Fluctuation tests are more sensitive than plate tests in detection of chemicals which cause enhanced excision of transposon Tn10 in Salmonella typhimurium

Precise excision of transposon Tn10, as judged by reversion of Salmonella typhimurium strain LT2 trp1014::Tn10 to Trp+, was not detectably enhanced following exposure to 9-aminoacridine, 5-azacytidine or mitomycin C in conventional treat-and-plate assays. By contrast, 7/13 chemicals, including 5-azacytidine and mitomycin C, were found to be capable of enhancing precise excision of Tn10 when tested in modified fluctuation assays. Despite earlier reports, precise excision is one activity of transposons which is not therefore refractory to enhancement by chemical mutagens.     

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.     

Mutants of Escherichia coli K-12 with altered excision efficiency of transposons Tn5 and Tn9

HFETn5, HFETn9 and LFETn9 mutants of Escherichia coli K-12 have been isolated. The frequency of Tn5 precise excision from the chromosomal lac operon is increased 3-660-fold in nine HFETn5 mutants. The majority of these mutations have no influence on the efficiency of precise excision of transposon Tn9, though hfeTn5-04 and hfeTn5-06 mutations decrease excision efficiency 2-13-fold. The Tn9 transposon is excised in HFETn9 mutant about 20-fold more efficiently than in the wild type strain. This mutation does not stimulate excision of Tn5 and Tn10. LfeTn9 mutation decreases excision frequency of Tn9 11-17-fold, but has no effect on Tn5 excision and increases that of Tn10 about 20-fold. The differences in genetic control and mechanisms of excision of the transposons with long and short inverted repeats are discussed.     

Translocation of transposons Tn10 and Tn5 in the Yersinia pestis chromosome

The possibility of translocation of the transposons Tn5 and Tn10 into the genome of Yersinia pestis, with the subsequent mutagenic effect was demonstrated. We revealed transposon harbouring clones at frequency 10(-4) to 10(-2). Derivatives of P1cml clr100ts phage served as vectors. Insertion of Tn10 transposon induced mutations in ilv, ser, arg, pur, pro, leu, nic, tyr, gua genes. The number of the insertion sites on the chromosome obtained for Tn5 was the same, these being arg, ade, pyr, leu, gua, trp, his, pan, ilv. The majority of auxotrophs did not revert. Occasionally, revertants were observed at frequencies 10(-8) to 10(-6). Unlike Escherichia coli, reversion was not accompanied by the loss of transposons. The rearrangements induced by transposons, presumably, near the insertion site, as well as duplications of transposons followed by incorporation of copies into novel sites, led to the appearance of additional defective genes, which made it possible to select various types of polyauxotrophs. Based on reiteration of coinciding double and triple mutant markers, we proposed a linkage group of genes within a segment of Y. pestis chromosome: lys ... tyr - ser - arg - ilv - leu - gua - ade(pur) - pro ... his ... pyr ... trp. The reasons for peculiarities of the behaviour of transposons in Y. pestis bacteria are discussed.     

Participation of the uvrD gene in the precise excision of the Tn9 transposon

HfeTn5-(04,06) and IfeTn9 mutations increase efficiency of precise excision of Tn5, Tn10 and decrease that of Tn9. These mutations have been mapped in uvrD gene. In LFETn9 and UVRE502 mutants, the multicopy plasmid pEM61 carrying the cloned uvrD gene complements LFETn9- phenotype (Low Frequency Excision of Tn9). These results indicate that the uvrD gene product plays different role in excision of transposons with long and short inverted repeats. The mechanism of this effect is discussed.     

Interaction of related Tn916-like transposons: analysis of excision events promoted by Tn916 and Tn5386 integrases

In recent work, we described the excision of a large genomic region from Enterococcus faecium D344R in which the sequence from "joint" regions suggested that excision resulted from the interaction of conjugative transposon Tn916 and the related mobile element Tn5386. In the present study, we examined the ability of integrases and integrase-excisase combinations from Tn916 and Tn5386 to promote the excision of constructs consisting of the termini of Tn916, Tn5386, and the VanB mobile element Tn5382. Integrases alone from either Tn916 or Tn5386 promoted the circularization of constructs from the three different transposons, even when the different termini used in the constructs were discordant in their transposon of origin. The termini of Tn916 and Tn5382 found in all joints were consistent with previously identified Tn916 and Tn5382 termini. Substantial variation was seen in the integrase terminus of Tn5386 used to form joints, regardless of the integrase that was responsible for circularization. Variability was observed in joints formed from Tn5386 constructs, in contrast to joints observed with the termini of Tn916 or Tn5382. The coexpression of excisase yielded some variability in the joint regions observed. These data confirm that integrases from some Tn916-like elements can promote circularization with termini derived from heterologous transposons and, as such, could promote excision of large genomic regions flanked by such elements. These findings also raise interesting questions about the sequence specificities of the C terminals of Tn916-like integrases, which bind to the ends and facilitate strand exchange.     

Three Tn10-associated excision events: relationship to transposition and role of direct and inverted repeats

We describe three related DNA alterations associated with transposon Tn10: precise excision of Tn10, nearly precise excision of Tn10 and precise excision of the nearly precise excision remnant. DNA sequence analysis shows that each of these alterations results in excision of all or part of the Tn10 element, and each involves specific repeat sequences at or near the ends of the element. Furthermore, all three events are structurally analogous: in each case, excision occurs between two short direct-repeat sequences, with resulting deletion of all intervening material plus one copy of the direct repeat; and in all three cases, the direct repeats involved occur at either end of an inverted repeat. Analysis of mutant Tn10 elements and characterization of bacterial host mutations suggest that all three types of excision events occur by pathways that are fundamentally distinct from the pathway(s) for Tn10-promoted transposition and other DNA rearrangements (deletions and inversions) actively promoted by the element. In addition, precise excision and nearly precise excision appear to occur by very closely related or identical pathways; and several lines of evidence suggest that the 1400 bp inverted repeats at the ends of Tn10 may play a structural role in both of these events. The third excision event appears to occur by yet another pathway.     

Characteristics of Escherichia coli K-12 mutants with altered effectiveness of excision of Tn5 and Tn9 transposons

The properties of Escherichia coli K-12 mutans HFETn5, HFETn9 and LFETn9 have been studied. The majority of mutations were shown to have pleiotropic effect. Some of them increase cell sensitivity to UV light and mitomycin C and affect efficiency of homologous recombination in transduction and conjugation. The level of spontaneous mutagenesis is increased in a number of mutants. None of the mutations isolated affect frequency of transposition of Tn5 from bacteriophage lambda::Tn5 into the chromosome. Based on analysis of properties of hfeTn5-09 and hfeTn9 mutations and on the date of preliminary mapping of hfeTn5-09 mutation, these mutations were considered to be novel. It is shown that the processes of precise excision of Tn5 and Tn9 transposons may be accomplished by at least two pathways, one of them being dependent on recA gene functions.     

Enhanced Tn10 and mini-Tn10 precise excision in DNA replication mutants of Escherichia coli K12

The precise excision of transposon Tn10 and a mini-Tn10 derivative, inserted in the gal or lac operons, was studied in dnaB252 and dnaE486 temperature-sensitive mutants of Escherichia coli. dnaB codes for a DNA replication helicase and dnaE for the alpha subunit of DNA polymerase III. Mutations in these genes were found to enhance, at the permissive temperature, the precise excision of both genetic elements. The increase factor was much more pronounced for the dnaB252 mutant with the transposons inserted in gal. The stimulated excision was only partially affected by a recA null mutation but was significantly reduced by introduction of recF null or ruvA mutations. A model involving template switching of the polymerase between the direct repeats flanking the transposons, on the same strand or between sister strands, could account for the observed results.     

RecA-dependent increased precise excision of Tn10 in Salmonella typhimurium.

UV irradiation induced the precise excision of Tn10 inserted in met, trp or srl in a Salmonella typhimurium strain; mitomycin C was also found to induce the frequency of precise excision of Tn10 from srl or met. Precise excision of Tn10 was not increased by either UV or mitomycin C in a recA mutant. Similarly, a recA mutant derived from a uvrD strain showed a drastic reduction in the high spontaneous levels of precise excision of Tn10 of this strain. These results indicate that recA is involved in the increased precise excision of Tn10. In contrast to point mutations excision of Tn10 was found to be UV inducible in a top mutant.     

Modulation of mutagenesis involving precise excision of transposon Tn10

Precise excision of transposon Tn10 results in reversion of the Trp- phenotype to Trp+ in a trp-1014::Tn10 strain of Salmonella typhimurium, and also occurs at a markedly higher frequency in a strain carrying the temperature-sensitive polA7 allele. The frequency with which precise excision events occurs can be modified by the plating medium, results indicating that the great majority of mutants which arise on broth-supplemented or tryptophan-supplemented minimal media actually arise on the selective plating medium. Trp+ revertants (1000) arising from excision of Tn10 were purified by re-streaking for single colonies; none were found to retain the Tn10 encoded resistance to tetracycline. Yields of Trp+ revertants of the polA7 strain were consistently higher when glycerol rather than glucose was used as sole carbon source in the selective medium. Clean excision of Tn10 can also be increased by ultraviolet irradiation in (R) plasmid-free strains, and is further increased in strains carrying an N-group plasmid (R205, R46 or pKM101). Ultraviolet-induced precise excision of Tn10 also occurs at a much enhanced frequency in a strain with a deletion through the uvrB gene; in this case, however, the addition of plasmid pKM101 leads to a decrease in yields of ultraviolet-induced precise excision events.     

Precise and nearly-precise excision of the symmetrical inverted repeats of Tn5; common features of recA-independent deletion events in Escherichia coli

The transposon Tn5 contains a unique central region bordered by 1.5-kb inverted repeats. The in vitro deletion of the centre of Tn5, with a restriction endonuclease (XhoI) which cuts within the inverted repeats leads to the production of a palindrome on subsequent ligation. This palindromic region is unstable on subsequent transformation into Escherichia coli (Collins, 1981). Precise excision of the Tn5 region plus one copy of the bracketing 9-bp direct repeat occurred in about one-third of the transformants. The rest of the transformants contain only remnants of the inverted repeat. Sequence analysis indicated that deletion had occurred between short direct repeats. The precise excision of these "nearly precise" excision products continued with high frequency and was found to be affected by mutations that interfere with the normal precise excision of transposons. In a recB, sbcB host precise excision was markedly reduced. A common mechanism is proposed for all recA-independent deletions occurring in E. coli.     

Postexcision transposition of the transposon Tn10 in Escherichia coli K12

An experimental analysis of the fate of transposon Tn10 after excision from a proA::Tn10 site localized on the plasmid F' leads to the conclusions: 1. The precise excision is a progressive process. Its probability is estimated per time unit. 2. An excised Tn10 is always integrated into a different genetic locus. 2. An excised Tn10 is always integrated into a different genetic locus. 3. The kinetics of postexcision transposition are sometimes very slow. The excised transposon is inherited in one cell line in spite of cell multiplication. 4. The processes of excision and secondary insertion have no absolute requirement for the recA+ genotype but they are strongly enhanced in recA+ cells. 5. The kinetics of postexcision transposition are strongly dependent on the genetic site from which the transposon was excised. 6. The probability of postexcision transposition is fully determined by the probability of excision and depends on the genotype of the host and many other factors.     

RecBC and RecF recombination pathways and the induced precise excision of Tn10 in Escherichia coli

Mitomycin C (MMC) treatment or mutations in uvrD enhance the frequency of Tn10 precise excision. We have shown previously that several repair-recombination genes, such as recA, ruv and recF are involved in the induced excision process. In this study, we find that other genes belonging to the RecBC and RecF sexual recombination pathways also participate in this process since mutations in recB, sbcB or recO diminish, though to different degrees, the frequency of Tn10 precise excision induced by MMC treatment or by uvrD mutants. Pairwise combinations of some of these mutations were also tested for Tn10 induced precise excision; most of these double mutants showed additive effects in reducing the frequency of the excision process. The results of these studies suggest that recombinational-repair genes, particularly recF, sbcB and recO have different roles in the induced excision of Tn10 than in recombinational mating.     

Characterization of Tn5386, a Tn916-related mobile element

In recent work, we described excision of a large genomic region from Enterococcus faecium D344R resulting from the interaction of Tn916 and a related transposon designated Tn5386. In the present study, we present and analyze the complete sequence of Tn5386. Tn5386 is 29,451 bp in length. Fifteen of its 30 open reading frames are analogous to ORFs found in Tn916. Significant differences include a series of ORFs with homology to lantibiotic immunity genes in the same location where tetM is found in Tn916, insertion of a Group II intron and an ORF with similarities to previously described surface exposed collagen adhesion proteins. Our results indicate that Tn5386 falls within the Tn916 family of transposons, and in place of tetM encodes a novel region that may confer resistance to lantibiotics.     

A multicomponent system is required for tetracycline-induced excision of Tn4555

Bacteroides spp. are the predominant organisms in the intestinal tract, and they also are important opportunistic pathogens. Antibiotic therapy of Bacteroides infections often is complicated by the prevalence of drug-resistant organisms which acquire resistance genes from a variety of mobile genetic elements including conjugative transposons (CTns) and mobilizable transposons (MTns). Tn4555 is an MTn that encodes beta-lactam resistance, and it is efficiently mobilized by the Bacteroides CTns via a tetracycline (TET)-inducible mechanism. In this study a model system with CTn341 and a Tn4555 minielement was used to examine Tn4555 excision from the chromosome. Using PCR and mobilization assays it was established that excision was stimulated by TET in the presence of CTn341. In order to determine which Tn4555 genes were required for excision, int, tnpA, tnpC, xis, and mobA mutants were examined. The results indicated that int plus two additional genes, tnpC and xis, were required for optimal excision. In addition, there was no requirement for the mobA gene, as had been shown for another MTn, NBU1. The Xis protein sequence is related to a family of plasmid excisionases, but the TnpC gene product did not match anything in the sequence databases. Evidence also was obtained that suggested that Xis is involved in the control of TET-induced excision and in control of mobilization by CTn341. Overall, these results indicate that excision of MTns is a complex process that requires multiple gene products.     

Characterization of the transposons Tn1822 (Tc) and Tn1824 (TpSm) and the light they throw on the natural spread of resistance genes

The transposons Tn1822 (Tc) and Tn1824 (TpSm) described in this paper are very similar or identical to the transposons Tn1771 and Tn1721 (Tc), and Tn7 (TpSm), respectively. They were isolated from different sources and replicons, however, suggesting a wide distribution of these types of transposons. The method used to isolate and characterize the transposons, involving a temperature-sensitive P1 phage as their transient vehicle, is particularly suitable for epidemiological studies on transposon distribution.     

pIF21, a versatile donor of transposons Tn1, Tn5 and Tn7 for tagging and mobilizing cryptic and non-selectable plasmids

A plasmid, pIF21, has been constructed that is able to donate transposons Tn1, Tn5 and Tn7. The transposons are located on a temperature-sensitive derivative of the incP1 plasmid pRP1, which is transferable to a wide range of Gram-negative genera.