Specificity of Tn9 insertion into the genome of bacteriophage lambda att80 depending on its preceding location

It was shown that the site of previous integration (the donor site) of Tn9 affects the specificity of its next integration into the target molecule--phage lambda att80 DNA. The transposon integration sites were mapped by restriction and heteroduplex analysis following Tn9 transposition from chromosomal sites of Escherichia coli K-12 differing in location and Tn9 stability. When transposed from chromosomal galT::IS1 gene, Tn9 inserted into the site with coordinates 44,5 +/- 2 kb of lambda att80; when transposed from chromosomal attTn9A site, the transposon inserted into the sites with coordinates 31 +/- 0,7 kb or 33,3 +/- 0,5 kb. In the course of transposition of Tn9 from chromosomal attTn9N site the transposon inserted into the lambda att80 site with coordinates 26,5 +/- 5 kb. In the latter case, the increase of Tn9 single-stranded loop and the appearance of two new HindIII cleavage sites were observed in heteroduplex experiments. The data were interpreted as indicating structural rearrangements of Tn9 or linked sequences in the course of transposition.     

Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones.

The 264 bp mini-transposon Tn5supF was constructed to sequence DNAs cloned in phage lambda without extensive shotgun subcloning or primer walking. Unique sequences near each transposon end serve as primer binding sites, and a supF gene is used to select transposition to lambda. We describe here PCR methods that facilitate Tn5supF-based sequencing. In a first pass, insertions are mapped relative to the ends of the cloned fragment using pairs of primers specific for vector DNA next to the cloning site and for a Tn5supF end. Most insertions not mapped in this step are near the center of the cloned fragment or in the vector arms, and are then mapped relative to the two innermost insertions by 'crossover' PCR. This involves amplification from primers on different DNA molecules, and generates hybrid DNA products whose lengths correspond to the distances between the two insertions. We routinely amplified more than 6 kb in direct PCR and 3 kb in crossover PCR; at the limit we amplified up to approximately 10 kb in direct PCR and approximately 6 kb in crossover PCR, but not reproducibly. Crossover PCR products were also obtained with insertions separated by only 200 bp, indicating that no rare sites are needed to switch templates. PCR products were purified by adsorption and then elution from glass slurry, and sequenced directly. Ladders of more than 400 bp were obtained from primer sites on each DNA strand; 2 kb was read from crossover PCR products, and showed that they were amplified with fidelity. In conclusion, direct and crossover PCR methods expedite transposon insertion mapping, and yield templates for accurate sequencing of both DNA strands.     

cAMP acceptor protein of Escherichia coli K-12 may be the repressor of the late promoter of bacteriophage lambda

By computer analysis of the lambda phage DNA putative cAMP-CAP binding site has been found. This site (44506-AcgTGTGAccgcatTCAAAaT-44486) is located 40 bp upstream to the late PR' promoter. The sequence denoted is located on the coding DNA strand, similar to the cAMP-CAP binding sites in all four genes known to be subject to cAMP-CAP repression. It is suggested that cAMP-CAP can serve as a repressor of the phage lambda late promoter PR'. Such repression can arrest the expression of late lambda genes and in this way increase the frequency of lysogenization.     

Specific DNA cleavage mediated by the integrase of conjugative transposon Tn916.

The conjugative transposon Tn916 encodes a protein called INT(Tn916) which, based on DNA sequence comparisons, is a member of the integrase family of site-specific recombinases. Integrase proteins such as INT(lambda), FLP, and XERC/D that promote site-specific recombination use characteristic, conserved amino acid residues to catalyze the cleavage and ligation of DNA substrates during recombination. The reaction proceeds by a two-step transesterification reaction requiring the formation of a covalent protein-DNA intermediate. Different requirements for homology between recombining DNA sites during integrase-mediated site-specific recombination and Tn916 transposition suggest that INT(Tn916) may use a reaction mechanism different from that used by other integrase recombinases. We show that purified INT(Tn916) mediates specific cleavage of duplex DNA substrates containing the Tn916 transposon ends and adjacent bacterial sequences. Staggered cleavages occur at both ends of the transposon, resulting in 5' hydroxyl protruding ends containing coupling sequences. These are sequences that are transferred with the transposon from donor to recipient during conjugative transposition. The nature of the cleavage products suggests that a covalent protein-DNA linkage occurs via a residue of INT(Tn916) and the 3'-phosphate group of the DNA. INT(Tn916) alone is capable of executing the strand cleavage step required for recombination during Tn916 transposition, and this reaction probably occurs by a mechanism similar to that of other integrase family site-specific recombinases.     

Location of ribosome-binding sites in the nin5 region of bacteriophage lambda

Seven ribosome-binding sites on DNA have been located within the region defined by the nin5 deletion as well as several ribosome-binding sites on each side of the nin5 region. These were mapped by electron microscopy relative to the end points of the nin5 deletion and two Tn903 transposons, one inserted into gene Rz and another inserted near gene Q. These ribosomes binding sites within the nin5 region may correspond to polypeptide initiation sites for up to seven new dispensible lambda genes.     

Transposition of the kanamycin resistance determinant (Tn601) from plasmid 5T to the genome of bacteriophage lambda and the expression of this gene after prophage induction

Tn601, determinging kanamycin resistance of Escherichia coli, has been transposed into the bacteriophage lambda genome from R6 plasmid. After curing lambda gtc1857 (Tn601) lysogenes on the kanamycin containing medium, the clones with stable and unstable integrations of the Tn6-1 into the chromosome were obtained. After the lysogenization of these clones with the phage lambda att80c1857S7, the phages lambda att80c1857S7 (Tn601) were obtained. These phages contained the Tn601 from the sites of stable or unstable integrations. The frequency of the Tn601 transposition from the sites of unstable integration was 10(-7), that was two order of magnitude higher than the frequency of the Tn601 transpostion from the site of stable integration. Temperature induction of the lambda att80c1857 (Tn601) prophage resulted in 10--15 times increase of the yeild of aminoglycoside-3'-phosphotransferase I, the enzyme coded by the aphA gene of the Tn601.     

Xis protein binding to the left arm stimulates excision of conjugative transposon Tn916

Tn916 and related conjugative transposons are clinically significant vectors for the transfer of antibiotic resistance among human pathogens, and they excise from their donor organisms using the transposon-encoded integrase ((Tn916)Int) and excisionase ((Tn916)Xis) proteins. In this study, we have investigated the role of the (Tn916)Xis protein in stimulating excisive recombination. The functional relevance of (Tn916)Xis binding sites on the arms of the transposon has been assessed in vivo using a transposon excision assay. Our results indicate that in Escherichia coli the stimulatory effect of the (Tn916)Xis protein is mediated by sequence-specific binding to either of its two binding sites on the left arm of the transposon. These sites lie in between the core and arm sites recognized by (Tn916)Int, suggesting that the (Tn916)Xis protein enhances excision in a manner similar to the excisionase protein of bacteriophage lambda, serving an architectural role in the stabilization of protein-nucleic acid structures required for strand synapsis. However, our finding that excision in E. coli is significantly enhanced by the host factor HU, but does not depend on the integration host factor or the factor for inversion stimulation, defines clear mechanistic differences between Tn916 and bacteriophage lambda recombination.     

Chromosome methylation and measurement of faithful, once and only once per cell cycle chromosome replication in Caulobacter crescentus

Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5Omega-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5Omega-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5Omega-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5Omega-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the "once and only once per cell cycle" rule of chromosome replication in C. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.     

DNA sequence analysis of Tn10 insertions: origin and role of 9 bp flanking repetitions during Tn10 translocation.

The sequences of insertions of the translocatable tetracycline-resistance element Tn10 into the repressor (cl) gene of bacteriophage lambda have been analyzed. Each insertion contains the same discrete set of Tn10 sequences flanked by a direct repetition of a 9 bp cl-gene sequence. The flanking repititions are generated by duplication of information present only in the target DNA molecule rather than by a Campbell-type recombination event between one 9 bp sequence on the target DNA and a second one provided on the incoming element. The repetitions do not contain genetic or structural information important for translocation. A genetically constructed Tn10 insertion which lacks flanking repetitions is fully functional in translocation to a new position. Tn10 insertions cluster at preferred positions along a target DNA (Kleckner et al., 1979). Sequence analysis shows that four independently isolated cl::Tn10 insertions occur at identical positions in the cl gene. We speculate that homology between Tn10 and its target, at some distance from the site of the actual recombination event, could be relevant to the preference of Tn10 for particular insertion sites.     

Transposon-induced non-motile mutants of Vibrio cholerae

Non-motile mutants of Vibrio cholerae were isolated after transposon insertion mutagenesis with either Tn5 on a plasmid or Tn10ptac mini-kan in bacteriophage lambda. The physical location and number of transposon insertions was determined. Eighteen Tn5 insertion mutants and 11 Tn10ptac mini-kan insertion mutants had single unique insertion sites. The 18 Tn5 insertions were contained within six different EcoRI fragments and the 11 Tn10ptac mini-kan insertions were contained within eight different fragments of V. cholerae chromosomal DNA. These data suggest that multiple genes are involved in motility. Immunoblot analysis of non-motile mutants with antibody to wild-type flagellar core protein indicated that two of the non-motile mutants made flagellar core protein. Three additional mutants reacted weakly with the antibodies. However, these mutants with immunopositive reactions did not produce any structures which resembled flagella by transmission electron microscopy. In addition, none of the other non-motile mutants produced wild-type flagella. However, five mutants which did not react in the immunoblot produced a structure which resembled a flagellar sheath without the internal flagellar core. In addition to having no filamentous core, the sheaths often extended from the sides of the bacteria, rather than from the poles where the flagellum is normally located. The data suggest that sheath formation is independent of flagellar filament formation, but that proper positioning of the sheath may require the flagellar filament.     

Generation of Tn5 insertions in streptococcal conjugative transposon Tn916

A method has been developed for the introduction of Tn5 into Escherichia coli plasmid chimeras containing Streptococcus faecalis DNA. Tn5 could be introduced via a lambda::Tn5 delivery vehicle. The system proved to be particularly efficient and facilitated insertions at numerous sites on DNA containing the 16-kilobase conjugative transposon Tn916. It was possible to introduce some of the resulting Tn916::Tn5 derivatives back into S. faecalis by using a recently developed protoplast transformation procedure. A presumed zygotic induction resulted in insertion of the Tn916 derivatives at multiple sites in the S. faecalis chromosome.     

Some Features of Base Pair Mismatch and Heterology Repair in Escherichia coli

We have used artificially constructed heteroallelic heteroduplex molecules of bacteriophage lambda DNA to transfect Escherichia coli, and E. coli mutants deficient in various functions involved in the adenine methylation-directed mismatch repair system, MutL, MutS, MutH, and UvrD (MutU). Analysis of the allele content of single infective centers shows that this repair system often acts on several mismatches, separated by as many as 2000 bp, on one of the strands of a heteroduplex molecule. When the methyl-directed mismatch repair system is disabled by mutH or uvrD mutations, localized mismatch repair becomes prominent. This prominent localized repair that can result in separation of very closely linked markers requires the functions MutL and MutS, is independent of adenine methylation, and appears to reflect another mechanism of mismatch repair. Heterology-containing heteroduplex molecules with a deletion in one strand often escape processing. However, when the heterology includes the stem and loop structure of a transposon, Tn10, the transposon is lost.     

Generation of Tn5 insertions in streptococcal conjugative transposon Tn916.

A method has been developed for the introduction of Tn5 into Escherichia coli plasmid chimeras containing Streptococcus faecalis DNA. Tn5 could be introduced via a lambda::Tn5 delivery vehicle. The system proved to be particularly efficient and facilitated insertions at numerous sites on DNA containing the 16-kilobase conjugative transposon Tn916. It was possible to introduce some of the resulting Tn916::Tn5 derivatives back into S. faecalis by using a recently developed protoplast transformation procedure. A presumed zygotic induction resulted in insertion of the Tn916 derivatives at multiple sites in the S. faecalis chromosome.     

Identification of citrate utilization transposon Tn3411 from a naturally occurring citrate utilization plasmid.

We have isolated a new transposon, Tn3411, encoding citrate-utilizing ability, from a naturally occurring citrate utilization (Cit) plasmid, pOH3001. Citrate transposon Tn3411 was transposed from pOH3001 to lambda b519 b515 cI857 S7 (abbreviated lambda bb) phage, and further from the resulting lambda bb:Tn3411 to a vector plasmid, pBR322, in recA-deficient strains. The Cit+ plasmids (pOH2 and pOH3) constructed by the integration of Tn3411 into pBR322 were examined by restriction endonuclease and heteroduplex analysis. The results obtained were as follows: (i) Tn3411 was 7.4 kilobases long and flanked by small inverted repeats, and it contained one more pair of inverted repeats at the opposite orientation in the internal region, thus making alternate repeats; and (ii) the Cit+ structure gene was located on the fragment (5.5 kilobases) between two SalI cleavage sites on Tn3411.     

Substrate recognition and induced DNA deformation by transposase at the target-capture stage of Tn10 transposition

The bacterial transposon Tn10 inserts preferentially into sites that conform to a 9 bp consensus sequence: 5' NGCTNAGCN 3'. However, this sequence is not on its own sufficient to confer target specificity as the base-pairs flanking this sequence also contribute significantly to target-site selection. We have performed a series of "contact-probing experiments" to define directly the protein-DNA interactions that govern target-site selection in the Tn10 system. The HisG1 hotspot for Tn10 insertion was the main focus here. We infer that there is a rather broad zone ( approximately 24 bp) of contact between transposase and target DNA in the target-capture complex. This includes base-specific contacts at all of the purine residues in the consensus positions of the target core and primarily backbone contacts out to 7-8 bp in the two flanking regions immediately adjacent to the core. Also, highly localized sites of chemical hypersensitivity are identified that reveal symmetrically disposed deformations in DNA structure in the target-capture complex. Furthermore, the level of strand transfer is shown to be reduced by phosphorothioate substitution of phosphate groups at or close to the sites of target DNA deformation. Interestingly, for one particular target DNA, a mutant form of HisG1 called MutF, the above phosphorothioate inhibition of strand transfer is suppressed by replacing Mg(2+) with Mn(2+). Based on these results a model for sequence-specific target capture is proposed which attempts to define possible relationships between transposase interactions with the target core and flanking sequences, transposase-induced DNA deformation of the target site and divalent metal ion binding to the target-capture complex.     

Characterisation of Tn1721, a new transposon containing tetracycline resistance genes capable of amplification.

R plasmid pRSD1 contains tetracycline resistance (tet) genes in a 3.55 Mdal-region capable of amplification by forming tandem repeats (Mattes, Burkardt and Schmitt, Molec. gen. Genet., 1979). The repetitious tet element is itself part of a 7.2 Mdal-transposon, named Tn1721, as demonstrated by the following criteria; (i) Tn1721 has been translocated to phage lambda. The resulting hybrid phage lambda tet contains the 7.2 Mdal-insertion to the right of the attachment site, but not continguous with it indicating translocation of the element by non-homologous recombination. In addition, lambda tet has sustained a 3.4 Mdal-deletion adjacent to the insertion. (ii) Further transposition of Tn1721 to the 21.5 Mdal-plasmid R388 resulted in R388::Tn1721 derivatives, two of which were characterised. They contain Tn1721 inserted into different sites but in the same orientation as shown by restriction and heteroduplex analyses. These translocation of Tn1721 were not accompanied by deletions of DNA. (iii) The insertion plasmid pRSD102(R388::Tn1721) has conserved the capacity of the original plasmid pRSD1 to amplify the 3.55 Mdal-tet region. It has been concluded that Tn1721 constitutes a novel transposon encompassing a tet region capable of selective amplification. The model proposed for Tn1721 contains three short repeats. Two direct repeats, flanking the 3.55 Mdal tet region, provide sequence homology for amplification. The third repeat (located distally to tet) is inverted and provides the basis for transposition of the 7.2 Mdal-element.     

Nearly precise excision: a new type of DNA alteration associated with the translocatable element Tn10.

We describe an unusual DNA alteration, "nearly precise excision," which has been identified among tetracycline-sensitive deletion derivatives of lambda phages carrying the translocatable tetracycline-resistance element Tn10. DNA sequence analysis of two such derivatives demonstrates that each retains exactly 50 bp of Tn10 material. The original junctions between lambda and Tn10 sequences remain intact; however, an internal deletion has occurred within Tn10 which eliminates all but the last few base pairs at each end of the element. This deletion occurs within a short A + T-rich inverted repeat which is present near each end of Tn10. Nearly precise excisions occur at frequencies comparable to Tn10-promoted deletions, inversions and translocations, and, like these other events, are independent of phage and bacterial functions for homologous recombination (recA, recB, red). It is not yet clear, however, whether nearly precise excisions are specifically promoted by Tn10 or whether they arise during the course of normal DNA replication processes as a consequence of unusual symmetries present in the DNA sequence at the ends of Tn10.     

Physical structures of Tn10-promoted deletions and inversions: role of 1400 bp inverted repetitions.

We report here the physical structures of deletions and inversions promoted by the translocatable tetracycline-resistance element Tn10. DNA/DNA heteroduplex and restriction enzyme analyses of alterations in the genome of bacteriophage lambda suggest that both types of DNA alterations almost always originate at the internal termini of the 1400 bp terminal inverted repetitions of Tn10. Tn10-promoted deletions remove a single contiguous DNA segment beginning at one such terminus; Tn10-promoted inversions are more complex, and involve both an inversion and a specific deletion of Tn10 DNA.     

The nucleoid binding protein H-NS acts as an anti-channeling factor to favor intermolecular Tn10 transposition and dissemination

Dissemination of the bacterial transposon Tn10 is limited by target site channeling, a process wherein the transposon ends are forced to interact with and insert into a target site located within the transposon. Integration host factor (IHF) promotes this self-destructive event by binding to the transpososome and forming a DNA loop close to one or both transposon ends; this loop imposes geometric and topological constraints that are responsible for channeling. We demonstrate that a second ‘host’ protein, histone-like nucleoid structuring protein (H-NS), acts as an anti-channeling factor to limit self-destructive intramolecular transposition events in vitro. Evidence that H-NS competes with IHF for binding to the Tn10 transpososome to block channeling and that this event is relatively insensitive to the level of DNA supercoiling present in the Tn10-containing substrate plasmid are presented. This latter observation is atypical for H-NS, as H-NS binding to other DNA sequences, such as promoters, is generally affected by subtle changes in DNA structure.