Molecular genetic analysis of the Tn5041 transposition system

A study was made of the transposition of the mercury resistance transposon Tn5041 which, together with the closely related toluene degradation transposon Tn4651, forms a separate group in the Tn3 family. Transposition of Tn5041 was host-dependent: the element transposed in its original host Pseudomonas sp. KHP41 but not in P. aeruginosa PAO-R and Escherichia coli K12. Transposition of Tn5041 in these strains proved to be complemented by the transposase gene (tnpA) of Tn4651. The gene region determining the host dependence of Tn5041 transposition was localized with the use of a series of hybrid (Tn5041 x Tn4651) tnpA genes. Its location in the 5'-terminal one-third of the transposase gene is consistent with the data that this region is involved in the formation of the transposition complex in transposons of the Tn3 family. As in other transposons of this family, transposition of Tn5041 occurred via cointegrate formation, suggesting its replicative mechanism. However, neither of the putative resolution proteins encoded by Tn5041 resolved the cointegrates formed during transposition or an artificial cointegrate in E. coli K12. Similar data were obtained with the mercury resistance transposons isolated from environmental Pseudomonas strains and closely related to Tn5041 (Tn5041 subgroup).     

Molecular structure and interrelationships of multiresistance beta-lactamase transposons

Transposons coding for beta-lactamases OXA-3, OXA-4, OXA-5, LCR-1, and CARB-3 have been isolated and compared functionally and structurally with transposons for TEM-1, OXA-1, PSE-1, PSE-2, and PSE-4 enzymes. Each beta-lactamase gene type occurred in a unit together with resistance to other antibiotics, particularly streptomycin and sulfonamide but also chloramphenicol, mercuric ion, or gentamicin, kanamycin, and tobramycin. Restriction mapping, gene cloning, and DNA hybridization were used to compare the transposons and to localize their functional components. Although the multiresistance beta-lactamase transposons varied in size from 8 to 25 kb, the similarity of some of their restriction maps suggested a common derivation. Six of 12 transposons contained DNA segments homologous to the tnpR gene of transposon Tn21 and could complement a tnpR- Tn21 derivative. Consequently, these six transposons appear to have evolved from a common progenitor by acquisition of DNA coding for various beta-lactamases and other resistance genes.     

Libraries of green fluorescent protein fusions generated by transposition in vitro

Two artificial transposons have been constructed that carry a gene encoding Green Fluorescent Protein and can be used for generating libraries of GFP fusions in a gene of interest. One such element, AT2GFP, can be used to generate GFP insertions in frame with the amino acid sequence of the protein of interest, with a stop codon at the end of the GFP coding sequence; AT2GFP also contains a selectable marker that confers trimethoprim resistance in bacteria. The second element, GS, can be used to generate tribrid GFP fusions because there is no stop codon in the GFP transposon, and the resulting fusion proteins contain the entire amino acid sequence encoded by the gene. The GS element consists of a gfp open reading frame and a supF amber suppressor tRNA gene; the supF portion of the GS transposon can be utilized as a selectable marker in bacteria. Its sequence contains a fortuitous open reading frame, and thus it can be translated continuously with the gfp amino acid sequence. As a target for GFP insertions, we used a plasmid carrying the native Ty1 retrotransposon of the yeast Sacharomyces cerevisiae. The resulting multiple GFP fusions to Ty1 capsid protein Gag and Ty1 integrase were useful in determining the cellular localization of these proteins. Libraries of GFP fusions generated by transposition in vitro represent a novel and potentially powerful method to study the cell distribution and cellular localization signals of proteins.     

Toluene transposons Tn4651 and Tn4653 are class II transposons

The toluene degradative transposon Tn4651 is included within another transposon, Tn4653, and both of these elements are members of the Tn3 family. The tnpA gene product of each element mediates formation of cointegrates as intermediate products of transposition, and the tnpS and tnpT gene products encoded by Tn4651 take part in resolution of both Tn4651- and Tn4653-mediated cointegrates. Sequence analysis demonstrated that Tn4651 and Tn4653 have 46- and 38-base-pair terminal inverted repeats, respectively, and that both elements generate 5-base-pair duplication of the target sequence upon transposition. Complementation tests of the Tn4651- and Tn4653-encoded transposition functions with those of Tn3, Tn21, and Tn1721 showed that (i) the trans-acting transposition functions encoded by Tn4651 were not interchangeable with those encoded by the four other transposons, (ii) the Tn4653 tnpA function was interchangeable with the Tn1721 function, and (iii) Tn4653 coded for a resolvase (tnpR gene product) that complemented the tnpR mutations of Tn21 and Tn1721. The Tn4653 tnpR gene was located just 5' upstream of the tnpA gene and shared extensive sequence homology with the Tn1721 tnpR gene. The res region was located adjacent to the tnpR gene, and sequence analysis indicated that failure of the Tn4653 tnpR product to resolve the Tn4653-mediated cointegrates is ascribed to an incomplete structure of the res region.     

Tn5053 family transposons are res site hunters sensing plasmidal res sites occupied by cognate resolvases

DNA sequence database search revealed that most of Tn5053/Tn402 family transposons inserted into natural plasmids were located in putative res regions upstream of genes encoding various resolvase-like proteins. Some of these resolvase genes belonged to Tn3 family transposons and were closely related to the tnpR genes of Tn1721 and a recently detected Tn5044. Using recombinant plasmids containing fragments of Tn1721 or Tn5044 as targets in transposition experiments, we have demonstrated that Tn5053 displays striking insertional preference for the res regions of these transposons: more than 70% of Tn5053 insertion events occur in clusters inside the target res regions, while most remaining insertion events occur no further than 200 base pairs away from both sides of the res regions. We demonstrate that Tn5053 insertions (both into and outside a res region of the target plasmid) require the presence of a functional cognate resolvase gene either in cis or in trans. To our knowledge, this is the first case when a site-specific recombination system outside a transposon has been shown to be involved in transposition.     

Genomic analysis of insertion behavior and target specificity of mini-Tn7 and Tn3 transposons in Saccharomyces cerevisiae

Transposons are widely employed as tools for gene disruption. Ideally, they should display unbiased insertion behavior, and incorporate readily into any genomic DNA to which they are exposed. However, many transposons preferentially insert at specific nucleotide sequences. It is unclear to what extent such bias affects their usefulness as mutagenesis tools. Here, we examine insertion site specificity and global insertion behavior of two mini-transposons previously used for large-scale gene disruption in Saccharomyces cerevisiae: Tn3 and Tn7. Using an expanded set of insertion data, we confirm that Tn3 displays marked preference for the AT-rich 5 bp consensus site TA[A/T]TA, whereas Tn7 displays negligible target site preference. On a genome level, both transposons display marked non-uniform insertion behavior: certain sites are targeted far more often than expected, and both distributions depart drastically from Poisson. Thus, to compare their insertion behavior on a genome level, we developed a windowed Kolmogorov–Smirnov (K–S) test to analyze transposon insertion distributions in sequence windows of various sizes. We find that when scored in large windows (>300 bp), both Tn3 and Tn7 distributions appear uniform, whereas in smaller windows, Tn7 appears uniform while Tn3 does not. Thus, both transposons are effective tools for gene disruption, but Tn7 does so with less duplication and a more uniform distribution, better approximating the behavior of the ideal transposon.     

Four genes, two ends, and a res region are involved in transposition of Tn5053: a paradigm for a novel family of transposons carrying either a mer operon or an integron

The complete nucleotide sequence of an 8447 bp-long mercury-resistance transposon (Tn 5053 ) has been determined. Tn 5053 is composed of two modules: (i) the mercury-resistance module and (ii) the transposition module. The mercury-resistance module carries a mer operon, merRTPFAD , and appears to be a single-ended relic of a transposon closely related to the classical mercury-resistance transposons Tn 21 and Tn 501 . The transposition module of Tn 5053 is bounded by 25 bp terminal inverted repeats and contains four genes involved in transposition, i.e. tniA, tniB, tniQ , and tniR . Transposition of Tn 5053 occurs via cointegrate formation mediated by the products of the tniABQ genes, followed by site-specific cointegrate resolution. This is catalysed by the product of the tniR gene at the res region, which is located upstream of tniR . The same pathway of transposition is used by Tn 402 (Tn 5090 ) which carries the integron of R751. Transposition genes of Tn 5053 and Tn 402 are interchangeable. Sequence analysis suggests that Tn 5053 and Tn 402 are representatives of a new family of transposable elements, which fall into a recently recognized superfamily of transposons including retroviruses, insertion sequences of the IS 3 family, and transposons Tn 552 and Tn 7 . We suggest that the tni genes were involved in the dissemination of integrons.     

Transposition of DNA fragments flanked by two inverted Tn1 sequences: translocation of the plasmid RP4::Tn1 region harboring the Tcr marker

We have demonstrated the possibility of transposition of the plasmid RP4::Tn1 fragment (21.2 kb) carrying the tetracycline resistance (Tcr) gene and flanked by two Tn1 copies. The new transposon, designated Tn1756, bears lethal genes that kill host cells. Therefore, its transposition can only be revealed in the presence of lethality-compensating helper regions of the plasmid RP4. Thus, RP4::Tn1 consists of two transposons, Tn1755 (Tn1-Kmr-Tn1) and Tn1756 (Tn1-Tcr-Tn1), sharing the Tn1 sequences. Both of these transposons are capable of recA-independent translocation to other plasmids. Therefore, transposition of DNA fragments flanked by two inverted Tn1 sequences does not depend on Tn1 orientation.     

Relationships among the streptothricin resistance transposons Tn1825 and Tn1826 and the trimethoprim resistance transposon Tn7

The streptothricin resistance transposons Tn1825 and Tn1826 are closely related, based on physical and genetic characteristics, to the trimethoprim resistance transposon Tn7. These transposons may be considered to be members of a transposon family sharing in common the transposition functions and a basic streptomycin/spectinomycin resistance determinant but differing from one another with respect to particular additional resistance genes inserted to the left of the aadA gene.     

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.     

Transposition mutagenesis in Streptomyces fradiae: identification of a neutral site for the stable insertion of DNA by transposon exchange

We explored transposition in Streptomyces fradiae (Sf) as a means to insert a second copy of the tylF gene to improve tylosin (Ty) production. Transposons Tn5096 and Tn5099 transposed relatively randomly in Sf, and many of the insertions caused no deleterious effects on Ty production yields. Tn5098, a derivative of Tn5096 containing tylF and tylJ genes, recombined into the chromosome into the tyl gene cluster and transposition was not observed. However, following the tagging of a neutral site (NS) by Tn5099 transposition, tylF was effectively inserted into the NS by homologous recombination (transposon exchange). Recombinants obtained by transposon exchange produced higher yields of Ty.     

Isolation and characteristics of compound transposons Tn1000::Tn5

Two types of compound transposons were derived. In the first case, transposon Tn5 is inserted into the gene responsible for Tn1000 transposase synthesis. In the other, Tn5 is inserted into the region near the left end of Tn1000, where no functionally significant genes were found. It is known that translocation of the compound transposons does not depend on their size and takes place with the efficiency close to that characteristic of the intact Tn1000. Insertion of Tn5 into the gene coding for Tn1000 transposase results in sharp decrease in the efficiency of Tn1000 translocations. This effect, however, may be eliminated by introduction into the cell of the intact Tn1000.     

Characterization of Tn3926, a new mercury-resistance transposon from Yersinia enterocolitica

A new transposon coding for mercury resistance (HgR), Tn3926, has been found in a strain of Yersinia enterocolitica, YE138A14. The element has a size of 7.8 kb and transposes to conjugative plasmids belonging to different incompatibility groups. A restriction map has been established. DNA-DNA hybridization indicates that Tn3926 displays homology with both Tn501 and Tn21; the greatest homology is shown with the regions of these transposons that encode HgR. Weaker homology is observed between Tn3926 sequences and those regions of Tn501 and Tn21 that encode transposition functions. Complementation experiments indicate that the Tn3926 transposase mediates transposition of Tn21, albeit somewhat inefficiently, but not of Tn501, while the resolvase mediates resolution of transposition cointegrates formed via Tn21, Tn501, or Tn1721.     

Genetic properties of transposons Tn6-1, Tn6-2 and Tn19-1

The level and range transposition of the transposons Tn6-1, Tn6-2, Tn19-1, and their ability to influence plasmid transfer has been studied. The widest range of transposition was shown for transposon Tn6-2. Insertions of each of the studied transposons into different conjugative plasmids genomes resulted in change of frequencies of plasmids transfer and change of plasmids mobilization activity.     

Bi-functional gfp- and gusA-containing mini-Tn5 transposon derivatives for combined gene expression and bacterial localization studies

The gfp gene, encoding the green fluorescent protein, was combined with the gusA gene, coding for the beta-glucuronidase enzyme, in mini-Tn5 transposon derivatives for use in Gram-negative bacteria. These mini-Tn5 elements allow simultaneously monitoring of gene expression and localization of the marked bacteria. Introduction of the resultant mini-Tn5 transposons into Rhizobium etli, Azospirillum brasilense and Pseudomonas stutzeri allowed us to visualise the interaction of these bacteria with their host plant. The dual-marker mini-Tn5 transposons constitute a powerful new tool for studying gene expression and ecology of bacteria in the environment and during the interaction with plants.     

Fine structure of transposition genes on Tn2603 and complementation of its tnpA and tnpR mutations by related transposons

The fine structure of the genes tnpA, tnpR and res of Tn2603 required for its own transposition, was determined. The order of the genes was tnpA-tnpR-res from the right end of the right hand side region in Tn2603, the tnpA and tnpR encoded gene products having molecular weights of 110,000 and 21,000, respectively. The 110,000 molecular weight polypeptides was absolutely required for replicon fusion as the first stage of transposition, and named transposase. On the other hand, the 21,000 molecular weight polypeptide was necessary for resolution of the cointegrate as the second stage of transposition, and named resolvase. We also examined the ability of various transposons, assumed to be closely related, to complement the tnpA and tnpR mutations of Tn2603. The results indicated that the mercury resistance transposon, Tn2613, and Tn501, can complement both genes, but TnAs and gamma delta cannot at all. Tn501 had much less efficiency of complementation for tnpA than Tn2613. We have also discovered that the transposition frequency of transposons in the tn2613 family systematically depend on their size of transposon.     

THE EVOLUTION OF TRANSPOSABLE ELEMENTS: CONDITIONS FOR ESTABLISHMENT IN BACTERIAL POPULATIONS

Previous theoretical studies have shown that bacterial transposons can become established in populations by infectious transfer, even if they reduce the fitness of their host cells. Conditions for the persistence of “parasitic” transposons are, however, restrictive: i) transposition must be replicative, rather than conservative; ii) the rate of transposition must be greater than the loss in host fitness caused by the transposon; and iii) cells must exchange plasmids at rates greater than the fitness cost of the transposon. I sought to test the validity of the model underlying this theory by performing experiments with laboratory populations of the bacterium Escherichia coli, the conjugative plasmid R100, and the transposons Tn3 and Tn5. A plasmid-borne transposon was introduced at low frequency into a population of bacteria carrying the same plasmid without the transposon in a habitat where the transposon offered no benefit to its host. The fate of the invading transposon was followed by tracking the various bacterial populations appearing in the cultures. Using independent estimates of the parameters of the model, predicted population changes were generated with numerical solutions of the model, and these were compared to experimental results. Plasmids transferred into new hosts as predicted by the model, and the resulting transconjugant populations either maintained a steady low density or rose slowly in abundance. Transposition appeared to play no role in population changes. Abundance of all cell types fit theoretical predictions of a system with no transposition, despite evidence that transposition was taking place. This is exactly what the model predicted. It thus appears unlikely that deleterious or neutral transposons have much impact on the genetics of bacterial populations. This is consistent with the hypothesis that most bacterial transposons are not parasitic DNA, but rather invade and persist in populations by providing a fitness advantage to cells carrying them.