This study was initiated to characterize a small Xylella fastidiosa (X. fastidiosa) plasmid and attempt to create a X. fastidosal Escherichia coli shuttle vector that was stable in planta. Restriction enzyme analysis of a 1.3 kb plasmid DNA from a grape-infecting strain of X. fastidiosa (UCLA) revealed the presence of three similar, but genetically distinct, plasmids, pUCLAs. Evidence that suggests the pUCLA plasmids replicate via a rolling-circle (RC) mechanism include: (i) the presence of ssDNA in X. fastidiosa cells; (ii) the presence of conserved motifs in the predicted ORF1 that are typical of initiator (Rep) proteins associated with RC replication; (iii) high amino acid identity between the putative Rep proteins of pUCLAs and Pf3, a filamentous bacteriophage of Pseudomonas aeruginosa that replicates by a RC mechanism; and (iv) the presence of a putative origin of replication upstream of ORF1 that has the potential to form secondary hairpin structures. One DNA motif present in pUCLA shared sequence similarity to known nicking sites in the origins of replication of other RC plasmids and phages. The shuttle vector, pXF001, successfully transformed grape X. fastidiosa strains and was found to be present as autonomous, structurally unchanged DNA molecules in X. fastidiosa. However, pXF001 was not stably maintained in X. fastidiosa without antibiotic selection. (c) 2005 Elsevier Inc. All rights reserved.