Translocation of transposons Tn10 and Tn5 in the Yersinia pestis chromosome

The possibility of translocation of the transposons Tn5 and Tn10 into the genome of Yersinia pestis, with the subsequent mutagenic effect was demonstrated. We revealed transposon harbouring clones at frequency 10(-4) to 10(-2). Derivatives of P1cml clr100ts phage served as vectors. Insertion of Tn10 transposon induced mutations in ilv, ser, arg, pur, pro, leu, nic, tyr, gua genes. The number of the insertion sites on the chromosome obtained for Tn5 was the same, these being arg, ade, pyr, leu, gua, trp, his, pan, ilv. The majority of auxotrophs did not revert. Occasionally, revertants were observed at frequencies 10(-8) to 10(-6). Unlike Escherichia coli, reversion was not accompanied by the loss of transposons. The rearrangements induced by transposons, presumably, near the insertion site, as well as duplications of transposons followed by incorporation of copies into novel sites, led to the appearance of additional defective genes, which made it possible to select various types of polyauxotrophs. Based on reiteration of coinciding double and triple mutant markers, we proposed a linkage group of genes within a segment of Y. pestis chromosome: lys ... tyr - ser - arg - ilv - leu - gua - ade(pur) - pro ... his ... pyr ... trp. The reasons for peculiarities of the behaviour of transposons in Y. pestis bacteria are discussed.     

Interaction of related Tn916-like transposons: analysis of excision events promoted by Tn916 and Tn5386 integrases

In recent work, we described the excision of a large genomic region from Enterococcus faecium D344R in which the sequence from "joint" regions suggested that excision resulted from the interaction of conjugative transposon Tn916 and the related mobile element Tn5386. In the present study, we examined the ability of integrases and integrase-excisase combinations from Tn916 and Tn5386 to promote the excision of constructs consisting of the termini of Tn916, Tn5386, and the VanB mobile element Tn5382. Integrases alone from either Tn916 or Tn5386 promoted the circularization of constructs from the three different transposons, even when the different termini used in the constructs were discordant in their transposon of origin. The termini of Tn916 and Tn5382 found in all joints were consistent with previously identified Tn916 and Tn5382 termini. Substantial variation was seen in the integrase terminus of Tn5386 used to form joints, regardless of the integrase that was responsible for circularization. Variability was observed in joints formed from Tn5386 constructs, in contrast to joints observed with the termini of Tn916 or Tn5382. The coexpression of excisase yielded some variability in the joint regions observed. These data confirm that integrases from some Tn916-like elements can promote circularization with termini derived from heterologous transposons and, as such, could promote excision of large genomic regions flanked by such elements. These findings also raise interesting questions about the sequence specificities of the C terminals of Tn916-like integrases, which bind to the ends and facilitate strand exchange.     

Tn10-derived transposons active in Bacillus subtilis.

Small derivatives of the Escherichia coli transposon Tn10, comprising IS10 ends and a chloramphenicol resistance gene, were introduced in Bacillus subtilis on a thermosensitive plasmid, pE194. In the presence of the Tn10 transposase gene fused to signals functional in B. subtilis, these derivatives transposed with a frequency of 10(-6) per element per generation. They had no highly preferred insertion site or region, as judged by restriction analysis of the chromosomal DNA, and generated auxotrophic and sporulation-deficient mutants with a frequency of about 1%. These results suggest that Tn10 derivatives might be a useful genetic tool in B. subtilis and possibly other gram-positive microorganisms.     

Composite transposons Tn5 and Tn10 in Escherichia coli K12: precise excision and recombination

The number of exconjugants having the transposon Tn5 excised precisely during the crosses of the Escherichia coli proA::Tn5 donor with the recipients F- rec+ or F- recA441 (tif) was 20-30 times higher for the crosses involving the latter recipient. The high recombinogenic activity is characteristic of the tif recipient. Precise excision from a tandem duplication is more efficient than from nonduplicated region of the genome. It is four orders higher, if a transposon is localized in an arm of a duplication. The effect is recA-dependent. The presented data permit us to suggest the participation of RecA protein (its synaptic function) in the formation of the intermediate "stem-loop" structure. The latter is predicted by the three mechanisms of transposon excision: "slippage", "correctional" and "recombinational". The latter two mechanisms were formulated in the paper. The experimental proof of the postexcision transposition presented in the paper, is a good support to the version of "recombinational" excision.     

Isolation and characteristics of compound transposons Tn1000::Tn5

Two types of compound transposons were derived. In the first case, transposon Tn5 is inserted into the gene responsible for Tn1000 transposase synthesis. In the other, Tn5 is inserted into the region near the left end of Tn1000, where no functionally significant genes were found. It is known that translocation of the compound transposons does not depend on their size and takes place with the efficiency close to that characteristic of the intact Tn1000. Insertion of Tn5 into the gene coding for Tn1000 transposase results in sharp decrease in the efficiency of Tn1000 translocations. This effect, however, may be eliminated by introduction into the cell of the intact Tn1000.     

A system for in vivo selection of genomic rearrangements with predetermined endpoints in Escherichia coli using modified Tn10 transposons

Using recombinant DNA techniques, the Tn10-specific tetA gene (coding for tetracycline resistance) has been mutagenized by insertion of a streptomycin-resistance or a kanamycin-resistance gene. The insertions occurred at loci separated by 920 bp. The mutated tetA fragments, respectively designated as Tes (for tetracycline-streptomycin) and Tek (for tetracycline-kanamycin), were subsequently cloned into a phage lambda cIII+cIts857cII+ in replacement of the att lambda region. The two recombinant phages are convenient delivery vehicles which permit the in vivo substitution of the tetA locus of any Tn10 insertion with the Tes or the Tek fragment. The procedure involves two selectable steps: (i) integration of a lambda-Tes (or lambda-Tek) prophage into the Tn10 of interest; (ii) excision of the prophage by a second exchange which leaves the extra resistance gene installed within the Tn10. A major interest of the system is that, once a bacterium carries both Tn10-Tes and Tn10-Tek insertions, a recombination event between the two Tn10 sequences can reconstitute an active tetA gene. This selectable event may be associated with the rearrangement of the sequences surrounding the transposons. This unique property of the "Tes and Tek" system makes it very useful for selection of genomic rearrangements using the Tn10-Tes and Tn10-Tek as predetermined endpoints. The successful isolation of a chromosomal inversion is reported.     

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.     

Genome rearrangements by nonlinear transposons in maize.

Transposable elements have long been considered as potential agents of large-scale genome reorganization by virtue of their ability to induce chromosomal rearrangements such as deletions, duplications, inversions, and reciprocal translocations. Previous researchers have shown that particular configurations of transposon termini can induce chromosome rearrangements at high frequencies. Here, we have analyzed chromosomal rearrangements derived from an unstable allele of the maize P1 (pericarp color) gene. The progenitor allele contains both a full-length Ac (Activator) transposable element and an Ac terminal fragment termed fAc (fractured Ac) inserted in the second intron of the P1-rr gene. Two rearranged alleles were derived from a classical maize ear twinned sector and were found to contain a large inverted duplication and a corresponding deficiency. The sequences at the junctions of the rearrangement breakpoints indicate that the duplication and deletion structures were produced by a single transposition event involving Ac and fAc termini located on sister chromatids. Because the transposition process we describe involves transposon ends located on different DNA molecules, it is termed nonlinear transposition (NLT). NLT can rapidly break and rejoin chromosomes and thus could have played an important role in generating structural heterogeneity during genome evolution.     

Mutants of Escherichia coli K-12 with altered excision efficiency of transposons Tn5 and Tn9

HFETn5, HFETn9 and LFETn9 mutants of Escherichia coli K-12 have been isolated. The frequency of Tn5 precise excision from the chromosomal lac operon is increased 3-660-fold in nine HFETn5 mutants. The majority of these mutations have no influence on the efficiency of precise excision of transposon Tn9, though hfeTn5-04 and hfeTn5-06 mutations decrease excision efficiency 2-13-fold. The Tn9 transposon is excised in HFETn9 mutant about 20-fold more efficiently than in the wild type strain. This mutation does not stimulate excision of Tn5 and Tn10. LfeTn9 mutation decreases excision frequency of Tn9 11-17-fold, but has no effect on Tn5 excision and increases that of Tn10 about 20-fold. The differences in genetic control and mechanisms of excision of the transposons with long and short inverted repeats are discussed.     

Identification of Tn4451 and Tn4452, chloramphenicol resistance transposons from Clostridium perfringens.

The recombinant plasmids pJIR45 and pJIR97 contain the chloramphenicol resistance determinants derived from the Clostridium perfringens R plasmids pIP401 and pJIR27, respectively. Escherichia coli cultures which harbored these recombinant plasmids rapidly became chloramphenicol sensitive when grown in the absence of chloramphenicol. The loss of resistance was associated with the loss of 6.2-kilobase (kb) segments from both plasmids. Detailed restriction analysis of E. coli- and C. perfringens-derived deletion plasmids indicated that deletion of these segments was essentially precise. Transposition of the 6.2-kb segments was demonstrated by cloning the determinants into a temperature-sensitive plasmid, curing the recombinant plasmids, and selecting chloramphenicol-resistant, plasmid-free clones. Southern hybridization analysis of chromosomal DNA isolated from these recA E. coli clones indicated that the 6.2-kb segments had transposed to different sites on the chromosome. Heteroduplex analysis and restriction mapping indicated that the transposons, Tn4451 (pIP401) and Tn4452 (pJIR27), were closely related and did not contain large inverted or directly repeated sequences. These transposons represent the first transposable elements from the clostridia to be identified and characterized.     

Phage-lambda-mediated transduction of non-conjugative plasmids is promoted by transposons

Transduction experiments using phage λ as a vector have shown that non-conjugative plasmids can be transduced from one cell to the other, provided the phage or the plasmid DNA carries a copy of a Tn3-like transposon. The transduction is a result of replicon fusion between the phage and the plasmid DNA occurring during the transposition event.     

Characteristics of Escherichia coli K-12 mutants with altered effectiveness of excision of Tn5 and Tn9 transposons

The properties of Escherichia coli K-12 mutans HFETn5, HFETn9 and LFETn9 have been studied. The majority of mutations were shown to have pleiotropic effect. Some of them increase cell sensitivity to UV light and mitomycin C and affect efficiency of homologous recombination in transduction and conjugation. The level of spontaneous mutagenesis is increased in a number of mutants. None of the mutations isolated affect frequency of transposition of Tn5 from bacteriophage lambda::Tn5 into the chromosome. Based on analysis of properties of hfeTn5-09 and hfeTn9 mutations and on the date of preliminary mapping of hfeTn5-09 mutation, these mutations were considered to be novel. It is shown that the processes of precise excision of Tn5 and Tn9 transposons may be accomplished by at least two pathways, one of them being dependent on recA gene functions.     

Inter- and intramolecular transposition of Tn903.

Summary We have performed a detailed analysis of intra-and intermolecular endproducts of transposition of the compound transposon Tn903 and we show that, in our system, the transposition activity is almost entirely driven by one of the flanking insertion sequences, IS903L. The relatively inactive state of IS903R can be conferred on IS903L by changing the orientation of the internal Tn region. IS903L mediates the formation of the majority of adjacent deletions, insertion/inversions nd cointegrates, all of which are representative of replicative transposition; only a very low level of conservative transposition can be observed. Our results are discussed in relation to those showing that Tn903 uses predominantly the conservative pathway.     

pIF21, a versatile donor of transposons Tn1, Tn5 and Tn7 for tagging and mobilizing cryptic and non-selectable plasmids

A plasmid, pIF21, has been constructed that is able to donate transposons Tn1, Tn5 and Tn7. The transposons are located on a temperature-sensitive derivative of the incP1 plasmid pRP1, which is transferable to a wide range of Gram-negative genera.