. Transposition is efficient and the whole procedure from start to verification of insertion events can be done in less than 5 d. This first chromosome integration system in B. mallei provides an important contribution to the genetic tools emerging for Burkholderia spp. Vectors are available for gene complementation and expression, and gene fusion analyses.     

Formation of a nucleoprotein complex containing Tn7 and its target DNA regulates transposition initiation

Tn7 insertion into its specific target site, attTn7, is mediated by the proteins TnsA, TnsB, TnsC and TnsD. The double-strand breaks that separate Tn7 from the donor DNA require the Tns proteins, the transposon and an attTn7 target DNA, suggesting that a prerequisite for transposition is the formation of a nucleoprotein complex containing TnsABC+D, and these DNAs. Here, we identify a TnsABC+D transposon-attTn7 complex, and demonstrate that it is a transposition intermediate. We demonstrate that an interaction between TnsB, the transposase subunit that binds to the transposon ends, and TnsC, the target DNA-binding protein that controls the activity of the transposase, is essential for assembly of the TnsABC+D transposon-attTn7 complex. We also show that certain TnsB residues are required for recombination because they mediate a TnsB-TnsC interaction critical to formation of the TnsABC+D transposon-attTn7 complex. We demonstrate that TnsA, the other transposase subunit, which also interacts with TnsC, greatly stabilizes the TnsABC+D transposon-attTn7 complex. Thus multiple interactions between the transposase subunits, TnsA and TnsB, and the target-binding transposase activator, TnsC, control Tn7 transposition.     

Tn7 transposition in vitro proceeds through an excised transposon intermediate generated by staggered breaks in DNA.

We have developed a cell-free system in which the bacterial transposon Tn7 inserts at high frequency into its preferred target site in the Escherichia coli chromosome, attTn7; Tn7 transposition in vitro requires ATP and Tn7-encoded proteins. Tn7 transposes via a cut and paste mechanism in which the element is excised from the donor DNA by staggered double-strand breaks and then inserted into attTn7 by the joining of 3' transposon ends to 5' target ends. Neither recombination intermediates nor products are observed in the absence of any protein component or DNA substrate. Thus, we suggest that Tn7 transposition occurs in a nucleoprotein complex containing several proteins and the substrate DNAs and that recognition of attTn7 within this complex provokes strand cleavages at the Tn7 ends.     

Host proteins can stimulate Tn7 transposition: a novel role for the ribosomal protein L29 and the acyl carrier protein.

The bacterial transposon Tn7 is distinguished by its ability to insert at a high frequency into a specific site in the Escherichia coli chromosome called attTn7. Tn7 insertion into attTn7 requires four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC and TnsD. The selection of attTn7 is determined by TnsD, a sequence-specific DNA-binding protein. TnsD binds attTn7 and interacts with TnsABC, the core transposition machinery, which facilitates the insertion of Tn7 into attTn7. In this work, we report the identification of two host proteins, the ribosomal protein L29 and the acyl carrier protein (ACP), which together stimulate the binding of TnsD to attTn7. The combination of L29 and ACP also stimulates Tn7 transposition in vitro. Interestingly, mutations in L29 drastically decrease Tn7 transposition in vivo, and this effect of L29 on Tn7 transposition is specific for TnsABC+D reactions.     

Gain-of-Function Mutations in Tnsc, an Atp-Dependent Transposition Protein That Activates the Bacterial Transposon Tn7

The bacterial transposon Tn7 encodes five genes whose protein products are used in different combinations to direct transposition to different types of target sites. TnsABC+D directs transposition to a specific site in the Escherichia coli chromosome called attTn7, whereas TnsABC+E directs transposition to non-attTn7 sites. These transposition reactions can also recognize and avoid ``immune' targets that already contain a copy of Tn7. TnsD and TnsE are required to activate TnsABC as well as to select a target site; no transposition occurs with wild-type TnsABC alone. Here, we describe the isolation of TnsC gain-of-function mutants that activate the TnsA+B transposase in the absence of TnsD or TnsE. Some of these TnsC mutants enable the TnsABC machinery to execute transposition without sacrificing its ability to discriminate between different types of targets. Other TnsC mutants appear to constitutively activate the TnsABC machinery so that it bypasses target signals. We also present experiments that suggest that target selection occurs early in the Tn7 transposition pathway in vivo: favorable attTn7 targets appear to promote the excision of Tn7 from the chromosome, whereas immune targets do not allow transposon excision to occur. This work supports the view that TnsC plays a central role in the evaluation and utilization of target DNAs.     

Alternative interactions between the Tn7 transposase and the Tn7 target DNA binding protein regulate target immunity and transposition

The Tn7 transposon avoids inserting into a target DNA that contains a pre-existing copy of Tn7. This phenomenon, known as 'target immunity', is established when TnsB, a Tn7 transposase subunit, binds to Tn7 sequences in the target DNA and mediates displacement of TnsC, a critical transposase activator, from the DNA. Paradoxically, TnsB-TnsC interactions are also required to promote transposon insertion. We have probed Tn7 target immunity by isolating TnsB mutants that mediate more frequent insertions into a potentially immune target DNA because they fail to provoke dissociation of TnsC from the DNA. We show that a single region of TnsB mediates the TnsB-TnsC interaction that underlies both target immunity and transposition, but that TnsA, the other transposase subunit, channels the TnsB-TnsC interaction toward transposition.     

Transposon Tn7 is widespread in diverse bacteria and forms genomic islands

We find that relatives of the bacterial transposon Tn7 are widespread in disparate environments and phylogenetically diverse species. These elements form functionally diverse genomic islands at the specific site of Tn7 insertion adjacent to glmS. This work presents the first example of genomic island formation by a DDE type transposon.     

Tn7: a target site-specific transposon

The bacterial transposon Tn7 is an unusual mobile DNA segment. Most transposable elements move at low-frequency and display little target site-selectivity. By contrast, Tn7 inserts at high-frequency into a single specific site in the chromosomes of many bacteria. In the absence of this specific site, called attTn7 in Escherichia coli where Tn7 has been most extensively studied, Tn7 transposes at low-frequency and inserts into many different sites. Much has recently been learned about Tn7 transposition from both genetic and biochemical studies. The Tn7 recombination machinery is elaborate and includes a large number of Tn7-encoded proteins, probably host-encoded proteins and also rather large cis-acting transposition sequences at the transposon termini and at the target site. Dissection of the Tn7 transposition mechanism has revealed that the DNA strand breakage and joining reactions that underlie the translocation of Tn7 have several unusual features.     

Selective recognition of pyrimidine motif triplexes by a protein encoded by the bacterial transposon Tn7

The bacterial transposon Tn7 is distinguished among mobile genetic elements by its targeting abilities. Recently, we reported that Tn7 is able to selectively insert adjacent to triple-helical DNA. The binding of TnsC, a Tn7-encoded protein, to the triplex DNA target leads to the specific transposition of Tn7 adjacent to both inter- and intramolecular pyrimidine motif triplexes. Here, we further probe how Tn7 targets triplex DNA. We report that TnsC discriminates between different types of triplexes, showing binding preference for pyrimidine but not for purine motif intermolecular triplex DNA. The binding preferences of TnsC and the Tn7 insertion profiles were obtained using psoralenated, triplex- forming oligonucleotides annealed to plasmid DNAs. Although the presence of psoralen is not required for targeting nor is it alone able to attract TnsC, we show that the location of psoralen within the pyrimidine motif triplex does alter the position of Tn7 insertion relative to the triplex. Comparison between the triplex-targeting pathway and the highly site-specific targeting pathway mediated by the binding of the Tn7-encoded protein, TnsD, to the unique site attTn7, suggests that similar structural features within each target DNA are recognized by TnsC, leading to site-specific transposition. This work demonstrates that a prokaryotic protein involved in the targeting and regulation of Tn7 translocation, TnsC, can selectively recognize pyrimidine motif triplexes.     

Site-specific Tn7 transposition into the human genome

The bacterial transposon, Tn7, inserts into a single site in the Escherichia coli chromosome termed attTn7 via the sequence-specific DNA binding of the target selector protein, TnsD. The target DNA sequence required for Tn7 transposition is located within the C-terminus of the glucosamine synthetase (glmS) gene, which is an essential, highly conserved gene found ubiquitously from bacteria to humans. Here, we show that Tn7 can transpose in vitro adjacent to two potential targets in the human genome: the gfpt-1 and gfpt-2 sequences, the human analogs of glmS. The frequency of transposition adjacent to the human gfpt-1 target is comparable with the E.coli glmS target; the human gfpt-2 target shows reduced transposition. The binding of TnsD to these sequences mirrors the transposition activity. In contrast to the human gfpt sequences, Tn7 does not transpose adjacent to the gfa-1 sequence, the glmS analog in Saccharomyces cerevisiae. We also report that a nucleosome core particle assembled on the human gfpt-1 sequence reduces Tn7 transposition by likely impairing the accessibility of target DNA to the Tns proteins. We discuss the implications of these findings for the potential use of Tn7 as a site-specific DNA delivery agent for gene therapy.     

The conjugative transposon Tn5397 has a strong preference for integration into its Clostridium difficile target site

Tn5397 is a conjugative transposon, originally isolated from Clostridium difficile. The Tn5397 transposase TndX is related to the phage-encoded serine integrases and the Clostridium perfringens Tn4451 transposase TnpX. TndX is required for the insertion and excision of the transposon. Tn5397 inserts at one locus, attB(Cd), in C. difficile but at multiple sites in Bacillus subtilis. Apart from a conserved 5' GA dinucleotide at the recombination site, there appears to be little sequence conservation between the known target sites. To test the target site preference of Tn5397, attB(Cd) was introduced into the B. subtilis genome. When Tn5397 was transferred into this strain, 100% of the 50 independent transconjugants tested had Tn5397 inserted into attB(Cd). This experiment was repeated using a 50-bp attB(Cd) with no loss of target preference. The mutation of the 5' GA to 5' TC in the attB(Cd) target site caused a switch in the polarity of insertion of Tn5397, which is consistent with this dinucleotide being at the crossover site and in keeping with the mechanism of other serine recombinases. Tn5397 could also transpose into 50-bp sequences encoding the end joints attL and attR but, surprisingly, could not recombine into the circular joint of Tn5397, attTn. Purified TndX was shown to bind specifically to 50-bp attB(Cd), attL, attR, attTn, and attB(Bs)(3) with relative binding affinities attTn approximately attR > attL > attB(Cd) > attB(Bs3). We conclude that TndX has a strong preference for attB(Cd) over other potential recombination sites in the B. subtilis genome and therefore behaves as a site-specific recombinase.