Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/1/2010
Publication Date: 10/8/2010
Publication URL: http://dx.doi.org/10.1128/AEM.01921-10
Citation: Lee, M.W., Stenger, D.C., Rogers, E.E. 2010. Functional characterization of replication and stability factors of an incP-1 plasmid from Xylella fastidiosa. Applied and Environmental Microbiology. 76: 7734-7740. Interpretive Summary: Xylella fastidiosa is the bacterial pathogen responsible for Pierce’s disease of grapevines. Certain strains of X. fastidiosa harbors plasmids that serve as accessory DNA molecules encoding genes not present on the bacterial chromosome and which may be exchanged among strains. Plasmid-encoded factors required for replication and stable maintenance of the X. fastidiosa plasmid pXF-RIV11 were identified and characterized. Plasmid replication in X. fastidiosa required only a small region encoding a replication initiator gene and flanking DNA sequences constituting the origin of replication. However, long term stable inheritance of the plasmid in X. fastidiosa required addition of a plasmid addiction system (also derived from pXF-RIV11) encoding a toxin and cognate anti-toxin. A shuttle vector was developed using the replication and stability factors of pXF-RIV11. Features of the shuttle vector include ability to replicate in both X. fastidiosa and Escherichia coli, a selectable marker, and ten unique sites for insertion of foreign DNA. As the shuttle vector is stable in the absence of antibiotic selection, this plasmid has utility as a basic research tool for mutant complementation assays conducted in plants infected with X. fastidiosa. Restoration of pathogencity and/or virulence of targeted X. fastidiosa mutants via plasmid complementation facilitates basic understanding of X. fastidiosa genes required for infection of plants and, therefore, expedites identification of key processes in X. fastidiosa that may be targeted for disruption in efforts to obtain disease resistance and/or amelioration of disease in infected plants of commercial value.
Technical Abstract: Xylella fastidiosa strain riv11 harbors a ~25 kbp plasmid (pXF-RIV11) belonging to the incP-1 incompatibility group. Replication and stability factors of pXF-RIV11 were identified and used to construct plasmids able to propagate in both X. fastidiosa and Escherichia coli. Replication in X. fastidiosa required a ~1.4 kbp region from pXF-RIV11 containing a replication initiation gene (trfA) and the adjacent origin of DNA replication (oriV). Constructs containing trfA and oriV from pVEIS01, a related incP-1 plasmid of the earthworm symbiont Verminephropbacter eiseniae, also were competent for replication in X. fastidiosa. Inc-P-1 replicons derived from pXF-RIV11 but not pVEIS01 replicated in Agrobacterium tumefaciens, Xanthomonas campestris, and Pseudomonas syringae. Although incP-1 derived replicons could be maintained in X. fastidiosa under antibiotic selection, removal of selection resulted in loss of the plasmids. A toxin/anti-toxin (pemI/pemK) addiction system of pXF-RIV11 improved stability of incP-1 replicons in X. fastidiosa in the absence of antibiotic selection. Over-expression of PemK in E. coli was toxic for cell growth but toxicity was nullified by co-expression of PemI anti-toxin. Deletion of N-terminal sequences of PemK containing the conserved toxin motif RGD abolished toxicity. In vitro assays revealed a direct interaction of PemI with PemK, suggesting that anti-toxin activity of PemI is mediated by toxin sequestration. IncP-1 plasmid replication and stability factors were added to a standard E. coli cloning vector to constitute a stable ~6 kbp shuttle vector(pXF20-PEMIK) bearing 10 unique endonuclease restriction sites suitable for cloning and delivery of foreign DNA to X. fastidiosa.