Bacterial Transposon Tn7 Research Articles

Natural and Engineered Guide RNA-Directed Transposition with CRISPR-Associated Tn7-Like Transposons.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated nuclease) defense systems have been naturally coopted for guide RNA-directed transposition on multiple occasions. In all cases, cooption occurred with diverse elements related to the bacterial transposon Tn7. Tn7 tightly controls transposition; the transposase is activated only when special targets are recognized by dedicated target-site selection proteins. Tn7 and the Tn7-like elements that coopted CRISPR-Cas systems evolved complementary targeting pathways: one that recognizes a highly conserved site in the chromosome and a second pathway that targets mobile plasmids capable of cell-to-cell transfer. Tn7 and Tn7-like elements deliver a single integration into the site they recognize and also control the orientation of the integration event, providing future potential for use as programmable gene-integration tools. Early work has shown that guide RNA-directed transposition systems can be adapted to diverse hosts, even within microbial communities, suggesting great potential for engineering these systems as powerful gene-editing tools.

Conformational toggling controls target site choice for the heteromeric transposase element Tn7.

The bacterial transposon Tn7 facilitates horizontal transfer by directing transposition into actively replicating DNA with the element-encoded protein TnsE. Structural analysis of the C-terminal domain of wild-type TnsE identified a novel protein fold including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-activity variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structure-based analysis of a series of TnsE mutants relates transposition activity to DNA binding stability. Wild-type TnsE appears to naturally form an unstable complex with a target DNA, whereas mutant combinations required for large changes in transposition frequency and targeting stabilized this interaction. Collectively, our work unveils a unique structural proofreading mechanism where toggling between two conformations regulates target commitment by limiting the stability of target DNA engagement until an appropriate insertion site is identified.

Tn7.

The bacterial transposon Tn7 is distinguished by the levels of control it displays over transposition and its capacity to utilize different kinds of target sites. Transposition is carried out using five transposon-encoded proteins, TnsA, TnsB, TnsC, TnsD, and TnsE, which facilitate transfer of the element while minimizing the chances of inactivating host genes by using two pathways of transposition. One of these pathways utilizes TnsD, which targets transposition into a single site found in bacteria (attTn7), and a second utilizes TnsE, which preferentially directs transposition into plasmids capable of moving between bacteria. Control of transposition involves a heteromeric transposase that consists of two proteins, TnsA and TnsB, and a regulator protein TnsC. Tn7 also has the ability to inhibit transposition into a region already occupied by the element in a process called target immunity. Considerable information is available about the functional interactions of the Tn7 proteins and many of the protein-DNA complexes involved in transposition. Tn7-like elements that encode homologs of all five of the proteins found in Tn7 are common in diverse bacteria, but a newly appreciated larger family of elements appears to use the same core TnsA, TnsB, and TnsC proteins with other putative target site selector proteins allowing different targeting pathways.

PBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes

BackgroundSince publication in 1977 of plasmid pBR322, many breakthroughs in Biology have depended on increasingly sophisticated vector platforms for analysis and engineering of given bacterial strains. Although restriction sites impose a certain format in the procedures for assembling cloned genes, every attempt thus far to standardize vector architecture and nomenclature has ended up in failure. While this state of affairs may still be tolerable for traditional one-at-a-time studies of single genes, the onset of systems and synthetic biology calls for a simplification -along with an optimization- of the currently unwieldy pool of genetic tools.ResultsThe functional DNA sequences present in the natural bacterial transposon Tn5 have been methodically edited and refactored for the production of a multi-purpose genetic tool named pBAM1, which allows a range of manipulations in the genome of Gram-negative bacteria. This all-synthetic construct enhances the power of mini-transposon vectors for either de-construction or re-construction of phenotypes á la carte by incorporating features inspired in systems engineering: modularity, re-usability, minimization, and compatibility with other genetic tools. pBAM1 bears an streamlined, restriction site-freed and narrow-host range replication frame bearing the sequences of R6K oriV, oriT and an ampicillin resistance marker. These go along with a business module that contains a host-independent and hyperactive transposition platform for in vivo or in vitro insertion of desired DNA into the genome of the target bacterium. All functional sequences were standardized for a straightforward replacement by equivalent counterparts, if required. pBAM1 can be delivered into recipient cells by either mating or electroporation, producing transposon insertion frequencies of 1.8 × 10-3 and 1.02 × 10-7, respectively in the soil bacterium Pseudomonas putida. Analyses of the resulting clones revealed a 100% of unique transposition events and virtually no-cointegration of the donor plasmid within the target genome.ConclusionsThis work reports the design and performance of an all-synthetic mini-transposon vector. The power of the new system for both identification of new functions or for the construction of desired phenotypes is shown in a genetic survey of hyper-expressed proteins and regulatory elements that influence the expression of the σ54-dependent Pu promoter of P. putida.

Characterization of the TnsD-attTn7 complex that promotes site-specific insertion of Tn7

The bacterial transposon Tn7 is distinguished by its ability to recognize a specific site called attTn7, and insert just downstream of the highly conserved chromosomal glmS gene. TnsD is one of four transposon-encoded polypeptides (TnsABC+D) required for site-specific insertion of Tn7 into attTn7, and is the target site-selector that binds to a highly conserved sequence in the end of the glmS protein coding region. In this study, we identified important nucleotides within this region that are crucial for TnsD-attTn7 interaction. We also probed the regions of TnsD that interact with attTn7 and found that there are important DNA-binding determinants throughout the entire length of the protein, including an amino-terminal CCCH zinc-finger motif. A key role of TnsD is to recruit the non-sequence specific DNA-binding protein TnsC to attTn7; TnsC also interacts with and controls both the TnsA and TnsB subunits of the Tn7 transposase. TnsC stimulates the binding of TnsD to attTn7 in vivo, and TnsCD and TnsD can also interact in the absence of DNA and localize their interaction domains to the N-terminal region of each protein.

Transposon Tn7 Preferentially Inserts into GAA•TTC Triplet Repeats under Conditions Conducive to Y•R•Y Triplex Formation

BackgroundExpansion of an unstable GAA•TTC repeat in the first intron of the FXN gene causes Friedreich ataxia by reducing frataxin expression. Structure formation by the repeat has been implicated in both frataxin repression and GAA•TTC instability. The GAA•TTC sequence is capable of adopting multiple non-B DNA structures including Y•R•Y and R•R•Y triplexes. Lower pH promotes the formation of Y•R•Y triplexes by GAA•TTC. Here we used the bacterial transposon Tn7 as an in vitro tool to probe whether GAA•TTC repeats can attract a well-characterized recombinase.Methodology/Principal FindingsTn7 showed a pH-dependent preference for insertion into uninterrupted regions of a Friedreich ataxia patient-derived repeat, inserting 48, 39 and 14 percent of the time at pH 7, pH 8 and pH 9, respectively. Moreover, Tn7 also showed orientation and region specific insertion within the repeat at pH 7 and pH 8, but not at pH 9. In contrast, transposon Tn5 showed no strong preference for or against the repeat during in vitro transposition at any pH tested. Y•R•Y triplex formation was reduced in predictable ways by transposon interruption of the GAA•TTC repeat. However, transposon interruptions in the GAA•TTC repeats did not increase the in vitro transcription efficiency of the templates.Conclusions/SignificanceWe have demonstrated that transposon Tn7 will recognize structures that form spontaneously in GAA•TTC repeats and insert in a specific orientation within the repeat. The conditions used for in vitro transposition span the physiologically relevant range suggesting that long GAA•TTC repeats can form triplex structures in vivo, attracting enzymes involved in DNA repair, recombination and chromatin modification.

Architecture of the Tn7 Posttransposition Complex: An Elaborate Nucleoprotein Structure

Four transposition proteins encoded by the bacterial transposon Tn7, TnsA, TnsB, TnsC, and TnsD, mediate its site- and orientation-specific insertion into the chromosomal site attTn7. To establish which Tns proteins are actually present in the transpososome that executes DNA breakage and joining, we have determined the proteins present in the nucleoprotein product of transposition, the posttransposition complex (PTC), using fluorescently labeled Tns proteins. All four required Tns proteins are present in the PTC in which we also find that the Tn7 ends are paired by protein–protein contacts between Tns proteins bound to the ends. Quantification of the relative amounts of the fluorescent Tns proteins in the PTC indicates that oligomers of TnsA, TnsB, and TnsC mediate Tn7 transposition. High-resolution DNA footprinting of the DNA product of transposition attTn7∷Tn7 revealed that about 350 bp of DNA on the transposon ends and on attTn7 contact the Tns proteins. All seven binding sites for TnsB, the component of the transposase that specifically binds the ends and mediates 3′ end breakage and joining, are occupied in the PTC. However, the protection pattern of the sites closest to the Tn7 ends in the PTC are different from that observed with TnsB alone, likely reflecting the pairing of the ends and their interaction with the target nucleoprotein complex necessary for activation of the breakage and joining steps. We also observe extensive protection of the attTn7 sequences in the PTC and that alternative DNA structures in substrate attTn7 that are imposed by TnsD are maintained in the PTC.

Transposition into Replicating DNA Occurs through Interaction with the Processivity Factor

The bacterial transposon Tn7 directs transposition into actively replicating DNA by a mechanism involving the transposon-encoded protein TnsE. Here we show that TnsE physically and functionally interacts with the processivity factor of the DNA replication machinery in vivo and in vitro. Our work establishes an in vitro TnsABC+E transposition reaction reconstituted from purified proteins and target DNA structures. Using the in vitro reaction we confirm that the processivity factor specifically reorders TnsE-mediated transposition events on target DNAs in a way that matches the bias with active DNA replication in vivo. The TnsE interaction with an essential and conserved component of the replication machinery, and a DNA structure reveals a mechanism by which Tn7, and probably other elements, selects target sites associated with DNA replication.

DNA Damage Differentially Activates Regional Chromosomal Loci for Tn7 Transposition in Escherichia coli

The bacterial transposon Tn7 recognizes replicating DNA as a target with a preference for the region where DNA replication terminates in the Escherichia coli chromosome. It was previously shown that DNA double-strand breaks in the chromosome stimulate Tn7 transposition where transposition events occur broadly around the point of the DNA break. We show that individual DNA breaks actually activate a series of small regional hotspots in the chromosome for Tn7 insertion. These hotspots are fixed and become active only when a DNA break occurs in the same region of the chromosome. We find that the distribution of insertions around the break is not explained by the exonuclease activity of RecBCD moving the position of the DNA break, and stimulation of Tn7 transposition is not dependent on RecBCD. We show that other forms of DNA damage, like exposure to UV light, mitomycin C, or phleomycin, also stimulate Tn7 transposition. However, inducing the SOS response does not stimulate transposition. Tn7 transposition is not dependent on any known specific pathway of replication fork reactivation as a means of recognizing DNA break repair. Our results are consistent with the idea that Tn7 recognizes DNA replication involved in DNA repair and reveals discrete regions of the chromosome that are differentially activated as transposition targets.

Large-scale mutagenesis of the yeast genome using a Tn7-derived multipurpose transposon.

We present here an unbiased and extremely versatile insertional library of yeast genomic DNA generated by in vitro mutagenesis with a multipurpose element derived from the bacterial transposon Tn7. This mini-Tn7 element has been engineered such that a single insertion can be used to generate a lacZ fusion, gene disruption, and epitope-tagged gene product. Using this transposon, we generated a plasmid-based library of approximately 300,000 mutant alleles; by high-throughput screening in yeast, we identified and sequenced 9032 insertions affecting 2613 genes (45% of the genome). From analysis of 7176 insertions, we found little bias in Tn7 target-site selection in vitro. In contrast, we also sequenced 10,174 Tn3 insertions and found a markedly stronger preference for an AT-rich 5-base pair target sequence. We further screened 1327 insertion alleles in yeast for hypersensitivity to the chemotherapeutic cisplatin. Fifty-one genes were identified, including four functionally uncharacterized genes and 25 genes involved in DNA repair, replication, transcription, and chromatin structure. In total, the collection reported here constitutes the largest plasmid-based set of sequenced yeast mutant alleles to date and, as such, should be singularly useful for gene and genome-wide functional analysis.

The bacterial transposon Tn7 causes premature polyadenylation of mRNA in eukaryotic organisms: TAGKO mutagenesis in filamentous fungi.

TAGKO is a Tn7-based transposition system for genome wide mutagenesis in filamentous fungi. The effects of transposon insertion on the expression of TAGKO alleles were examined in Magnaporthe grisea and Mycosphaerella graminicola. Northern analysis showed that stable, truncated transcripts were expressed in the TAGKO mutants. Mapping of the 3'-ends of TAGKO cDNAs revealed that they all contain Tn7 end sequences, regardless of the transposon orientation. Polyadenylation signals characteristic of eukaryotic genes, preceded by stop codons in all frames, are located in both ends of the bacterial transposon. Thus, TAGKO transcripts are prematurely polyadenylated, and truncated proteins are predicted to be translated in the fungal mutants. Depending on the extent of protein truncation, TAGKO mutations in HPD4 (encoding p-hydroxyphenylpyruvate dioxygenase) resulted in tyrosine sensitivity in the two fungi. Similarly, a particular M.grisea CBS1 (encoding cystathionine beta-synthase) TAGKO cDNA failed to complement cysteine auxotrophy in a yeast CBS mutant. TAGKO, therefore, represents a useful tool for in vivo study of truncated gene products in filamentous fungi.

Tn7-based genome-wide random insertional mutagenesis of Candida glabrata.

We describe and characterize a method for insertional mutagenesis of the yeast pathogen Candida glabrata using the bacterial transposon Tn7. Tn7 was used to mutagenize a C. glabrata genomic fosmid library. Pools of random Tn7 insertions in individual fosmids were recovered by transformation into Escherichia coli. Subsequently, these were introduced by recombination into the C. glabrata genome. We found that C. glabrata genomic fragments carrying a Tn7 insertion could integrate into the genome by nonhomologous recombination, by single crossover (generating a duplication of the insertionally mutagenized locus), and by double crossover, yielding an allele replacement. We were able to generate a highly representative set of approximately 10(4) allele replacements in C. glabrata, and an initial characterization of these shows that a wide diversity of genes were targeted in the mutagenesis. Because the identity of disrupted genes for any mutant of interest can be rapidly identified, this method should be of general utility in functional genomic characterization of this important yeast pathogen. In addition, the method might be broadly applicable to mutational analysis of other organisms.

A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast.

Heterologous markers are important tools required for the molecular dissection of gene function in many organisms, including Saccharomyces cerevisiae. Moreover, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene. We recently introduced a new and efficient gene disruption cassette for repeated use in budding yeast, which combines the heterologous dominant kan(r) resistance marker with a Cre/loxP-mediated marker removal procedure. Here we describe an additional set of four completely heterologous loxP-flanked marker cassettes carrying the genes URA3 and LEU2 from Kluyveromyces lactis, his5(+) from Schizosaccharomyces pombe and the dominant resistance marker ble(r) from the bacterial transposon Tn5, which confers resistance to the antibiotic phleomycin. All five loxP--marker gene--loxP gene disruption cassettes can be generated using the same pair of oligonucleotides and all can be used for gene disruption with high efficiency. For marker rescue we have created three additional Cre expression vectors carrying HIS3, TRP1 or ble(r) as the yeast selection marker. The set of disruption cassettes and Cre expression plasmids described here represents a significant further development of the marker rescue system, which is ideally suited to functional analysis of the yeast genome.

Tn7-mediated mutagenesis of Saccharomyces cerevisiae genomic DNA in vitro

Publisher Summary This chapter describes in vitro insertional mutagenesis of the complete genome of Saccharomyces cerevisiae by the bacterial transposon Tn7. The in vitro transposition reaction of Tn7 is accomplished with purified components, allowing the mutagenesis of any purified DNA fragment. The chapter reviews the application of this in vitro transposition technique using total yeast genomic DNA to create a library of insertional mutants, which are incorporated into the genome by transformation and homologous recombination. After phenotypic analysis of the resulting mutant collection, the mutagenic insertion site can be determined rapidly and easily. The randomness, coverage, and efficiency of this method are also discussed. Tn7 is a bacterial DNA transposon that transposes via a cut-and-paste mechanism. The Tn7 reaction is mediated by the transposon-encoded proteins, TnsA, TnsB, TnsC, TnsD, and TnsE. TnsABC form the core recombination machinery, and TnsD and E are alternative target site selecting proteins. If insertion is mediated by TnsABC+D in vivo, transposition occurs into the single attTn7 site in the E. coli genome.

Tn7: smarter than we thought.

A notable feature of transposable elements--segments of DNA that can move from one position to another in genomes--is that they are highly prevalent, despite the fact that their translocation can result in mutation. The bacterial transposon Tn7 uses an elaborate system of target-site selection pathways that favours the dispersal of Tn7 in diverse hosts as well as minimizing its negative effects.

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.

Tn7 recognizes transposition target structures associated with DNA replication using the DNA-binding protein TnsE.

We report that the bacterial transposon Tn7 selects targets by recognizing features associated with DNA replication using the transposon-encoded DNA-binding protein TnsE. We show that Tn7 transposition directed by TnsE occurs in one orientation with respect to chromosomal DNA replication, indicating that a structure or complex involved in DNA replication is likely to be a critical determinant of TnsE insertion. We find that mutant TnsE proteins that allow higher levels of transposition also bind DNA better than the wild-type protein. The increased binding affinity displayed by the TnsE high-activity mutants indicates that DNA binding is relevant to transposition activity and suggests that TnsE interacts directly with target DNAs. In vitro, TnsE interacts preferentially with certain DNA structures, indicating a mechanism for the TnsE-mediated orientation and insertion preference. The pattern of TnsE-mediated insertion events around the Escherichia coli chromosome provides insight into how DNA replication forks proceed in vivo.

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.

Analysis of gain-of-function mutants of an ATP-dependent regulator of Tn7 transposition

The bacterial transposon Tn7 is distinguished by its unusal discrimination among targets, being particularly attracted to certain target DNA and actively avoiding other DNA. Tn7 transposition is mediated by the interaction of two alternative transposon-encoded target selection proteins, TnsD and TnsE, with a common core transposition machinery composed of the transposase (TnsAB) and an ATP-dependent DNA-binding protein TnsC. No transposition is observed with wild-type TnsABC. Here, we analyze the properties of two gain-of-function TnsC mutants that allow transposition in the absence of TnsD or TnsE. We find that these TnsC mutants have altered interactions with ATP and DNA that can account for their gain-of-function phenotype. We also show that TnsC is an ATPase and that it directly interacts with the TnsAB transposase. This work provides strong support to the view that TnsC and its ATP state are central to the control of Tn7 transposition.

Tn7 Transposes Proximal to DNA Double-Strand Breaks and into Regions Where Chromosomal DNA Replication Terminates

We report that the bacterial transposon Tn7 can preferentially transpose into regions where chromosomal DNA replication terminates. DNA double-strand breaks are associated with the termination of chromosomal replication; therefore, we directly tested the effect of DNA breaks on Tn7 transposition. When DNA double-strand breaks are induced at specific sites in the chromosome, Tn7 transposition is stimulated and insertions are directed proximal to the induced break. The targeting preference for the terminus of replication and DNA double-strand breaks is dependent on the Tn7-encoded protein TnsE. The results presented in this study could also explain the previous observation that Tn7 is attracted to events associated with conjugal DNA replication during plasmid DNA transfer.