Tn4400, a compound transposon isolated from Bacteroides fragilis, functions in Escherichia coli.

Transfer factor pBFTM10, isolated from the obligate anaerobic bacterium Bacteroides fragilis, carries a clindamycin resistance determinant which we have suggested is part of a transposable element. DNA homologous to this determinant is found in many Clnr Bacteroides isolates, either in the chromosome or on plasmids. We have now established that Ccr resides on a transposon, Tn4400. In addition to the Ccr determinant that functions under anaerobic conditions in B. fragilis, Tn4400 also carries a determinant for tetracycline resistance (Tcr) which only functions in Escherichia coli under aerobic conditions. The presence of Tn4400 on pBFTM10 does not confer tetracycline resistance on B. fragilis cells containing it. DNA from pBFTM10 was cloned in E. coli, with pDG5 as the cloning vector, to form pGAT500. Using a mobilization assay involving pGAT500 and an F factor derivative, pOX38, we determined that a 5.6-kilobase region of pBFTM10 DNA was capable of mediating replicon fusion and transposition. Most of the mobilization products resulted from inverse transposition reactions, while some were the result of true cointegrate formation. Analysis of the cointegrate molecules showed that three were formed by the action of one of the ends of Tn4400 (IS4400), and one was formed by the action of the whole element (Tn4400). The cointegrate molecule carrying intact copies of Tn4400 at the junction of the two plasmids could resolve to yield an unaltered donor plasmid (pGAT500) and a conjugal plasmid containing a copy of Tn4400 or a copy of one insertion sequence element (pOX38::Tn4400 or pOX38::IS4400). Thus, Tn4400 is a compound transposon containing active insertion sequence elements as directly repeated sequences at its ends.     

Frequency of IS1-mediated molecular events in different members of the family Enterobacteriaceae.

The numbers of chromosomal copies of the insertion sequence IS1 in strains of Salmonella typhimurium (0 to 8 copies), Shigella sonnei (56 copies), and Shigella flexneri (41 copies) isolated in Mexico City, Mexico, were similar to those reported for these genera isolated in other countries. Of the 11 Shigella strains studied, all carried several small plasmids; however, in only one of these strains did a small plasmid contain IS1, IS1 recombination, cointegrate formation mediated by IS1 or by the IS1-flanked transposon Tn9, and transposition of Tn9 occurred at a higher frequency in S. typhimurium than in either Escherichia coli or S. sonnei strains. The frequencies of IS1 recombination in S. typhimurium strains containing either zero or eight copies of IS1 were similar.     

Identification of a second endogenous Porphyromonas gingivalis insertion element.

In this study a second endogenous Porphyromonas gingivalis insertion element (IS element) that is capable of transposition within P. gingivalis was identified. Nucleotide sequence analysis of the Tn4351 insertion site in a P. gingivalis Tn4351-generated transconjugant showed that a complete copy of the previously unidentified IS element, designated PGIS2, had inserted into IS4351R in Tn4351. PGIS2 is 1,207 bp in length with 19-bp imperfect terminal inverted repeats, and insertion resulted in a duplicated 10-bp target sequence. Results of Southern hybridization of chromosomal DNA isolated from several P. gingivalis strains with a PGIS2-specific probe demonstrated that the number of copies of PGIS2 per genome varies among different P. gingivalis strains. Computer analysis of the putative polypeptide encoded by PGIS2 revealed strong homologies to the products encoded by IS1358 from Vibrio cholerae, ISAS1 from Aeromonas salmonicida, and H-rpt in Escherichia coli K-12.     

Transposition of IS10 from the host Escherichia coli genome to a plasmid may lead to cloning artefacts

During recloning of Nicotiana tabacum L. repetitive sequence R8.3 in Escherichia coli, a modified clone that differed from the original by the insertion of an IS10 sequence was unintentionally produced. The insert was flanked by a 9-bp direct repeat derived from the R8.3 sequence, the 9-bp duplication of acceptor DNA in the site of insertion being a characteristic of IS10 transposition events. A database search using the FASTA program showed IS10 and other prokaryotic IS elements inserted into numerous eukaryotic clones. Unexpectedly, the IS10, which is not a natural component of the E. coli genome, appeared to be by far the most frequent contaminant of DNA databases among several IS sequences tested. In the GenEMBL database, the IS10 query sequence yielded positive scores with more than 500 eukaryotic clones. Insertions of shortened IS10 sequences having only one intact terminal inverted repeat were commonly found. Most full-length IS10 insertions (32 out of 40 analyzed) were flanked by 9-bp direct repeats having the consensus 5'-NPuCNN-NGPyN-3' with a strong preference for 5'-TGCTNA-GNN-3'. One insertion was flanked by an inverted repeat of more than 400 bp in length. PCR amplification and Southern analysis revealed the presence of IS10 sequences in E. coli strains commonly used for DNA cloning, including some reported to be Tn10-free. No IS10-specific PCR product was obtained with N. tabacum or human DNA. Our data suggest that transposition of IS10 elements may accompany cloning steps, particularly into large BAC vectors. This might lead to the relatively frequent contamination of DNA databases by this bacterial sequence. It is estimated that one in approximately every thousand eukaryotic clone in the databases is contaminated by IS-derived sequences. We recommend checking submitted sequences for the presence of IS10 and other IS elements. In addition, DNA databases should be corrected by removing contaminating IS sequences.     

Copy Number Control of Tn5 Transposition

Transposition of Tn5 in Escherichia coli strains containing one or multiple copies of the transposable element was investigated. It was found that the overall frequency of transposition within a cell remained constant regardless of the number of copies of Tn5 present in that cell. Experiments measuring the transposition frequency of differentially marked Tn5s confirmed that the frequency of transposition of an individual Tn5 decreased proportionally with the total number of copies of the element present in a cell. The IS50R -encoded function, protein 2, which has previously been shown to be an inhibitor of transposition, is sufficient to mediate this inhibitory effect. The concentration of protein 2 in a cell appears to modulate the transposition of individual Tn5 elements in such a way that the overall transposition of Tn5 in a cell remains constant.     

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.     

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.     

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.     

Diversity of Tn4001 Transposition Products: the Flanking IS256 Elements Can Form Tandem Dimers and IS Circles

We show that both flanking IS256 elements carried by transposon Tn4001 are capable of generating head-to-tail tandem copies and free circular forms, implying that both are active. Our results suggest that the tandem structures arise from dimeric copies of the donor or vector plasmid present in the population by a mechanism in which an IS256 belonging to one Tn4001 copy attacks an IS256 end carried by the second Tn4001 copy. The resulting structures carry abutted left (inverted left repeat [IRL]) and right (inverted right repeat [IRR]) IS256 ends. Examination of the junction sequence suggested that it may form a relatively good promoter capable of driving transposase synthesis in Escherichia coli. This behavior resembles that of an increasing number of bacterial insertion sequences which generate integrative junctions as part of the transposition cycle. Sequence analysis of the IRL-IRR junctions demonstrated that attack of one end by the other is largely oriented (IRL attacks IRR). Our experiments also defined the functional tips of IS256 as the tips predicted from sequence alignments, confirming that the terminal 4 bp at each end are indeed different. The appearance of these multiple plasmid and transposon forms indicates that care should be exercised when Tn4001 is used in transposition mutagenesis. This is especially true when it is used with naturally transformable hosts, such as Streptococcus pneumoniae, in which reconstitution of the donor plasmid may select for higher-order multimers.     

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.     

Interactions between the transposable element IS21 on R68.45 and TN7 in Pseudomonas aeruginosa PAO

Tn7 transposes from the chromosome of Pseudomonas aeruginosa into the plasmid R68.45 with tandem IS21, at up to 400 times the frequency that it transposes into R68, which has only one copy of IS21. While R68::TN7 derivatives are stable, R68.45::Tn7 isolates undergo frequent deletions. Instability of R68.45 occurs whether Tn7 is inserted into the plasmid (cis configuration) or into the bacterial chromosome (trans configuration). The deletions of R68.45 start at the junction between the tandem IS21 copies and proceed clockwise, ending in the region of oriT. It appears that Tn7 and IS21 can mutually stimulate transposition of each other.     

Lack of hotspot targets: a constraint for IS30 transposition in Salmonella

IS30 is an insertion element common in E. coli strains but rare or absent in Salmonella. Transfer of the IS30-flanked transposon Tn2700 to Salmonella typhimurium was assayed using standard delivery procedures of bacterial genetics (conjugation and transduction). Tn2700 'hops' were rare and required transposase overproduction, suggesting the existence of host constraints for IS30 activity. Sequencing of three Tn2700 insertions in the genome of S. typhimurium revealed that the transposon had been inserted into sites with a low homology to the IS30 consensus target, suggesting that inefficient Tn2700 transposition to the Salmonella genome might be caused by a lack of hotspot targets. This view was confirmed by the introduction of an IS30 'hot target sequence', whose sole presence permitted Tn2700 transposition without transposase overproduction. Detection of IS30-induced DNA rearrangements in S. typhimurium provided further evidence that the element undergoes similar activities in E. coli and S. typhimurium. Thus, hotspot absence may be the main (if not the only) limitation for IS30 activity in the latter species. If these observations faithfully reproduce the scenario of natural populations, establishment of IS30 in the Salmonella genome may have been prevented by a lack of DNA sequences closely related to the unusually long (24 bp) IS30 consensus target.     

Is103, a New Insertion Element in Escherichia Coli: Characterization and Distribution in Natural Populations

IS103 is a previously unknown insertion sequence found in Escherichia coli K12. We have sequenced IS103 and find that it is a 1441-bp element that consists of a 1395-bp core flanked by imperfect 23-bp inverted repeats. IS103 causes a 6-bp duplication of the target sequence into which it inserts. There is a single copy of IS103 present in wild-type E. coli K12 strain HfrC. In strain X342 and its descendents there are two additional copies, one of which is located within the bglF gene. IS103 is capable of excising from within bglF and restoring function of that gene. IS103 exhibits 44% sequence identity with IS3, suggesting that the two insertion sequences are probably derived from a common ancestor. We have examined the distribution of IS103 in the chromosomes and plasmids of the ECOR collection of natural isolates of E. coli. IS103 is found in 36 of the 71 strains examined, and it strongly tends to inhabit plasmids rather than chromosomes. Comparison of the observed distribution of IS103 with distributions predicted by nine different models for the regulation of transposition according to copy number and of the effects of copy number on fitness suggest that transposition of IS103 is strongly regulated and that it has only minor effects on fitness. The strong clustering of IS103 within one phylogenetic subgroup of the E. coli population despite its presence on plasmids suggests that plasmids tend to remain within closely related strains and that transfer to distantly related strains is inhibited.     

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.     

The admid system: generation of recombinant adenoviruses by Tn7-mediated transposition in E. coli

A new system has been developed for generating recombinant adenoviruses by Tn7-mediated transposition in E. coli. Low copy number E. coli plasmids containing a full-length adenoviral genome with lacZattTn7 replacing E1 have been constructed. The adenovirus plasmid or admid, as well as high copy number progenitors, were stably maintained in E. coli strain DH10B. Several transfer vectors containing a mammalian expression cassette flanked by Tn7R and Tn7L were used as donors to transpose the mini-Tn7 into the E1 region of the adenoviral genome. Transposed recombinant admids are readily identified by their beta-galactosidase phenotype. Transfection of admid DNA into producer cells resulted in the efficient production of infectious adenovirus. This easy-to-use, efficient system generates pure, clonal stocks of recombinant adenovirus without successive rounds of plaque purification.     

Distribution and Abundance of Insertion Sequences among Natural Isolates of Escherichia coli

A reference collection of 71 natural isolates of Escherichia coli (the ECOR collection) has been studied with respect to the distribution and abundance of transposable insertion sequences using DNA hybridization. The data include 1173 occurrences of six unrelated insertion sequences (IS1, IS2, IS3, IS4, IS5 and IS30). The number of insertion elements per strain, and the sizes of DNA restriction fragments containing them, is highly variable and can be used to discriminate even among closely related strains. The occurrence and abundance of pairs of unrelated insertion sequences are apparently statistically independent, but significant correlations result from stratifications in the reference collection. However, there is a highly significant positive association among the insertion sequences considered in the aggregate. Nine branching process models, which differ in assumptions regarding the regulation of transposition and the effect of copy number on fitness, have been evaluated with regard to their fit of the observed distributions. No single model fits all copy number distributions. The best models incorporate no regulation of transposition and a moderate to strong decrease in fitness with increasing copy number for IS1 and IS5, strong regulation of transposition and a negligible to weak decrease in fitness with increasing copy number for IS3, and less than strong regulation of transposition for IS2, IS4 and 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.     

Amplification of drug resistance genes flanked by inversely repeated IS1 elements: involvement of IS1-promoted DNA rearrangements before amplification.

Tn2653 contains one copy of the tet gene and two copies of the cat gene derived from plasmid pBR325 and is flanked by inverted repeats of IS1. Transposed onto the P1-15 prophage, it confers a chloramphenicol resistance phenotype to the Escherichia coli host. Because the prophage is perpetuated as a plasmid at about one copy per host chromosome, the host cell is still tetracycline sensitive even though P1-15 is carrying one copy of the tet gene. We isolated P1-15::Tn2653 mutants conferring a tetracycline resistance phenotype, in which the whole transposon and variable flanking P1-15 DNA segments were amplified. Amplification was most probably preceded by IS1-mediated DNA rearrangements which led to long direct repeats containing Tn2653 sequences and P1-15 DNA. Subsequent recombination events between these direct repeats led to amplification of a segment containing the tetracycline resistance gene in tandem arrays.