Transposition effect of adenine (Dam) methylation on activity of O end mutants of IS50

The two ends of insertion sequence IS50 (from Tn5) differ in sequence and in activity during transposition: the IS50 I end contains DNA adenine methylation (Dam) sites and is affected directly by Dam methylation, whereas the O end lacks Dam sites. The effect of Dam methylation on the transposition of IS50-derived elements with base substitution mutations in their O ends was assayed to understand better how the divergent O and I ends interact. Of 31 O end mutations tested, ten impaired transposition less, and two impaired transposition more in Dam- than in Dam+ cells. These results suggest that the interaction between the two ends in a transposition complex is affected by the sequence or the extent of methylation of one end.     

Orientation of IS50 transposase gene and IS50 transposition.

Reversal of transposase gene orientation with respect to the nonidentical ends of IS50 strongly decreased IS50 transposition in both Dam- and Dam+ hosts. In either orientation, IS50 transposase expression was unaffected. These effects were independent of the surrounding DNA context. This shows that the efficiency of IS50 transposition is dependent on transposase gene orientation. The transposition frequencies of transposons utilizing inverted IS50 inside ends (IE), IE-IE transposons, were lower than either outside end (OE)-IE or OE-OE transposons.     

Fis plays a role in Tn5 and IS50 transposition.

The Fis (factor for inversion stimulation) protein of Escherichia coli was found to influence the frequency of transposon Tn5 and insertion sequence IS50 transposition. Fis stimulated both Tn5 and IS50 transposition events and also inhibited IS50 transposition in Dam-bacteria. This influence was not due to regulation by Fis of the expression of the Tn5 transposition proteins. We localized, by DNase I footprinting, one Fis site overlapping the inside end of IS50 and give evidence to strongly suggest that when Fis binds to this site, IS50 transposition is inhibited. The Fis site at the inside end overlaps three Dam GATC sites, and Fis bound efficiently only to the unmethylated substrate. Using a mobility shift assay, we also identified another potential Fis site within IS50. Given the growth phase-dependent expression of Fis and its differential effect on Tn5 versus IS50 transposition in Dam-bacteria, we propose that the high levels of Fis present during exponential growth stimulate transposition events and might bias those events toward Tn5 and away from IS50 transposition.     

Escherichia coli integration host factor binds specifically to the ends of the insertion sequence IS1 and to its major insertion hot-spot in pBR322

We report here that the ends of IS1 are bound and protected in vitro by the heterodimeric protein integration host factor (IHF). Under identical conditions, RNA polymerase binds to one of these ends (IRL) and protects a region that includes the sequences protected by IHF. Other potential sites within IS1, identified by their homology to the apparent consensus sequence, are not protected. Footprinting analysis of deletion derivatives of the ends demonstrates a correspondence between the ability of the end sequence to bind IHF and its ability to function as an end in transposition. Nonetheless, some transposition occurs in IHF- cells, indicating that IHF is not an essential component of the transposition apparatus. IHF also binds and protects four closely spaced regions within the major hot-spot for insertion of IS1 in the plasmid pBR322. This striking correlation of hot-spot and IHF-binding sites suggests a possible role for IHF in IS1 insertion specificity.     

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.     

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.     

Use of a Tn5 derivative that creates lacZ translational fusions to obtain a transposition mutant

We constructed a derivative of Tn5, Tn5 ORFlac, that is capable of creating lacZ translational fusions upon transposition. Lac- strains carrying this construct formed red papillae when plated on MacConkey-lactose media. Lac+ cells isolated from independent papillae expressed distinct beta-galactosidase fusion proteins, suggesting that the Lac+ phenotype resulted from transposition. In support of this, analysis of plasmids carrying Tn5 ORFlac prepared from these cells indicated that the Lac+ phenotypes arose as a result of intermolecular rearrangements. Furthermore, a derivative of Tn5 ORFlac that contains an ochre mutation in the transposase gene formed papillae only in a supB strain. Tn5 ORFlac is useful for obtaining mutants that affect Tn5 transposition and for creating lacZ fusions. We used the papillation phenotype to isolate a spontaneous revertant of IS50L that promotes transposition at a 3.6-fold higher rate than IS50R. The mutation altered the amino acid sequence of both transposase and inhibitor.     

Spontaneous transposition in the bacteriophage lambda cro gene residing on a plasmid.

A new mutagenesis assay system based on the phage lambda cro repressor gene residing on a plasmid was developed. The assay detects mutations in cro that decrease the binding of the repressor to the OR operator in an OR PR-lacZ fusion present in a lambda prophage. Mutations arose spontaneously during growth of E. coli cells harboring cro plasmids at a frequency of 3-6 x 10(-6). Analysis of some 200 cro mutants from several 'wild-type' strains revealed a substantial fraction of 25-70% insertion events caused by transposition of IS elements. Most of the insertions were caused by IS1, but IS5 insertions were observed too. In strains harboring Tn10, IS10 was responsible for most insertions. Restriction nuclease digestion analysis revealed a preference for insertion of IS10 into the C-terminal half of cro, despite the absence of sequences which are known hot spots for Tn10 insertions. The frequency of IS1 insertions into cro decreased 25-60-fold and that of IS10 insertions decreased 200-fold in cells carrying the recA56 mutation, suggesting that RecA is involved in transposition of these elements. During the logarithmic phase of growth, the mutation frequency was constant for at least 22 generations; however, upon continuous incubation at the stationary phase, the mutation frequency gradually increased, yielding a 3-fold increase in the frequency of insertion and a 4-5-fold increase in point mutation. Genomic Southern analysis of chromosomal IS elements in cells which underwent a transposition from the chromosome into the cro plasmid revealed that the number and distribution of IS1 and IS5 were usually unaltered compared to cells which did not undergo a transposition event. In contrast, essentially each IS10 transposition was accompanied by multiple events which led to changes in the number and distribution of chromosomal IS10 elements.     

Tn10 transposition and circle formation in vitro

We describe a cell-free system that promotes Tn10 transposition and transposon circle formation, a related intramolecular event. Tn10 circle formation in vitro has been characterized in detail, and is shown to require a supercoiled substrate and to proceed in the absence of ATP. The reaction requires Tn10 transposase protein, and either of two E. coli proteins, integration host factor (IHF) and HU, which are small DNA binding proteins that change the conformation of DNA. Tn10 is composed of inverted repeats of insertion sequence IS10. Pair-wise combinations of the IS10 "outside" and "inside" ends mediate distinct classes of rearrangements in vivo, and they exhibit different reaction requirements in vitro. In contrast to the Tn10 reaction, which involves two outside ends, circle formation with two inside ends proceeds with a transposase fraction alone, in the absence of added host factors, and is inhibited by methylation of the dam site within each terminus.     

Integration host factor increases the transpositional immunity conferred by gamma delta ends.

The ends of the bacterial transposon gamma delta contain adjacent binding sites for gamma delta transposase and integration host factor (IHF). IHF+ and IHF- strains were used in conjunction with gamma delta transposon ends containing or lacking the site for IHF binding to determine the role that IHF plays in various gamma delta-mediated transposition events. IHF was not essential for the transposition of gamma delta and seemed to decrease its frequency of transposition about threefold. IHF played no role in determining the distribution of gamma delta inserts into a target replicon, nor did it significantly alter the frequency of simple transpositions. The only clear role discerned for IHF and the terminal IHF-binding sites was in transposition immunity. IHF stimulated the immunity of those plasmids that contain an end of gamma delta, provided the end included the terminal IHF-binding site. For both ends, the degree of stimulation of immunity was similar to the stimulation of binding of transposase by IHF.     

dnaA, an essential host gene, and Tn5 transposition.

Mutations in dnaA, an essential gene in Escherichia coli, decrease the frequency of transposition of Tn5. An insertion mutation in the dnaA gene does not affect Tn5 gene expression. Therefore, the DnaA protein plays a role either in the transposition reaction itself or in some type of cellular regulation of transposition. Analysis of a mutation in the DnaA box, found at the outside end of IS50, is consistent with a direct interaction of the protein through these bases. IS50 transposition, which utilizes only one end containing a DnaA box, is not affected by dnaA mutations. Overproduction of the DnaA protein does not increase transposition frequencies in wild-type cells, even when the transposase is also overproduced.     

Transposition of IS50L activates downstream genes.

A transposition system constructed to detect the transposition of Tn5 to a site upstream of the lacZ gene has revealed that transposition of IS50L can activate downstream genes. Expression is apparently mediated by the NPTII promoter. Transposase produced either by IS50R or by the suppressed IS50L catalyzed transposition of IS50L.     

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.     

Functional organization of the ends of IS 1: specific binding site for an IS1-encoded protein

The IS 1-encoded protein InsA binds specifically to both ends of IS1, and acts as a repressor of IS1 gene expression and may be a direct inhibitor of the transposition process. We show here, using DNasel 'foot-printing' and gel retardation, that the InsA binding sites are located within the 24/25 bp minimal active ends of IS1 and that InsA induces DNA bending upon binding. Conformational modification of the ends of IS1 as a result of binding of the host protein integration host factor (IHF) to its site within the minimal ends has been previously observed. Using a collection of synthetic mutant ends we have mapped some of the nucleotide sequence requirements for InsA binding and for transposition activity. We show that sequences necessary for InsA binding are also essential for transposition activity. We demonstrate that InsA and IHF binding sites overlap since some sequence determinants are shared by both InsA and IHF. The data suggest that these ends contain two functional domains: one for binding of InsA and IHF, and the other for transposition activity. A third region, when present, may enhance transposition activity with an intact right end. This 'architecture' of the ends of IS1 is remarkably similar to that of IS elements IS10, IS50 and IS903.     

A sequence at the inside end of IS50 down regulates transposition.

Deletion analysis has shown that the segment at the IS50 inside (I) end that is needed for efficient transposition is approximately 19 bp long. Dam methylation at two 5' GATC sequences within this segment decreases I-end transposition activity. A third 5' GATC sequence is present at bp 21-24 of the I end. The comparisons presented here show that extension of the I end from 19 to 24 bp decreases its transposition activity in dam cells 5- to 50-fold, depending on the overall transposon structure.     

A derivative of Tn5 with direct terminal repeats can transpose

The 5.7 kb⁴ transposable kanamycin resistance determinant Tn5 contains 1.5 kb terminal inverted repeats which we here call arms. Tn5's arms contain the genes and sites necessary for Tn5 transposition, and are not homologous to previously described transposable elements. To determine whether one or both arms is a transposable (IS) element, we transposed Tn5 to pBR322 and used restriction endonuclease digestion and ligation in vitro to generate plasmid derivatives designated pTn5-DR1 and pTn5-DR2 in which Tn5's arms were present in direct rather than in inverted orientation. Analysis of transposition products from dimeric forms of the pTn5-DR1 plasmid to phage λ showed that the outside and inside termini of right and of left arms could function in transposition. We conclude that both of Tn5's arms are transposable elements and name them IS50L (left) and IS50R (right). IS50R, which encodes transposase, was used several-fold more frequently than IS50L, which contain an ochre mutant allele of transposase: this implies that Tn5's transposase acts preferentially on the DNA segment which encodes it. Analysis of transpositions of the amp^rkan^r element Tn5-DR2 to the lac operon showed that Tn5-DR2, like Tn5 wild-type, exhibits regional preference without strict site specificity in the choice of insertion sites.     

IHF is the limiting host factor in transposition of Pseudomonas putida transposon Tn4652 in stationary phase

Transpositional activity of mobile elements is not constant. Conditional regulation of host factors involved in transposition may severely change the activity of mobile elements. We have demonstrated previously that transposition of Tn4652 in Pseudomonas putida is a stationary phase-specific event, which requires functional sigma S (Ilves et al., 2001, J Bacteriol 183: 5445-5448). We hypothesized that integration host factor (IHF), the concentration of which is increased in starving P. putida, might contribute to the transposition of Tn4652 as well. Here, we demonstrate that transposition of Tn4652 in stationary phase P. putida is essentially limited by the amount of IHF. No transposition of Tn4652 occurs in a P. putida ihfA-defective strain. Moreover, overexpression of IHF results in significant enhancement of transposition compared with the wild-type strain. This indicates that the amount of IHF is a bottleneck in Tn4652 transposition. Gel mobility shift and DNase I footprinting studies revealed that IHF is necessary for the binding of transposase to both transposon ends. In vitro, transposase can bind to inverted repeats of transposon only after the binding of IHF. The results obtained in this study indicate that, besides sigma S, IHF is another host factor that is implicated in the elevation of transposition in stationary phase.     

Excision and Transposition of Tn5 as an Sos Activity in Escherichia Coli

Excision and transposition of the Tn5 element in Escherichia coli ordinarily appear to occur by recA-independent mechanisms. However, recA(Prtc) genes, which encode RecA proteins that are constitutively activated to the protease state, greatly enhanced excision and transposition; both events appeared to occur concomitantly and without destruction of the donor DNA. The recombinase function of the RecA protein was not required. Transposition was accompanied by partial, and occasionally full, restoration of the functional integrity of the gene vacated by the excised Tn5. The stimulation of transposition was inhibited by an uncleavable LexA protein and was strongly enhanced by an additional role of the RecA(Prtc) protein besides its mediation of LexA cleavage. To account for the enhanced transposition, we suggest that (i) there may be a LexA binding site within the promoter for the IS50 transposase, (ii) activated RecA may cleave the IS50 transposition inhibitor, and (iii) the transposase may be formed by RecA cleavage of a precursor molecule.     

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.