Figure 1.
The genetic organization of the Paracoccus spp. plasmids analyzed in this study.
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 Paracoccus spp. have been included: pAMI3 of P. aminophilus JCM 7686 [10] and pWKS1 of P. pantotrophus DSM 11072 [12].
Figure 2.
Schematic structure of the REP modules analyzed in this study.
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 rep 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.
Figure 3.
Comparison of the structure and G+C sequence profile of P. marcusii plasmids pMARC1 and pMOS7.
Arrows show the transcriptional orientation of the genes and the color code indicates their predicted functions (as shown in Figure 1). 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).
Figure 4.
Distribution of the REP modules analyzed in this study in the Paracoccus spp. genomes.
A specific DNA probe (fragment of a rep 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 Paracoccus spp. The results are presented as a matrix. The relatedness of the tested Paracoccus 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 Paracoccus strains from which the plasmids were isolated are denoted by red text.
Figure 5.
The plasmids containing the DIY cassettes constructed in this study.
A. pKRP-DIY plasmids. B. pVIV mobilizable shuttle vectors. The plasmids contain DIYAES7 and DIYMOS6 cassettes composed of REP modules (of plasmids pAES7 and pMOS6, respectively), a Kmr 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.