|Lee, Min Woo|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 25, 2011
Publication Date: February 4, 2011
Citation: Stenger, D.C., Lee, M.W. 2011. Phylogeny of replication initiator protein TrfA reveals a highly divergent clade of incompatibility group P1 plasmids. Applied and Environmental Microbiology. 77:2522-2526. Interpretive Summary: The causal agent of Pierce’s Disease of grapevine, Xylella fastidiosa, exists in nature as a variety of strains that differ in host range, virulence, and genetic complement. Strains of X. fastidiosa isolated from mulberry contain extrachromosomal DNA molecules classified in the incP-1 group of plasmids. The plasmid-encoded gene trfA is required for DNA replication and was used to define evolutionary relationships of the X. fastidiosa incP-1 plasmid pXF-RIV11. Results indicated that pXF-RIV11 is classified within the gamma subgroup of incP-1 plasmids which is shown in this work to be comprised of an unusually diverse array of recently discovered plasmids from an equally diverse range of bacteria isolated from a wide range of habitats. Genetically engineered derivatives of pXF-RIV11 and a related plasmid from the earthworm symbiont Verminephrobacter eiseniae were evaluated for trfA specificity with respect to initiation of plasmid replication. As derivatives of pXF-RIV11 can be reintroduced into X. fastidiosa strains lacking plasmid, these molecules have utility as gene vectors to deliver foreign DNA to X. fastidiosa.
Technical Abstract: Incompatibility group P-1 (incP-1) includes broad host range plasmids of Gram negative bacteria and are classified into five subgroups (alpha, beta, gamma, delta, and epsilon). The incP-1 replication module consists of the trfA gene, encoding the replication initiator protein TrfA, and the origin of replication (oriV). BLAST searches using TrfA of pXF-RIV11 from Xylella fastidiosa as query identified a diverse range of TrfA homologues, many of which were discovered over the past decade in prokaryotic genome projects. Phylogenetic analysis indicated that 18 recently discovered TrfA proteins share a common ancestor with TrfA of the subgroup gamma archetype member (pQKH54) such that diversity of subgroup gamma-(expanded) as defined here is much greater than that of other incP-1 subgroups. Plasmid replication assays demonstrated that the trfA gene from two new members (pVEIS01 from Verminephrobacter eiseniae and pXF-RIV11) of subgroup gamma-(expanded) were functionally interchangeable with respect to plasmid replication initiation using the heterologous oriV.