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.
Highlights
Plasmids are extrachromosomal elements that colonize a vast majority of bacteria and other organisms, often carrying genes that confer an adaptive advantage to the host
We could distinguish two well-defined structural regions in this minimal replicating fragment; based on their conservation and functionality, we have defined these regions as plasmid replicon exchangeable (REx) modules: the Rex-C module contains the putative replication control system, whereas the Rex-R module comprises the replication system
Incompatibility mediated by the native replicon from plasmid B and the B-C chimera was evident in plasmid profile gels, where they either cointegrated with p1448A-B or induced its loss, or appeared with an apparently reduced copy number (Figure 5). These results indicate that strong replicon incompatibility between RepA-PFP replicons is associated to the REx-C module and, unlike what happens with ColE2 replicons (Tajima et al, 1988; Yasueda et al, 1994), not to the replication initiator (Rep) protein or the origin of replication and that it can be overcome by only a few nucleotide changes in this module
Summary
Plasmids are extrachromosomal elements that colonize a vast majority of bacteria and other organisms, often carrying genes that confer an adaptive advantage to the host (del Solar et al, 1998; Jackson et al, 2011; Ruiz-Masó et al, 2015). The basic replicon is the fundamental element for plasmid survival, ensuring timely duplication in coordination with cell division (Nordström, 1993; Summers, 1996; del Solar et al, 1998). Plasmid replication is controlled by either directly repeated sequences (iterons) or by antisense RNAs, which can act alone or in coordination with a protein repressing transcription of the rep gene, and is tightly regulated so as to maintain the number of plasmid molecules in the cell within acceptable limits (Summers, 1996; del Solar and Espinosa, 2000). An immediate consequence of this is that plasmids sharing elements for replication or replication control cannot coexist in the same cell and are incompatible (Novick, 1987)
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