Tn5 Transposition Research Articles

Control of Tn5 transposition in Escherichia coli is mediated by protein from the right repeat

The right repeat in Tn5, which encodes protein absolutely required for transposition, is also capable of inhibiting Tn5 transposition. Analysis of Tn5 mutants indicates that the left repeat is defective in supplying the transposition-inhibition function because of the sequence difference between the repeats located at nucleotide 1443; that the transposition-inhibition activity is a function of the quantity of right-repeat protein synthesis; that the smaller of the right-repeat proteins, protein 2, is sufficient for supplying the transposition-inhibition function (but not for the transposase activity); and that the transposition-inhibition function can act in trans, as opposed to the transposase activity, which functions efficiently only in cis. Gene fusion experiments indicate that the transposition-inhibition activity cannot be explained by autogenous regulation of right-repeat protein synthesis. Finally, immunoprecipitation assays of right-repeat protein- lacZ fusion proteins indicate that protein 2 is synthesized in significantly greater amounts than protein 1 in whole cells. This synthetic ratio may be important with respect to the control of Tn5 transposition.

Intermolecular and intramolecular transposition and transposition immunity in Tn3 and Tn2660.

Intermolecular transposition of Tn2660 into pCR1 was measured at 30 degrees C in recA- and recA+ hosts as between 2.6 and 5.5 X 10(-3), a similar value to that previously found for Tn3. No cointegrate structures were found under conditions where 10(4) transposition events occurred. Immunity to intermolecular transposition of Tn2660, similar to that found for Tn3 was demonstrated by showing that the above transposition frequency was reduced by a factor of between 10(-3) and 10(-4) when a mutant Tn2660 (resulting in the synthesis of a temperature-sensitive beta-lactamase) was present in the recipient plasmid. Intramolecular transposition of Tn3 was found to occur under the same conditions as previously demonstrated for Tn2660 giving rise to similar end products, in which the newly introduced Tn3 is oriented inversely to the resident Tn3 and the DNA sequence between the two transposons has been inverted. Thus, in all respects functional identity of the transposition activities of Tn3 and Tn2660 is shown, thereby identifying characteristics of intramolecular transposition that are not readily accommodated by current models of transposition.

Transposon Tn 3-mediated replicon fusion

To investigate inter-replicon transposition of Tn3, we used the cosmid-phage λ packaging system coupled with density gradient fractionation and isolated recombinant molecules of different sizes. Cosmids derived from ampicillin-resistance-transducing phage were classified into four groups: (1) cosmid-Tn3 donor cointegrates considered as Tn3 transposition intermediates, (2) similar cointegrates carrying deletions of one copy of Tn3 and of adjacent cosmid DNA sequences, (3) cosmids carrying a single Tn3 insertion, and (4) cosmids carrying two independent Tn3 insertions. Genetic and biochemical studies indicated that cosmids isolated from ampicillin-resistance transductants were derived from the authentic cosmid-Tn3 donor cointegrate intermediates.

Cointegrate formation by Tn5, but not transposition, is dependent on recA.

We have studied the effect of the recA-dependent homologous recombination system of Escherichia coli on both Tn5-mediated cointegrate formation and Tn5 transposition. We demonstrate here that, whereas transposition of Tn5 is independent of the recA gene product (as has been shown by other workers), Tn5-mediated cointegrate formation is strongly dependent on recA. The structures of both the simple transposition products and the cointegrates formed in a recA- background seem to be the same as those produced in a recA+ background. These results provide strong evidence that Tn5 does not transpose via an obligate cointegrate intermediate and suggest that the recA effect on cointegrate formation is exerted during the process of transposition.

Transposition of Tn7 occurs at a single site on the Caulobacter crescentus chromosome.

Insertion of Tn7 in nine independently derived isolates occurred in a single small region of the Caulobacter crescentus chromosome, suggesting that Tn7 insertion occurs at or near a single site. The order of markers in this region of the Caulobacter chromosome was shown to be flaA-argG-Tn7.

Structure and stability of transposon 5-mediated cointegrates

We have determined the structure of a set of independently derived, Tn5-mediated cointegrates and examined the stability of several examples. A variety of cointegrate structures was found, including those mediated by the entire compound transposon, and those mediated by a single flanking IS50 element, which was always IS50-R, and never IS50-L. IS50-R but not IS50-L is reported to code for a protein(s) required for transposition. This finding confirms that IS50-L is relatively inactive and suggests that the active transposition protein(s) acts largely in cis on IS50-R. Another class of cointegrate was created by inverse transposition of Tn5 (using the inside ends of the flanking elements). In addition, we found an unexpectedly large set of cointegrates, in which the joint between the two plasmids was not adjacent to the transposon. All cointegrates analysed were found to be stable. This suggests that Tn5, unlike the transposon Tn3, does not transpose via an obligate cointegrate intermediate. This finding is compared to previous results with Tn5 and Tn9, and is discussed in terms of current models of transposition.

DNA gyrase is a host factor required for transposition of Tn5

We show that DNA gyrase is required for transposition of Tn5. Coumermycin, a potent inhibitor of DNA gyrase subunit B, inhibits transposition in a wild-type strain, but has no effect on strains carry ing a coumermycin-resistant allele in gyrB. In addition, strains containing a thermolabile subunit A of gyrase ( gyrA43) are defective for transposition at a nonpermissive temperature. The requirement for gyrase is due to a requirement for supercoiled DNA. We showed this by introducing into the gyrA43 strain a deletion of the gene encoding topoisomerase I. The introduction of the second mutation caused an increase in the superhelical density of DNA as well as an increase in the transposition frequency. This also implies that if the DNA is supercoiled there is no further requirement for gyrase. Experiments with coumermycin support this, because the drug does not inhibit transposition if the recipient DNA remains supercoiled. This indicates that if the DNA acting as recipient of the transposon is deficient in supercoils, it will be a poor substrate for transposition. We also describe a system in which a gene on a multicopy plasmid can be efficiently introduced into the Escherichia coli chromosome.

A derivative of Tn5 with direct terminal repeats can transpose

The 5.7 kb § § Abbreviation used: kb, 10 3 bases or base-pairs where appropriate; bp, base-pairs. 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 r kan 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.

The effect of tnm mutations of Escherichia coli K12 on transposition of various movable genetic elements.

The effects of different tnm mutations on the transposition of Tn3, Tn5, Tn10, Tn601, and bacteriophage Mu were studied. Five tnm mutations were placed into three phenotypic classes. The representatives of the first class, tnm-1 and tnm-2, caused a complete block of transposition of all Tn-elements studied; the representatives of the second class, tnm-3 and tnm-4, specifically affected Tn9 transposition while the tnm-5 mutation attributed to the third class caused formation of cointegrates between the donor genome of phage lambda carrying Tn-elements Tn3 or Tn5 and the recipient genome (bacterial chromosome).

Site-specific properties of Tn7 transposition into the E. coli chromosome.

A study has been made of the insertional properties of transposon Tn7, a 14 kilobase transposable element encoding resistances to trimethoprim, streptomycin and spectinomycin. It has previously been shown that Tn7 transposes at a low frequency and with low specificity into multiple sites in large transmissible plasmids. However, Tn7 transposes with extreme specificity and at high efficiency into the E. coli chromosome. In all cases we have studied, insertion of Tn7 into the chromosome has occurred at a unique site and with a unique orientation. A combination of genetic and biochemical techniques have been used to precisely locate this site on the E. coli chromosome to minute 82 on the linkage map between markers glmS and uncA. To investigate the nature of this highly specific transpositional event, a small region of the E. coli chromosome that includes the unique site, was cloned into the plasmid vector pBR322. Subsequently a lkb restriction fragment, including the Tn7 insertion site, was sub-cloned from this plasmid into the plasmid pACYC184. We show that Tn7 transposes into both these plasmid recombinants with the frequency and specificity characteristic of the E. coli chromosome.

Replicon fusions promoted by the inverted repeats of Tn5: The right repeat is an insertion sequence

Several transposable elements are able to promote replicon fusions in Escherichia coli. We show here that the transposon, Tn5, also has this property. Tn5 is able to promote fusions between the plasmid pBR322 (on which it resides) and bacteriophage λ at 2% of the frequency of Tn5 transposition onto λ. Plasmid/phage replicon fusions formed in strains that are totally deficient in homologous and integrative recombination always consist of direct duplications of the insertion element at the junctions between λ and pBR322. In order to define those regions within the Tn5 necessary for causing replicon fusions, a series of deletions of Tn5 generated in vitro and in vivo were constructed. Deletion derivatives that remove all but the 1500-base-pair right inverted repetition (stem) are competent to promote fusions. The left inverted repetition is unable to supply this activity, but can be complemented in trans to form replicon fusions. We conclude that both repeats have the sites necessary for replicon fusions, but only the right stem has the required protein(s). This establishes that the right repeat is an insertion sequence.

The requirement for both DNA polymerase and 5' to 3' exonuclease activities of DNA polymerase I during Tn5 transposition.

By assaying transposition of Tn5 from lambda b221 cI857 rex::Tn5 (Berg 1977) in polA-proficient and deficient cells, both the polymerase activity and 5' to 3' exonuclease activity of DNA polymerase I have been shown to be required for transposition. This requirement could not be observed in three other systems in which the transposon donor replicon had existed in the PolA-proficient and deficient cells before the transposition event to be assayed occurred. By analogy to Tn3, this may indicate that the repressor encoded by Tn5 has already been expressed and hence become rate-limiting in the overall transposition process, even PolA-deficient cells still possessing a residual activity. One PolA mutant was found among more than 50 transposition deficient (tnp) mutants isolated by the use of lambda b221 cI857 rex::Tn5.

Mutations in the gene coding for Escherichia coli DNA topoisomerase I affect transcription and transposition.

Mutations in top, the structural gene for Escherichia coli DNA topoisomerase I, have been identified and mapped at 28 min on the chromosome, near cysB. Strains carrying deletions of the top gene are viable. The top mutations, however, do exert pleiotropic effects on transcription and transposition. Mutants lacking DNA topoisomerase I have a more rapid rate of induction and a higher level of catabolite-sensitive enzymes including tryptophanase and beta-galactosidase. This general activation of transcription by top mutations can be attributed to an increase in the negative superhelicity of the DNA in vivo when the topoisomerase activity is abolished. The frequency of transposition of Tn5, a transposon carrying kanamycin resistance, is decreased by a factor of 40 or more in top mutants. A direct or indirect role of the topoisomerase in transposition is discussed. The transposition frequency of Tn3, however, is not dependent on top. Based on the studies of the E. coli top mutants, it appears that the supX gene, which was originally studied in Salmonella typhimurium [Dubnau, E. & Margolin, P. (1972) Mol. Gen. Genet. 117, 91-112] is likely to be the structural gene for DNA topoisomerase I.

Genetic organization of transposon Tn10

Transposon Tn10 is 9300 bp in length, with 1400 bp inverted repeats at its ends. The inverted repeats are structurally intact IS-like sequences (Ross et al., 1979). Analysis of deletion mutants and structural variants of Tn10, reported below, shows that the two IS10 segments contain all of the Tn10-encoded genetic determinants, both sites and functions, that are required for transposition. Furthermore, the two repeats (IS10-Right and IS10-Left) are not functionally equivalent: IS10-Right is fully functional and is capable by itself of promoting normal levels of Tn10 transposition; IS10-Left functions only poorly by itself, promoting transposition at a very low level when IS10-Right is inactivated. Complementation analysis shows that IS10-Right encodes at least one function, required for Tn10 transposition, which can act in trans and which works at the ends of the element. Also, all of the sites specifically required for normal Tn10 transposition have been localized to the outermost 70 bp at each end of the element; there is no evidence that specific sites internal to the element play an essential role. Finally, Tn10 modulates its own transposition in such a way that transposition-defective point mutants, unlike deletion mutants, are not complemented by functions provided in trans; and wild-type Tn10, unlike deletion mutants, is not affected by functions provided in trans from a “high hopper” Tn10 element.

Regulation of Tn5 transposition.

Regulation of Tn5 transposition.

Regulation of Tn5 transposition in Salmonella typhimurium.

The drug-resistance element Tn5 transposes with high frequency immediately after entry into a cell. Establishment of Tn5 within a cell results in a decrease in this transposition frequency. This phenomenon resembles "zygotic induction" of repressible operons and prophages. Evidence is presented that Tn5 transposition is under negative control by a factor encoded within the element itself. Established Tn5 elements (that contain point mutations inactivating the resistance gene) are able to inhibit transposition of an incoming Tn5 element by a factor of 12- to 70-fold. Several deletion derivatives of Tn5 lack the ability to inhibit transposition.

Transposon-mediated conjugational transmission of nonconjugative plasmids.

When coresident with conjugative plasmid pNC21, the nonconjugative deletion F-prime pJC59, which retains the F transfer origin oriT, was transmitted to transconjugants at a frequency comparable to that of pNC21. In addition, pJC59 was transmitted as an independent plasmid, physically separate from pNC21, an example of plasmid donation. In contrast, two plasmids that are derived from F and deleted for the oriT site, pJC61 and pML31, were transmitted at frequencies 10(4) lower than that of pNC21. This low-frequency transmission was associated with the appearance of a new plasmid in the transconjugants. In the case of pML31, we determined that this new plasmid was a recombinant composed of pNC21 and pML31, the latter flanked by two copies of transposable element Tn3. We believe that this recombinant plasmid was formed as an intermediate in the transposition of Tn3 from pNC21 to pML31 and was the vehicle for conjugational transmission of pML31 genes by a process known as plasmid conduction.

Isolation of symbiotically defective mutants in Rhizobium leguminosarum by insertion of the transposon Tn5 into a transmissible plasmid

Selection was made for the transposition of the kanamycin resistance transposon Tn5 from a location on the chromosome of R. leguminosarum into a transmissible, bacteriocinogenic plasmid that also carries genes required for the induction of nitrogen-fixing nodules on peas. One hundred and sixty independent insertions into transmissible plasmids were isolated. When these plasmids were transferred by conjugation into a non-nodulating strain, which carries a deletion in one of its resident plasmids, of the 160 isolates tested 14 yielded transconjugants that formed nodules that did not fix nitrogen (Fix-) and in a further 15 cases the transconjugants were unable to form nodules (were Nod-). When transferred to a symbiotically proficient strain (i.e. Nod+ Fix+) none of the transconjugants was symbiotically defective; thus the mutations were not dominant. When kan was transduced from the clones that generated Fix- transconjugants into a Fix+ recipient the majority of transductants inherited Fix- indicating that the insertion of Tn5 had induced the symbiotic mutations. Transduction of kan, from the clones that failed to donate Nod+ by conjugation to strain 6015, occurred at barely detectable frequencies and it was not possible to demonstrate transduction of Nod-. kan was co-transduced with Nod+ from some of the clones and some of these transductants also inherited the ability to produce medium bacteriocin and to transfer at high frequency by conjugation. Thus the genes for all these characters are closely linked.

The inverted repeats of Tn 5 are functionally different

The inverted repeats of Tn5, which have identical restriction endonuclease cleavage patterns, have different functional properties. They differ with respect to RNA polymerase binding, full promotion of neomycin resistance, the polypeptides coded for by the repeats and their function in the transposition process. There is a week RNA polymerase binding site present in one repeat and not in the other which seems to be important for neomycin resistance. The two inverted repeats code for polypeptides of different molecular weights, with each repeat appearing to encode two polypeptides. The polypeptides from only one of the repeats of Tn5 appear to be absolutely required for Tn5 transposition.

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 β-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.