Table 1.
Characteristics of transposable elements identified in this study.
Figure 1.
Non-autonomous and autonomous transposable elements of the Tn3 transposon family identified in Pseudomonas spp. strains.
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 (Tn4662a), as well as LM7 (Tn5563a). 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. res 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 Tn3-family transposons, Tn5563a and Tn5044. 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.
Figure 2.
Comparison of the nucleotide sequences of TIME elements.
A. Multiple alignment of the nucleotide sequences of TIMEs identified in bacterial genomes (NCBI database) by in silico 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 Table S3. 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.
Figure 3.
Predicted secondary structures of TIMEs at the RNA level.
RNA secondary structures predicted by in silico 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 ARMphe and IS101 (see Discussion for details). The minimum folding energy (ΔG) of the predicted secondary structures was calculated by Mfold.
Figure 4.
Genetic structure of Pseudomonas spp. plasmids containing non-autonomous TEs of the Tn3 transposon family.
Circular plasmids pZM1P1 (A) and pLM8P2 (B) originate from Pseudomonas 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 (ttgH and orf5 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.
Figure 5.
Possible mechanism for the generation of diverse non-autonomous and autonomous elements of the Tn3 transposon family.
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: IS1071 (accession no. M65135), Tn3434 (accession no. AY232820), Tn5393 (accession no. M96392), TnPpa1 (accession no. DQ149577) and Tn5271 (accession no. U18133).