Intramolecular transposition by a synthetic IS50 (Tn5) derivative.

We report the formation of deletions and inversions by intramolecular transposition of Tn5-derived mobile elements. The synthetic transposons used contained the IS50 O and I end segments and the transposase gene, a contraselectable gene encoding sucrose sensitivity (sacB), antibiotic resistance genes, and a plasmid replication origin. Both deletions and inversions were associated with loss of a 300-bp segment that is designated the vector because it is outside of the transposon. Deletions were severalfold more frequent than inversions, perhaps reflecting constraints on DNA twisting or abortive transposition. Restriction and DNA sequence analyses showed that both types of rearrangements extended from one transposon end to many different sites in target DNA. In the case of inversions, transposition generated 9-bp direct repeats of target sequences.     

Saturation mutagenesis of the inside end of insertion sequence IS50

A 19-bp segment at the inside (I) end of IS50 (Tn5) is needed for efficient transposition. The importance of each position was assayed by making at least one base substitution at each position by either chemical-or oligodeoxyribonucleotide-directed mutagenesis. Mutant I ends were paired with a wild-type (wt) segment from the outside (O) end of IS50 and the transposase (tnp) gene was placed either between the ends or 1200 bp from the O end. The frequency of transposition of the resultant elements to bacteriophage lambda was measured. At least one substitution at each of the 19 I-end positions decreased transposition activity to less than 25% of wt, and most substitutions (25 of 28) decreased it to less than 5% of wt from one or both donor plasmids. These results show that each position in the I end is important during transposition.     

Characterization of a class II defective transposon carrying two haloacetate dehalogenase genes from Delftia acidovorans plasmid pUO1

The two haloacetate dehalogenase genes, dehH1 and dehH2, on the 65-kb plasmid pUO1 from Delftia acidovorans strain B were found to be located on transposable elements. The dehH2 gene was carried on an 8.9-kb class I composite transposon (TnHad1) that was flanked by two directly repeated copies of IS1071, IS1071L and IS1071R. The dehH1 gene was also flanked by IS1071L and a truncated version of IS1071 (IS1071N). TnHad1, dehH1, and IS1071N were located on a 15.6-kb class II transposon (TnHad2) whose terminal inverted repeats and res site showed high homology with those of the Tn21-related transposons. TnHad2 was defective in transposition because of its lacking the transposase and resolvase genes. TnHad2 could transpose when the Tn21-encoded transposase and resolvase were supplied in trans. These results demonstrated that Tn Had2 is a defective Tn21-related transposon carrying another class I catabolic transposon.     

IS50-mediated inverse transposition: specificity and precision

The IS50 elements, which are present as inverted repeats in the kanamycin-resistance transposon, Tn5, can move in unison carrying with them any interstitial DNA segment. In consequence, DNA molecules such as a lambda::Tn5 phage genome are composed of two overlapping transposons - the kan segment bracketed by IS50 elements (Tn5), and lambda bracketed by IS50 elements. During direct transposition, mediated by IS50 "O" (outside) ends, the kan gene is moved and the lambda vector is left behind. During inverse transposition, mediated by the "I" (inside) ends of the IS50 elements, the lambda vector segment is moved and the kan gene is left behind. Direct transposition is several orders of magnitude more frequent than inverse transposition (Isberg and Syvanen, 1981; Sasakawa and Berg, 1982). We assessed the specificity and precision of the rare events mediated by pairs of I ends by mapping and sequencing independent inverse transpositions from a lambda::Tn5 phage into the amp and tet genes of plasmid pBR322. Using restriction analyses, 32 and 40 distinct sites of insertion were found among 46 and 72 independent inverse transpositions into the amp and tet genes, respectively. Eleven sites were used in two or more insertion events, and the two sites in tet used most frequently corresponded to major hotspots for the insertion of the Tn5 (by direct transposition). The sequences of 22 sites of inverse transposition (including each of the sites used more than once) were determined, in eleven cases by analyzing both pBR322-IS50 junctions, and in eleven others by sequencing one junction. The sequence of the "I" end of IS50 was preserved and 9-bp target sequence duplications were present in every case analyzed. GC pairs were found at each end of the target sequence duplication in ten of the eleven sites used more than once, and also in seven of the other eleven sites. Our data indicate that transposition mediated by pairs of "I" ends is similar in its specificity and precision to the more frequent transposition mediated by IS50 "O" ends.     

Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257

Trimethoprim resistance mediated by the Staphylococcus aureus multi-resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789-790 bp), the outside two of which are flanked by directly repeated 8-bp target sequences. IS257 has imperfect terminal inverted repeats of 27-28 bp and encodes for a putative transposase with two potential alpha-helix-turn-alpha-helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram-negative bacteria and with ISS1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim-resistant DHFRs from Gram-negative bacteria and with chromosomally encoded DHFRs from Gram-positive and Gram-negative bacteria. 5' to dfrA is a thymidylate synthetase gene, designated thyE.     

Orientation of IS50 transposase gene and IS50 transposition.

Reversal of transposase gene orientation with respect to the nonidentical ends of IS50 strongly decreased IS50 transposition in both Dam- and Dam+ hosts. In either orientation, IS50 transposase expression was unaffected. These effects were independent of the surrounding DNA context. This shows that the efficiency of IS50 transposition is dependent on transposase gene orientation. The transposition frequencies of transposons utilizing inverted IS50 inside ends (IE), IE-IE transposons, were lower than either outside end (OE)-IE or OE-OE transposons.     

Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA.

Summary The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots). Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites. We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences. Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 by away from this hotspot. Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam ^– mutation did not affect Tn5 insertion relative to an I end sequence in target DNA. These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.     

A novel type of transposon generated by insertion element Is102 present in a psc101 derivative

We describe a novel type of transposon in the tetracycline resistance plasmid pYM103, a derivative of pSC101 carrying a single copy of an insertion element IS102. The new transposons we found were identified as DNA segments, approximately 6 kb (Tn1021) and 10 kb (Tn1022) in length, able to mediate the cointegration of pYM1O3 with plasmid Col E1. The resulting cointegrate contains either of these pYM1O3 segments duplicated in a direct orientation at the junctions of the parent plasmids. A direct duplication of a 9 bp sequence at the target site in Col E1 is found at the junctions for cointegration. Both transposons have IS1O2 at one end and also contain different lengths of the pYM103 DNA adjacent to IS102, including the tetracycline resistance gene. Each transposon contains terminal inverted repeats of a short nucleotide sequence. These results and the fact that IS102 can itself mediate plasmid cointegration, giving rise to a duplication of a 9 bp target sequence, indicate that IS102 is responsible for generation of Tn1021 and Tn1022. They are quite different from the common IS-associated transposons, which are always flanked by two copies of an IS element, and may be similar to transposons such as those of the Tn3 family and phage Mu.     

MMTS, a new subfamily of Tc1-like transposons

A novel Tc1-like transposable element has been identified as a new DNA transposon in the mud loach, Misgurnus mizolepis. The M. mizolepis Tc1-like transposon (MMTS) is comprised of inverted terminal repeats and a single gene that codes Tc1-like transposase. The deduced amino acid sequence of the transposase-encoding region of MMTS transposon contains motifs including DDE motif, which was previously recognized in other Tc1-like transposons. However, putative MMTS transposase has only 34-37% identity with well-known Tc1, PPTN, and S elements at the amino acid level. In dot-hybridization analysis used to measure the copy numbers of the MMTS transposon in genomes of the mud loach, it was shown that the MMTS transposon is present at about 3.36 x 104 copies per 2 x 109 bp, and accounts for approximately 0.027% of the mud loach genome. Here, we also describe novel MMTS-like transposons from the genomes of carp-like fishes, flatfish species, and cichlid fishes, which bear conserved inverted repeats flanking an apparently intact transposase gene. Additionally, BLAST searches and phylogenetic analysis indicated that MMTS-like transposons evolved uniquely in fishes, and comprise a new subfamily of Tc1-like transposons, with only modest similarity to Drosophila melanogaster (foldback element FB4, HB2, HB1), Xenopus laevis, Xenopus tropicalis, and Anopheles gambiae (Frisky).     

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.     

Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria.

A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into the chromosome of a variety of gram-negative bacteria, including the enteric bacteria and typical soil bacteria like Pseudomonas species. The minitransposons consist of genes specifying resistance to kanamycin, chloramphenicol, streptomycin-spectinomycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. Further derivatives also contain lacZ, phoA, luxAB, or xylE genes devoid of their native promoters located next to the terminal repeats in an orientation that affords the generation of gene-operon fusions. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor.     

Evidence of recent lateral gene transfer among hyperthermophilic archaea

A total of 153 nucleotide differences were found over a contiguous 16 kb region between two hyperthermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis. The 16 kb region in P. furiosus is flanked by insertion sequence (IS) elements with inverted and direct repeats. Both IS elements contain a single open reading frame (ORF) encoding a putative protein of 233 amino acids identified as a transposase. This 16 kb region has the features of a typical bacterial composite transposon and represents a possible mechanism for lateral gene transfer between Archaea or possibly between Archaea and Bacteria. A total of 23 homologous IS elements was found in the genome sequence of P. furiosus, whereas no full-length IS elements were identified in the genomes of Pyrococcus abyssi and Pyrococcus horikoshii. Only one IS element was found in T. litoralis. In P. furiosus and T. litoralis, the 16 kb region contains an ABC transport system for maltose and trehalose that was characterized biochemically for T. litoralis. Regulation of expression studies showed that the malE gene, located on the transposon, and the encoded trehalose/maltose-binding protein (TMBP) are induced in the presence of maltose and trehalose in both P. furiosus and T. litoralis. The implications of transposition as a mechanism for lateral gene transfer among Archaea are discussed.     

Transitory Cis Complementation: A Method for Providing Transposition Functions to Defective Transposons

A genetic complementation system is described in which the complementing components are close together in a single linear DNA fragment; the complementation situation is temporary. This system is useful for providing transposition functions to transposition-defective transposons, since transposition functions act preferentially in cis. The basic procedure involves placing a transposition-defective transposon near the gene(s) for its transposition functions on a single DNA fragment. This fragment is introduced, here by general transduction, into a new host. The transposase acts in cis to permit the defective element to transpose from the introduced fragment into the recipient chromosome. The helper genes do not transpose and are lost by degradation and segregation. The method yields single insertion mutants that lack transposase and are not subject to further transposition or chromosome rearrangement. The general procedure is applicable to other sorts of transposable elements and could be modified for use in other genetic systems.     

IS50L as a non-self transposable vector used to integrate the Bacillus thuringiensis delta-endotoxin gene into the chromosome of root-colonizing pseudomonads

Insertion sequence IS50L of transposon Tn5 was used as a non-self transposable vector to integrate the delta-endotoxin gene (tox) from Bacillus thuringiensis subsp. kurstaki HD-1 into the chromosome of two corn-root colonizing strains of Pseudomonas fluorescens (112-12 and Ps3732-3-7). A DNA fragment containing the KmR gene from Tn5 and tox was inserted into an IS50L element (IS50L-tox) contained on a suicide plasmid. Transposition of IS50L-tox into the chromosome of P. fluorescens 112-12 and Ps3732-3-7 occurred by selecting for KmR transconjugants and supplying transposase in cis from a linked IS50R element. A frameshift mutation in the transposase gene of the IS50L-tox element was also constructed to decrease the likelihood that suppression or a spontaneous reversion at the UAA (ochre) termination codon of IS50L would create an active transposase. The inability of IS50L-tox to transpose further minimizes the potential for horizontal gene transfer of the tox gene to other bacterial species. Expression of the Tox protein in strains 112-12 and Ps3732-3-7 was demonstrated by an immunological assay (Western blot) and toxicity against larvae of the tobacco hornworm (Manduca sexta).     

Artificial transposable elements in the study of the ends of IS1

We have constructed artificial IS1-based transposons by attaching synthetic oligodeoxynucleotides, corresponding to the sequence of the ends of IS1, to a selectable DNA segment ['omega' fragment; Prentki and Krisch, Gene 29 (1984) 303-313]. These transposons were used to examine the sequence requirements at the ends for IS1 transposition. We show here that a 24- to 28-bp sequence from the left or right ends of IS1 is capable of transposition when present at both ends of the omega fragment in the correct orientation. Transposition activity requires the presence of an intact IS1 in cis on the same plasmid molecule. In trans, however, neither resident genomic copies of IS1, nor copies carried by a compatible, high-copy-number plasmid present in the same cell, complement the artificial transposons efficiently. Transposition frequencies in the presence of a cis-complementing IS1 are, however, similar to those of the naturally occurring IS1-based transposon, Tn9. In addition, transposition results in a 9-bp duplication in the target DNA molecule as is usually the case for insertion of the intact IS1. Using this system, we have obtained evidence indicating that the activity of a synthetic IS1 end is not determined exclusively by its sequence, but can be strongly enhanced by a second, wild-type end used in the transposition event. The data also show that single base pair mutations can exhibit a cumulative effect in reducing transposition activity.     

Early intermediates of mariner transposition: catalysis without synapsis of the transposon ends suggests a novel architecture of the synaptic complex

The mariner family is probably the most widely distributed family of transposons in nature. Although these transposons are related to the well-studied bacterial insertion elements, there is evidence for major differences in their reaction mechanisms. We report the identification and characterization of complexes that contain the Himar1 transposase bound to a single transposon end. Titrations and mixing experiments with the native transposase and transposase fusions suggested that they contain different numbers of transposase monomers. However, the DNA protection footprints of the two most abundant single-end complexes are identical. This indicates that some transposase monomers may be bound to the transposon end solely by protein-protein interactions. This would mean that the Himar1 transposase can dimerize independently of the second transposon end and that the architecture of the synaptic complex has more in common with V(D)J recombination than with bacterial insertion elements. Like V(D)J recombination and in contrast to the case for bacterial elements, Himar1 catalysis does not appear to depend on synapsis of the transposon ends, and the single-end complexes are active for nicking and probably for cleavage. We discuss the role of this single-end activity in generating the mutations that inactivate the vast majority of mariner elements in eukaryotes.     

The bacteriophage Mu transposase protein can form high-affinity protein-DNA complexes with the ends of transposable elements of the Tn 3 family

The 37 kb transposable bacteriophage Mu genome encodes a transposase protein which can recognize and bind to a consensus sequence repeated three times at each extremity of its genome. A subset of this consensus sequence (5'-PuCGAAA(A)-3') is found in the ends of many class II prokaryotic transposable elements. These elements, like phage Mu, cause 5 bp duplications at the site of element insertion, and transpose by a cointegrate mechanism. Using the band retardation assay, we have found that crude protein extracts containing overexpressed Mu transposase can form high-affinity protein-DNA complexes with Mu att R and the ends of the class II elements Tn 3 (right) and IS101. No significant protein-DNA complex formation was observed with DNA fragments containing the right end of the element IS102, or a non-specific pBR322 fragment of similar size. These results suggest that the Mu transposase protein can specifically recognize the ends of other class II transposable elements and that these elements may be evolutionarily related.     

Detection and characterisation of pr1 virulent gene deficiencies in the insect pathogenic fungus Metarhizium anisopliae

The method of suppressive subtractive hybridization was employed to map out genomic differences between the highly pathogenic Yersinia enterocolitica (Ye) biogroup 1B, serotype O:8 strain (WA-314) and the closely related apathogenic Y. enterocolitica biogroup 1A, serotype O:5 strain (NF-O). A novel IS10-like element, IS1330, uncovered by this technique was found to be uniquely present in high copy numbers among the highly pathogenic Y. enterocolitica 1B strains, while a single copy of the element was found in the low pathogenic Ye biogroup 4 serotype O:3 strain. The 1321-bp repetitive element has 19-bp imperfect inverted terminal repeats and is bracketed by a 10-bp duplication of the target sequence. The predicted transposase shares high homology with the IS10 open reading frame of the large virulence plasmid pWR501, of Shigella flexneri, with IS10 transposase of Salmonella typhi, and with IS1999 (tnpA) of Pseudomonas aeruginosa. The IS1330 tnp gene is transcribed in vitro and in vivo in HeLa cells. At least one copy of IS1330 flanks the recently described chromosomal type III secretion cluster in Y. enterocolitica WA-314, O:8, and future studies should shed light on whether this novel transposase mediates transposition events in highly pathogenic Y. enterocolitica strains, thus enhancing the genetic plasticity of this species.