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.     

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.     

Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions.

Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained.     

Tn5cos: a transposon for restriction mapping of large plasmids using phage lambda terminase.

A method for the rapid restriction mapping of large plasmids has been developed. A 400-bp fragment of phage lambda DNA containing the cos region has been inserted into Tn5. After in vivo transposition of this Tn5cos element into the plasmid of choice, the plasmid is isolated and linearized at its cos site with phage lambda terminase (Ter). Such Ter linearization was about 70% efficient. After partial digestion of the linear molecules with the appropriate restriction enzyme, the products are selectively labelled at the right or left cohesive phage lambda DNA termini by hybridization with digoxygenin (DIG)-11-dUTP-labelled (using terminal transferase) oligodeoxyribonucleotides complementary to the single-stranded cos ends. After pulsed field gel electrophoresis, the labelled fragments are visualized in the dried gel using a DIG-detection kit. The restriction map can be directly determined from the 'ladder' of partial digestion products.     

Isolation of transposon Tn551 insertions near chromosomal markers of interest in Staphylococcus aureus.

A procedure was developed to isolate insertions of transposon Tn551 near other markers of interest on the chromosome of Staphylococcus aureus NCTC 8325. When an inoculum of strain 8325-4 carrying a thermosensitive mutant of plasmid pI258 (on which Tn551 resides) was inoculated into brain heart infusion agar plus erythromycin and grown to saturation at 43 degrees C, the transforming DNA extracted from this population of cells contained a random collection of different chromosomal insertions of Tn551; this DNA is referred to as pooled Tn551 DNA. When erythromycin-sensitive recipient strains containing chromosomal markers of interest were transformed with pooled Tn551 DNA, and the resulting Emr transformants were screened for coinheritance of the donor allele of the marker of interest, insertions of Tn551 were isolated near several markers, including fus-149, tet-3490, mec-4916, pig-131, ilv-129, pur-140, and uraA141. Many of the insertions were within the linkage groups that contained these markers, and several insertions occupied different positions between the linkage groups in heretofore undefined regions of the circular chromosomal map of S. aureus. These insertions of transposon Tn551 extend the known limits of the existing linkage groups, provide linkage data and map locations for markers not previously mapped, and provide a means to map markers which cannot be directly selected.     

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.     

Physical mapping of transposon Tn5 insertions defines a gene cluster functional in nitrous oxide respiration by Pseudomonas stutzeri.

By transposon Tn5 mutagenesis, 19 strains of Pseudomonas stutzeri were acquired that had defects in nitrous oxide respiration (Nos- phenotype). A physical map of the mutants showed nearly random Tn5 insertions into genomic DNA within a single region ca. 8 kilobases long. Mutants were characterized immunochemically, enzymatically, and chemically. Several functions related to the synthesis and regulation of nitrous oxide reductase were associated with this DNA region, indicating that in P. stutzeri part of the genetic information necessary to respire nitrous oxide is clustered.     

Insertional specificity of transposon Tn5 in Acinetobacter sp.

Suicide plasmid pJB4JI, containing transposon Tn5 and phage Mu, was introduced from Escherichia coli 1830 into Acinetobacter sp. strain HO1-N and Acinetobacter calcoaceticus BD413. Kanamycin-resistant (Kmr) exconjugants of HO1-N and BD413, isolated on complex medium, were screened for auxotrophic requirements. Over 10,000 Kmr clones were examined, but no auxotrophs were detected. Several Kmr exconjugants of BD413 and HO1-N, obtained from independent matings, were chosen for further study. All Tn5-containing strains exhibited kanamycin phosphotransferase activity. Kmr strains lacked plasmid DNA as determined by three plasmid screening procedures, and the Kmr phenotype was not transferable by conjugal matings to kanamycin-sensitive BD413, HO1-N, or E. coli HB101. The chromosomal location of Tn5 insertions in independently isolated Kmr exconjugants of BD413 and HO1-N was characterized by restriction endonuclease mapping and hybridization studies. Results obtained from Southern hybridization studies were consistent with a single Tn5-specific insertion site in HO1-N and two such sites in BD413. Phage Mu sequences were not detected in Tn5-containing Acinetobacter sp.     

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

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

An alternative inverse PCR (IPCR) method to amplify DNA sequences flanking Tn5 transposon insertions

We have developed an alternative method to amplify DNA sequences flanking Tn5 transposon insertions. This method relies on the identical sequences of inverted terminal repeats, located at the 5' and 3' ends of Tn5, to determine the location and orientation of a transposon insertion within a restriction endonuclease fragment. From this information, PCR primers can be designed to selectively amplify by inverse PCR the DNA flanking one side of the transposon. This method avoids the problem of amplifying or cloning long sequences flanking Tn5. To demonstrate the applicability of this method, we generated Tn5 transposon mutants of Pseudomonas abietaniphila BKME-9 which no longer grew on dehydroabietic acid (DhA). The flanking sequence of one of the mutant (strain BKME-941) which accumulated 7-oxoDhA, was amplified.     

Tn5 transposase loops DNA in the absence of Tn5 transposon end sequences

Transposases mediate transposition first by binding specific DNA end sequences that define a transposable element and then by organizing protein and DNA into a highly structured and stable nucleoprotein 'synaptic' complex. Synaptic complex assembly is a central checkpoint in many transposition mechanisms. The Tn5 synaptic complex contains two Tn5 transposase subunits and two Tn5 transposon end sequences, exhibits extensive protein-end sequence DNA contacts and is the node of a DNA loop. Using single-molecule and bulk biochemical approaches, we found that Tn5 transposase assembles a stable nucleoprotein complex in the absence of Tn5 transposon end sequences. Surprisingly, this end sequence-independent complex has structural similarities to the synaptic complex. This complex is the node of a DNA loop; transposase dimerization and DNA specificity mutants affect its assembly; and it likely has the same number of proteins and DNA molecules as the synaptic complex. Furthermore, our results indicate that Tn5 transposase preferentially binds and loops a subset of non-Tn5 end sequences. Assembly of end sequence-independent nucleoprotein complexes likely plays a role in the in vivo downregulation of transposition and the cis-transposition bias of many bacterial transposases.     

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

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

Bacterial transposon Tn5: evolutionary inferences

Transposable elements induce spontaneous mutations, promote genome rearrangements, regulate gene expression, and participate in the horizontal spread of genes encoding traits such as antibiotic resistance among bacterial genera too distantly related to undergo homologous recombination. Here we review the bacterial transposon Tn5 and focus on those aspects of its functional organization and transposition which provide insights into how it and other elements may have arisen, proliferated, and evolved.      

Tn5-rpsL: a new derivative of transposon Tn5 useful in plasmid curing

The rpsL gene of Escherichia coli was inserted into the BamHI site of transposon Tn5. This transposon was called Tn5-rpsL. Tn5-rpsL may be useful in microbiological studies when one wants to cure various bacterial genera of certain plasmid(s). A streptomycin-resistant (SmR) derivative of the host bacterial strain is first isolated. The plasmid(s) later to be cured are then labelled with Tn5-rpsL, which makes the cells Sm-sensitive. These cells can regain their resistance to Sm if they lose the Tn5-rpsL-tagged plasmid. Thus, plasmid-free bacteria are easily selected among SmR survivors. The frequency of occurrence of the plasmid-less variants of plasmid-containing wild-type Salmonella typhimurium measured by this method is given as an example.     

Precise excision of bacterial transposon Tn5 in yeast

Summary We have demonstrated that precise excision of bacterial transposon Tn5 can occur in the yeast, Saccharomyces cerevisiae. Tn5 insertions in the yeast gene LYS2 were generated by transposon mutagenesis made in Escherichia coli by means of a ::Tn5 vector. Nine insertions of Tn5 into the structural part of the yeast LYS2 gene situated in a shuttle epsiomal plasmid were selected. All the plasmids with a Tn5 insertion were used to transform yeast strains carrying a deletion of the entire LYS2 gene or a deletion of the part of LYS2 overlapping the point of insertion.All insertions inactivated the LYS2 gene and were able to revert with low (about 10^-8) frequencies to lysine prototrophy. Restriction analysis of revertant plasmids revealed them to be indistinguishable from the original plasmid without Tn5 insertion. DNA sequencing of the regions containing the points of insertions, made for two revertants, proved that Tn5 excision was completely precise.     

Transposon Tn5supF-based reverse genetic method for mutational analysis of Escherichia coli with DNAs cloned in lambda phage.

An efficient method for systematic mutational analysis of the Escherichia coli genome was developed. It entails Tn5supF transposition to lambda-E. coli hybrid phage clones (Kohara library) and then transduction of recipient cells to Sup+. Essential and nonessential genes are distinguished by the ability of insertion mutant phage to form haploid versus only heterozygous partial diploid bacterial recombinants.     

Cosmid cloning and transposon mutagenesis in Salmonella typhimurium using phage lambda vehicles

Summary We have constructed a strain of Salmonella typhimurium which contains the malB region from Escherichia coli and carries the bacteriophage receptor protein in its outer membrane. Phage adsorbs to this strain but cannot grow, thus providing a very useful system for transposon mutagenesis of S. typhimurium using vehicles carrying transposons. This system can also be used for cosmid cloning.     

The organization of the outside end of transposon Tn5.

The end sequences of the IS50 insertion sequence are known as the outside end (OE) and inside end. These complex ends are related but nonidentical 19-bp sequences that serve as substrates for the activity of the Tn5 transposase. Besides providing the binding site of the transposase, the end sequences of a transposon contain additional types of information necessary for transposition. These additional properties include but are not limited to host protein interaction sites and sites that program synapsis and cleavage events. In order to delineate the properties of the IS50 ends,the base pairs involved in the transposase binding site have been defined. This has been approached through performing a variety of in vitro analyses: a ++hydroxyl radical missing-nucleoside interference experiment, a dimethyl sulfate interference experiment, and an examination of the relative binding affinities of single-site end substitutions. These approaches have led to the conclusion that the transposase binds to two nonsymmetrical regions of the OE, including positions 6 to 9 and 13 to 19. Proper binding occurs along one face of the helix, over two major and minor grooves, and appears to result in a significant bending of the DNA centered approximately 3 bp from the donor DNA-OE junction.