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.     

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.     

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.     

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.     

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.     

Structural and functional organization of the transposon Tn2555 carrying saccharose utilization genes

Transposon Tn2555 was isolated from a clinical E. coli strain carries the genes for sucrose utilization. Previously it was shown that Tn2555 is very unstable and undergoes structural rearrangements with a high frequency. Several deletion derivatives of Tn2555 and one with an inversion of the internal segment were found. They form the Tn2555 transposon family. This paper describes further structural and functional analysis of Tn2555. In the course of the experiments on pBR325 (Mob-) mobilization by conjugative RP4 derivatives, containing Tn2555 family elements, it was found, that all of them induce cointegrate formation. Some of these cointegrates were able to dissociate in rec+ and recA E. coli cells. Restriction endonuclease analysis of the resulting plasmids have shown, that among them were the end products of the Tn2555 transposition from RP4 to pBR325. Besides, the pBR325 derivatives, containing a discrete DNA segment of approximately 800 b.p., originating from Tn2555, were found. The segment can transpose from pBR325 to RP4 indicating that it is an insertion sequence. This new IS-element was designated IS286. The size and the genetic properties of IS286 resemble those of the IS1 element. However restriction analysis and Southern hybridization data show no significant homology between IS286 and IS1. It was found that the Tn2555 family elements are flanked by directly repeated IS286. One of them (Tn2555.3) contains an additional copy of IS286 in its internal region.     

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.     

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.     

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.     

Interaction (integration and excision) of the pRP19.6 plasmid, a derivative of the RP1 plasmid containing duplicated sequence IS21, with the chromosome of Escherichia coli K12

A pRP19.6 plasmid is the derivative of the temperature sensitive RPlts12 plasmid and contains a duplicated IS21 (IS8) element. Using temperature sensitive pRP19.6 replication, Hfr strains have been obtained by integration of the plasmid into the chromosome of E. coli rec+ and recA- cells and their properties were studied. According to the results obtained, pRP19.6 insertion into the genome of the rec+ bacteria IS reversible, and its integration into the chromosome of the recA- bacteria produced the stable Hfr strains. To elucidate the mechanism of pRP19.6 excision from the bacterial chromosome, plasmids of R+ transconjugates generated with a low frequency in the crosses between the stable Hfr strains and the rec+ recipients were analyzed. It was shown that the stable Hfr clones might produce stable R1 plasmids as well as a family of deletion KmsTra- derivatives of the pRP19.6. The structure of the KmsTra- was investigated and the mechanism of their formation was proposed. In the light of the data obtained, prospects of pRP19.6 practical application are discussed.     

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.     

recA-independent recombination between repeated IS50 elements is not caused by an IS50-encoded function.

Certain pBR322-related plasmids containing direct repeats of the insertion element IS50 appear to be unstable in recA Escherichia coli because smaller recombinant derivatives accumulate rapidly in plasmid DNA populations. We show here that (i) this instability is plasmid specific, but not IS50 specific; (ii) it is due to a detrimental effect exerted by these plasmids on bacterial growth; and (iii) the growth impairment is alleviated in cells harboring the smaller recombinant plasmids. Although a recent report had concluded that accumulation of recombinants reflected an IS50-specific recombination function, when correction is made for the relative growth rates of cells containing the parental and recombinant plasmids the evidence for such a recombination function disappears.     

Transposition behavior of IS15 and its progenitor IS15-Δ: Are cointegrates exclusive end products?

We report that the major product of IS15-promoted transposition is a cointegrate. When present in the multicopy plasmid pBR322, IS15 and its progenitor IS15-delta mediate the formation of cointegrates at frequencies of 3.5 X 10(-4) and 2.9 X 10(-5), respectively. We have studied the stability of the cointegrates generated by IS15 and IS15-delta. While these structures are resolved in a rec+ host, they were stable in a rec- host. These observations suggest that neither IS15 nor IS15-delta encode a resolvase and that cointegration is an end product of their transposition process. These properties of IS15-delta and IS15 can explain the transitions from IS15-delta to IS15 and from IS15 to IS15-delta observed in vivo.     

Convenient construction of strains useful for transducing recA mutations with bacteriophage P1

Abstract The generalized transducing phage P1 grew well on heterozygous Escherichia coli carrying recA srlC 300::Tn 10 on the chromosome and recA ⁺ on a pBR322-derived plasmid. Because of the close linkage of Tn 10 to recA mutations, including recA 1, recA 13, recA 56, recA deletion and recA allele of E. coli BNG30, the latter can be moved to other strains in transductional crosses for selective resistance to tetracycline.     

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.     

The site-specific deletion in plasmid pBR322

The formation of a deletion derivative of plasmid pBR322, designated pBR322 delta 1, was observed during cloning of various eukaryotic DNAs, when the BamHI site of the plasmid vector was used for construction of the recombinant molecules. The restriction analysis of six independently isolated pBR322 delta 1 plasmids allowed establishment of their complete identity. Similar deletion derivatives were also formed as a result of transformation of Escherichia coli cells by the linear form of vector pBR322 produced by BamHI cleavage, but not by SalI or HindIII. The endpoints of the deletion in one of the pBR322 delta 1 plasmids occurred at positions 375 and 16666 bp from the EcoRI site, as determined by sequence analysis. Formation of pBR322 delta 1 is most probably due to site-specific recombination between the sequence in the 1666-1670 bp region and the BamHI end of the linear pBR322 molecule. THe deletion was not controlled by the recA system of the host bacteria.