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.     

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.     

Intramolecular transposition of insertion sequence IS91 results in second-site simple insertions

A series of plasmids carrying an IRL-kan-IRR transposable cassette, in which IRL and IRR are the left- and right-terminal sequences of IS91, have been constructed. These cassettes could be complemented for transposition with similar efficiency when IS91 transposase was provided either in cis or in trans. A total of 87% of IS91 transposition products were simple insertions of the element, while the remaining 13% were plasmid fusions and co-integrates. When transposase expression was induced from an upstream lac promoter, transposition frequency increased approximately 100-fold. An open reading frame (ORF) present upstream of the transposase gene, ORF121, could be involved in target selection, as mutations affecting this ORF were altered in their insertion specificity. Intramolecular rearrangements were analysed by looking at transposition events disrupting a chloramphenicol resistance gene (cat ) located outside the transposable cassette. Plasmid instability resulting from insertion of an extra copy of IRL-kan-IRR within the cat gene was observed; transposition products contained a second copy of the cassette inserted either as a direct or as an inverted repeat. No deletion or inversion of the intervening DNA was observed. These results could be explained as a consequence of intramolecular transposition of IS91 according to a model of rolling-circle transposition.     

Transposition of IS1-lambdaBIO-IS1 from a bacteriophage lambda derivative carrying the IS1-cat-IS1 transposon (Tn9)

Summary Tn9 is a transposable element in which a gene (cat) determining chloramphenicol resistance is flanked by directly repeated sequences that are homologous to the insertion sequence IS1. We show here that infection of Escherichia coli K12 (under Rec^- Red^- Int^- conditions) with a bio transducing phage carrying Tn9 results in the formation of bio transductants as frequently as cat transductants (about 1 per 10⁶ to 10⁷ infected cells). Most of the bio transductants do not carry cat, just as most of the cat transductants do not carry bio. In spite of the absence of cat, the bio prophage can transpose a second time, from the E. coli chromosome to different sites on an F gal plasmid. Analysis of the structure of the transposed bio element, by restriction nuclease digestion and by electron microscopy, demonstrates that the integrated bio prophage is flanked by directly repeated IS1 elements. We conclude that there is no genetic information for the ability to transpose encoded in the non-repeated portion of Tn9, i.e. that the directly repeated IS1 elements alone are responsible for Tn9 transposition.     

Cloning of a lac+, bamHI fragment into transposon Tn3 and transposition of the Tn3[lac] element

By use of recombinant DNA techniques, we have inserted the lac⁺ operon into a transposon (Tn3). We constructed the recombinant in such a way that the essential step in assaying for transposition consisted of screening for bacteria with a thermostable Lac⁺ phenotype. Our results showed that transposition of the Tn3[lac⁺] element occurred and that its frequency was derepressed compared to frequencies reported by others for wild-type Tn3 transposition.     

Duplication of insertion element IS50 associated with Tn5 transposition in Azospirillum brasilense

The characterization of a DNA fragment with a Tn5 insertion in a regulatory nif gene of Azospirillum brasilense is reported. Restriction endonuclease mapping, Southern hybridization with a Tn5 probe, and nucleotide sequencing revealed that IS50 had duplicated in Tn5. The duplication of an IS50 element suggests the occurrence of a replicative mechanism of transposition. A strategy, based on the bacterial ability of homologous recombination that was used to precisely eliminate Tn5 along with the duplicated IS50 element, is presented.     

Nature of the plasmids determining the unstable inheritance of transposon Tn9 in Escherichia coli K-12

Unstable inheritance of transposon Tn9 in the Escherichia coli strain KS7201 had been connected with its integration into a certain bacterial chromosome site (attTn9A). However, the present work shows that the transposon is situated within an unstable plasmid in this strain. A possibility of such plasmid's formation, as a result of a deletion of a part of bacteriophage lambda DNA, is shown.     

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.     

Postexcision transposition of the transposon Tn10 in Escherichia coli K12

An experimental analysis of the fate of transposon Tn10 after excision from a proA::Tn10 site localized on the plasmid F' leads to the conclusions: 1. The precise excision is a progressive process. Its probability is estimated per time unit. 2. An excised Tn10 is always integrated into a different genetic locus. 2. An excised Tn10 is always integrated into a different genetic locus. 3. The kinetics of postexcision transposition are sometimes very slow. The excised transposon is inherited in one cell line in spite of cell multiplication. 4. The processes of excision and secondary insertion have no absolute requirement for the recA+ genotype but they are strongly enhanced in recA+ cells. 5. The kinetics of postexcision transposition are strongly dependent on the genetic site from which the transposon was excised. 6. The probability of postexcision transposition is fully determined by the probability of excision and depends on the genotype of the host and many other factors.     

IS8- and Tn2353-mediated cointegration of the plasmids R15 and RP4::Tn1

Summary The plasmids R15 and RP4:: Tn1 form fused structures (85 Md and 92 Md cointegrates). The cointegrates do not resolve practically in recA ^– Escherichia coli cells and have a mean life-time of more than 50 generations in a recA ⁺ background.The 85 Md cointegrates were generated at a frequency of 4×10^–4 per R15 transconjugant during a mating between E. coli [R15; RP4:: Tn1] and E. coli [FColVBtrp:: Tn1755]. These plasmids carry two directly repeated copies of the mobile element IS8 at the junctions between R15 and RP4:: Tn1. The transposition of IS8 from RP4:: Tn1 to the R15 plasmid and the formation of hybrid molecules promoted by this process appear to be induced by the IS8 element of the Tn1755 structure during or after conjugal transfer of FColVBtrp:: Tn1755 into E. coli [R15; RP4:: Tn1] cells.The formation of the 92 Md cointegrates occurs at a frequency of 2×10^–5. The fused molecules of R15 and RP4:: Tn1 carry two direct copies of an 8.65 Md R15 fragment at the junctions between these replicons. The fragment has specific features of a new transposon. This element designated Tn2353 determines resistance to Hg, Sm and Su and contains two sites for each BamHI, BglII and SalI and three sites for both EcoRI and PstI. The physical map and some other characteristics of Tn2353 are presented.Abbreviations Ap ampicillin - EtBr ethidium bromide - Km kanamycin - Md megadaltons - Sm streptomycin - Su sulfanilamide - Tc tetracycline - [] brackets indicate plasmid-carrier state     

Deletions and an inversion induced by a resident IS1 of the lactose transposon Tn951

Summary DNA-DNA filter binding tests, Southern blotting experiments and DNA heteroduplex analysis clearly show that Tn951 contains an IS1 element. This IS1-951 sequence is peculiar in that it does not contain the PstI cleavage site which is usually observed on E. coli derived IS1 elements. Nonetheless, IS1-951 induces deletions. This process is temperature dependent. One instance of an IS1-951 induced inversion was observed, the structure of which is compatible with the current models of transposition of IS elements.     

Altered imino diacid synthesis and transcription in crown gall tumors with transposon Tn5 insertions in the 3' end of the octopine synthase gene

Octopine synthase encoded by the T-DNA (transferred DNA) locus ocs synthesizes N2-(D-1-carboxyethyl)-L-amino acids in octopine-type crown gall tumors. So far, derivatives of only basic amino acids have been isolated. We have detected a glutamine derivative and called it heliopine. Tumors induced by several Ti plasmids with transposon Tn5 insertions in the 3' end of ocs still synthesized small quantities of N2-(1-carboxyethyl)-arginine and N2-(1-carboxyethyl)-glutamine. In addition, N2-(1,3-dicarboxypropyl)-asparagine, which is absent in wild-type octopine tumors, was detected in these tumors. These three imino diacids (octopine, heliopine, and asparaginopine, respectively, or their isomers) were undetectable in tumors induced by Ti plasmids harboring deletions of the ocs gene. Poly(A)+ RNAs which hybridize to the ocs sequence can also be detected in the ocs::Tn5 tumors; these RNAs, however, were heterogeneous in size and shorter in length than the normal ocs mRNA. These results indicate that mutant ocs products synthesize imino diacids in these ocs::Tn5 tumors.     

H-NS mediates the dissociation of a refractory protein-DNA complex during Tn10/IS10 transposition

Tn10/IS10 transposition takes place in the context of a protein-DNA complex called a transpososome. During the reaction, the transpososome undergoes several conformational changes. The host proteins IHF and H-NS, which also are global regulators of gene expression, play important roles in directing these architectural changes. IHF binds tightly to only one of two transposon ends within the transpososome, folding this end into a DNA loop structure. Unfolding this DNA loop is necessary for excising the transposon from flanking donor DNA and preventing integration of the transposon into itself. We show here that efficient DNA loop unfolding relies on the continuity of the flanking donor DNA on the side of the transpososome opposite to the folded transposon end. We also show this same donor DNA is a preferred binding site for H-NS, which promotes opening of the IHF-loop, which is required for productive target interactions. This is counter to the usual mode of H-NS action, which is repressive due to its propensity to coat DNA. The interplay between IHF and H-NS likely serves to couple the rate of transposition to the host cell physiology as both of these proteins are integrated into cellular stress response pathways.     

Ordering of the flagellar genes in Pseudomonas aeruginosa by insertions of mercury transposon Tn501.

The flagellar genes of Pseudomonas aeruginosa PAO cluster on the chromosome at two distinct regions, region I and region II. The order of the flagellar cistrons in this organism was established by using transducing phage G101 and plasmids FP5 and R68.45. A method to insert transposon Tn501 near the fla genes was devised. We obtained two strains in which Tn501 was inserted at sites close to the flagellar cistrons in region II. We isolated Fla mutants in which the chromosomal segment between the two Tn501 insertion sites was deleted. Using Tn501-encoded mercury resistance as an outside marker, we determined the order of 9 of the 11 flagellar cistrons in region II as follows: puuF-region I-flaG-flaC-flaI-flaH-flaD-flaB-flaA-flaF-flaE-pur-67. By using phage G101-mediated transduction, the mutation converting monoflagellated bacteria into the multiflagellated (mfl) form was closely linked to the five fla cistrons in region I. Using mfl as an outside marker, we determined the order of the five cistrons as follows: puuF-flaV-flaZ-flaW-flaX-flaY-region II. The mfl mutation was shown to be either located within the flaV cistron or linked very closely to this cistron. No linkage was observed in transductions between any of the fla cistrons in region I and any of the fla cistrons in region II.     

Transduction analysis of transposon Tn551 insertions in the trp-thy region of the Staphylococcus aureus chromosome.

Previous studies have shown that Tn551, a 5.2-kilobase-pair transposon that determines constitutive resistance to erythromycin, can occupy a variety of chromosomal sites between thy-101 and trp-103 in Staphylococcus aureus 8325. Although many of these insertions were "silent," many others, including lys, thr, met, tyr, and trp, resulted in auxotrophic mutations. The close proximity and erythromycin-resistant phenotypes of the insertions in this region have made their mapping by transformation difficult. Analysis of these sites and similar chemically induced mutations by generalized transduction with phage 80 alpha have defined the order and relationship of these insertion sites and provided a detailed map of this region of the chromosome, including the orientation of the trp operon. The results of this study and a limited phenotypic characterization of the mutants have shown that the divergent pathway from aspartate to lysine, threonine, and methionine, several reactions in tyrosine biosynthesis, and the entire tryptophan operon are determined by this region of the chromosome. The linkage results obtained by transduction have been compared with similar data obtained previously by transformation; this comparison suggests the existence, between thy and lys, of a preferred headful cutting site for transducing phage DNA morphogenesis from the host chromosome.     

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 beta-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.