Factors that affect transposition mediated by the Tn21 transposase

The frequencies of one-ended transposition mediated by the Tn21 transposase acting on plasmids containing 38-bp inverted repeat sequences (IRs) of both Tn21 and of Tn501/Tn1721 and Tn2501 were measured. The enzyme acted on all these IRs, but more efficiently on the homologous sequences. These differences were magnified when the enzyme acted on plasmids containing two copies of the IRs, inverted with respect to each other. The Tn21 enzyme did not recognize the IR of Tn3. The Tn501 transposase did not mediate measurable one-ended transposition of any of the plasmids used, including those containing an IR of Tn501.     

Toluene transposons Tn4651 and Tn4653 are class II transposons

The toluene degradative transposon Tn4651 is included within another transposon, Tn4653, and both of these elements are members of the Tn3 family. The tnpA gene product of each element mediates formation of cointegrates as intermediate products of transposition, and the tnpS and tnpT gene products encoded by Tn4651 take part in resolution of both Tn4651- and Tn4653-mediated cointegrates. Sequence analysis demonstrated that Tn4651 and Tn4653 have 46- and 38-base-pair terminal inverted repeats, respectively, and that both elements generate 5-base-pair duplication of the target sequence upon transposition. Complementation tests of the Tn4651- and Tn4653-encoded transposition functions with those of Tn3, Tn21, and Tn1721 showed that (i) the trans-acting transposition functions encoded by Tn4651 were not interchangeable with those encoded by the four other transposons, (ii) the Tn4653 tnpA function was interchangeable with the Tn1721 function, and (iii) Tn4653 coded for a resolvase (tnpR gene product) that complemented the tnpR mutations of Tn21 and Tn1721. The Tn4653 tnpR gene was located just 5' upstream of the tnpA gene and shared extensive sequence homology with the Tn1721 tnpR gene. The res region was located adjacent to the tnpR gene, and sequence analysis indicated that failure of the Tn4653 tnpR product to resolve the Tn4653-mediated cointegrates is ascribed to an incomplete structure of the res region.     

Characterisation of Tn1721, a new transposon containing tetracycline resistance genes capable of amplification.

R plasmid pRSD1 contains tetracycline resistance (tet) genes in a 3.55 Mdal-region capable of amplification by forming tandem repeats (Mattes, Burkardt and Schmitt, Molec. gen. Genet., 1979). The repetitious tet element is itself part of a 7.2 Mdal-transposon, named Tn1721, as demonstrated by the following criteria; (i) Tn1721 has been translocated to phage lambda. The resulting hybrid phage lambda tet contains the 7.2 Mdal-insertion to the right of the attachment site, but not continguous with it indicating translocation of the element by non-homologous recombination. In addition, lambda tet has sustained a 3.4 Mdal-deletion adjacent to the insertion. (ii) Further transposition of Tn1721 to the 21.5 Mdal-plasmid R388 resulted in R388::Tn1721 derivatives, two of which were characterised. They contain Tn1721 inserted into different sites but in the same orientation as shown by restriction and heteroduplex analyses. These translocation of Tn1721 were not accompanied by deletions of DNA. (iii) The insertion plasmid pRSD102(R388::Tn1721) has conserved the capacity of the original plasmid pRSD1 to amplify the 3.55 Mdal-tet region. It has been concluded that Tn1721 constitutes a novel transposon encompassing a tet region capable of selective amplification. The model proposed for Tn1721 contains three short repeats. Two direct repeats, flanking the 3.55 Mdal tet region, provide sequence homology for amplification. The third repeat (located distally to tet) is inverted and provides the basis for transposition of the 7.2 Mdal-element.     

Analysis of the variable endpoints generated by one-ended transposition of Tn21.

One-ended transposition of Tn21 generates recombinants usually containing a whole copy of the donor replicon plus a short duplication of it (S. M?tsch, R. Schmitt, P. Avila, F. de la Crue, E. Ward, and J. Grinsted, Nucleic Acids Res. 13:3335-3342, 1985). This work shows that recombinants containing less than a whole copy of the donor replicon (hereafter called short recombinants) could also be detected when plasmid donors which contained two selectable genetic markers were used. Short recombinants were produced at the same frequency from TnpR+ donor molecules as from TnpR- donor molecules in a RecA- background. Therefore, they were not resolution products of larger recombinants. This result invalidates a previous hypothesis to explain one-ended transposition, that is, that one-ended transposition arises from the use of secondary ends by the transposition apparatus. On the other hand, it suggests that one-ended transposition of Tn21 occurs via a simple insertion mechanism.     

Tn5053 family transposons are res site hunters sensing plasmidal res sites occupied by cognate resolvases

DNA sequence database search revealed that most of Tn5053/Tn402 family transposons inserted into natural plasmids were located in putative res regions upstream of genes encoding various resolvase-like proteins. Some of these resolvase genes belonged to Tn3 family transposons and were closely related to the tnpR genes of Tn1721 and a recently detected Tn5044. Using recombinant plasmids containing fragments of Tn1721 or Tn5044 as targets in transposition experiments, we have demonstrated that Tn5053 displays striking insertional preference for the res regions of these transposons: more than 70% of Tn5053 insertion events occur in clusters inside the target res regions, while most remaining insertion events occur no further than 200 base pairs away from both sides of the res regions. We demonstrate that Tn5053 insertions (both into and outside a res region of the target plasmid) require the presence of a functional cognate resolvase gene either in cis or in trans. To our knowledge, this is the first case when a site-specific recombination system outside a transposon has been shown to be involved in transposition.     

A Tn21 terminal sequence within Tn501: complementation of tnpA gene function and transposon evolution

Summary The prokaryotic mercury-resistance transposon Tn501 contains a sequence, 80 nucleotides from one end, which is identical with an inverted terminal repeat (IR) of Tn21. This Tn21 IR sequence is used when Tn21 complements a TnpA^- derivative of Tn501, but not when Tn501 is used for the complementation. Complementation by Tn1721 shows a preference for the normal Tn501 IRs. The element (Tn820) transposed when Tn21 is used to complement a Hg^- TnpR^- TnpA^- Res^- deletion mutant of Tn501 contains the Tn21 IR sequence at one terminus and a Tn501 IR at the other. Transposition of Tn820 can be complemented by Tn501 and Tn1721, but at a much lower frequency than transposition of the parental element (Tn819) which has two Tn501 IRs. The relationship between the transposition functions of Tn501, Tn21 and Tn1721, and available nucleotide sequence data suggest that Tn501 evolved by the transposition of a Tn21-like element into another transposable element (similar to that found within Tn1721) followed by deletion of the Tn21-like transposition functions.Abbreviations used (IR) Inverted repeat - (Cb) carbenicillin - (Cm) chloramphenicol - (Sm) streptomycin - (Su) sulphonamide - (Tc) tetracycline - (Tp) trimethoprim     

Two domains in the terminal inverted-repeat sequence of transposon Tn3

Tn3 and related transposons have terminal inverted repeats (IR) of about 38 bp that are needed as sites for transposition. We made mini-Tn3 derivatives which had a wild-type IR of Tn3 at one end and either the divergent IR of the Tn3-related transposon, gamma delta or IS101, or a mutant IR of Tn3 at the other end. We then examined both in vivo transposition (cointegration between transposition donor and target molecules) of these mini-Tn3 elements and in vitro binding of Tn3-encoded transposase to their IRs. None of the elements with an IR of gamma delta or IS101 mediated cointegration efficiently. This was due to inefficient binding of transposase to these IR. Most mutant IR also interfered with cointegration, even though transposase bound to some mutant IR as efficiently as it did to wild type. This permitted the Tn3 IR sequence to be divided into two domains, named A and B, with respect to transposase binding. Domain B, at positions 13-38, was involved in transposase binding, whereas domain A, at positions 1-10, was not. The A domain may contain the sequence recognized by some other (e.g., host) factor(s) to precede the actual cointegration event.     

Genetic and molecular characterization of Tn21, a multiple resistance transposon from R100.1.

Tge transposon Tn21 has been transposed from R100.1 to plasmid pACYC184 and, from the resulting recombinants, to plasmid R388. The sites of insertion and the orientation of the element in several pACYC184::Tn21 recombinants have been examined. Restriction enzyme analysis of these recombinants has resulted in a detailed map of Tn21; this is compared with the published maps of the relevant part of R100.1. Heteroduplex analysis has shown short inverted repeat sequences at the ends of the element. With various in vitro-generated deletion mutants of Tn21, the internal gene necessary for transposition (tnpA) was localized within the terminal 4.3 kilobases of the right-hand end of the element. Genetic analysis of transposition of Tn21 suggests that the process proceeds via cointegrates. Since the end products of transposition are simple recombinants of the element and the recipient replicon, Tn21 must contain a gene that codes for a resolvase type of activity (tnpR gene).     

Characterization of Tn3926, a new mercury-resistance transposon from Yersinia enterocolitica

A new transposon coding for mercury resistance (HgR), Tn3926, has been found in a strain of Yersinia enterocolitica, YE138A14. The element has a size of 7.8 kb and transposes to conjugative plasmids belonging to different incompatibility groups. A restriction map has been established. DNA-DNA hybridization indicates that Tn3926 displays homology with both Tn501 and Tn21; the greatest homology is shown with the regions of these transposons that encode HgR. Weaker homology is observed between Tn3926 sequences and those regions of Tn501 and Tn21 that encode transposition functions. Complementation experiments indicate that the Tn3926 transposase mediates transposition of Tn21, albeit somewhat inefficiently, but not of Tn501, while the resolvase mediates resolution of transposition cointegrates formed via Tn21, Tn501, or Tn1721.     

Tn7 Transposition Creates a Hotspot for Homologous Recombination at the Transposon Donor Site

Homologous recombination at the bacterial transposon Tn7 donor site is stimulated 10-fold when Tn7 is activated to transpose at high frequency in RecD(-) Escherichia coli, where recombination is focused near the ends of double-chain breaks. This is observed as an increase in recombination between two lacZ heteroalleles when one copy of lacZ carries within it a Tn7 that is transposing at high frequency. This stimulation of recombination is dependent upon the presence of homology with the donor site, is independent of SOS induction, and is not due to a global stimulation of recombination. When stimulated by Tn7 transposition, the conversion events giving rise to Lac(+) recombinants occur preferentially at the site of Tn7, suggesting that transposition is stimulating gene conversion at the donor site. These results support the model that Tn7 transposition occurs by a ``cut and paste' mechanism, leaving a double-chain break at the donor site that is repaired by the host homologous recombination machinery; normally, repair would use homology in a sister chromosome to regenerate a copy of the transposon. This proposed series of events allows transposition that is nonreplicative, per se, to be effectively replicative.     

Complementation of transposition of tnpA mutants of Tn3, Tn21, Tn501, and Tn1721

The transposons Tn21, Tn501, and Tn1721 are related to Tn3. Transposition-deficient mutants (tnpA) of these elements were used to test for complementation of transpostion. Transposition of tnpA mutants of Tn501 and Tn1721 was restored by the presence in trans of Tn21, Tn501, and Tn1721, but transposition of a tnpA mutant of Tn21 was restored in trans only by Tn21 itself. Tn3 did not complement transposition of Tn21, Tn501, or Tn1721, and these elements did not complement transposition of Tn3.     

Transposition of Tn1000: activity of single or directly repeated termini.

Tn1000 (gamma delta) termini IRR and IRL, or direct repetitions of IRR-IRL carried by pBR322 derivatives mediate cointegration with pOX38 at similar rates. Structures of product plasmids indicate that the transposed segments correspond to DNA bounded by IR segments in the donor plasmid. Such structures could arise by symmetric transposition from a replication intermediate.     

Temperature sensitivity of transposition of class II transposons

It has been reported that transposition of Tn3 is temperature-sensitive. The effect of temperature on the transposition of other class II bacterial transposable elements is reported here: Tn21, Tn501, Tn1721, Tn2501 and Tn3926 all also display temperature-sensitivity of transposition. The temperature at which the highest transposition frequency was observed varied between room temperature and 30 degrees C.     

Transposon Tn951 (TnLac) is defective and related to Tn3

Summary Tn951 is flanked by two perfect inverted repeats of 41 bp which include the 38 bp sequence of the IR of Tn3. Tn951 also contains the last 100 bp of the tnpA gene but with at least two mutations. However, beyond nucleotide 137 the sequences diverge and hybridization experiments show that Tn951 lacks at least the first two thirds of the tnpA gene.In agreement with these observations Tn951 does not transpose by itself at a detectable frequency but can be complemented by the tnpA gene of Tn801 or Tn3. Tn501, Tn1721 and gamma delta do not complement Tn951 transposition.Transposition of Tn951 duplicates 5 bp of target DNA sequence.     

Bacteroides fragilis transfer factor Tn5520: the smallest bacterial mobilizable transposon containing single integrase and mobilization genes that function in Escherichia coli

Many bacterial genera, including Bacteroides spp., harbor mobilizable transposons, a class of transfer factors that carry genes for conjugal DNA transfer and, in some cases, antibiotic resistance. Mobilizable transposons are capable of inserting into and mobilizing other, nontransferable plasmids and are implicated in the dissemination of antibiotic resistance. This paper presents the isolation and characterization of Tn5520, a new mobilizable transposon from Bacteroides fragilis LV23. At 4,692 bp, it is the smallest mobilizable transposon reported from any bacterial genus. Tn5520 was captured from B. fragilis LV23 by using the transfer-deficient shuttle vector pGAT400DeltaBglII. The termini of Tn5520 contain a 22-bp imperfect inverted repeat, and transposition does not result in a target site repeat. Tn5520 also demonstrates insertion site sequence preferences characterized by A-T-rich nucleotide sequences. Tn5520 has been sequenced in its entirety, and two large open reading frames whose predicted protein products exhibit strong sequence similarity to recombinase-integrase enzymes and mobilization proteins, respectively, have been identified. The transfer, mobilization, and transposition properties of Tn5520 have been studied, revealing that Tn5520 mobilizes plasmids in both B. fragilis and Escherichia coli at high frequency and also transposes in E. coli.     

DNA sequence analysis of host range mutants of the promiscuous IncP-1 plasmids R18 and R68 with Tn7 insertions in oriV

Transposon Tn7 insertions in the origin of vegetative replication (oriV) result in host range mutants of the promiscuous IncP-1 plasmids R18 and R68 which affect plasmid replication in Escherichia coli but not in Pseudomonas aeruginosa. The sites of these insertions have been analyzed by DNA sequence analysis. In two mutants, the insertions generated direct duplications of 5′GTATT3′ at the target site which included the first base at the 5′ end of the fourth 17-bp direct repeat in oriV. In a third mutant the duplication of 5′GACAC3′ also involved the same direct repeat also at the 5′ end but contiguous with the previous duplication. DNA sequence analysis of another Tn7-induced host range mutant of R18, characterized by reduced conjugational transmissibility into P. stutzeri while retaining normal transmissibility within P. aeruginosa, showed that the insertion generated a 474-bp deletion which brought the insertion 20 bp 5′ to the 17-bp direct repeat between oriV and the oxytetracycline hydrochloride-resistant gene. The analysis of the DNA sequence data at the site of the Tn7 insertions shows that particular segments of the DNA sequence in oriV are differentially required for the replication of these plasmids in different bacterial hosts and thus of importance to the promiscuity of these plasmids.     

Definition of three resolvase binding sites at the res loci of Tn21 and Tn1721.

The dual functions of resolvase, site-specific recombination and the regulation of its own expression from tnpR, both require the interaction of this protein with the DNA sequence at res, but the specificity of this interaction differs between groups of Tn3-like elements. In this study, DNA fragments that contained res from Tn21 or Tn1721 were subjected to either cleavage by DNase I or methylation by dimethyl sulphate in the presence of the purified resolvase from Tn21 or Tn1721. These experiments showed that each resolvase bound to the same three sites (I, II and III) within res from Tn1721 and to an equivalent series of three sites on Tn21: the differences in the amino acid sequences of the two proteins did not affect their interaction with either DNA. The DNA sequences at each site had some similarities and, in conjunction with data from the related transposon Tn501, a consensus was established. However, the three sites are functionally distinct: site I (tnpR-distal) spans the recombination cross-over point and sites II and III (tnpR-proximal) overlap the promoter of tnpR. The binding sites on these transposons were compared with those in the gamma delta/Tn3 system: the similarities between the two groups of transposons revealed some general features of resolvase-DNA interactions while the differences in fine structure elucidated the specificity of each resolvase.     

The minimum internal and external sequence requirements for transposition of the eukaryotic transformation vector piggyBac

The piggyBac element from Trichoplusia ni is recognized as a useful vector for transgenesis of a wide variety of species. This transposable element is 2472 bp in length, and has a complex repeat configuration consisting of an internal repeat (IR), spacer, and terminal repeat (TR) at both ends, and a single ORF encoding the transposase. Excision assays performed in microinjected T. ni embryos using plasmids deleted for progressively larger portions of the piggyBac internal sequence reveal that the 5' and 3' IR, spacer, and TR configuration is sufficient for precise excision of piggyBac when transposase is provided in trans. Interplasmid transposition assays using plasmids carrying varying lengths of intervening sequence between the piggyBac termini in T. ni demonstrate that a minimum of 55 bp of intervening sequence is required for optimal transposition, while lengths less than 40 bp result in a dramatic decrease in transposition frequency. These results suggest that the piggyBac transposase may bind both termini simultaneously before cleavage can occur, and/or that the formation of a transposition complex requires DNA bending between the two termini. Based on these results we constructed a 702-bp cartridge with minimal piggyBac 5' and 3' terminal regions separated by an intervening sequence of optimal length. Interplasmid transposition assays demonstrate that the minimal terminal configuration is sufficient to mediate transposition, and also verify that simply inserting this cartridge into an existing plasmid converts that plasmid into a non-autonomous piggyBac transposon. We also constructed a minimal piggyBac vector, pXL-Bac, that contains an internal multiple cloning site sequence between the minimal terminal regions. These vectors should greatly facilitate the utilization of the piggyBac transposon in a wide range of hosts.