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

The plasmids containing the DIY cassettes constructed in this study.

<p>A. pKRP-DIY plasmids. B. pVIV mobilizable shuttle vectors. The plasmids contain DIY_AES7 and DIY_MOS6 cassettes composed of REP modules (of plasmids pAES7 and pMOS6, respectively), a Km^r gene and a MOB module derived from BHR plasmid RK2. B – BamHI, E – EcoRI, H – HindIII, K – KpnI, P – PstI, Sc – SacI, Sh – SphI, Sl – SalI, Sm – SmaI, X – XbaI. </p

Schematic structure of the REP modules analyzed in this study.

<p>The color-coded keys show the species and strain of origin of each plasmid (circles) and identified direct repeats (DRs), inverted repeats (IRs) as well as predicted DnaA and IHF binding sites (mixed shapes). The sequences of the iteron-like DRs are presented next to the relevant diagrams with a consensus sequence shown for DRs of plasmids with related REP modules. Blue arrows indicate the <i>rep</i> genes and their transcriptional orientation. Specific motifs identified within the aa sequences of the Rep proteins are indicated by colored rounded bars. A+T and G+C indicate DNA regions of lower or higher than average G+C content, respectively. The components of the REP modules are not shown to scale.</p

Distribution of the REP modules analyzed in this study in the <i>Paracoccus</i> spp. genomes.

<p>A specific DNA probe (fragment of a <i>rep</i> gene amplified by PCR and DIG-labeled) was prepared for each analyzed REP module and used in dot blot hybridization analysis with total DNA isolated from 20 strains of <i>Paracoccus</i> spp. The results are presented as a matrix. The relatedness of the tested <i>Paracoccus</i> strains is shown beneath by a phylogenetic tree based on their 16S rDNA sequences. The tree was constructed by the neighbor-joining algorithm with Kimura corrected distances. The statistical support for the internal nodes was determined by 1000 bootstrap replicates and values of >50% are shown. The <i>Paracoccus</i> strains from which the plasmids were isolated are denoted by red text. </p

Comparison of the structure and G+C sequence profile of <i>P. marcusii</i> plasmids pMARC1 and pMOS7.

<p>Arrows show the transcriptional orientation of the genes and the color code indicates their predicted functions (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080258#pone-0080258-g001" target="_blank">Figure 1</a>). Shaded areas connect homologous DNA regions. The PI regions (with indicated G+C content) are marked by yellow rectangles and dashed lines. The plot shows the G+C content of pMARC1 and pMOS7 sequences (the average values are given to the right).</p

The genetic organization of the <i>Paracoccus</i> spp. plasmids analyzed in this study.

<p>Arrows indicate the transcriptional orientation of the ORF2. The color-coded keys show the species and strain of origin of each plasmid (circles) and the likely plasmid maintenance/transfer processes in which the genes are involved (squares). Plasmid islets (PI) of lower than average G+C content, insertion sequences (IS) and restriction and modification systems (R-M) are indicated by the use of different boxes (see figure). Shaded areas connect genes of plasmids that encode orthologous proteins. For comparative analysis, two other related plasmids of <i>Paracoccus</i> spp. have been included: pAMI3 of <i>P. aminophilus</i> JCM 7686 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080258#B10" target="_blank">10</a>] and pWKS1 of <i>P. pantotrophus</i> DSM 11072 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080258#B12" target="_blank">12</a>].</p

Plasmids of Carotenoid-Producing <i>Paracoccus</i> spp. (<i>Alphaproteobacteria</i>) - Structure, Diversity and Evolution

<div><p>Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in <i>Alphaproteobacteria</i>. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus <i>Paracoccus</i> (<i>Alphaproteobacteria</i>): <i>P. aestuarii</i> DSM 19484, <i>P. haeundaensis</i> LG P-21903, <i>P. marcusii</i> DSM 11574 and <i>P. marcusii</i> OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the <i>relBE</i>/<i>parDE</i>, <i>tad</i>-<i>ata</i>, <i>higBA</i>, <i>mazEF</i> and <i>toxBA</i> families) and (iii) mobilization for conjugal transfer (encoding relaxases of the Mob_Q, Mob_P or Mob_V families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of <i>exc1</i>-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of <i>Paracoccus</i> spp., and of the entire <i>Alphaproteobacteria</i>. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of <i>Paracoccus</i> spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria. </p> </div

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