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. 2008 Sep;190(17):5797-805.
doi: 10.1128/JB.00468-08. Epub 2008 Jul 7.

Evolution of the Rhodococcus Equi Vap Pathogenicity Island Seen Through Comparison of Host-Associated vapA and vapB Virulence Plasmids

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Free PMC article

Evolution of the Rhodococcus Equi Vap Pathogenicity Island Seen Through Comparison of Host-Associated vapA and vapB Virulence Plasmids

Michal Letek et al. J Bacteriol. .
Free PMC article

Abstract

The pathogenic actinomycete Rhodococcus equi harbors different types of virulence plasmids associated with specific nonhuman hosts. We determined the complete DNA sequence of a vapB(+) plasmid, typically associated with pig isolates, and compared it with that of the horse-specific vapA(+) plasmid type. pVAPB1593, a circular 79,251-bp element, had the same housekeeping backbone as the vapA(+) plasmid but differed over an approximately 22-kb region. This variable region encompassed the vap pathogenicity island (PAI), was clearly subject to selective pressures different from those affecting the backbone, and showed major genetic rearrangements involving the vap genes. The pVAPB1593 PAI harbored five different vap genes (vapB and vapJ to -M, with vapK present in two copies), which encoded products differing by 24 to 84% in amino acid sequence from the six full-length vapA(+) plasmid-encoded Vap proteins, consistent with a role for the specific vap gene complement in R. equi host tropism. Sequence analyses, including interpolated variable-order motifs for detection of alien DNA and reconstruction of Vap family phylogenetic relationships, suggested that the vap PAI was acquired by an ancestor plasmid via lateral gene transfer, subsequently evolving by vap gene duplication and sequence diversification to give different (host-adapted) plasmids. The R. equi virulence plasmids belong to a new family of actinobacterial circular replicons characterized by an ancient conjugative backbone and a horizontally acquired niche-adaptive plasticity region.

Figures

FIG. 1.
FIG. 1.
Genetic structure and ACT analysis of the VR of the R. equi virulence plasmids pVAPB1593 and pVAPA1037. Regions with significant similarity (tBLASTx) are connected by colored lines (red, sequences in direct orientation; blue, sequences in reverse orientation). The intensity of the color indicates the strength of the sequence homology (pink/light blue, lowest; red/deep blue, highest). The virulence-associated vap genes are shown in black, and the mobility-related resA- and invA-like genes are shown in gray. The vap PAI, as defined based on Alien Hunter output (see Fig. S1 in the supplemental material), is boxed. Gene designations for pVAPB1593 are according to standardized annotation nomenclature adopted in this study for R. equi virulence plasmids (pVAP) (see Table S1 in the supplemental material), and those for pVAPA1037 are according to the nomenclature used by Takai et al. (50) (except for the newly identified genes, in which standardized nomenclature has been used) (see Table S1 in the supplemental material). The reference bar at the top right indicates 1 kb.
FIG. 2.
FIG. 2.
Neighbor-joining unrooted phylogenetic tree of the vap multigene family constructed from a ClustalW alignment of the mature Vap proteins encoded by pVAPB1593 and pVAPA1037. For the analyses, a full-length VapF protein was reconstructed by correcting the two frameshift mutations in the 3′ region of the gene. Bootstrap values (10,000 replicates) are indicated at the nodes. Outgroup, putative DnaB protein from pVAPB1593 (pVAPB_0390). The bar indicates genetic distance.
FIG. 3.
FIG. 3.
Model of the evolutionary dynamics of the vap multigene family. On a schematic representation of the vap PAIs from pVAPB1593 and pVAPA1037, vap genes (in black) presumably derived from vertical evolution of a preexisting common ancestral determinant are connected by straight arrows, and those probably originated by gene duplication in the specific plasmid are connected by curved arrows (see the text for details). The connection between vapM and the vapX pseudogene was deduced from a phylogenetic tree constructed with the 28-residue VapX product aligned with the corresponding protein fragments from other members of the Vap family (not shown). Asterisks indicate degenerate vap genes; note in pVAPA1037 that one gene in each of the duplicated vapIE and vapCF tandems is undergoing decay.
FIG. 4.
FIG. 4.
ACT analysis of the rhodococcal pVAP and pREC1 plasmids and Gordonia westfalica pKB1 plasmid (8). See the legends to Fig. 1 and to Fig. S1 in the supplemental material for an explanation of gene color codes and ACT output. To facilitate the visualization of the colinearity and gene synteny, the plasmids were aligned on the first conserved gene of the backbone. Arrowheads indicate the mobility genes present in pKB1 (the black arrowhead shows the conserved phage excisionase [phage exc.] gene also present in the rhodococcal plasmids) (see Fig. S1 and Table S4 in the supplemental material).
FIG. 5.
FIG. 5.
ACT analysis of the conserved signature region of the CURV plasmids, a new family of large, circular actinobacterial plasmids. This highly syntenic region includes the conserved gene encoding a putative conjugal transfer protein, TraG/TraD (indicated by an arrow), and spans the second half of the conjugation and the first half of the unknown-function plasmid modules (see the text for details). An ≈15-kb fragment of each of the indicated plasmids from Arthrobacter and Micrococcus spp. (total size in brackets) was compared with the corresponding fragment from pVAPB1593 (above). See also Table S4 in the supplemental material.

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