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.     

Molecular characterization of erm(B)- and mef(E)-mediated erythromycin-resistant Streptococcus pneumoniae in China and complete DNA sequence of Tn2010

Aims: To characterize the erm(B)‐ and mef(E)‐mediated erythromycin‐resistant Streptococcus pneumoniae clinical isolates obtained from ten hospitals located different cities in China. Methods and Results: Totally 83 S. pneumoniae were collected, and eighteen representative strains of 66 strains that exhibited erythromycin resistance were used for further characterization by antibiograms, serotyping, PFGE, MLST, DNA sequencing of the macrolide‐resistance elements and mapping of the elements on the chromosome. Twelve isolates showed a high‐level resistance to erythromycin, and six other isolates showed a low‐level resistance to erythromycin. Thirteen isolates harboured a Tn2010 transposon (26·4 kbp) encoding the erm(B), tet(M) and mef(E) genes and were classified into three types by Tn2010 structures. The remaining five isolates harboured a Tn6002 transposon (20·9 kbp) encoding the erm(B) and tet(M) genes and were classified into three types by Tn6002 locations on the chromosome. Three of the Tn6002 elements were located within the Tn5252‐like element, implying that these composed a large mobile element. The MLST analyses showed that several clones had been disseminated and that the CC271 strains carrying the Tn2010 element expressing the high‐level resistance to erythromycin were predominant in China. Four new MLST strains, which were designated as ST3262, ST3263, ST3397 and ST3398 were also identified. Conclusions: The erythromycin resistance determinant of S. pneumoniae that had been isolated in China was located in Tn2010 or the Tn6002 element and several clones had been disseminated, and the CC271 strains carrying the Tn2010 element expressing the high‐level resistance to erythromycin were predominant in China. Significance and Impact of the Study: This is the first molecular analysis of erythromycin‐resistant Streptococcus pneumoniae clinical isolates in China, and the first report of the complete nucleotide sequence of Tn2010 (26 390 bp).     

Control of Tn7 transposition

Summary Our isolate of Tn7 (named Tn7S) contains an IS1 insertion, and this IS1 can be converted into Tn9. In vitro and in vivo deletions of Tn7S and Tn7S:: Tn9 define regions of the transposon required for antibiotic resistance and transposition. Complementation of deletion mutants by cloned Tn7 fragments indicates the existence of two regions, denoted tnp7A and tnp7B, required for all transposition events. Another region, denoted tnp7C, is required for transposition from the chromosome to RP1 but not for transposition from a small IncP-1 replicon to the chromosome. The presence of Tn7S terminal sequences in an RP1 replicon reduces the transposition of a second Tn7S derivative from the chromosome by about one order of magnitude. The measured frequency of Tn7S transpositions from a small IncP-1 replicon to the chromosome depends on the particular incompatibility system used to eliminate that replicon. Genetic and physical data indicate that high frequencies of Tn7S transposition to the chromosome (40%) are triggered by the IncP-1 incompatibility reaction, thus suggesting the existence of a Tn7 mechanism for sensing the state of the carrier replicon.     

Identification of Tn4430, a transposon of Bacillus thuringiensis functional in Escherichia coli

Summary The mobile genetic element Tn4430, originating from the gram-positive bacterium, Bacillus thuringiensis, and previously described as the Th-sequence, is the first transposon isolated from the genus Bacillus. In the present work a gene (APH-III) conferring resistance to kanamycin was inserted into this 4.2 kb transposon. Transposition experiments showed that Tn4430APH-III could transpose in the gram-negative host Escherichia coli when its insertion functions were supplied by an intact copy of Tn4430. By transposing Tn4430APH-III directly onto pBR322, it was possible to determine the nucleotide sequence of the terminal inverted repeats of Tn4430 and of the target DNA site. Identical 38 bp in inverted orientation are situated at each end of the transposon and there is a direct duplication of 5 bp at the insertion site. Thus, it is clear that Tn4430 is closely related to the transposons belonging to the Tn3 family (class II elements).     

Nucleotide sequence of the ends of the conjugative shuttle transposon Tn1545

Summary The conjugative shuttle transposon Tn1545 from Streptococcus pneumoniae transposes in various gram-positive bacterial genera following self-transfer and in Escherichia coli after cloning. Analysis of the junction fragments and of the targets before insertion and after excision of the element by DNA hybridization and sequencing indicated that Tn1545 (1) is not flanked by terminal repeated sequences in either direct or opposite orientation, (2) is flanked, in an asymmetric fashion, by terminal variable base pairs, one at the left and three at the right of the element, (3) inserts in a target DNA consensus sequence, (4) does not generate duplication of the target DNA upon insertion, and (5) excises precisely.     

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     

Site-specific integration of genes into hot spots for recombination flanking aadA in Tn21 transposons.

Summary Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA ^– background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5 CTAAAACAAAGTTA 3 (hs2). The double-site-specific recombination occurred with a frequency of 10^–5–10^–6. The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a trans-acting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations.     

Identification of a new transposon Tn5403 in aKlebsiella pneumoniae strain isolated from a polluted aquatic environment

AKlebsiella pneumoniae strain having mobilization helper potential has been isolated from the river Rhine. Analysis of the transconjugants resulting from the mobilization of nonconjugative pBR-type plasmids and RSF1010 derivatives showed that the transfer-helper capacity of theK. pneumoniae strain is related to the presence of a Tn3-like transposable element, Tn5403. This element has been identified and localized in a plasmid.     

Evolutionary relationship between Tn21-like elements and pBP201, a plasmid from Klebsiella pneumoniae mediating resistance to gentamicin and eight other drugs

Summary We have characterized pBP201 one of the plasmids from a collection of 46 strains producing adenylyltransferase ANT (2) (Schmidt 1984). It confers resistance to sulphonamides and produces aminoglycoside adenylyltransferases AAD (3) and ANT (2) and -lactamase TEM-1. Plasmid pBP201 has a size of 24.8 kilobases (kb) and contains TnA and a Tn21-related element, Tn4000, with deletions in mer and the termini and a substitution at tnpR. In complementation assays with transposition-deficient mutants of Tn21 the element in pBP201 appears to be TnpA⁺ but TnpR^-. It represents a naturally occurring defective transposon. The sequence organization of pBP201 has been compared with that of Tn21-related elements such as Tn2410, Tn2603, Tn2424, Tn1696, and Tn4000. In these transposons the integration sites of resistance genes cat, bla, aacA, aacC or aadB have been identified at two preferential locations; these are at the termini of the streptomycin resistance gene aadA. Two additional sites have been localized in the Tn21 backbone to the right of the mer operon and at res (internal resolution site) and are probably involved in the evolution of these elements. Based on these results a model for the possible genealogy of class II transposons is presented.     

Nisin biosynthesis genes are encoded by a novel conjugative transposon

Summary Genes for biosynthesis of the lactococcal peptide antibiotic nisin were shown to be encoded by a novel chromosomally located transposon Tn5301. The element is 70 kb in size and lacks inverted repeats at its termini. Although a copy of the insertion sequence IS904 is located near to one end, this did not appear to be involved in the transposition process. The integrated element is flanked by the directly repeated sequence 5-TTTTTG-3. Analysis of ten independent transconjugants revealed that Tn5301 integration is site-specific; two chromosomal targets were identified and shown to have some sequence homology. The element shares features with the Tn916 family of conjugative transposons and with Tn554 but is also exhibits some unique properties. Tn5301 is thus considered to be the prototype of a novel class of conjugative transposon.