Insights into the Transposable Mobilome of Paracoccus spp. (Alphaproteobacteria)

Several trap plasmids (enabling positive selection of transposition events) were used to identify a pool of functional transposable elements (TEs) residing in bacteria of the genus Paracoccus (Alphaproteobacteria). Complex analysis of 25 strains representing 20 species of this genus led to the capture and characterization of (i) 37 insertion sequences (ISs) representing 9 IS families (IS3, IS5, IS6, IS21, IS66, IS256, IS1182, IS1380 and IS1634), (ii) a composite transposon Tn6097 generated by two copies of the ISPfe2 (IS1634 family) containing two predicted genetic modules, involved in the arginine deiminase pathway and daunorubicin/doxorubicin resistance, (iii) 3 non-composite transposons of the Tn3 family, including Tn5393 carrying streptomycin resistance and (iv) a transposable genomic island TnPpa1 (45 kb). Some of the elements (e.g. Tn5393, Tn6097 and ISs of the IS903 group of the IS5 family) were shown to contain strong promoters able to drive transcription of genes placed downstream of the target site of transposition. Through the application of trap plasmid pCM132TC, containing a promoterless tetracycline resistance reporter gene, we identified five ways in which transposition can supply promoters to transcriptionally silent genes. Besides highlighting the diversity and specific features of several TEs, the analyses performed in this study have provided novel and interesting information on (i) the dynamics of the process of transposition (e.g. the unusually high frequency of transposition of TnPpa1) and (ii) structural changes in DNA mediated by transposition (e.g. the generation of large deletions in the recipient molecule upon transposition of ISPve1 of the IS21 family). We also demonstrated the great potential of TEs and transposition in the generation of diverse phenotypes as well as in the natural amplification and dissemination of genetic information (of adaptative value) by horizontal gene transfer, which is considered the driving force of bacterial evolution

Molecular Analysis of VanA Outbreak of Enterococcus faecium in Two Warsaw Hospitals: The Importance of Mobile Genetic Elements

Vancomycin-resistant Enterococcus faecium represents a growing threat in hospital-acquired infections. Two outbreaks of this pathogen from neighboring Warsaw hospitals have been analyzed in this study. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA, multilocus VNTR analysis (MLVA), and multilocus sequence typing (MLST) revealed a clonal variability of isolates which belonged to three main lineages (17, 18, and 78) of nosocomial E. faecium. All isolates were multidrug resistant and carried several resistance, virulence, and plasmid-specific genes. Almost all isolates shared the same variant of Tn1546 transposon, characterized by the presence of insertion sequence ISEf1 and a point mutation in the vanA gene. In the majority of cases, this transposon was located on 50 kb or 100 kb pRUM-related plasmids, which lacked, however, the axe-txe toxin-antitoxin genes. 100 kb plasmid was easily transferred by conjugation and was found in various clonal backgrounds in both institutions, while 50 kb plasmid was not transferable and occurred solely in MT159/ST78 strains that disseminated clonally in one institution. Although molecular data indicated the spread of VRE between two institutions or a potential common source of this alert pathogen, epidemiological investigations did not reveal the possible route by which outbreak strains disseminated

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

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

Genetic organization of the Tn<i>3</i> family transposons captured in <i>Paracoccus</i> spp. using trap plasmids.

<p>The cryptic transposon Tn<i>3434</i>a (A), Tn<i>5393</i> carrying streptomycin resistance genes (B) and the composite element Tn<i>Ppa1</i> flanked by two identical copies of Tn<i>3434</i> (C). Predicted coding regions are represented by arrows indicating the direction of transcription. Gray arrows indicate TPase genes (<i>tnpA</i>) and additional genes (including resolvase genes – <i>tnpR</i>) are indicated by white arrows. Inverted repeats (IRs) flanking transposons are marked by black arrowheads (IRL – left IR; IRR – right IR) and <i>res</i> sites are shown by black squares. Plots of the G+C content of the Tn<i>3434</i>a and Tn<i>5393</i> sequences are shown in panels A and B (the average value is given on the right).</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

<i>Paracoccus</i> spp. strains used in this study.

<p><i>Paracoccus</i> spp. strains used in this study.</p

The distribution of TEs (identified in our studies) in the genomes of strains of <i>Paracoccus</i> spp.

<p>A specific DNA probe (fragment of a TPase gene amplified by PCR and DIG-labeled) was prepared for each TE and used in dot blot hybridization analysis with total DNA isolated from the <i>Paracoccus</i> spp. strains.</p

Genetic organization of the insertion sequences (A, B, C, D, E) and composite transposon Tn<i>6097</i> (F) of <i>Paracoccus</i> spp. identified in this study.

<p>The families and names of the identified ISs are shown on the panel. Inverted repeats (IRL – left IR; IRR – right IR) flanking ISs are marked by black arrowheads. Predicted coding regions are represented by arrows indicating the direction of transcription. Gray arrows indicate TPase genes and white arrows indicate additional ORFs present within the ISs and within the core of Tn<i>6097</i>. Two of the ORFs of Tn<i>6097</i> (ORF2 and ORF13) are truncated (the disruptions are most probably remnants of the ancient IS<i>Pfe2</i> transposition events that led to the formation of Tn<i>6097</i> in its native host). The predicted genetic modules of the transposon are indicated.</p