Possible mechanism for the generation of diverse non-autonomous and autonomous elements of the Tn<i>3</i> transposon family.

<p>The elements of the Tn3 family originate from progenitor insertion sequences (IS). Tns – diverse autonomous non-composite or composite transposons generated by acquisition of foreign DNA and mobilization for transposition of genomic DNA results. TIME - non-autonomous elements resulting from a reduction in the number of transposon-encoded genes, able to form mosaic elements resembling non-composite and composite Tns in structure. Elements representative of the different types are shown as examples: IS<i>1071</i> (accession no. M65135), Tn<i>3434</i> (accession no. AY232820), Tn<i>5393</i> (accession no. M96392), Tn<i>Ppa1</i> (accession no. DQ149577) and Tn<i>5271</i> (accession no. U18133).</p

Characteristics of transposable elements identified in this study.

a<p>The length of the IRs/the number of identical residues. nd - not determined.</p

Predicted secondary structures of TIMEs at the RNA level.

<p>RNA secondary structures predicted by <i>in silico</i> folding using Mfold software for TIME1 (class 1), two TIMEs representing class 3 and 4 elements shown in Fig. 3 (accession nos. AF020724 and AE016855, respectively), and the TIME-like elements ARM<i>phe</i> and IS<i>101</i> (see Discussion for details). The minimum folding energy (ΔG) of the predicted secondary structures was calculated by Mfold.</p

Genetic structure of <i>Pseudomonas</i> spp. plasmids containing non-autonomous TEs of the Tn<i>3</i> transposon family.

<p>Circular plasmids pZM1P1 (A) and pLM8P2 (B) originate from <i>Pseudomonas</i> spp. strains ZM1 and LM8, respectively. Predicted genetic modules involved in plasmid replication (REP), mobilization for conjugal transfer (MOB) and toluene resistance (TtgGHI efflux pump - truncated operon) are boxed and appropriately labeled. Predicted coding regions are represented by thick arrows indicating the direction of transcription. Broken arrows indicated truncated genes (<i>ttgH</i> and <i>orf5</i> of pLM8P2). Shaded areas connect DNA regions of pZM1P1 included in non-autonomous TIME-COMP transposons. Note that TIMEs are not drawn to scale. Thin black arrows above TIMEs indicate the orientation of the elements. A plot of the G+C content of pZM1P1 is shown above the structure diagram and the average G+C value is given to the right.</p

Mobility and Generation of Mosaic Non-Autonomous Transposons by Tn<i>3</i>-Derived Inverted-Repeat Miniature Elements (TIMEs)

<div><p>Functional transposable elements (TEs) of several <i>Pseudomonas</i> spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS<i>3</i>, IS<i>5</i>, IS<i>L3</i>, IS<i>30</i> and IS<i>1380</i>), (ii) isoforms of two Tn<i>3</i>-family transposons – Tn<i>5563</i>a and Tn<i>4662</i>a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn<i>5563</i>a and many other Tn<i>3</i>-family transposons. The simplest elements of this type, designated TIMEs (Tn<i>3</i> family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of <i>Pseudomonas</i> spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element “captured” with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a <i>res</i> site typical for many Tn<i>3</i>-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements.</p></div

Non-autonomous and autonomous transposable elements of the Tn<i>3</i> transposon family identified in <i>Pseudomonas</i> spp. strains.

<p>A. The genetic organization of TEs identified with trap plasmid pMAT1 in strains ZM1 (TIME, TIME-COMP1, TIME-COMP2), LM8 (TIME, TIME-RES), LM10 and LM13 (Tn<i>4662</i>a), as well as LM7 (Tn<i>5563</i>a). Inverted repeats (IRs) flanking the elements are marked by red triangles. Predicted coding regions are represented by thick arrows indicating the direction of transcription. Thin black arrows above TIMEs indicate the orientation of the elements. Shaded areas connect homologous DNA regions. <i>res</i> indicates the cointegrate resolution site of the site-specific recombination systems. The predicted toxin-antitoxin system is boxed and denoted by TA. Note that TIMEs are not drawn to scale. B. Alignment of the terminal inverted repeat nucleotide sequences (IRL – left IR; IRR – right IR) of TIMEs and two autonomous Tn<i>3</i>-family transposons, Tn<i>5563</i>a and Tn<i>5044</i>. Identical residues are indicated by gray shading. C. Nucleotide sequences of direct repeats (DRs) generated by TIME elements during transposition into the selective cartridge of pMAT1.</p

Comparison of the nucleotide sequences of TIME elements.

<p>A. Multiple alignment of the nucleotide sequences of TIMEs identified in bacterial genomes (NCBI database) by <i>in silico</i> comparative analyses. Accession numbers of the sequences are given on the left. A detailed description of the sequences (including the host strain, replicon and nucleotide positions) is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105010#pone.0105010.s003" target="_blank">Table S3</a>. TIME1-TIME4– functional non-autonomous elements identified in this study (stars indicate nucleotide substitutions in the TIME2-TIME4 sequences in relation to TIME1). Dots indicate gaps introduced to optimize the alignment. Only the sequence of TIME-RES homologous to TIME was used for the comparative analysis. Residues conserved in all predicted elements are shaded light violet, residues conserved in TIME1-TIME4 and other elements are shaded yellow, and those conserved in other elements (but not in TIME1-TIME4) are shaded green and red. The IRL and IRR sequences are marked. Four classes of TIME, distinguished by sequence similarity, are indicated on the right. B. Comparative analysis of the TIME and TIME-RES IR sequences shown as a pictogram (font size corresponds to the relative frequencies of the bases at each position within the termini). Numbers at right indicate the position in nucleotide sequence of each element.</p

Deletions within the trap plasmid pMEC1 generated in <i>P. versutus</i> UW400 upon transposition of IS<i>Pve1</i>.

<p>The range of the deletion within four individual derivatives of pMEC1 is shown by curved gray lines.</p

Transposable elements of <i>Paracoccus</i> spp. identified in this study.

a<p>The length of the IRs/the number of identical residues.</p>b<p>Location in plasmids, if applicable.</p

Schematic overview of five ways in which transposition can deliver promoters to the transcriptionally silent tetA (tetracycline resistance) gene of the trap plasmid pCM132TC.

<p>The location of promoters in the plasmids pCM132TC::TE, conferring a Tc^r phenotype, are appropriately indicated: an outwardly oriented promoter in the terminal parts of a TE (A), a hybrid promoter composed of a −35 hexamer (delivered by the TE) and a −10 hexamer located in close proximity to the target site of transposition (B), the promoter of a TPase gene (C), a promoter present in the core region of a composite transposon (D), and a promoter derived from another plasmid delivered by the generation of transient co-integrates resulting from replicative transposition (E). DNA fragments used in the localization of the promoters are shown as open thin boxes below each panel. The activity of promoters (tested in <i>Paracoccus</i> spp.) accompanying the presence of the DNA fragments is indicated on the right: (+) promoter activity, (−) lack of promoter activity.</p