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. 2014 Aug;16(8):2374-88.
doi: 10.1111/1462-2920.12345. Epub 2014 Jan 7.

The rulB Gene of Plasmid pWW0 Is a Hotspot for the Site-Specific Insertion of Integron-Like Elements Found in the Chromosomes of Environmental Pseudomonas Fluorescens Group Bacteria

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Free PMC article

The rulB Gene of Plasmid pWW0 Is a Hotspot for the Site-Specific Insertion of Integron-Like Elements Found in the Chromosomes of Environmental Pseudomonas Fluorescens Group Bacteria

Glenn Rhodes et al. Environ Microbiol. .
Free PMC article

Abstract

The rulAB operon of Pseudomonas spp. confers fitness traits on the host and has been suggested to be a hotspot for insertion of mobile elements that carry avirulence genes. Here, for the first time, we show that rulB on plasmid pWW0 is a hotspot for the active site-specific integration of related integron-like elements (ILEs) found in six environmental pseudomonads (strains FH1-FH6). Integration into rulB on pWW0 occurred at position 6488 generating a 3 bp direct repeat. ILEs from FH1 and FH5 were 9403 bp in length and contained eight open reading frames (ORFs), while the ILE from FH4 was 16 233 bp in length and contained 16 ORFs. In all three ILEs, the first 5.1 kb (containing ORFs 1-4) were structurally conserved and contained three predicted site-specific recombinases/integrases and a tetR homologue. Downstream of these resided ORFs of the 'variable side' with structural and sequence similarity to those encoding survival traits on the fitness enhancing plasmid pGRT1 (ILE(FH1) and ILE(FH5)) and the NR-II virulence region of genomic island PAGI-5 (ILE(FH4)). Collectively, these ILEs share features with the previously described type III protein secretion system effector ILEs and are considered important to host survival and transfer of fitness enhancing and (a)virulence genes between bacteria.

Figures

Fig. 1
Fig. 1
RFLP profiles of HindIII digested pWW0 plasmid variants from strains FH1–FH6. Lanes 1–6 = pWW0FH1–FH6. Lane 7 = pWW0. The size of fragments generated from in silico digestion of pWW0 are shown for comparison.
Fig. 2
Fig. 2
Insertion point and orientation of ILEs in pWW0. ILEs (orange) inserted into the rulB gene (blue) at position 6488 on pWW0, generating the truncated rulB′ and a new predicted ORF rulB(2). The direct repeat created by insertion is illustrated.
Fig. 3
Fig. 3
The structure of ILEs from FH1/FH5 and FH4 aligned with regions of closest similarity. (A) The general structure of ILEs inserted into rulB on pWW0 with ORFs flanking the insertion point on pWW0 is illustrated. (B) The detailed structure of chromosomally located ILEFH1–FH5 alongside predicted ILEs in P. putida DOT-1E plasmid pGRT1 and P. aeruginosa genomic islands PAGI-5 and RGP63. ILEs are shown inside black rectangles with interrupted flanking rulB-like regions (light blue). Related regions are linked by adjoining black lines. When not specifically indicated, other colours indicate the following: Blue: rulB-like regions; purple: rulA-like gene; red: site-specific recombinase/phage integrase; pink: transposase, dark green, fitness-related; pale green: helicase; orange: mercury resistance genes; grey: hypothetical proteins (HP); yellow: hypothetical proteins with domains of unknown function. Predicted ORFs for ILEFH1/5 and ILEFH4 are numbered inside arrows, whereas those of relatives are shown above the sequence and are numbered in accordance with deposited sequences. ORFs 87a and b and ORF 88a in RGP63 are predicted in the present study and not in the original genome sequence. The sequences of pGRT1, PA7 (RGP63) and PAGI-5 are HM626202, CP000744 and EF611301 respectively.
Fig. 4
Fig. 4
Alignment of intergenic regions found immediately upstream and downstream of rulAB′ on integron-like elements.A. Alignment of the predicted promoter region and LexA binding site upstream of rulA. The conserved CTG-N10-CAG LexA binding site motif (yellow), the −35 box (bold and underlined) and the −10 box (underlined bold italics) are highlighted.B. The 118–119 bp intergenic region between the known 5′-GAT-3′ insertion point in pWW0 and the predicted ATG start codon of ORF1 (xerC/xerD) aligned with chromosomal locations in strains FH1, FH4 and FH5, and other close relatives. aNo ATG start codon for rulA in FH5 chromosome.

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