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.     

Binding of the Tn3 transposase to the inverted repeats of Tn3

The transposase protein and the inverted repeat sequences of Tn3 are both essential for Tn3 cointegrate formation and transposition. We have developed two assays to detect site-specific binding of transposase to the inverted repeats: (1) a nitrocellulose filter binding assay in which transposase preferentially retains DNA fragments containing inverted repeat sequences, and (2) a DNase 1 protection assay in which transposase prevents digestion of the inverted repeats by DNase 1. Both assays show that transposase binds directly to linear, duplex DNA containing the inverted repeats. The right inverted repeat of Tn3 binds slightly more strongly than the left one. Site-specific binding requires magnesium but does not require a high energy cofactor.     

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.     

Junction sequences generated by ''one-ended transposition''.

In the presence of the cognate transposase, plasmids containing a single inverted repeat (IR) sequence of Tn21 or of Tn1721 can fuse efficiently with other plasmids ('one-ended transposition'). The junctions across the sequences of donor and recipient DNA in recombinants generated by this process have been determined. These show that the segment of donor DNA starts precisely at the IR sequence (it is variable at the other end), and is flanked by a direct repeat of host DNA (usually 5bp) that was present only once in the original host sequence. These are characteristics of recombinants generated by transposition of Tn21 and Tn1721 themselves, suggesting that the mechanism of one-ended transposition is very similar to that of the corresponding entire elements.     

Physical structures of Tn10-promoted deletions and inversions: role of 1400 bp inverted repetitions.

We report here the physical structures of deletions and inversions promoted by the translocatable tetracycline-resistance element Tn10. DNA/DNA heteroduplex and restriction enzyme analyses of alterations in the genome of bacteriophage lambda suggest that both types of DNA alterations almost always originate at the internal termini of the 1400 bp terminal inverted repetitions of Tn10. Tn10-promoted deletions remove a single contiguous DNA segment beginning at one such terminus; Tn10-promoted inversions are more complex, and involve both an inversion and a specific deletion of Tn10 DNA.     

Complete sequence determination combined with analysis of transposition/site-specific recombination events to explain genetic organization of IncP-7 TOL plasmid pWW53 and related mobile genetic elements

Recent studies have indicated that the evolutionarily common catabolic gene clusters are loaded on structurally diverse toluene-catabolic (TOL) plasmids and their residing transposons. To elucidate the mechanisms supporting the diversification of catabolic plasmids and transposons, we determined here the complete 107,929 bp sequence of pWW53, a TOL plasmid from Pseudomonas putida MT53. pWW53 was found to belong to the IncP-7 incompatibility group that play important roles in the catabolism of several xenobiotics. pWW53 carried two distinct transposase-resolvase gene clusters (tnpAR modules), five short terminal inverted repeats (IRs), and three site-specific resolution (res) sites that are all typical of class II transposons. This organization of pWW53 suggested the four possible transposable regions, Tn4657 to Tn4660. The largest 86 kb region (Tn4657) spanned the three other regions, and Tn4657 and Tn4660 (62 kb) covered all of the 36 xyl genes for toluene catabolism. Our subsequent transposition experiments clarified that the three transposons, Tn4657 to Tn4659, indeed exhibit their transposability, and that pWW53 also generated another 37 kb toluene-catabolic transposon, Tn4656, which carried the two separated and inversely oriented segments of pWW53: the tnpRA-IR module of Tn4658 and a part of xyl gene clusters on Tn4657. The Tn4658 transposase was able to mediate the transposition of Tn4658, Tn4657, and Tn4656, while the Tn4659 transposase catalyzed only the transposition of Tn4659. Tn4656 was formed by the Tn4658 resolvase-mediated site-specific inversion between the two inversely oriented res sites on pWW53. These findings and comparison with other catabolic plasmids clearly indicate multiple copies of transposition-related genes and sites on one plasmid and their recombination activities contribute greatly to the diversification of plasmid structures as well as wide dissemination of the evolutionary common gene clusters in various plasmids.     

Involvement of E. coli dcm methylase in Tn3 transposition

The effects of DNA methyltransferases on Tn3 transposition were investigated. The E. coli dam (deoxyadenosine methylase) gene was found to have no effect on Tn3 transposition. In contrast, Tn3 was found to transpose more frequently in dcm+ (deoxycytosine methylase) cells than in dcm- mutants. When the EcoRII methylase gene was introduced into dcm- cells (E. coli strain GM208), the frequency of Tn3 transposition in GM208 was dramatically increased. The EcoRII methylase recognizes and methylates the same sequence as does the dcm methylase. These results suggest that deoxycytosine methylase modified DNA may be a preferred target for Tn3 transposition. Experiments were also performed to determine whether the Tn3 transposase was involved in DNA modification. Plasmid DNA isolated from dcm- E. coli containing the Tn3 transposase gene was susceptible to ApyI digestion but resistant to EcoRI digestion, suggesting that Tn3 transposase modified the dcm recognition sequence. In addition, restriction enzymes TaqI, AvaII, BglI and HpaII did not digest this DNA completely, suggesting that the recognition sequences of TaqI, AvaII, BglI and HpaII were modified by Tn3 transposase to a certain degree. The type(s), the extent and mechanism(s) of this modification remain to be investigated.     

Molecular structures of packageable ColE1 hybrids and the generation of deletion mutants from one of the hybrids

ColE1 derivatives carrying cohesive end sites of lambda phage genome (= cos lambda) can be packaged within lambda phage particles. The DNA structure of the prototype ColE1-cos lambda derivative named pKY2257 was studied because of its potential usefulness in various fields in molecular biology. pKY2257, which carries an intact galactose operon of E. coli, is a convenient replicon to detect Tn3 translocation. It was found that one of the PK2257::Tn3 derivatives, pKY2113, generated various small plasmids in E. coli. The molecular structures of some of these deletion mutants were compared with each other and with those of parental plasmid DNAs by heteroduplex analysis and restriction enzyme digestion. A possible mechanism, which seems to be unique to this kind of deletions, is discussed on the basis of the present results.     

Nucleotide sequence of diatom plasmids: identification of open reading frames with similarity to site-specific recombinases

We have determined the nucleotide sequence of two small circular DNA plasmids, pCf1 and pCf2 [22], from the marine diatom Cylindrotheca fusiformis. pCf1 is 4273 bp, and pCf2 is 4079 bp in size. In each plasmid, all of the major open reading frames (ORFs) are encoded on the same DNA strand. Two ORFs are similar, comparing the two plasmids. ORF218 (pCf1) and ORF217 (pCf2) share 80% amino acid identity and ORF482 (pCf1) and ORF484 (pCf2) share 54% amino acid identity. ORF218/217 shows significant similarity (28–31% amino acid identity) to the Tn3 class of resolvases. Resolvases are most commonly found in bacterial transposons. However, two other features found in the Tn3 class of transposon are missing in the plasmids; an ORF encoding a transposase and terminal inverted repeat sequences. This, and data mapping the portions of the plasmids that hybridize to genomic chloroplast DNA, suggest that the plasmids do not contain active transposons. By analogy with the R46 plasmid from Enterobacter [5, 6], another potential role for the resolvases encoded by pCf1 and pCf2 is the conversion of multimeric forms of the plasmid to monomers. The similarity of ORF218/217 to resolvases documents the first identification of a potential coding function in an algal plasmid.     

Equimolar generation of the four possible arrangements of adjacent L components in herpes simplex virus type 1 replicative intermediates.

Herpes simplex virus type 1 (HSV-1) replication generates high-molecular-weight intermediates containing branched DNA and concatemers carrying adjacent genomes with inverted L components. We have studied replicative intermediates generated by (i) wild-type HSV-1; (ii) 5dl1.2, an ICP27 null mutant which fails to synthesize normal amounts of DNA and late proteins; (iii) RBMu3, a mutant containing a deletion in the inverted repeats which fails to generate genomic isomers; and (iv) amplicon plasmids and vectors which contain no inverted sequences. Replication intermediates were analyzed by pulsed-field gel electrophoresis, after restriction enzyme digestion of infected-cell DNA, followed by blot hybridization. DNA fragments were statistically quantified after phosphorimaging. We observed that (i) the four possible configurations of L components of two adjacent genomes in the concatemers are present at equimolar amounts at any time during virus replication, (ii) ICP27 is not required for inversions or for branched DNA to occur, and (iii) replication intermediates of both RBMu3 mutant and amplicon plasmids or vectors do contain branched structures, although the concatemers they generate contain no inversions. These data indicate that inversions are generated by a mechanism intrinsically linked to virus DNA replication, most likely homologous recombination between inverted repeats. Branched structures are detected in all replicating molecules, including those that do not invert, suggesting that they are constitutively linked to virus DNA synthesis. Our results are consistent with the notion that the four HSV-1 genomic isomers are generated by alternative cleavage frames of replication concatemers containing equimolar amounts of L-component inversions.     

Unexpected structural diversity in DNA recombination: the restriction endonuclease connection

Transposition requires a coordinated series of DNA breakage and joining reactions. The Tn7 transposase contains two proteins: TnsA, which carries out DNA breakage at the 5' ends of the transposon, and TnsB, which carries out breakage and joining at the 3' ends of the transposon. TnsB is a member of the retroviral integrase superfamily whose hallmark is a conserved DDE motif. We report here the structure of TnsA at 2.4 A resolution. Surprisingly, the TnsA fold is that of a type II restriction endonuclease. Thus, Tn7 transposition involves a collaboration between polypeptides, one containing a DDE motif and one that does not. This result indicates that the range of biological processes that utilize restriction enzyme-like folds also includes DNA transposition.     

Plasmid-borne cadmium resistance genes in Listeria monocytogenes are present on Tn5422, a novel transposon closely related to Tn917.

The complete (6,449-bp) nucleotide sequence of the first-described natural transposon of Listeria monocytogenes, designated Tn5422, was determined. Tn5422 is a transposon of the Tn3 family delineated by imperfect inverted repeats (IRs) of 40 bp. It contains two genes which confer cadmium resistance (M. Lebrun, A. Audurier, and P. Cossart, J. Bacteriol. 176:3040-3048, 1994) and two open reading frames that encode a transposase (TnpA) and a resolvase (TnpR) of 971 and 184 amino acids, respectively. The cadmium resistance genes and the transposition genes are transcribed in opposite directions and are separated by a putative recombination site (res). The structural elements presumed to be involved in transposition of Tn5422 (IRs, transposase, resolvase, and res) are very similar to those of Tn917, suggesting a common origin. The transposition genes were not induced by cadmium. Analysis of sequences surrounding Tn5422 in nine different plasmids of L. monocytogenes indicated that Tn5422 is a functional transposon, capable of intramolecular replicative transposition, generating deletions. This transposition process is probably the reason for the size diversity of the L. monocytogenes plasmids. Restriction analysis and Southern hybridization revealed the presence of Tn5422 in all the plasmid-mediated cadmium-resistant L. monocytogenes strains tested but not in strains encoding cadmium resistance on the chromosome.     

Structure and function of Tn5467, a Tn21-like transposon located on the Thiobacillus ferrooxidans broad-host-range plasmid pTF-FC2.

A 3.5-kb region of plasmid pTF-FC2, which contains a transposon-like element designated Tn5467, has been sequenced, and its biological activity has been investigated. The transposon is bordered by two 38-bp inverted repeat sequences which have sequence identity in 37 of 38 and in 38 of 39 bp to the tnpA distal and tnpA proximal inverted repeats of Tn21, respectively. Within these borders, open reading frames with amino acid similarity to a glutaredoxin-like protein, a MerR regulatory protein, and a multidrug-resistant-membrane transport-like protein were found. The gene for the glutaredoxin-like protein was expressed in Escherichia coli and enabled growth of a glutathione-requiring E. coli trxA gshA mutant on minimal medium and the reduction of methionine sulfoxide to methionine. In addition, there were two regions which, when translated, had homology to 85% of the N-terminal region of the Tn21 resolvase (tnpR) and to 15% of the C terminus of the Tn21 transposase (tnpA). A region containing res-like sites was located immediately upstream of the partial tnpR gene. Neither the partial transposase nor the resolvase genes of Tn5467 were biologically active, but Tn5467 was transposed and resolved when the Tn21 transposase and resolvase were provided in trans. Tn5467 appears to be a defective transposon which belongs to the Tn21 subgroup of the Tn3 family.     

A derivative of Tn5 with direct terminal repeats can transpose

The 5.7 kb⁴ transposable kanamycin resistance determinant Tn5 contains 1.5 kb terminal inverted repeats which we here call arms. Tn5's arms contain the genes and sites necessary for Tn5 transposition, and are not homologous to previously described transposable elements. To determine whether one or both arms is a transposable (IS) element, we transposed Tn5 to pBR322 and used restriction endonuclease digestion and ligation in vitro to generate plasmid derivatives designated pTn5-DR1 and pTn5-DR2 in which Tn5's arms were present in direct rather than in inverted orientation. Analysis of transposition products from dimeric forms of the pTn5-DR1 plasmid to phage λ showed that the outside and inside termini of right and of left arms could function in transposition. We conclude that both of Tn5's arms are transposable elements and name them IS50L (left) and IS50R (right). IS50R, which encodes transposase, was used several-fold more frequently than IS50L, which contain an ochre mutant allele of transposase: this implies that Tn5's transposase acts preferentially on the DNA segment which encodes it. Analysis of transpositions of the amp^rkan^r element Tn5-DR2 to the lac operon showed that Tn5-DR2, like Tn5 wild-type, exhibits regional preference without strict site specificity in the choice of insertion sites.     

Site-specific relaxation and recombination by the Tn3 resolvase: recognition of the DNA path between oriented res sites

We studied the dynamics of site-specific recombination by the resolvase encoded by the Escherichia coli transposon Tn3. The pure enzyme recombined supercoiled plasmids containing two directly repeated recombination sites, called res sites. Resolvase is the first strictly site-specific topoisomerase. It relaxed only plasmids containing directly repeated res sites; substrates with zero, one or two inverted sites were inert. Even when the proximity of res sites was ensured by catenation of plasmids with a single site, neither relaxation nor recombination occurred. The two circular products of recombination were catenanes interlinked only once. These properties of resolvase require that the path of the DNA between res sites be clearly defined and that strand exchange occur with a unique geometry. A model in which one subunit of a dimeric resolvase is bound at one res site, while the other searches along adjacent DNA until it encounters the second site, would account for the ability of resolvase to distinguish intramolecular from intermolecular sites, to sense the relative orientation of sites and to produce singly interlinked catenanes. Because resolvase is a type 1 topoisomerase, we infer that it makes the required duplex bDNA breaks of recombination one strand at a time.     

Novel one-step cloning vector with a transposable element: application to the Myxococcus xanthus genome.

A new strategy was developed for rapid cloning of genes with a transposon mutation library. We constructed a transposon designated TnV that was derived from Tn5 and consists of the gene coding for neomycin phosphotransferase II as well as the replication origin of an Escherichia coli plasmid, pSC101, flanked by Tn5 inverted repeats (IS50L and IS50R). TnV can transpose to many different sites of DNA in E. coli and Myxococcus xanthus and confers kanamycin resistance (Kmr) to the cells. From the Kmr cells, one-step cloning of a gene which is mutated as a result of TnV insertion can be achieved as follows. Chromosomal DNA isolated from TnV-mutagenized cells is digested with an appropriate restriction enzyme, ligated, and transformed into E. coli cells with selection for Kmr. The plasmids isolated contain TnV in the target gene. The plasmid DNA can then be used as a probe for characterization of the gene and screening of clones from a genomic library. We used this vector to clone DNA fragments containing genes involved in the development of M. xanthus.     

Cointegrates carrying two copies of a Tn3 derivative in an inverted orientation

We constructed a mutant of Tn3, Tn3 #2, that contains a 55-bp direct repeat of sequences near the amino-terminal coding region of the transposase, and an 8-bp EcoRI linker. This mutant transposase is functional. The plasmid carrying Tn3 #2, pMB8::Tn3 #2, recombines with the plasmid pHS1 at a frequency of 2.8 X 10(-7) recombinants per division cycle. This is similar to the recombination frequency of pHS1 and pMB8::Tn3+ (wild-type) which is 4.5 X 10(-6) recombinants per division cycle. One-third of the recombinants between pMB8::Tn3 #2 and pHS1 were approx. 22 kb in length. Restriction analysis and nucleotide sequencing showed that these large plasmids were Tn3 #2-mediated cointegrates formed by integration of pMB8::Tn3 #2 into pHS1. However, unlike Tn3 tnpR- -mediated cointegrates that contain direct repeats of the incoming element, Tn3 #2-mediated cointegrates carry two copies of Tn3 #2 in the form of inverted repeats. Like the tnpR- repeats, the Tn3 #2 repeats occur at both junctions between the parental plasmids, and are associated with a 5-bp direct duplication of the pHS1 target site. Furthermore, these recombinants contain a small deletion starting precisely at the end of Tn3 #2 and extending into pMB8 sequences. We propose a model for the generation of Tn3 #2-mediated cointegrates.     

Characterization of incompatibility group HI1 plasmids from Salmonella typhi by restriction endonuclease digestion and hybridization of DNA probes for Tn3, Tn9, and Tn10

Chloramphenicol resistance in Salmonella typhi is medicated by plasmids of the incompatibility group H, subgroup 1 (IncHI1). Eight IncHI1 plasmids from S. typhi strains originating in Mexico, Vietnam, Thailand, and India were examined by restriction enzyme digestion. The restriction enzymes, Apal, Xbal, and PstI were found to be most useful for comparison of plasmid DNAs. Four plasmids from S. typhi isolated in Mexico, Vietnam, and Thailand between 1972 and 1974 had identical restriction patterns with all three enzymes. The other IncHI1 plasmids showed only minor differences. However, some significant differences were noted between these IncHI1 plasmids and the prototype IncHI1 plasmid R27, which was isolated from S. typhimurium in 1961 and for which a restriction map has been constructed. Southern transfer hybridization with a nick-translated HI1 plasmid as a probe confirmed that there is a great deal of sequence homology among the IncHI1 plasmids. DNA probes were used to locate DNA sequences for ampicillin resistance (Tn3), chloramphenicol resistance (Tn9), tetracycline resistance (Tn10), and the one-way incompatibility between IncHI1 plasmids and the F factor, a characteristic property of IncHI1 plasmids. The results demonstrate that IncHI1 plasmids isolated from S. typhi from widely different geographic sources are very similar. Comparisons between the S. typhi plasmids and R27 indicated that conserved regions of DNA were those involved in conjugative transfer.