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Abstract
Pseudomonas syringae pv. savastanoi NCPPB 3335 contains three virulence plasmids of the
widespread PFP family, and here we sought to investigate the mechanisms involved in their stable inheritance. Plasmid pPsv48C is extremely stable, and we previously showed that it contains two independent functional replicons (repA and repJ) and 29.5% of its sequence occupied by putative mobile elements. Here we show that this plasmid spontaneously suffers the deletion of an 8.3 kb fragment, with a frequency 4 ± 0.3 × 10-3, by recombination between two direct copies of
MITEPsy2. Likewise, we showed that insertion sequence IS801 promotes the occurrence of deletions of pPsv48C by one-ended transposition with an average frequency of 1.8±0.7 × 10-4,
half of them resulting in the loss of a virulence gene. These deletion derivatives were stably maintained in the population by replication mediated by repJ, which is adjacent to IS801. We demonstrated that IS801 also promotes deletions in plasmid pPsv48A, either by recombination or failed transposition. The accumulation of these type of deletions in vivo is prevented by the occurrence in these plasmids of functional postsegregational killing systems, which also increased in two orders of magnitude the stability of pPsv48A. Together, our results indicate that maintenance of pathogenicity genes in bacterial populations of P. syringae results from the combination of diverse antagonistic evolutionary forces that are unrelated and independent of the pathogenicity process.
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Introduction
Plasmids are dispensable extrachromosomal elements widely distributed in bacteria, facilitating their survival and the colonization of eukaryotic hosts (Vivian et al., 2001; Norman et al., 2009; Sundin and Murillo, 2009). Their plasticity and transmissibility contributes to the rapid dissemination of resistance and virulence genes in the environment, thus promoting the emergence of uncontrollable bacterial diseases both in clinical and agricultural settings (Sundin, 2007; Kelly et al., 2009; Carattoli, 2013; Aviv et
al., 2016; Porse et al., 2016). However, plasmids are usually large and exists in several
copies per cell. This can impose a significant metabolic burden to the cell that might facilitate the emergence of plasmid-free derivatives in the absence of selection for plasmid-borne characters (Dahlberg and Chao, 2003; Harrison and Brockhurst, 2012; Romanchuk et al., 2014; Porse et al., 2016). This metabolic cost can be lowered by diverse plasmid-host adaptations, including specific deletions of the plasmid backbone, mutations in the plasmid replication machinery, or chromosomal mutations, among others (Dahlberg and Chao, 2003; Harrison and Brockhurst, 2012; Porse et al., 2016). Additionally, plasmids can increase their stability by conjugal transfer and/or carry an additional battery of determinants specifically dedicated to ensure their stable inheritance and maintenance in the bacterial population, which can be classified into three categories (De Gelder et al., 2007; Sengupta and Austin, 2011). The first category are partition determinants, which direct the active segregation of plasmid molecules during cell division. All low-copy plasmids appear to contain a partition system, which usually consists of an operon of two genes plus a specific DNA sequence for recognition. Multimer resolution systems comprise the second category and include recombinases that, through site-specific recombination, resolve plasmid cointegrates into the corresponding monomers, thus maximizing the number of copies available at cell division. The postsegregational killing systems, in the third category, ensure plasmid maintenance by selective killing of plasmid-free cells and include toxin-antitoxin (TA) systems and, with less prominence, restriction-modification loci.
The Pseudomonas syringae complex is considered the most important bacterial plant pathogen in the world (Mansfield et al., 2012a). Most P. syringae strains contain plasmids carrying a diverse array of adaptive genes, including virulence genes, contributing to increase aggressiveness or expand host range, and genes for resistance to antibacterials and UV light (Jackson et al., 1999; Sundin and Murillo, 1999; Vivian et
al., 2001; Sundin, 2007; Gutiérrez-Barranquero et al., 2017). Most of these plasmids
Plasmid stability determinants
79 PFP replicon; these replicons are highly plastic and adaptable, and strains often contain two or more stably co-existing PFP plasmids (Murillo and Keen, 1994; Stavrinides and Guttman, 2004; Sundin, 2007; Bardaji et al., 2011b). PFP plasmids also often contain several types of insertion sequences, transposons and miniature inverted-repeat transposable elements (MITEs), which can account for up to at least a third of the plasmid, and that participate in the acquisition and exchange of adaptive characters (Alarcón-Chaidez et al., 1999; Rohmer et al., 2003; Stavrinides and Guttman, 2004; Bardaji et al., 2011a; Bardaji et al., 2011b). Insertion sequence IS801 (1.5 kb), and its isoforms, is particularly significant for its relatively high transposition frequency, its common association with virulence genes and its ability to undergo one-ended transposition, whereby the element can mobilize adjacent DNA (Alarcón-Chaidez et al., 1999; Garcillán-Barcia and de la Cruz, 2002; Bardaji et al., 2011a). Additionally, plasmids of P. syringae have a mosaic structure and often share extensive regions of similarity, suggesting their evolution through a multistep process involving the acquisition and loss of large DNA regions (Murillo and Keen, 1994; Sesma et al., 1998; Jackson et al., 1999; Rohmer et al., 2003; Stavrinides and Guttman, 2004; Ma et al., 2007; Bardaji et al., 2011b; O'Brien et al., 2011; Gutiérrez-Barranquero et al., 2017). Despite this, plasmid profiles of individual strains appear to be in general characteristic and stable, although certain plasmids can be lost with high frequency depending on the culture conditions (Murillo et al., 1994; Sundin and Bender, 1994; Sundin et al., 1994; Vivian et al., 2001; Pérez-Martínez et al., 2008). Although diverse potential stability determinants were identified among PFP plasmids (Gibbon et al., 1999; Stavrinides and Guttman, 2004; Sundin et al., 2004; Joardar et al., 2005; Bardaji et al., 2011b; Gutiérrez-Barranquero et
al., 2017), it is not yet clear if they are functional and what is their relative role in the
bacterial life cycle.
P. syringae pv. savastanoi NCPPB 3335 causes aerial tumors in olive (Olea europaea) and is a prominent model for the study of the molecular basis of pathogenicity
on woody hosts (Ramos et al., 2012). This strain contains three native PFP plasmids pPsv48A (80 kb), pPsv48B (45 kb) and pPsv48C (42 kb). Plasmid pPsv48A is essential for full virulence, probably because it carries gene ptz, which is potentially involved in the biosynthesis of cytokinins (Pérez-Martínez et al., 2008; Bardaji et al., 2011b); likewise, pPsv48B carries the effector gene hopAO1, contributing to fitness and virulence in olive plants (Castañeda-Ojeda et al., 2017a). Plasmid pPsv48C also carries a gene potentially involved in cytokinin biosynthesis, gene idi, and was shown to be remarkably stable, perhaps because it carries two different replicons (Bardaji et al., 2017a). We were interested in the identification and characterization of the stability determinants of the
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plasmid complement of strain NCPPB 3335, to gain insights into the mechanisms allowing the coexistence of PFP plasmids and the dynamics of virulence genes, as well as to develop better strategies to obtain plasmid-cured strains. In this work we determined that the stability of plasmids pPsv48A and pPsv48C is modulated by diverse mechanisms. The presence of two replicons in pPsv48C contributes to its very high stability, whereas three TA systems in both plasmids A and C are necessary to avoid the occurrence of high frequency deletions and rearrangements mediated by IS801 isoforms and MITEPsy2. The TA systems also favored the maintenance in pPsv48C of the virulence gene idi, potentially involved in the biosynthesis of cytokinins, while outside the plant. Together, our results indicate that maintenance of pathogenicity genes in bacterial populations of P. syringae results from the combination of diverse antagonistic evolutionary forces that are unrelated and independent of the pathogenicity process.