Tn916 delta E: a Tn916 transposon derivative expressing erythromycin resistance

The tetracycline resistance gene encoded within the transposon Tn916 was replaced with the gene encoding erythromycin resistance from the plasmid pVA838. The derivative transposon of Tn916 was designated Tn916 delta E and was introduced into the Streptococcus faecalis chromosome by protoplast transformation. The conjugation/transposition functions of Tn916 delta E were similar to those observed for Tn916 in S. faecalis and Tn916 delta E was capable of self-conjugation at frequencies similar to those of other S. faecalis and Group B Streptococcus. This transposon will be useful for mutagenesis studies in gram-positive organisms, especially in those species where erythromycin resistance is a more desirable selectable marker.     

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.      

Chromatin structure of transposon Tn903 cloned into a yeast plasmid

Transposon Tn903 contains the APH gene for kanamycin resistance, which is active in yeast [A. Jiménez and J. Davies (1980) Nature (London) 287, 869-871] and is flanked by two inverted repeats (IR) 1057 bp long. When plasmid pAJ50, carrying Tn903 and the 2-microns circle origin of replication, is cloned into Saccharomyces cerevisiae, nucleosomes are assembled in vivo on the prokaryotic DNA of the transposon. Indirect end labeling revealed that three nucleosomes are preferentially positioned on symmetrical sequences from both IRs. DNase I digestion also confirmed that the chromatin structure is symmetrical in both IRs. This suggests that sequence determinants are decisive for chromatin structure in these regions. We have calculated the rotational and translational fits [H. R. Drew and C. R. Calladine (1987) J. Mol. Biol. 195, 143-173] for the Tn903 sequence and the results indicate that the nucleosome positioning on the IRs is sequence-directed. Nucleosome deposition on the APH gene also occurs, but no clear positioning exists. Some sequence preference for positioning nucleosomes on the promoter can be predicted, especially from the translational fit. Experimental data indicate, however, that nucleosomes are absent from the promoter. Therefore, chromatin can be organized on prokaryotic DNA in a manner that resembles the typical eukaryotic chromatin structure.     

The transposon Tn5 carries a bleomycin-resistance determinant

Transposon Tn5 carries a determinant for resistance to bleomycin (Bm). Deletion mapping and cloning experiments have shown that this determinant, gene ble, is located between the determinant for kanamycin (Km) and neomycin resistance (gene neo) and the determinant for streptomycin resistance (gene str). Genes neo, ble, and str belong to an operon controlled by the common promoter. The Mr of the ble product, as determined by polyacrylamide gel electrophoresis, is 12000 to 13000.     

Tn951: A new transposon carrying a lactose operon

Summary A new transposon, Tn951, is described, which derives from plasmid pGC1, originally isolated from Yersinia enterocolitica. Tn951 is 16.6 kb long and presumably flanked by small inverted repeats. It carries the lac genes i, z and y. This lac system is homologous to the E. coli lac operon. However, homology is restricted to 5.6 kb. The DNA sequences surrounding the lac operons on Tn951 and E. coli are nonhomologous. This leads to speculations about the origin of the E. coli lac operon itself.     

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.     

Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives

Summary Two derivatives of the prokaryotic transposon Tn5 were constructed in vitro. In Tn5-233, the central area of Tn5, which carries resistance to kanamycin/neomycin, bleomycin and streptomycin, is replaced by a fragment carrying resistance to the aminocyclitol antibiotics gentamycin/kanamycin and streptomycin/spectinomycin. In Tn5-235, the Escherichia coli -galactosidase gene is inserted within the streptomycin resistance gene of Tn5, and constitutively expressed from a Tn5 promoter. Both constructs transpose with about the same frequency as Tn5 in Escherichia coli and Rhizobium meliloti. When a Tn5-derivative is introduced into an R. meliloti strain which already contains a different Tn5-derivative, in situ transposon replacement is obtained at high frequency, presumably by a pair of crossovers between the IS50 sequences at the ends of the incoming and resident transposons. In this way we converted a previously isolated recA::Tn5 mutant into the corresponding recA::Tn5-233 strain, which can now be used as a genetic background in the study of complementation of other Tn5-induced mutations. We also replaced the drug markers of several Tn5-induced exo mutants, which we were then able to map relative to each other by transduction with phage M12. In a strain carrying Tn5-235 located near Tn5-233, we were able to isolate deletions of the intervening markers, presumably resulting from general recombination between the two transposons, by screening for loss of the Lac⁺ phenotype. Unlike Tn5 itself, resident Tn5-233 does not appear to suppress transposition of another incoming Tn5-derivative.Abbreviations bp base pairs - Nm neomycin - Km kanamycin - Sm streptomycin - Sp spectinomycin - Gm gentamycin - Tc tetracycline - Tp trimethoprim - Ot oxytetracycline - Rf rifampicin - Xgal 5-bromo-4-chloro-3-indolyl--d-galactoside     

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.     

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.     

Pseudo-transposition of a Tn5 derivative in Neisseria gonorrhoeae

Abstract We constructed a Tn5 derivative for potential use in transposon mutagenesis of Neisseria gonorrhoeae . It was incorporated into the chromosome apparently at random following transformation, but the insertion events were dependent on a functional RecA and independent of a functional transposase. Furthermore, in most cases there was an incomplete transposon inserted with little or no IS50 insertion sequence. These observations suggest that TnJ transposition may not be possible in N. gonorrhoeae and that this organism may have an unexplored illegitimate recombination system.     

Tn1545: a conjugative shuttle transposon

Summary Tn1545, from Streptococcus pneumoniae BM4200, confers resistance to kanamycin (aphA-3), erythromycin (ermAM) and tetracycline (tetM). The 25.3 kb element is self-transferable to various Gram-positive bacterial genera where it transposes. Tn1545 was cloned in its entirety in the recombination deficient Escherichia coli HB101 where it was unstable. The three resistance genes aphA-3, ermAM and tetM were expressed but were not transferable to other E. coli cells. Tn1545 transposed from the hybrid plasmid to multiple sites of the chromosome of its new host. The element re-transposed, at a frequency of 5×10^-9, from the chromosome to various sites of a conjugative plasmid where it could be lost by apparently clean excision. The element transformed and transposed to the chromosome of Bacillus subtilis. The properties of the conjugative shuttle transposon Tn1545 may account for the recent emergence of genes from Gram-positive bacteria in Gramnegative organisms.     

Streptococcus faecalis R plasmid pJH1 contains an erythromycin resistance transposon (Tn3871) similar to transposon Tn917

The R plasmid pJH1 contains a 5.1-kilobase transposon ( Tn3871 ) that mediates inducible resistance to erythromycin. Three AvaI digestion fragments from this transposon are identical in size to and homologous with three AvaI-derived fragments from the previously described erythromycin resistance transposon Tn917 . These three DNA fragments account for greater than 90% of both transposons.     

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.     

Tn7: a target site-specific transposon

The bacterial transposon Tn7 is an unusual mobile DNA segment. Most transposable elements move at low-frequency and display little target site-selectivity. By contrast, Tn7 inserts at high-frequency into a single specific site in the chromosomes of many bacteria. In the absence of this specific site, called attTn7 in Escherichia coli where Tn7 has been most extensively studied, Tn7 transposes at low-frequency and inserts into many different sites. Much has recently been learned about Tn7 transposition from both genetic and biochemical studies. The Tn7 recombination machinery is elaborate and includes a large number of Tn7-encoded proteins, probably host-encoded proteins and also rather large cis-acting transposition sequences at the transposon termini and at the target site. Dissection of the Tn7 transposition mechanism has revealed that the DNA strand breakage and joining reactions that underlie the translocation of Tn7 have several unusual features.     

Mobilization and transfer of Azospirillum lipoferum plasmid by the Tn5-Mob transposon into a plasmid-free Agrobacterium tumefaciens strain

Azospirillum lipoferum 4B harbors five cryptic plasmids. Several suicide plasmids were used to transfer Tn5-Mob to A. lipoferum 4B. Tn5-Mob insertion mutations of this strain could be obtained at frequencies of 10(-8)-10(-7) per recipient cell. One hundred Tn5-Mob A. lipoferum 4B mutants were used in bacterial matings with a plasmid-free Agrobacterium tumefaciens recipient strain. This is the first report of mobilization, transfer, and replication of an Azospirillum plasmid in Agrobacterium tumefaciens. One transconjugant was found which had lost an indigenous plasmid.     

Isolation of symbiotically defective mutants in Rhizobium leguminosarum by insertion of the transposon Tn5 into a transmissible plasmid

Summary Selection was made for the transposition of the kanamycin resistance transposon Tn5 from a location on the chromosome of R. leguminosarum into a transmissible, bacteriocinogenic plasmid that also carries genes required for the induction of nitrogen-fixing nodules on peas.One hundred and sixty independent insertions into transmissible plasmids were isolated. When these plasmids were transferred by conjugation into a non-nodulating strain, which carries a deletion in one of its resident plasmids, of the 160 isolates tested 14 yielded transconjugants that formed nodules that did not fix nitrogen (Fix^-) and in a further 15 cases the transconjugants were unable to form nodules (were Nod^-). When transferred to a symbiotically proficient strain (i.e. Nod⁺ Fix⁺) none of the transconjugants was symbiotically defective; thus the mutations were not dominant.When kan was transduced from the clones that generated Fix^- transconjugants into a Fix⁺ recipient the majority of transductants inherited Fix^- indicating that the insertion of Tn5 had induced the symbiotic mutations. Transduction of kan, from the clones that failed to donate Nod⁺ by conjugation to strain 6015, occurred at barely detectable frequencies and it was not possible to demonstrate transduction of Nod^-. kan was co-transduced with Nod⁺ from some of the clones and some of these transductants also inherited the ability to produce medium bacteriocin and to transfer at high frequency by conjugation. Thus the genes for all these characters are closely linked.