Function of the InsA gene in the IS1 element of the Tn9' transposon: influence of oligonucleotide inserts in the InsA gene on formation of simple insertions and plasmid cointegrates]

To elucidate the role of the insA reading frame in transposition of the IS1 element of the Tn9' transposon, the derivatives of plasmids pUC19::Tn9' and pUC19::IS1 have been obtained using oligonucleotide inserts of the length equal or exceeding 9 bp and equal to 10 bp. The ability of mutant variants of the Tn9' transposon and the IS1 element to form simple insertions and plasmid cointegrates was studied. To this end, experiments were performed on mobilization of the derivatives of pUC19 containing mutant variants of the IS1 element and Tn9' as well as of the plasmids pUC19::Tn9' by the conjugative plasmid pRP3.1. According to the data obtained, mutations (inserts) in the insA gene have no influence on the frequency of transposition of the IS1 element and Tn9' from the plasmid pUC19 to pRP3.1. At the same time, the frequency of transposition events of mutant variants of Tn9' from the plasmid pRP3.1 to pBR322 is more than 10 times lower in comparison with the wild type transposon. The data obtained are in accordance with the assumption that the insA gene is not essential for transposition. A hypothesis is put forward explaining the role of the insA gene product in the process of bringing together short inverted repeats of the IS1, which are the sites for the transposase to be recognized at first stages of transposition.     

Genetic evidence for absence of transposition functions from the internal part of Tn981 a relative of Tn9

Summary Inverse transposition of the DNA of pBR322 was found to be mediated by the small transposon Tn981 a relative of Tn9 flanked by direct repeats of IS1. Since the resulting structure IS1:: pBR322::IS1 (Tn983) is transposed in a second step in the absence of Tn981, it is concluded that all the functions necessary for transposition of IS1 flanked transposons are coded for by IS1 itself or the E. coli chromosome, respectively.     

Mechanism of Is1 Transposition in E. COLI: Choice between Simple Insertion and Cointegration

Insertion element IS1 and IS1-based transposon Tn9 generate cointegrates (containing vector and target DNAs joined by duplicate copies of IS1 or Tn9) and simple insertions (containing IS1 or Tn9 detached from vector sequences). Based on studies of transposon Tn5 we had proposed a conservative (non-replicative) model for simple insertion. Others had proposed that all transposition is replicative, occurring in a rolling circle structure, and that the way DNA strands are joined when replication terminates determines whether a simple insertion or a cointegrate is formed.--We selected for the transposition of amp and cam resistance markers from pBR322::Tn9 plasmids to an F factor in recA-E. coli and identified products containing three and four copies of IS1, corresponding to true cointegrates (from monomeric plasmids), and simple insertions (from dimeric plasmids). The simple insertions with four copies of IS1 outnumbered those with three by a ratio of about 3:1, whereas true cointegrates containing three copies of IS1 were more numerous than those with four.--A straightforward rolling circle model had predicted that the simple insertions containing three copies of IS1 should be more frequent than those with four. Because we obtained the opposite result we propose that simple insertions only arise when the element fails to replicate or if replication starts but then terminates prematurely. The two classes of products, simple insertions and cointegrates, reflect alternative conservative and replicative fates, respectively, of an early intermediate in transposition.     

Characterization of in vitro constructed IS30-flanked transposons

In order to facilitate functional studies on the mobile genetic element IS30, a resident of the Escherichia coli chromosome, transposon structures with two copies of IS30 flanking the chloramphenicol-resistance gene cat were constructed in vitro. Transposons containing IS30 as direct repeats (Tn2700 and Tn2702) transpose from multicopy plasmids into the genome of phage P1-15, thus giving rise to special transduction for cat with frequencies between 10(-5) and 10(-8)/plaque-forming unit. In contrast, transposon structures with IS30 in inverted repeat (Tn2701 and Tn2703) showed no detectable (less than 10(-9] transposition activity in vivo. By restriction analysis, two insertion sites of Tn2700 and Tn2702 on the phage P1-15 genome were indistinguishable from those observed earlier with a single copy of the IS30 element. These two insertion sites were used several times independently by Tn2700 and Tn2702. This confirms the non-random target selection by the element and it indicates that transposition of Tn2700 and Tn2702 follows the same rules as that of IS30.     

Structure and stability of cointegrating plasmids mediated by the IS1 elements in transposon delta Tn9'

In order to elucidate the structural features of the transposon Tn9', representative of the Tn9 family, which define the ability of the transposon to produce unstable cointegrates, we have obtained a derivative of this transposon carrying a deletion in its central region. The deletion in the obtained transposon delta Tn9' covers a DNA segment of about 50 bp in length, occupying the most distal position in relation to the cat gene, at its junction with the right copy of the IS1. The structure and stability of the IS1/delta Tn9'-mediated cointegrates between the plasmids pDK57.1 (pBR322::delta Tn9') and pRP3.1, a deletion derivative of RP1, have been studied. The three types of cointegrates were found. Those of the type I are predominantly formed, due to the left copy of the IS1 which in delta Tn9' occupies proximal position to the promoter of the cat gene. These cointegrates contain three copies of IS1 and are of high stability. The cointegrates of the type II contain two entire copies of delta Tn9' (i.e. four copies of IS1) as well as the structures of the type II, representing the cointegrate equivalent of inverse transposition and also containing four copies of IS1. Cointegrates of the type II and III dissociate efficiently in the rec+ cells but, in contrast to the cointegrates mediated by the original transposon Tn9', are unable to dissociate efficiently in the recA- cells. It was concluded that a DNA segment in the central region of Tn9' may be essential for the expression of the IS1-specific resolvase encoded by the right copy of IS1.     

Structure and stability of Tn9-mediated cointegrates. Evidence for two pathways of transposition

We have measured the frequency of Tn9 transposition and cointegrate formation in several different ways and have examined the stability of the cointegrates. We have also physically analyzed the structure of 40 independently derived cointegrate molecules. We present evidence here that Tn9, unlike the transposable element Tn3, does not transpose via an obligate cointegrate intermediate. We suggest that transposition of Tn9 leads to two, mutually exclusive, end-products: either direct insertion of the element into a recipient replicon (transposition), or fusion between donor and recipient replicons (cointegrate formation). This conclusion is based on our observations that, while Tn9-mediated cointegrates are very stable, they are formed at a rate lower than the transposition frequency. This finding is discussed in terms of current models for transposition.We also present evidence that clearly demonstrates the compound nature of Tn9. We find that the individual flanking IS1 elements are more active than the entire Tn9 transposon in cointegrate formation. In addition, we find that one IS1 element that is proximal to the cam gene promoter, is more active than the other, and suggest that the difference in activity might be due to differences in nucleotide sequence at their extremities.     

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.     

Study of the role of the insA open reading frame in the IS1 insertion element structure during transposition and resolution of the IS1 mediated cointegrates

In order to elucidate the function of the IS1 insA gene derivatives of plasmid pUC19::Tn9' with insertions of synthetic oligonucleotides were obtained. The latter are equal or multiple of 9 b.p. in length and are located in the Pst1 site within each of the two IS1 copies of the Tn9' transposon. The insertions of the nine base oligonucleotides code for the neutral amino acids and do not shift the reading frame. One of the mutant transposon obtained - Tn9'/X was studied on the ability to form simple insertions and plasmid cointegrates. For this purpose the pUC19 derivatives carrying the wild type and mutant transposon were mobilized by conjugative plasmid pRP3.1. It was found that the damage of the insA gene does not influence the ability of transposon to form simple insertions and plasmid cointegrates in both recA - and rec+ cells of E. coli. However, the frequency of the cointegrate formation in the subsequent transposition of the mutant transposon from pRP3.1::Tn9'/X to pBR322 was by 10-20 times lower in comparison to the wild type transposon. Instable (dissociating) Tn9'/X-mediated plasmid cointegrates formed by interaction pUC19::Tn9'/X and pRP3.1 were obtained. It was shown that in the E. coli recA-cells such cointegrates dissociate, as a rule, "correctly", i.e. they segregate mainly plasmids of types pUC19::Tn9'/X and pUC19::IS1/X. The data obtained correspond with the notion that the gene insA product is not essential for transposition, but is, possibly, involved in the formation of the IS1-generated deletions.     

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.     

Spontaneous deletion of citrate-utilizing ability promoted by insertion sequences.

The citrate utilization (Cit+) transposon Tn3411 was shown to be flanked by directly repeated sequences (IS3411L and IS3411R) by restriction enzyme analysis and electron microscope observation. Cit- deletion mutants were frequently found to be generated in pBR322::Tn3411 by intramolecular recombination between the two copies of IS3411. The flanking IS3411 elements of Tn3411 were shown to be functional insertion sequences by Tn3411-mediated direct and inverse transposition. Tn3411-mediated inverse transposition from pBR322::Tn3411 to the F-plasmid derivative pED100 occurred more efficiently than that of direct transposition of the Cit+ determinant. This was thought to be due to the differential transposability of IS3411L and IS3411R in the transposition process. The frequency of transposition of IS3411 marked with a chloramphenicol resistance determinant was much higher than IS3411-mediated cointegrate formation, suggesting that replicon fusions are not essential intermediates in the transposition process of Tn3411 or IS3411. Spontaneous deletions occurred with high frequency in recA hosts. The spontaneous deletion promoted by homologous recombination between two IS3411 elements in Tn3411 was examined with deletion mutants.     

Integration host factor plays a role in IS50 and Tn5 transposition.

In Escherichia coli, the frequencies of IS50 and Tn5 transposition are greater in Dam- cells than in isogenic Dam+ cells. IS50 transposition is increased approximately 1,000-fold and Tn5 transposition frequencies are increased about 5- to 10-fold in the absence of Dam methylation. However, in cells that are deficient for both integration host factor (IHF) and Dam methylase, the transposition frequencies of IS50 and Tn5 approximate those found in wild-type cells. The absence of IHF alone has no effect on either IS50 or Tn5 transposition. These results suggest that IHF is required for the increased transposition frequencies of IS50 and Tn5 that are observed in Dam- cells. It is also shown that the level of expression of IS50-encoded proteins, P1 and P2, required for IS50 and Tn5 transposition and its regulation does not decrease in IHF- or in IHF- Dam- cells. This result suggests that the effects of IHF on IS50 and Tn5 transposition are not at the level of IS50 gene expression. Finally, IHF is demonstrated to significantly retard the electrophoretic mobility of a 289-base-pair segment of IS50 DNA that contains a putative IHF protein-binding site. The physiological role of this IHF binding site remains to be determined.     

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.     

Characteristic features of the structure of co-integrating plasmids and simple insertions formed during transposition of the IS1 element in transposon Tn9'

Earlier we have studied unstable dissociating IS1/Tn9'-mediated cointegrates between the plasmids pDK57 (pBR322::Tn9') and pRP3.1, a deletion derivative of RP1, and two types of such cointegrates containing three and four copies of IS1 were revealed. In the present paper we studied the structure of stable IS1/Tn9'-mediates cointegrates and simple insertions formed by interaction between the plasmids pDK57 and pRP3.1 in the E. coli recA- cells. It was shown, that the stable cointegrates were formed by insertion of pDK57 in different loci of pRP3.1 and these cointegrates contain three copies of IS1, i.e. one copy of IS1 and a copy of Tn9' at the junction of the two replicons. The cointegrates are formed predominantly due to the activity of the left copy of Tn9', which occupies a proximal position in regard to the promoter of the cat gene. It was found that the integration of pDK57 into the kan gene region of pRP3.1 leading to the formation of the KmS cointegrates occurs only in one of the two possible orientations. Meanwhile the insertions of the transposon Tn9' into the kan region of pRP3.1 leading to simple insertions occurs in the orientation opposite to the orientation of the transposon in the KmS cointegrates. It is proposed that simple insertions are not the products of direct transposition of Tn9', but they are formed from unstable cointegrates under the action of IS1-specific resolvase.     

Evidence for Campbell's mechanism for fine excision of Tn9 type transposons in Escherichia coli K12]

The plasmid-transposon Tn9-322 was constructed by inverted transposition from the pBR322::Tn9 plasmid. The precise excision of the Tn9-322 transposon from the proB gene site can proceed by the Campbell's model. This fact was demonstrated by appearance of the plasmid-transposons after their precise excision. They contain two IS1 elements flanking a short direct repeat of the target DNA. The recombinational mechanism of precise excision of Tn9 type transposons seems not to be alternative but looks as an additional one to a well-known slippage mechanism proved for Tn5 and Tn10.     

Regulation of Tn5 by the right-repeat proteins: Control at the level of the transposition reaction?

The transposon Tn5 consists of inverted repeats, called IS50R and IS50L, each of which encode two proteins. We show here that the larger protein encoded on IS50R, protein 1, is absolutely required for transposition. Deletion or insertion mutants that fail to make this protein fail to promote gene movement. In addition, this protein acts in cis preferentially. We also show that the smaller protein encoded on IS50R, protein 2, is competent to inhibit transposition of a Tn5 freshly introduced into the cell on a λ phage. In contrast, the proteins from IS50L possess neither of these two activities. By assaying expression of proteins that are hybrids between β-galactosidase and IS50R proteins, we find that the regulation of transposition cannot be due to the inhibitor repressing synthesis of Tn5 proteins. Control experiments, in which we assay synthesis of IS50 proteins synthesized from a λ::IS50R that has been infected into cells carrying the transposition inhibitor, confirm this conclusion.     

Specificity of Tn9 insertion into the genome of bacteriophage lambda att80 depending on its preceding location

It was shown that the site of previous integration (the donor site) of Tn9 affects the specificity of its next integration into the target molecule--phage lambda att80 DNA. The transposon integration sites were mapped by restriction and heteroduplex analysis following Tn9 transposition from chromosomal sites of Escherichia coli K-12 differing in location and Tn9 stability. When transposed from chromosomal galT::IS1 gene, Tn9 inserted into the site with coordinates 44,5 +/- 2 kb of lambda att80; when transposed from chromosomal attTn9A site, the transposon inserted into the sites with coordinates 31 +/- 0,7 kb or 33,3 +/- 0,5 kb. In the course of transposition of Tn9 from chromosomal attTn9N site the transposon inserted into the lambda att80 site with coordinates 26,5 +/- 5 kb. In the latter case, the increase of Tn9 single-stranded loop and the appearance of two new HindIII cleavage sites were observed in heteroduplex experiments. The data were interpreted as indicating structural rearrangements of Tn9 or linked sequences in the course of transposition.     

The mobile element IS1207 of Brevibacterium lactofermentum ATCC21086: isolation and use in the construction of Tn5531, a versatile transposon for insertional mutagenesis of Corynebacterium glutamicum

IS1207 is the insertion most frequently found among the spontaneous mutations that abolish the activity of an Escherichia coli phage lambda cI gene integrated in the Corynebacterium Brevibacterium lactofermentum ATCC21086 genome. We examined the transposition of transposon-like structures composed of a selective kanamycin resistance gene (aph3), and one or two IS1207 sequences. One of these, the Tn5531 transposon, transposed efficiently in Corynebacterium glutamicum. A replicative and a non-replicative Tn5531 delivery vector were used in Tn5531 mutagenesis. As IS1207, transposon Tn5531 shows a high frequency of transposition and mutagenesis, and a low target specificity. These features make of Tn5531 an adequate choice for gene identification and gene tagging experiments.     

Role of the IS50 R proteins in the promotion and control of Tn5 transposition

IS50R, the inverted repeat sequence of Tn5 which is responsible for supplying functions that promote and control Tn5 transposition, encodes two polypeptides that differ at their N terminus. Frameshift, in-frame deletion, nonsense, and missense mutations within the N terminus of protein 1 (which is not present in protein 2) were isolated and characterized. The properties of these mutations demonstrate that protein 1 is absolutely required for Tn5 transposition. None of these mutations affected the inhibitory activity of IS50, confirming that protein 2 is sufficient to mediate inhibition of Tn5 transposition. The effects on transposition of increasing the amount of protein 2 (the inhibitor) relative to protein 1 (the transposase) were also analyzed. Relatively large amounts of protein 2 were required to see a significant decrease in the transposition frequency of an element. In addition, varying the co-ordinate synthesis of the IS50 R proteins over a 30-fold range had little effect on the transposition frequency. These studies suggest that neither the wild-type synthesis rate of protein 2 relative to protein 1 nor the amount of synthesis of both IS50 R proteins is the only factor responsible for controlling the transposition frequency of a wild-type Tn5 element in Escherichia coli.     

Physical Analysis of Tn10- and Is10-Promoted Transpositions and Rearrangements

We have investigated by Southern blot hybridization the rate of IS10 transposition and other Tn10/IS10-promoted rearrangements in Escherichia coli and Salmonella strains bearing single chromosomal insertions of Tn10 or a related Tn10 derivative. We present evidence for three primary conclusions. First, the rate of IS10 transposition is approximately 10(-4) per cell per bacterial generation when overnight cultures are grown and plated on minimal media and is at least ten times more frequent than any other Tn10/IS10-promoted DNA alteration. Second, all of the chromosomal rearrangements observed can be accounted for by two previously characterized Tn10-promoted rearrangements: deletion/inversions and deletions. Together these rearrangements occur at about 10% the rate of IS10 transposition. Third, the data suggest that intramolecular Tn10-promoted rearrangements preferentially use nearby target sites, while the target sites for IS10 transposition events are scattered randomly around the chromosome.