Composite transposons Tn5 and Tn10 in Escherichia coli K12: precise excision and recombination

The number of exconjugants having the transposon Tn5 excised precisely during the crosses of the Escherichia coli proA::Tn5 donor with the recipients F- rec+ or F- recA441 (tif) was 20-30 times higher for the crosses involving the latter recipient. The high recombinogenic activity is characteristic of the tif recipient. Precise excision from a tandem duplication is more efficient than from nonduplicated region of the genome. It is four orders higher, if a transposon is localized in an arm of a duplication. The effect is recA-dependent. The presented data permit us to suggest the participation of RecA protein (its synaptic function) in the formation of the intermediate "stem-loop" structure. The latter is predicted by the three mechanisms of transposon excision: "slippage", "correctional" and "recombinational". The latter two mechanisms were formulated in the paper. The experimental proof of the postexcision transposition presented in the paper, is a good support to the version of "recombinational" excision.     

Transposition of Tn1545-delta 3 in the pathogenic Neisseriae: a genetic tool for mutagenesis.

The ability to study the virulence of pathogenic Neisseria spp. has been greatly limited by the absence of genetic tools which allow the construction of defined mutants. We have engineered a transposon system which allows random mutagenesis of the Neisseria genome at relatively high frequency. Tn1545-delta 3 is a 3.4-kb derivative of the gram-positive transposon Tn1545 encoding resistance to kanamycin. Tn1545-delta 3 was subcloned into an erythromycin-resistant derivative of the mobilizable shuttle vector pLES2 to yield the plasmid pMGC20. This latter plasmid was introduced by conjugation from Escherichia coli S17-1 into Neisseria meningitidis 8013N and Neisseria gonorrhoeae 15063G. Kanamycin-resistant 8013N and 15063G transconjugants appeared at frequencies of 10(-5) and 10(-6), respectively. Restriction enzyme analysis and Southern blot hybridization of these transconjugants showed that, in Neisseria spp., the transposon excised spontaneously from pMGC20 and integrated into chromosomal DNA. Our studies revealed that (i) transposition of Tn1545-delta 3 was in numerous, apparently distinct sites, (ii) in most cases, for each transconjugant a single copy of Tn1545-delta 3 was integrated into the chromosome, and (iii) several passages on selective media did not induce secondary transposition. The kanamycin resistance marker expressed by the transconjugants was subsequently transformed into a parental background without change in the chromosomal location of the transposon. To assess the role of the general recombination system in the transposition of Tn1545-delta 3, the recA gene of N. meningitidis has been cloned and a rec derivative of 8013N has been engineered. Similar results were obtained when this latter strain was used as recipient, suggesting that recA function were not required for Tn1545-delta 3 transposition in N. meningitidis. Transposition with Tn1545-delta 3 may be an important technique for mutagenesis of the pathogenic neisseriae.     

A specific class of IS10 transposase mutants are blocked for target site interactions and promote formation of an excised transposon fragment

We report the identification and characterization of a class of IS10 transposase mutants that carry out only some of the steps required for transposition. These mutants were identified among transposition-defective mutants as a specific subclass that retains the wild-type ability to induce SOS functions in the presence of transposon ends. Mutants of this class successfully promote excision of the element from its donor site, but do not promote transfer of the transposon sequences to a target site. SOS induction presumably results from the degradation of the donor site. Uniquely among transposition-defective mutants, SOS+ Tnsp- mutants promote the formation of a new product, the excised transposon fragment (ETF), which consists of the transposon excised from the original donor molecule by double-strand breaks at the transposon ends. SOS+ Tnsp- mutants identified thus far define two patches of amino acids that might correspond to regions of different function. A single additional mutation maps within a region that is highly conserved among IS element transposases. The existence of SOS+ Tnsp- mutants and the structure of the ETF provide strong support for the previously proposed nonreplicative model of Tn10/IS10 transposition.     

Induction of transposon Tn1 translocations as affected by different mutagens

Transposition of ampicillin Tn1 transposon is repressed under normal conditions and occurs with low frequency (10(-4) per cell). Treatment of Escherichia coli cells with 22 c+mytomycin C, nitrosoguanidine and UV light induced transposition of Tn1 into different replicons. The degree of induction depended upon the strain of bacteria and the replicon which the transposon was inserted into. Mutation recA did not influence spontaneous translocation of the transposon but fully repressed induction of transposition during the period of mutagen treatment. In the present paper, the connection of inducible transposition process with the recA and lexA inducible functions of E. coli is discussed.     

All three residues of the Tn 10 transposase DDE catalytic triad function in divalent metal ion binding.

Tn 10/IS 10 transposition involves excision of the transposon from a donor site and subsequent joining of the excised transposon to a new target site. These steps are catalyzed by the Tn 10 -encoded transposase protein and require the presence of a suitable divalent metal ion. Like other transposase and retroviral integrase proteins, Tn 10 transposase appears to contain a single active site which includes a triad of acidic amino acid residues generally referred to as the DDE motif. In addition to its role in catalysis, the Tn 10 transposase DDE motif also functions in target capture, a step that in vitro is greatly facilitated by the presence of a suitable divalent metal ion. We show that cysteine residue substitutions at each of the DDE motif residues in Tn 10 transposase result in a change in the divalent metal ion requirements for catalysis, such that Mn2+but not Mg2+can be used. This switch in metal ion specificity provides evidence that each of the DDE motif residues functions directly in metal ion binding. We also show differential effects of DDE mutations on metal ion-assisted target capture. A number of models, including a two metal ion active site, are considered to explain these effects.     

Studies on Tn10 transposition and excision in DNA-repair mutants of Salmonella typhimurium

Transposition of Tn10 in polA, recA, uvrB, mutH and uvrD mutants of Salmonella typhimurium was studied by a mating-out assay mediated by R plasmid pKM101. A decrease in transposition frequency was observed with polA, recA and uvrD mutants; uvrB and mutH mutants showed frequencies somewhat higher than control values. No effect of dimethyl sulfoxide, sodium acetate or nitrofurazone on Tn10 transposition was observed with this assay. Precise excision of Tn10 from srl202::Tn10 in these DNA-repair mutants was also studied. An increase in excision frequency of about 20 or 150 times in 2 different polA mutants, and a smaller increase, of about 2 or 15 times over control values, was detected in mutH and uvrD mutants, respectively.     

Translocation of ampicillin transposon Tn1 in Escherichia coli cells during sexual reproduction

The efficiency of Tn1 transposition has been shown to increase considerably in course of bacterial conjugation. Usually, the frequency of Tn1 transposition from plasmid pSA2001, a derivative of RP4, into the chromosome never exceeded 0.1% per cell. Percentage of His+ transconjugants, marked by transposon Tn1 during conjugation between Hfr donor, carrying plasmid pSA2001, and auxotrophic recipient, was about 30%. Transposon Tn1 transfer into the recipient cells does not depend on the recA+ gene function in donor cells or on conjugative transfer of plasmid pSA2001. The transfer requires the recA+ gene function in recipients as well as the Hfr function in donor cells. Southern's blot-hybridization revealed the insertion of transposon Tn1 into the different sites of the chromosome of His+ transconjugants. The transposon inserted during conjugation retains the ability to potential further translocation into new sites on the chromosomal DNA.     

Genetic Evidence against Intramolecular Rejoining of the Donor DNA Molecule following Is10 Transposition

Tn10 and IS10 transpose by a nonreplicative mechanism in which the transposon is excised from the donor molecule and integrated into a target DNA site, leaving behind a break at the original donor site. The fate of this broken donor DNA molecule is not known. We describe here two experiments that address this issue. One experiment demonstrates that a polar IS10 element gives rise to polarity-relief revertants at less than 1% the frequency of transposition of the same element in the same culture. In a second experiment, transpositions of an IS10 element from one site in the bacterial genome to another are selected and the resulting isolates examined for alterations at the donor site; none of 1088 such isolates exhibited a detectable change at the donor locus. These results are compatible with two possible fates of the transposon donor molecule: degradation (``donor suicide'), or restoration of the original information at the donor site by a recombinational repair mechanism analogous to double-strand break repair. These results argue against the possibility that the donor molecule gap is simply resealed by intramolecular rejoining.     

Gain-of-Function Mutations in Tnsc, an Atp-Dependent Transposition Protein That Activates the Bacterial Transposon Tn7

The bacterial transposon Tn7 encodes five genes whose protein products are used in different combinations to direct transposition to different types of target sites. TnsABC+D directs transposition to a specific site in the Escherichia coli chromosome called attTn7, whereas TnsABC+E directs transposition to non-attTn7 sites. These transposition reactions can also recognize and avoid ``immune' targets that already contain a copy of Tn7. TnsD and TnsE are required to activate TnsABC as well as to select a target site; no transposition occurs with wild-type TnsABC alone. Here, we describe the isolation of TnsC gain-of-function mutants that activate the TnsA+B transposase in the absence of TnsD or TnsE. Some of these TnsC mutants enable the TnsABC machinery to execute transposition without sacrificing its ability to discriminate between different types of targets. Other TnsC mutants appear to constitutively activate the TnsABC machinery so that it bypasses target signals. We also present experiments that suggest that target selection occurs early in the Tn7 transposition pathway in vivo: favorable attTn7 targets appear to promote the excision of Tn7 from the chromosome, whereas immune targets do not allow transposon excision to occur. This work supports the view that TnsC plays a central role in the evaluation and utilization of target DNAs.     

Tn 10 transposition in vivo: temporal separation of cleavages at the two transposon ends and roles of terminal basepairs subsequent to interaction of ends.

During Tn10 transposition, the transposon is fully excised from the donor site by double strand cleavages at the two ends of the element prior to integration at a new target site. Results presented here demonstrate that an interaction between the two transposon ends is required for double strand cleavage at either end. Furthermore, despite this essential interaction of ends, subsequent cleavages at the two ends can occur at observably distinct times prior to occurrence of strand transfer at either end. Moreover, the time between cleavages at the two ends is exaggerated by the presence of an appropriate mutation at one end of the element. Biological rationales for this constellation of mechanistic features are suggested. Additional results demonstrate that mutations at the three terminal basepairs of Tn10 confer defects subsequent to interaction of ends, in confirmation of inferences from genetic analysis. More specifically, mutations in bp 1-3 confer strong defects during conversion of the full excision intermediate to a complete strand transfer product; mutations in bp 1 and 2 also confer more subtle defects subsequent to interaction of ends but prior to full excision. Such defects might reflect roles for these basepairs in the chemical steps of transposition per se, the positioning of terminal residues for those chemical steps, and/or the coupling of cleavage(s) to subsequent conformational changes.     

Isolation and Characterization of Escherichia Coli Mutants with Altered Rates of Deletion Formation

Using site-specific mutagenesis in vitro we constructed a genetic system to detect mutants with altered rates of deletion formation between short repeated sequences in Escherichia coli. After in vivo mutagenesis with chemical mutagens and transposons, the system allowed the identification of mutants with either increased or decreased deletion frequencies. One mutational locus, termed mutR, that results in an increase in deletion formation, was studied in detail. The mutR gene maps at 38.5 min on the E. coli genetic map. Since the precise excision of many transposable elements is also mediated at short repeated sequences, we investigated the effects of the mutant alleles, as well as recA, on precise excision of the transposon Tn9. Neither mutR nor recA affect precise excision of the transposon Tn9, from three different insertions in lacI, whereas these alleles do affect other spontaneous deletions in the same system. These results indicate that deletion events leading to precise excision occur principally via a different pathway than other random spontaneous deletions. It is suggested that, whereas precise excision occurs predominantly via a pathway involving replication enzymes (for instance template strand slippage), deletions on an F'factor are stimulated by recombination enzymes.     

Regularities of excising transposon Tn10 in rec-mutant E. coli cells exposed to gamma radiation

The regularities of gamma-induced excision of transposon Tn10 in different rec-strains of E. coli cells after gamma-irradiation have been studied. The survival of cells and relative frequency of the Tn10 elimination as a function of the 137Cs gamma-radiation doses were investigated. RecN and recA-mutants of E. coli were used for study of the role of rec-genes in the gamma-induced transposon excision. It was shown that the induced excision in the recN mutant was reduced. The transposon excision in the recA mutant was not revealed. The obtained results let to conclude that recA, and recN genes are involved not only in DNA repair processes but also in the gamma-induced transposon excision in bacteria.     

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.     

Evidence for Campbell's mechanism for fine excision of Tn9 type transposons in Escherichia coli K12]

The plasmid-transposon Tn9-322 was constructed by inverted transposition from the pBR322::Tn9 plasmid. The precise excision of the Tn9-322 transposon from the proB gene site can proceed by the Campbell's model. This fact was demonstrated by appearance of the plasmid-transposons after their precise excision. They contain two IS1 elements flanking a short direct repeat of the target DNA. The recombinational mechanism of precise excision of Tn9 type transposons seems not to be alternative but looks as an additional one to a well-known slippage mechanism proved for Tn5 and Tn10.     

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.     

Properties of transposon Tn2555 carrying the genes for sucrose utilization

Tn2555, a new transposon coding for genes of sucrose utilization was studied. Tn2555 was shown to integrate into the plasmids RP4 and R6K, phage P1CmClr100 and Escherichia coli K12 chromosome. Tn2555 frequency of transposition to RP4 and R6K DNA is (2-5) X 10(-7) in Rec+-strain, (3-6) X 10(-8) in Rec--strain. Tn2555 integration site in phage P1CmClr100 Sac+-derivative studied has been localised within the C-segment of P1 DNA. In three independent cases of Tn2555 transposition to the chromosome the transposon was found to be integrated in the region between 29 and 32 min of Escherichia coli K12 linkage map. The restriction endonuclease analysis of seven independent isolates of RP4::Tn2555 has shown the grouping of Tn2555 integration sites in the Tn1 region of RP4. Frequent rearrangements occurring within Tn2555 have been revealed by the analysis. However, an invertible DNA segment of about 6-7 kb was preserved in all transposon structures.     

The resolvase/invertase domain of the site-specific recombinase TnpX is functional and recognizes a target sequence that resembles the junction of the circular form of the Clostridium perfringens transposon Tn4451.

Tn4451 is a 6.3-kb chloramphenicol resistance transposon from Clostridium perfringens and is found on the conjugative plasmid pIP401. The element undergoes spontaneous excision from multicopy plasmids in Escherichia coli and C. perfringens and conjugative excision from pIP401 in C. perfringens. Tn4451 is excised as a circular molecule which is probably the transposition intermediate. Excision of Tn4451 is dependent upon the site-specific recombinase TnpX, which contains potential motifs associated with both the resolvase/invertase and integrase families of recombinases. Site-directed mutagenesis of conserved amino acid residues within these domains was used to show that the resolvase/invertase domain was essential for TnpX-mediated excision of Tn4451 from multicopy plasmids in E. coli. An analysis of Tn4451 target sites revealed that the transposition process showed target site specificity. The Tn4451 target sequence resembled the junction of the circular form, and insertion occurred at a GA dinucleotide. Tn4451 insertions were flanked by directly repeated GA dinucleotides, and there was also a GA at the junction of the circular form, where the left and right termini of Tn4451 were fused. We propose a model for Tn4451 excision and insertion in which the resolvase/invertase domain of TnpX introduces 2-bp staggered cuts at these GA dinucleotides. Analysis of Tn4451 derivatives with altered GA dinucleotides provided experimental evidence to support the model.     

Kinetic and structural analysis of a cleaved donor intermediate and a strand transfer intermediate in Tn10 transposition.

Tn10 transposes by a nonreplicative "cut and paste" mechanism. We describe here two protein-DNA complexes that are reaction intermediates in the Tn10 transposition process: a cleaved donor complex whose DNA component consists of transposon sequences cleanly excised from flanking donor DNA, and a strand transfer complex whose DNA component contains transposon termini specifically joined to a target site. The kinetic behavior of the first species suggests that it is an early intermediate in the transposition reaction. These two Tn10 complexes are closely analogous to complexes identified in the pathway for replicative "cointegrate" formation by bacteriophage Mu and thus represent intermediates that may be common to both nonreplicative and replicative transposition. These and other results suggest that the Tn10 and Mu reactions are fundamentally very similar despite their very different biological outcomes. The critical difference between the two reactions is the fate of the DNA strand that is not joined to target DNA.     

Inhibition of Tn554 transposition: Deletion analysis

Tn554, a transposon in Staphylococcus aureus that specifies resistance to erythromycin and spectinomycin, exhibits a high preference for a single chromosomal insertion site. If this site is already occupied by a copy of Tn554, the transposition of a second element is inhibited 100- to 1000-fold. This report defines the locus of the inhibitory activity and presents both a functional and a restriction map of Tn554. Fragments containing parts of Tn554 were cloned on an autonomously replicating plasmid. Those clones containing the "left" end of Tn554 strongly inhibited the transposition of an incoming, intact copy of Tn554. Analysis of deleted derivatives of these clones defined a locus tnpI, which is both necessary and sufficient for transpositional inhibition. This locus consists of the terminal 89 bp of the "left" end of Tn554. It is suggested that this terminal sequence acts to titrate a factor required for transposition.