Construction of a shuttle vector and transformation of Xylella fastidiosa with plasmid DNA

We have isolated, cloned, and sequenced a 5823-bp cryptic plasmid from a st rain of;Xylella fastidiosa. This plasmid encodes five open reading frames ( ORF) greater than 400 nucleotides each. ORF 2 encodes a protein with 37% am ino acid identity to the replication initiator protein of plasmid pECB2 fro m Pseudomonas alcaligenes. This RepA protein from X. fastidiosa contains bo th a leucine zipper and helix turn helix motif characteristic of proteins i nvolved in DNA replication. The sequence 5' of ORF 2 has all of the feature s characteristic of plasmid origins of replication as well as regulatory el ements required for transcription of ORF 2. Open reading frame 2, along wit h the upstream origin of replication, was cloned as an EcoRI fragment into pUC19 to create a shuttle vector. This construct was introduced into Xyllel la fastidiosa by electroporation, with selection for carbenicillin resistan ce. Transformation was verified by both PCR and Southern hybridization expe riments. Frequency of transformation was low, but increased ten-fold when t he plasmid was grown in X. fastidiosa rather than Escherichia coli prior to transformation. This work represents the first step towards the developmen t of a system for genetic analysis of this important plant pathogen of citr us, grapevines, and other horticultural crops.