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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.

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The REx-C modules are functionally exchangeable within and among REx-R families. (A) Schema of the DNA fragments exchanged for the construction of chimeras, which were cloned in vector pKMAG immediately after the transcriptional terminator. X, XmnI restriction site. (B) Autonomous replication in P. syringae pv. syringae B728a of wild type and chimeric DNA regions: +, autonomous replication; -, no replication; nd, not determined. (C) Undigested plasmid profile gels of B728a harboring the different wild type and chimeric clones, with numbers indicating the combination of SaL and rep fragments as in (B); discrete plasmid bands evidence autonomous replication, with multiple bands being different topological forms and/or multimers. Lane (-) is the wild type strain B728a, plasmidless; lane M, size markers, in kb; Clp, chromosome and high-molecular-weight plasmid multimers.
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Figure 4: The REx-C modules are functionally exchangeable within and among REx-R families. (A) Schema of the DNA fragments exchanged for the construction of chimeras, which were cloned in vector pKMAG immediately after the transcriptional terminator. X, XmnI restriction site. (B) Autonomous replication in P. syringae pv. syringae B728a of wild type and chimeric DNA regions: +, autonomous replication; -, no replication; nd, not determined. (C) Undigested plasmid profile gels of B728a harboring the different wild type and chimeric clones, with numbers indicating the combination of SaL and rep fragments as in (B); discrete plasmid bands evidence autonomous replication, with multiple bands being different topological forms and/or multimers. Lane (-) is the wild type strain B728a, plasmidless; lane M, size markers, in kb; Clp, chromosome and high-molecular-weight plasmid multimers.

Mentions: Our comparative analyses of extant sequences (Figure 3) suggest that REx-C modules are freely exchanged among different replicons. However, previous works postulated that compatibility of coexisting RepA-PFP replicons was due to specificity between the C-terminal part of RepA and the loop sequence of SaL 2 (Figures S3, S6), which are highly variable and could coevolve for complementarity (Murillo and Keen, 1994; Gibbon et al., 1999; Stavrinides and Guttman, 2004; Ma et al., 2007). Additionally, the same pattern of variation is seen in a comparison of RepJ replicons (Figure S3). We therefore tested this putative specificity by swapping the respective SaL structures from the REx-C module (SaL fragment in Figure 4) and the rep fragments (partial repI plus REx-R, see Figure 4) from plasmids p1448A-B (RepA-PFP) and pPsv48C (RepA-PFP and RepJ) (Figure 4 and Figure S6). These two RepA-PFP initiator proteins are 88% identical (92% similar; Table S1 and Figure S6), while they are not homologous to RepJ (Table S1). Likewise, the three replicons show a different loop in SaL 2 and 1–3 nt changes within SaL 3 (Figure S6). In spite of the differences in sequence, all the analyzed chimeras were able to sustain autonomous replication in the plasmidless strain P. syringae pv. syringae B728a (Figure 4), indicating a lack of specificity between the SaL structures from the REx-C module and the rest of the replicon.


Plasmid Replicons from Pseudomonas Are Natural Chimeras of Functional, Exchangeable Modules
The REx-C modules are functionally exchangeable within and among REx-R families. (A) Schema of the DNA fragments exchanged for the construction of chimeras, which were cloned in vector pKMAG immediately after the transcriptional terminator. X, XmnI restriction site. (B) Autonomous replication in P. syringae pv. syringae B728a of wild type and chimeric DNA regions: +, autonomous replication; -, no replication; nd, not determined. (C) Undigested plasmid profile gels of B728a harboring the different wild type and chimeric clones, with numbers indicating the combination of SaL and rep fragments as in (B); discrete plasmid bands evidence autonomous replication, with multiple bands being different topological forms and/or multimers. Lane (-) is the wild type strain B728a, plasmidless; lane M, size markers, in kb; Clp, chromosome and high-molecular-weight plasmid multimers.
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Figure 4: The REx-C modules are functionally exchangeable within and among REx-R families. (A) Schema of the DNA fragments exchanged for the construction of chimeras, which were cloned in vector pKMAG immediately after the transcriptional terminator. X, XmnI restriction site. (B) Autonomous replication in P. syringae pv. syringae B728a of wild type and chimeric DNA regions: +, autonomous replication; -, no replication; nd, not determined. (C) Undigested plasmid profile gels of B728a harboring the different wild type and chimeric clones, with numbers indicating the combination of SaL and rep fragments as in (B); discrete plasmid bands evidence autonomous replication, with multiple bands being different topological forms and/or multimers. Lane (-) is the wild type strain B728a, plasmidless; lane M, size markers, in kb; Clp, chromosome and high-molecular-weight plasmid multimers.
Mentions: Our comparative analyses of extant sequences (Figure 3) suggest that REx-C modules are freely exchanged among different replicons. However, previous works postulated that compatibility of coexisting RepA-PFP replicons was due to specificity between the C-terminal part of RepA and the loop sequence of SaL 2 (Figures S3, S6), which are highly variable and could coevolve for complementarity (Murillo and Keen, 1994; Gibbon et al., 1999; Stavrinides and Guttman, 2004; Ma et al., 2007). Additionally, the same pattern of variation is seen in a comparison of RepJ replicons (Figure S3). We therefore tested this putative specificity by swapping the respective SaL structures from the REx-C module (SaL fragment in Figure 4) and the rep fragments (partial repI plus REx-R, see Figure 4) from plasmids p1448A-B (RepA-PFP) and pPsv48C (RepA-PFP and RepJ) (Figure 4 and Figure S6). These two RepA-PFP initiator proteins are 88% identical (92% similar; Table S1 and Figure S6), while they are not homologous to RepJ (Table S1). Likewise, the three replicons show a different loop in SaL 2 and 1–3 nt changes within SaL 3 (Figure S6). In spite of the differences in sequence, all the analyzed chimeras were able to sustain autonomous replication in the plasmidless strain P. syringae pv. syringae B728a (Figure 4), indicating a lack of specificity between the SaL structures from the REx-C module and the rest of the replicon.

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.


Related in: MedlinePlus