Insertion sites and the terminal nucleotide sequences of the Tn4 transposon.

The nucleotide sequences at the ends of the Tn4 transposon (mercury spectinomycin and sulfonamide resistance) have been determined. They are inverted repeated sequences of 38 nucleotides with three mismatched base pairs. These sequences are strongly homologous with the terminal sequences of Tn501 (mercury resistance) but less so with those of Tn3 (ampicillin resistance). The Tn4 transposon generates pentanucleotide members (Tn3, Tn1000, Tn501, Tn551, IS2) with the exception of Tn1721 and bacteriophage Mu. Among the three Tn4 insertion sites examined here, two of them occurred near a nonanucleotide sequence in perfect homology with part of the terminal inverted-repeat sequence of Tn4 and the third insertion occurred near a sequence of partial homology to one end of Tn4. All three insertions were in the same orientation such that IRb is proximal to its homologous sequence on the recipient DNA.     

DNA sequences of the integration sites and inverted repeated structure of transposon Tn3.

The nucleotide sequence of the "inverted repeat" structure of the transposon Tn3 was determined by the DNA sequencing procedure developed by Maxam and Gilbert(1). The sequence, 38 base pairs long, is as follows: 5'-GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG..(Tn3) 3'-CCCCAGACTGCGAGTCACCTTGCTTTTGAGTGCAATTC.. The integration of Tn3 is associated with a directly repeated sequence of 5 nucleotides appearing at each end of Tn3. The two directly repeated sequences so far determined are not the same. Furthermore, there is no homologous structure around the integration point of Tn3.     

Transposon Tn4556 of Streptomyces fradiae: nucleotide sequence of the ends and the target sites.

A transposon, Tn4556, has recently been isolated from Streptomyces fradiae (S.-T. Chung, J. Bacteriol. 169:4436-4441, 1987). The ends of Tn4556 were found to contain inverted repeats of 38 base pairs with 70% sequence identity with the ends of Tn3. Insertion of Tn4556 into a Streptomyces plasmid resulted in a 5-base-pair duplication of the target site.     

Conduction of pEC22, a plasmid coding for MR.EcoT22I, mediated by a resident Tn3-like transposon, Tn5396.

pEC22 is a small plasmid that encodes the restriction-modification system MR.EcoT22I. Restriction and functional analysis of the plasmid identified the positions of genes encoding that system. The plasmid is able to be conducted by conjugal plasmids, a process mediated by a transposon contained within pEC22. This cryptic transposon, called Tn5396, was isolated from pEC22 and partially sequenced. The sequence of Tn5396 is for the most part typical of transposons of the Tn3 family and is most similar to that of Tn1000. The transposon differs from closely related transposons in that it lacks well-conserved sequences in the inverted-repeat region and has an unusually long terminal inverted repeat. Consideration of regions of internal sequence similarity in this and other transposons in the Tn3 family supports a theory of the mechanism by which the ends of Tn3-like transposons may maintain substantial identity between their inverted repeats over the course of evolutionary time.     

Nucleotide sequences at the ends of the mercury resistance transposon, Tn501.

The nucleotide sequences at the ends of the mercury-resistance transposon, Tn501, have been determined. The terminal sequences are inverted repeated sequences 38 nucleotide pairs in length, which differ in 3 nucleotide pairs. The transposon is flanked by directly repeated sequences of 5 nucleotide pairs, originating from a single pentanucleotide sequence in the recipient replicon. There is no obvious homology between recipient replicons at the site of insertion of the transposon. The structures of the ends of Tn501 are compared with those of other transposons and insertion sequences. The use of Tn501 to locate an EcoRI site within a genetically defined sequence of interest is discussed.     

An alternative inverse PCR (IPCR) method to amplify DNA sequences flanking Tn5 transposon insertions

We have developed an alternative method to amplify DNA sequences flanking Tn5 transposon insertions. This method relies on the identical sequences of inverted terminal repeats, located at the 5' and 3' ends of Tn5, to determine the location and orientation of a transposon insertion within a restriction endonuclease fragment. From this information, PCR primers can be designed to selectively amplify by inverse PCR the DNA flanking one side of the transposon. This method avoids the problem of amplifying or cloning long sequences flanking Tn5. To demonstrate the applicability of this method, we generated Tn5 transposon mutants of Pseudomonas abietaniphila BKME-9 which no longer grew on dehydroabietic acid (DhA). The flanking sequence of one of the mutant (strain BKME-941) which accumulated 7-oxoDhA, was amplified.     

A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction.

We have developed a strategy to rapidly construct DNA hybridization probes for the isolation of genes disrupted by transposon Tn5 insertions. A single oligonucleotide complementary to and extending outward from the ends of the inverted repeat of Tn5 was used to prime DNA synthesis in the polymerase chain reaction. The amplified product consisted of DNA sequences adjacent to both ends of the transposon insertion. The general feasibility of the approach was tested by amplifying pBR322 sequences from a derivative of pBR322 containing a Tn5 insertion. To amplify genomic DNA sequences flanking a Tn5 insertion in the chromosome of a Pseudomonas syringae strain, circular substrates were generated by ligating EcoRI-digested genomic DNA. Tn5 was contained intact within one such circular molecule, as the transposon does not contain sites for cleavage by EcoRI. The amplified product (approximately 2.5 kb) was used as a DNA hybridization probe to isolate the homologous fragment from a cosmid library of wild-type Pseudomonas syringae genomic DNA. This approach may be applied to the efficient isolation of sequences flanking any Tn5 insertion.     

Nucleotide sequence of insertion sequence IS3411, which flanks the citrate utilization determinant of transposon Tn3411.

The nucleotide sequences of insertion sequences IS3411L (left) and IS3411R (right), present as direct terminal repeats in the citrate utilization of citrate utilization transposon Tn3411, and of IS3411 (generated by intramolecular recombination between IS3411L and IS3411R) were determined. The three IS3411 elements (IS3411R, IS3411L, and IS3411) were 1,309 base pairs long and identical in DNA sequence. IS3411 had 27-base-pair terminal inverted repeats with three bases mismatched and one long open reading frame (240 amino acids) that was proposed to be a transposase. Three polypeptides of 29,000, 27,000, and about 10,000 molecular weight, determined by IS3411, were identified in minicells. Since Tn3411 generates a 3-base-pair repeat upon integration, the nucleotide sequences of IS3411 were compared with those of IS3.     

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.     

Three Tn10-associated excision events: relationship to transposition and role of direct and inverted repeats

We describe three related DNA alterations associated with transposon Tn10: precise excision of Tn10, nearly precise excision of Tn10 and precise excision of the nearly precise excision remnant. DNA sequence analysis shows that each of these alterations results in excision of all or part of the Tn10 element, and each involves specific repeat sequences at or near the ends of the element. Furthermore, all three events are structurally analogous: in each case, excision occurs between two short direct-repeat sequences, with resulting deletion of all intervening material plus one copy of the direct repeat; and in all three cases, the direct repeats involved occur at either end of an inverted repeat. Analysis of mutant Tn10 elements and characterization of bacterial host mutations suggest that all three types of excision events occur by pathways that are fundamentally distinct from the pathway(s) for Tn10-promoted transposition and other DNA rearrangements (deletions and inversions) actively promoted by the element. In addition, precise excision and nearly precise excision appear to occur by very closely related or identical pathways; and several lines of evidence suggest that the 1400 bp inverted repeats at the ends of Tn10 may play a structural role in both of these events. The third excision event appears to occur by yet another pathway.     

Sequence analysis of termini of conjugative transposon Tn916.

Transposon Tn916 is a 16.4-kilobase, broad-host-range, conjugative transposon originally identified on the chromosome of Enterococcus (Streptococcus) faecalis DS16. Its termini have been sequenced along with the junction regions for two different insertions. The ends were found to contain imperfect inverted repeat sequences with identity at 20 of 26 nucleotides. Further in from the ends, imperfect directly repeated sequences were present, with 24 of 27 nucleotides matching. The transposon junction regions contained homologous segments but of a nature not consistent with a direct duplication of the target sequence. Within the right terminus was a potential outwardly reading promoter. Tn916 is believed to transpose via an excision-insertion mechanism; based on the analyses of the termini, as well as two target sequences (before insertion and after excision), a possible model is suggested.     

Location of an ampicillin resistance transposon, Tn1701, in a group of small, nontransferring plasmids.

By restriction endonuclease cleavage mapping and electron microscopic examination of heteroduplexes, we have identified an ampicillin resistance determinant transposon, designated Tn1701, in a group of small, nontransferring plasmids which confer resistance to ampicillin (Ap), sulfonamide (Su), and streptomycin (Sm). Plasmid NTP1, which mediates Ap resistance, contains Tn1701. Recombinant plasmids NTP3 (Ap Su) and NTP4 (Ap Su Sm) contain Tn1701, indicating that they were derived by transposition of Tn1701 from NTP1 to an unrelated plasmid, NTP2 (Su Sm). The transposon Tn1701 is very similar to the known ampicillin resistance transposons Tn1, Tn2, and Tn3 in its size (3.2 x 10(6) daltons), base sequence homology observed by heteroduplex formation, restriction endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. Like the other TnA elements, Tn1701 also specifies a type TEM beta-lactamase.     

Detection and characterization of Tn2501, a transposon included within the lactose transposon Tn951

The DNA sequence spanning coordinates 9.9 to 16.4 kilobases of the lactose transposon Tn951 ( Cornelis et al., Mol. Gen. Genet. 160:215-224, 1978) constitutes a transposable element by itself. Unlike Tn951 ( Cornelis et al., Mol. Gen. Genet. 184:241-248, 1981), this element, called Tn2501 , transposes in the absence of any other transposon. Transposition of Tn2501 proceeds through transient cointegration and duplicates 5 base pairs of host DNA. Tn2501 is flanked by nearly perfect inverted repeats (44 of 48), related to the inverted repeats of Tn21 ( Zheng et al., Nucleic Acids Res. 9:6265-6278, 1982). Unlike Tn21 , Tn2501 does not confer mercury resistance.     

Diversity of Tn4001 Transposition Products: the Flanking IS256 Elements Can Form Tandem Dimers and IS Circles

We show that both flanking IS256 elements carried by transposon Tn4001 are capable of generating head-to-tail tandem copies and free circular forms, implying that both are active. Our results suggest that the tandem structures arise from dimeric copies of the donor or vector plasmid present in the population by a mechanism in which an IS256 belonging to one Tn4001 copy attacks an IS256 end carried by the second Tn4001 copy. The resulting structures carry abutted left (inverted left repeat [IRL]) and right (inverted right repeat [IRR]) IS256 ends. Examination of the junction sequence suggested that it may form a relatively good promoter capable of driving transposase synthesis in Escherichia coli. This behavior resembles that of an increasing number of bacterial insertion sequences which generate integrative junctions as part of the transposition cycle. Sequence analysis of the IRL-IRR junctions demonstrated that attack of one end by the other is largely oriented (IRL attacks IRR). Our experiments also defined the functional tips of IS256 as the tips predicted from sequence alignments, confirming that the terminal 4 bp at each end are indeed different. The appearance of these multiple plasmid and transposon forms indicates that care should be exercised when Tn4001 is used in transposition mutagenesis. This is especially true when it is used with naturally transformable hosts, such as Streptococcus pneumoniae, in which reconstitution of the donor plasmid may select for higher-order multimers.     

Transposition of the kanamycin-resistance transposon Tn903

Summary The insertion of the kanamycin-resistance transposon, Tn903, into the Escherichia coli chromosome was studied. Tn903 is similar in structure to the well known transposons Tn5 and Tn10 in that it has a unique central sequence flanked by inverted repeat sequences extending more than a thousand base pairs. However, the central region of Tn903 has enough single-frame coding capacity only for the drug modifying enzyme, whereas Tn5 and Tn10 carry multigenic unique sequences. In this paper we demonstrate that two different classes of insertion event occur: (1) the first class is a complex event in which all or part of the genome of the bacteriophage lambda vector is co-inserted near the purE locus on the E. coli chromosome (11.7 min); (2) the second class appears to be a simple transposition event in which the transposon alone is inserted at relatively nonspecific sites in the chromosome, as has been described for Tn5 and Tn10. Furthermore both classes show dependency on homology-requiring recombination systems. We suggest that Tn903 transposes infrequently because it must utilize a recA-controlled host function, whereas Tn5 and Tn10 are recA-independent and encode similar but more active functions on the transposon DNA.     

Tn2610, a transposon involved in the spread of the carbenicillin-hydrolyzing beta-lactamase gene

We have found a new transposon, Tn2610, on pCS200 in clinical isolates of Escherichia coli, which encodes the carbenicillin-hydrolyzing beta-lactamase gene in combination with the resistance determinants to streptomycin and sulfonamide. Tn2610 has a molecular size of 24 kilobase pairs and is flanked by long inverted repeat sequences of 3 kilobase pairs in length. Genetical and physical analyses indicate that Tn2610 is a single transposable unit encoding the multiple resistance determinants and that is different from any previously described transposon. The characteristic DNA structure observed in various complex resistance transposons involved in the transposition of the carbenicillin-hydrolyzing beta-lactamase gene is discussed.     

Evidence for Campbell's mechanism for fine excision of Tn9 type transposons in Escherichia coli K12]

The plasmid-transposon Tn9-322 was constructed by inverted transposition from the pBR322::Tn9 plasmid. The precise excision of the Tn9-322 transposon from the proB gene site can proceed by the Campbell's model. This fact was demonstrated by appearance of the plasmid-transposons after their precise excision. They contain two IS1 elements flanking a short direct repeat of the target DNA. The recombinational mechanism of precise excision of Tn9 type transposons seems not to be alternative but looks as an additional one to a well-known slippage mechanism proved for Tn5 and Tn10.     

IS10/Tn10 transposition efficiently accommodates diverse transposon end configurations.

Transposon Tn10 and its component insertion sequence IS10 move by non-replicative transposition. We have studied the array of reaction intermediates and products in a high efficiency in vitro IS10/Tn10 transposition reaction. Synapsis of two transposon ends, followed by cleavage and strand transfer, can occur very efficiently irrespective of the relative locations and orientations of the two ends. The two participating ends can occur in inverted or direct orientation on the same molecule or, most importantly, on two different molecules. This behavior contrasts sharply with that of Mu, in which transposition is strongly biased in favor of inverted repeat synapsis. Mechanistically, the absence of discrimination amongst various end configurations implies that the architecture within the IS10/Tn10 synaptic complex is relatively simple, i.e. lacking any significant intertwining of component DNA strands. Biologically these observations are important because they suggest that the IS10 insertion sequence module has considerable flexibility in the types of DNA rearrangements that it can promote. Most importantly, it now seems highly probable that a single non-replicative IS10 element can promote DNA rearrangements usually attributed to replicative transposition, i.e. adjacent deletions and cointegrates, by utilizing transposon ends on two sister chromosomes. Other events which probably also contribute to the diversity of IS10/Tn10-promoted rearrangements are discussed.