DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3.

The complete nucleotide sequence of the transposon Tn3 and of 20 mutations which affect its transposition are reported. The mutations, generated in vitro by random insertion of synthetic restriction sites, proved to contain small duplications or deletions immediately adjacent to the new restriction site. By determining the phenotype and DNA sequence of these mutations we were able to generate an overlapping phenotypic and nucleotide map. This 4957 bp transposon encodes three polypeptides which account for all but 350 bp of its total coding capacity. These proteins are the transposase, a high molecular weight polypeptide (1015 amino acids) encoded by the tnpA gene; the Tn3-specific repressor, a low molecular weight polypeptide (185 amino acids) encoded by the tnpR gene; and the 286 amino acid beta-lactamase. The 38 bp inverted repeats flanking Tn3 appear to be absolutely required in cis for Tn3 to transpose. Genetic data suggest that Tn3 contains a third site (Gill et al., 1978), designated IRS (internal resolution site), whose absence results in the insertion of two complete copies of Tn3 as direct repeats into the recipient DNA. We suggest that these direct repeats of complete copies of Tn3 are intermediates in transposition, and that the IRS site is required for recombination and subsequent segregation of the direct repeats to leave a single copy of Tn3 (Gill et al., 1978). A 23 nucleotide sequence within the amino terminus of the transposase which shares strong sequence homology with the inverted repeat may be the internal resolution site.     

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.     

A gene and its product required for transposition of resistance transposon Tn2603.

Tn2603 is a multiple-resistance transposon encoding resistance to ampicillin, streptomycin, sulfonamide, and mercury and having a molecular size of 20 kilobase pairs, with 200-base-pair inverted repeats at both ends. The essential sites and functions of Tn2603 which are required for its transposition were determined through construction and characterization of various deletion mutants affecting the efficiency of transposition. Deletions were introduced in plasmid pMK1::Tn2603 by partial digestion with restriction endonuclease EcoRI in vitro. Analysis of deletion mutants showed that the inverted repeat segments at both ends of the trans-acting diffusible product(s) encoded in the right-hand side of the central portion were required for the transposition of Tn2603. An essential gene product was revealed as a protein having a molecular weight of 110,000 by analysis of polypeptides synthesized in Escherichia coli minicells. This protein was assumed to be the so-called transposase.     

Molecular genetic analysis of the Tn5041 transposition system

A study was made of the transposition of the mercury resistance transposon Tn5041 which, together with the closely related toluene degradation transposon Tn4651, forms a separate group in the Tn3 family. Transposition of Tn5041 was host-dependent: the element transposed in its original host Pseudomonas sp. KHP41 but not in P. aeruginosa PAO-R and Escherichia coli K12. Transposition of Tn5041 in these strains proved to be complemented by the transposase gene (tnpA) of Tn4651. The gene region determining the host dependence of Tn5041 transposition was localized with the use of a series of hybrid (Tn5041 x Tn4651) tnpA genes. Its location in the 5'-terminal one-third of the transposase gene is consistent with the data that this region is involved in the formation of the transposition complex in transposons of the Tn3 family. As in other transposons of this family, transposition of Tn5041 occurred via cointegrate formation, suggesting its replicative mechanism. However, neither of the putative resolution proteins encoded by Tn5041 resolved the cointegrates formed during transposition or an artificial cointegrate in E. coli K12. Similar data were obtained with the mercury resistance transposons isolated from environmental Pseudomonas strains and closely related to Tn5041 (Tn5041 subgroup).     

Fine structure of transposition genes on Tn2603 and complementation of its tnpA and tnpR mutations by related transposons

The fine structure of the genes tnpA, tnpR and res of Tn2603 required for its own transposition, was determined. The order of the genes was tnpA-tnpR-res from the right end of the right hand side region in Tn2603, the tnpA and tnpR encoded gene products having molecular weights of 110,000 and 21,000, respectively. The 110,000 molecular weight polypeptides was absolutely required for replicon fusion as the first stage of transposition, and named transposase. On the other hand, the 21,000 molecular weight polypeptide was necessary for resolution of the cointegrate as the second stage of transposition, and named resolvase. We also examined the ability of various transposons, assumed to be closely related, to complement the tnpA and tnpR mutations of Tn2603. The results indicated that the mercury resistance transposon, Tn2613, and Tn501, can complement both genes, but TnAs and gamma delta cannot at all. Tn501 had much less efficiency of complementation for tnpA than Tn2613. We have also discovered that the transposition frequency of transposons in the tn2613 family systematically depend on their size of transposon.     

Structural and functional characterization of tnpI, a recombinase locus in Tn21 and related beta-lactamase transposons

A novel discrete mobile DNA element from Tn21 from the plasmid R100.1 is described, and its mobilization function was confirmed experimentally. In addition, the element behaves as a recombinase-active locus (tnpI) which facilitates insertions of antibiotic resistance genes as modules or cassettes at defined hot spots or integration sites. A similar tnpI sequence was detected by DNA hybridization in a series of beta-lactamase transposons and plasmids and localized on their physical maps. The genetic function of the locus cloned from Tn21 into pACYC184 was tested for conduction and integration into the plasmids R388 and pOX38Km, and the results suggested recombinase-integrase activity and recA independence. DNA sequence analysis of the tnpI locus revealed no inverted or direct terminal repeats or transposition features of class I and class II transposons. The coding capacity revealed three putative open reading frames encoding 131, 134, and 337 amino acids. Orf3 encoded a putative polypeptide product of 337 amino acids that shared highly significant identity with the carboxyl region of integrase proteins. A comparison and an alignment of the tnpI locus from Tn21 and its flanking sequences identified similar sequences in plasmids and in transposons. The alignment revealed discrete nucleotide changes in these tnpI-like loci and a conserved 3' and 5' GTTA/G hot spot as a duplicated target site. Our data confirm the remarkable ubiquity of tnpI associated with antibiotic resistance genes. We present a model of transposon modular evolution into more complex multiresistant units via tnpI and site-specific insertions, deletions, and DNA rearrangements at this locus.     

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.     

Restriction endonuclease cleavage maps of the ampicillin transposons Tn2601 and Tn2602

This paper reports the cleavage maps of ampicillin transposons Tn2601 and Tn2602, for restriction endonucleases BamHI, PvuII, AvaI, HincII, and HaeII. Both of the transposons are very similar to the well-known ampicillin transposon Tn3 in size, endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. A slight difference in the cleavage pattern among these three transposons was observed in the region around the BamHI site which was assumed to be a part of the repressor gene for transposition.     

Nucleotide sequence of the kanamycin resistance transposon Tn903

The entire nucleotide sequence of the kanamycin resistance transposon Tn903 was determined by analyzing a mini-ColE1 derivative carrying Tn903. Tn903 was 3094 base-pairs in length and at both extremities possessed two identical inverted 1057 base-pair sequences. Furthermore, 18 bases at the ends of the 1057 base-pair sequence are themselves present in an invertedly repeated order as has been described for various insertion sequences. Analysis of initiation and termination codons in the Tn903 sequence indicated that Tn903 could possibly code for at least three high molecular weight polypeptides. One in the region between the two 1057 base-pair sequences is suggested to be the kanamycin resistance determinant (aminoglycoside 3′-phosphotransferase) from its location and size. The other polypeptides were located within the 1057 base-pair sequence and may be associated with transposition functions of Tn903.     

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.     

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.     

Evolution of Tn21-related transposons: isolation of Tn2425, which harbours IS161

The isolation of two multi-resistance transposons, Tn2425 and Tn1831, and their relation to Tn21 and Tn2424, is described. A 1.7 kb segment present in Tn2424 and Tn2425 was identified as an IS element by rec-independent transposition, resulting in a cointegrate structure that carries two direct repeated copies of the IS element. By the isolation of this IS element we demonstrated that transposition is one mechanism leading to sequence variations in Tn21-like structures, especially in the region between the mer operon and the sul gene.     

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.     

Analysis of sequences transposed by complementation of two classes of transposition-deficient mutants of Tn3.

The Tn1 and Tn3 elements are closely related transposons which carry the structural gene for ampicillin resistance. Two classes of deletion mutants of the plasmid pMB8::Tn3 (RSF1050) are unable to transpose ampicillin resistance but can be complemented in trans by a coresident Tn1 or Tn3 element. The analysis of the sequences transposed upon complementation of one class of mutants (type I) showed that the mutant element had undergone bona fide transposition. Complementation of the type II mutants led to the transposition of a sequence analogous to bacteriophage mu-promoted integration of non-mu DNA. The transposed sequence consisted of two Tn3 elements which flanked a single copy of the pMB8 portion of the RSF1050 genome. Complementation data indicated that the type II mutants are defective in at least one trans-acting function which must be supplied for transposition to occur. The nature of sequence transposed from the type II mutant is the consequence of a defective cis-acting function (or site). In addition, the type II mutants were defective in a trans-acting function which regulated the frequency of transposition.     

Tn951 derivatives designed for high-frequency plasmid-specific transposition and deletion mutagenesis

We describe the construction of a system allowing high-frequency transposition and deletion mutagenesis with class-II transposons containing a kanamycin or a chloramphenicol-resistance marker. The system utilizes the transposition function of Tn3 and the resolution function of Tn951/Tn2501 which leads to an uncoupling of the resolution and repression functions. It consists of defective transposons inserted into conjugative, replication thermosensitive plasmids. The properties of the system are: easily selectable resistance markers, high transposition frequencies onto plasmids, low transposition frequencies onto the host chromosome, placement of the tnpA gene outside the transposons so that "second-generation" transposition does not occur, possibility to transpose the whole system onto other plasmid vectors with different selection strategies, consecutive use of two transposons for deletion mutagenesis and restriction mapping.     

Genetic properties of transposons Tn6-1, Tn6-2 and Tn19-1

The level and range transposition of the transposons Tn6-1, Tn6-2, Tn19-1, and their ability to influence plasmid transfer has been studied. The widest range of transposition was shown for transposon Tn6-2. Insertions of each of the studied transposons into different conjugative plasmids genomes resulted in change of frequencies of plasmids transfer and change of plasmids mobilization activity.     

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.     

Identification and characterization of Tn4656, a novel class II transposon carrying a set of toluene-degrading genes from TOL plasmid pWW53

It has been reported that the toluene-degrading (xyl) genes from Pseudomonas putida plasmid pWW53 are able to translocate to broad-host-range drug resistance plasmid RP4, and pWW53-4 is one of the smallest RP4 derivatives (H. Keil, S. Keil, R. W. Pickup, and P. A. Williams, J. Bacteriol. 164:887-895, 1985). Our investigation of pWW53-4 in this study demonstrated that such a translocated region that is 39 kb long is a transposon. This mobile element, Tn4656, was classified as a class II transposon since its transposition occurred by a two-step process: transposase (TnpA)-mediated formation of the cointegrate and resolvase (TnpR)-mediated site-specific resolution of the cointegrate at the two copies of the res site. The Tn4656 TnpA and TnpR functions encoded in the rightmost 4-kb region were found to be exchangeable with those specified by other Tn1721-related class II transposons, including another toluene transposon, Tn4653. Sequence analysis of the transposition-related genes and sites of Tn4656 also supported the hypothesis that this transposon is closely related to the Tn1721-related transposons. The lower transposition frequency of Tn4656 has been suggested to be due to the unique nucleotide sequence of one of the terminal 39-bp inverted repeats.