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Plasmid Replicons from Pseudomonas Are Natural Chimeras of Functional, Exchangeable Modules

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ABSTRACT

Plasmids are a main factor for the evolution of bacteria through horizontal gene exchange, including the dissemination of pathogenicity genes, resistance to antibiotics and degradation of pollutants. Their capacity to duplicate is dependent on their replication determinants (replicon), which also define their bacterial host range and the inability to coexist with related replicons. We characterize a second replicon from the virulence plasmid pPsv48C, from Pseudomonas syringae pv. savastanoi, which appears to be a natural chimera between the gene encoding a newly described replication protein and a putative replication control region present in the widespread family of PFP virulence plasmids. We present extensive evidence of this type of chimerism in structurally similar replicons from species of Pseudomonas, including environmental bacteria as well as plant, animal and human pathogens. We establish that these replicons consist of two functional modules corresponding to putative control (REx-C module) and replication (REx-R module) regions. These modules are functionally separable, do not show specificity for each other, and are dynamically exchanged among replicons of four distinct plasmid families. Only the REx-C module displays strong incompatibility, which is overcome by a few nucleotide changes clustered in a stem-and-loop structure of a putative antisense RNA. Additionally, a REx-C module from pPsv48C conferred replication ability to a non-replicative chromosomal DNA region containing features associated to replicons. Thus, the organization of plasmid replicons as independent and exchangeable functional modules is likely facilitating rapid replicon evolution, fostering their diversification and survival, besides allowing the potential co-option of appropriate genes into novel replicons and the artificial construction of new replicon specificities.

No MeSH data available.


The REx-C module is conserved among four non-homologous replicon families and subjected to recombination among them. Graphical view of NCBI blastn comparisons of the 600 nt region preceding the start codon of replication initiator protein genes (black bars, ticks every 100 nt), displayed with the ACT software; default algorithm parameters were used, except for a word size of 7. Red and blue bars indicate collinear and inverted regions of identity, respectively, with color intensity proportional to identity; only matches larger than 15 nt are shown. Symbols are followed by their corresponding plasmid name and are identical for rep genes of the same homology family. Sequence accession no. are pA506, NC_021361; plasmid Drgb7, KT351738; pMP-R124, NZ_CM001562; pP27494_2, CP015602; pPA7790, CP015000; pPsv48C, FR820587; pRA2, U88088. A and J refer to repA and repJ, respectively, from pPsv48C.
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Figure 3: The REx-C module is conserved among four non-homologous replicon families and subjected to recombination among them. Graphical view of NCBI blastn comparisons of the 600 nt region preceding the start codon of replication initiator protein genes (black bars, ticks every 100 nt), displayed with the ACT software; default algorithm parameters were used, except for a word size of 7. Red and blue bars indicate collinear and inverted regions of identity, respectively, with color intensity proportional to identity; only matches larger than 15 nt are shown. Symbols are followed by their corresponding plasmid name and are identical for rep genes of the same homology family. Sequence accession no. are pA506, NC_021361; plasmid Drgb7, KT351738; pMP-R124, NZ_CM001562; pP27494_2, CP015602; pPA7790, CP015000; pPsv48C, FR820587; pRA2, U88088. A and J refer to repA and repJ, respectively, from pPsv48C.

Mentions: Blastn comparisons showed that the REx-C module is present, with varying degrees of conservation, preceding the rep genes from at least four non-homologous replicon families from Pseudomonas (Figure 3; Table S1), whereas nucleotide identity is rapidly lost shortly after the CDS start codon. These families include the RepA-PFP family from P. syringae and other bacteria (exemplified by repA from pPsv48C) (Bardaji et al., 2011), the RepJ family (among others, repJ from pPsv48C and pA506) (Bardaji et al., 2011; Stockwell et al., 2013), and what we designated the RepA-RA2 family (named after pRA2) (Kato and Mizobuchi, 1994) and the RepA-Pa family (named here after the P. antarctica PAMC 27494 plasmid pP27494_2). The highest level of sequence conservation of the REx-C module among representatives of the four families is in a 175–194 nt fragment that spans SaL structures 1–3 (Figure S5). The stem sequences from SaL 1 (the putative transcriptional terminator for the antisense RNA), as well as the stretch of adenines in either side of them (Gibbon et al., 1999), are almost perfectly conserved and changes in one arm of the stems are usually compensated with complementary changes in the other arm (Figure S5) (Gibbon et al., 1999). The other structures are also well-conserved among the four replicon families; however, the number and position of palindromes, which could form SaL structures, are variable among replicons (Figure 3; Figure S5). Therefore, the stark conservation of the REx-C module suggests that it contains features universally essential for plasmid replication in species of Pseudomonas. Remarkably, we did not find REx-C sequences conserved in the control region of ColE2 replicons, which contain a replication initiator protein homologous to that from RepA-PFP replicons, or in any other plasmid outside of the genus Pseudomonas, indicating a diversity of REx-C modules among homologous theta replicons.


Plasmid Replicons from Pseudomonas Are Natural Chimeras of Functional, Exchangeable Modules
The REx-C module is conserved among four non-homologous replicon families and subjected to recombination among them. Graphical view of NCBI blastn comparisons of the 600 nt region preceding the start codon of replication initiator protein genes (black bars, ticks every 100 nt), displayed with the ACT software; default algorithm parameters were used, except for a word size of 7. Red and blue bars indicate collinear and inverted regions of identity, respectively, with color intensity proportional to identity; only matches larger than 15 nt are shown. Symbols are followed by their corresponding plasmid name and are identical for rep genes of the same homology family. Sequence accession no. are pA506, NC_021361; plasmid Drgb7, KT351738; pMP-R124, NZ_CM001562; pP27494_2, CP015602; pPA7790, CP015000; pPsv48C, FR820587; pRA2, U88088. A and J refer to repA and repJ, respectively, from pPsv48C.
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Figure 3: The REx-C module is conserved among four non-homologous replicon families and subjected to recombination among them. Graphical view of NCBI blastn comparisons of the 600 nt region preceding the start codon of replication initiator protein genes (black bars, ticks every 100 nt), displayed with the ACT software; default algorithm parameters were used, except for a word size of 7. Red and blue bars indicate collinear and inverted regions of identity, respectively, with color intensity proportional to identity; only matches larger than 15 nt are shown. Symbols are followed by their corresponding plasmid name and are identical for rep genes of the same homology family. Sequence accession no. are pA506, NC_021361; plasmid Drgb7, KT351738; pMP-R124, NZ_CM001562; pP27494_2, CP015602; pPA7790, CP015000; pPsv48C, FR820587; pRA2, U88088. A and J refer to repA and repJ, respectively, from pPsv48C.
Mentions: Blastn comparisons showed that the REx-C module is present, with varying degrees of conservation, preceding the rep genes from at least four non-homologous replicon families from Pseudomonas (Figure 3; Table S1), whereas nucleotide identity is rapidly lost shortly after the CDS start codon. These families include the RepA-PFP family from P. syringae and other bacteria (exemplified by repA from pPsv48C) (Bardaji et al., 2011), the RepJ family (among others, repJ from pPsv48C and pA506) (Bardaji et al., 2011; Stockwell et al., 2013), and what we designated the RepA-RA2 family (named after pRA2) (Kato and Mizobuchi, 1994) and the RepA-Pa family (named here after the P. antarctica PAMC 27494 plasmid pP27494_2). The highest level of sequence conservation of the REx-C module among representatives of the four families is in a 175–194 nt fragment that spans SaL structures 1–3 (Figure S5). The stem sequences from SaL 1 (the putative transcriptional terminator for the antisense RNA), as well as the stretch of adenines in either side of them (Gibbon et al., 1999), are almost perfectly conserved and changes in one arm of the stems are usually compensated with complementary changes in the other arm (Figure S5) (Gibbon et al., 1999). The other structures are also well-conserved among the four replicon families; however, the number and position of palindromes, which could form SaL structures, are variable among replicons (Figure 3; Figure S5). Therefore, the stark conservation of the REx-C module suggests that it contains features universally essential for plasmid replication in species of Pseudomonas. Remarkably, we did not find REx-C sequences conserved in the control region of ColE2 replicons, which contain a replication initiator protein homologous to that from RepA-PFP replicons, or in any other plasmid outside of the genus Pseudomonas, indicating a diversity of REx-C modules among homologous theta replicons.

View Article: PubMed Central - PubMed

ABSTRACT

Plasmids are a main factor for the evolution of bacteria through horizontal gene exchange, including the dissemination of pathogenicity genes, resistance to antibiotics and degradation of pollutants. Their capacity to duplicate is dependent on their replication determinants (replicon), which also define their bacterial host range and the inability to coexist with related replicons. We characterize a second replicon from the virulence plasmid pPsv48C, from Pseudomonas syringae pv. savastanoi, which appears to be a natural chimera between the gene encoding a newly described replication protein and a putative replication control region present in the widespread family of PFP virulence plasmids. We present extensive evidence of this type of chimerism in structurally similar replicons from species of Pseudomonas, including environmental bacteria as well as plant, animal and human pathogens. We establish that these replicons consist of two functional modules corresponding to putative control (REx-C module) and replication (REx-R module) regions. These modules are functionally separable, do not show specificity for each other, and are dynamically exchanged among replicons of four distinct plasmid families. Only the REx-C module displays strong incompatibility, which is overcome by a few nucleotide changes clustered in a stem-and-loop structure of a putative antisense RNA. Additionally, a REx-C module from pPsv48C conferred replication ability to a non-replicative chromosomal DNA region containing features associated to replicons. Thus, the organization of plasmid replicons as independent and exchangeable functional modules is likely facilitating rapid replicon evolution, fostering their diversification and survival, besides allowing the potential co-option of appropriate genes into novel replicons and the artificial construction of new replicon specificities.

No MeSH data available.