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.     

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.     

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.     

Dissociation of unstable cointegrate plasmids formed during transposition of IS1 element: the role of IS1 encoded recombinase

The properties of IS1/Tn9'-mediated cointegrates between plasmids pDK57 (pBR322:: :: Tn9') and pRP3.1--the deletion derivative of RP1 were investigated. It was found that IS1/Tn9'-mediated integration of pDK57 into the active transcribed regions of pRP3.1 (in particular kan and tet genes) leads to formation of unstable cointegrates capable of resolving in E. coli K-12 rec+ and recA cells. The structure of dissociation products of unstable cointegrates was studied. According to the data received in rec+ cells, the unstable cointegrates mainly produced plasmids pDK57 and pBR322::IS1--Cms-derivative of pDK57 as resolution products. In recA cells the cointegrates dissociate in different ways, and this process leads to the formation of not only pDK57 and pBR322::IS1, but also to the production of the deletion derivatives of these plasmids as well as to the derivatives of pDK57 and pBR322::IS1, containing duplications of IS1 or separate parts of Tn9'. It was concluded that the IS1-specific recombinase is involved in the dissociation (resolution) of unstable IS1/Tn9'-mediated cointegrates. This recombinase recognizes the sites localized in both inverted termini of IS1 as well as in the adjacent DNA segments. Hence, it is possible, that the IS1 recombinase is involved also in the generation of IS1-adjacent delations.     

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.     

Recombination in recA cells between direct repeats of insertion element IS1.

The IS1 sequences that flank the Tn9 chloramphenicol acetyltransferase gene as direct repeats recombine after transformation into an Escherichia coli recA strain. The recombination requires the lambda pL promoter on the plasmid. A plasmid that contains mutant IS1 elements does not recombine. These results indicate that this recombination requires an IS1-specific gene product. The recombinational activity of IS1 may resolve transient cointegrates formed during the transposition of IS1. I discuss a possible role for the lambda pL promoter.     

ISPpy1, a novel mobile element of the ancient Psychrobacter maritimus permafrost strain: translocations in Escherichia coli K-12 cells and formation of composite transposons

It was shown that IS element ISPpyl isolated earlier in the permafrost strain Psychrobacter maritimus MR29-12 has a high level of functional activity in cells of the heterologous host Escherichia coli K-12. ISPpyl can be translocated in E. coli cells by itself and mobilize adjacent genes and can also form composite transposons flanked by two copies of this element. Apart from translocations between different plasmids, the composite ISPpyl-containing transposon Tn5080a is capable of translocation from the plasmid into the E. coli chromosome with high frequency and from the chromosome into the plasmid. Among products of Tn5080a transposition into plasmid R388, simple insertions were predominantly formed together with cointegrates. Upon mobilization of adjacent genes with the use of one ISPpyl copy, only cointegrates arise.     

Regulation of IS1 transposition by the insA gene product

The IS1 element contains two adjacent genes called insA and insB, both required for IS1 transposition and IS1-mediated plasmid cointegration. These two genes are transcribed polycistronically from the promoter in the left terminal inverted repeat of IS1 (insL). We constructed overexpression systems of these genes with the tac promoter, which are regulated by an exogenous inducer, isopropyl-beta-D-thiogalactopyranoside (IPTG). Then we have examined, under various conditions of induction with IPTG, how overexpression of these genes affects IS1 transposition, using an assay based on plasmid cointegration. When the insA and insB genes were organized identically to the wild-type IS1 genes and simultaneously expressed using low concentrations of IPTG, activity of a mutant IS1 in cis was restored, but not in trans. Higher IPTG concentrations resulted in lower transposition activity. Expression in trans of insA and insB results in a 50 to 100-fold reduction of the frequency of cointegration mediated by wild-type IS1. Such a reduction is also observed when only the insA gene is overexpressed in trans. Overexpression of either mutant insA or insB does not affect the cointegration event. Tests with the insA-lacZ fusion gene showed that the InsA product inhibits the expression of IS1 genes directed by its own promoter in insL. These results suggest that the InsA product regulates IS1 transposition by inhibiting expression of IS1 transposition genes in addition to acting as part of a transposase complex.     

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.     

Immunity to repeated transposition of the insertion sequence IS21

The ability of pBR325 derivatives carrying a copy of IS21-element to accept the second copy of this element from plasmid pRP19.6, a temperature-sensitive for replication mutant of RPI containing the duplicated IS21 was studied. It was shown that the frequency of IS21 transposition into plasmids pBR32S::IS21 differing by localization IS21 was lower by two orders of magnitude as compared to that of pBR325. The restriction endonuclease analysis revealed that the insertion of the second copy of IS21 resulted in the formation of pBR325 derivatives carrying the tandem repeated copies of IS21. It was also shown that the plasmids pBR325::IS21 were capable of increasing the frequency of pRP19.6 insertion into the bacterial chromosome from 3-9 to 200-300 times depending on IS21 localization. On the basis of the results obtained and literature data the possible mechanism of the transposition immunity is discussed.     

IS1-mediated tandem duplication of plasmid pBR322. Dependence on recA and on DNA polymerase I

Transposable-element-mediated fusion of the conjugal plasmid pOX38::Tn9 with pBR322 results in the appearance of cointegrates composed of a single copy of each plasmid, and cointegrates which carry a single copy of pOX38 but multiple tandem copies of pBR322. These plasmids are separated by directly repeated copies of the transposable element. We demonstrate here that such multimers can be generated from monomeric cointegrates, probably by unequal crossing over between the flanking Tn9(IS1) elements. Their appearance is thus not necessarily associated with the original transposition (fusion) event. Our study demonstrates that the process of duplication is strongly dependent on the homologous recombination system of Escherichia coli, since it is undetectable by our methods in recA- strains. It is also strongly dependent on the presence of a functional DNA polymerase I in the cell. The major pathway(s) for this duplication thus appears to rely on both the homologous recombination system and the replication of the duplicated segment.     

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.     

A comparison of intramolecular rearrangements promoted by transposons Tn5 and Tn10.

The bacterial transposon Tn10 has previously been shown to move to other genomic sites by a conservative mechanism, whereby the transposon is excised by double-strand breaks and inserted between a pair of staggered nicks at the target. Other transposons, like Tn3, have been shown to transpose by a replicative mechanism that involves symmetrical nicking of the element and formation of the 'Shapiro intermediate', which can mature into either a cointegrate or a simple insert. The situation with respect to Tn5 is unclear; it was originally reported to use a conservative mechanism, but other evidence suggests that the mechanism might be replicative. In this paper, rearrangements of adjacent DNA promoted by Tn10 and Tn5 have been compared using positive selection for galactose-resistance to detect such rearrangements. Tn10 promoted the formation of adjacent deletions (that started from an inside end of Tn10), deletion/inversions and simple IS10 insertions, but no cointegrates. This behaviour is fully consistent with a conservative mechanism. In contrast, Tn5 was found to promote formation of adjacent deletions (that started mainly from an outside end of Tn5), IS50 insertions (that were frequently accompanied by inversions of adjacent DNA) and cointegrates. These characteristics seem compatible with a replicative, rather than a conservative, mode of transposition. Clearly, Tn5 and Tn10 exhibit some significant differences in their transposition. These results, and results of some previous experiments, have been interpreted to mean that Tn5 could use a replicative mechanism for its transposition.     

Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917

Plasmids pAD1 (37.8 megadaltons) and pAD2 (17.1 megadaltons) of Streptococcus faecalis strain DS16 have been mapped with restriction enzymes. The location of a hemolysin-bacteriocin determinant on the conjugative pAD1 plasmid was derived from analyses of transposon insertions. Electron microscope and hybridization analyses located Tn917(Em) and the streptomycin (Sm) and kanamycin (Km) resistance determinants on the nonconjugative pAD2 plasmid. It was shown previously that the erythromycin (Em) resistance associated with Tn917 is inducible and that transposition from pAD2 to pAD1 is also stimulated by exposure of cells to low concentrations of Em. Here we show that inducing concentrations of Em also increase the conjugative transfer potential of pAD1; this is possibly related to a mild and short-lived inhibitory stress placed on the cells before full induction of resistance. Selection of Em-resistant transconjugants arising from matings between DS16 and a plasmid-free recipient gave rise to transconjugants which primarily harbor stable pAD1::pAD2 cointegrates. A 30-min exposure of donors to Em (0.5 microgram/ml) before mating resulted in a severalfold increase in the number of such transconjugants. However, a small fraction (e.g., 3 of 40) of these Emr Smr Kmr transconjugants harbored pAD1::Tn917 and pAD2 molecules. Since we believe pAD2 is incapable of being mobilized by pAD1 without being covalently linked, it is likely that transfer in these cases involved cointegrates representing structural intermediates in the transposition of Tn917 from pAD2 to pAD1. It follows that such intermediates probably had two copies of Tn917 and readily resolved after transfer. (These cointegrates are different from the stable cointegrates which were shown to have only a single copy of Tn917; the latter are assumed not to be related to transposition.) Two variants with altered Tn917 transposition properties were derived. One of them transposed at an elevated frequency, whereas the other showed no detectabel transposition. In neither case was transposition influenced by Em exposure; however, both remained inducible for Em resistance.     

Structural instability of co-integrates formed during interaction of plasmid R57 with pB322 and RP1. Possible role of IS1 element in the degradation of co-integrates

The conjugative plasmid R57 determines resistance to ampicillin and chloramphenicol. Earlier it was shown that R57 encodes site-specific recA-independent recombinase, which acts in cis and resolves IS1-mediated cointegrates arising in the Escherichia coli recA cells between R57 and pBR322. In the present work the properties of the cointegrates between R57 and pBR322 or RP1 arising in the E. coli rec+ strains were studied. It was found that the cointegrates between R57 and pBR322, obtained by mating of the respective biplasmid donors of E. coli rec+ and the rec+ recipients, lost as a result of deletion a large DNA segment of R57 containing determinant Cmr. The resulting hybrid replicons preserved determinants Apr and Tcr of pBR322 and the R57 conjugative properties and were structurally identical. By using plasmid RP1ts12, which is temperature-sensitive in replication, it was demonstrated that in cells rec+ the cointegrates between R57 and RP1 are extremely unstable. On storage they undergo structural degradation mainly affecting the RP1 replicon. The degradation products of the hydrid complex had lost their RP1 genes but preserved the R57 functional determinants. For elucidation of the observed phenomena the properties of the IS1-mediated cointegrates between pBR322:Tn9 and plasmid pBR3.1--deletion derivative of RP1 were studied. It was found that insertion of IS1 sometimes resulted in formation of unstable cointegrates capable of resolving and loosing determinant Cmr with a high frequency. It was suggested that IS1 encodes the site-specific recombinase responsible for resolution of the IS1-mediated cointegrates and deletion generation. Expression of this recombinase appears to be dependent on structure of the insertion sites. The possible role of IS1 and recombinase encoded by it in resolution and structural instability of the cointegrates between R57 and pBR322 or RP1 is discussed.     

IS10/Tn10 transposition efficiently accommodates diverse transposon end configurations.

Transposon Tn10 and its component insertion sequence IS10 move by non-replicative transposition. We have studied the array of reaction intermediates and products in a high efficiency in vitro IS10/Tn10 transposition reaction. Synapsis of two transposon ends, followed by cleavage and strand transfer, can occur very efficiently irrespective of the relative locations and orientations of the two ends. The two participating ends can occur in inverted or direct orientation on the same molecule or, most importantly, on two different molecules. This behavior contrasts sharply with that of Mu, in which transposition is strongly biased in favor of inverted repeat synapsis. Mechanistically, the absence of discrimination amongst various end configurations implies that the architecture within the IS10/Tn10 synaptic complex is relatively simple, i.e. lacking any significant intertwining of component DNA strands. Biologically these observations are important because they suggest that the IS10 insertion sequence module has considerable flexibility in the types of DNA rearrangements that it can promote. Most importantly, it now seems highly probable that a single non-replicative IS10 element can promote DNA rearrangements usually attributed to replicative transposition, i.e. adjacent deletions and cointegrates, by utilizing transposon ends on two sister chromosomes. Other events which probably also contribute to the diversity of IS10/Tn10-promoted rearrangements are discussed.