Plasmids of Xylella fastidiosa Mulberry-Infecting Strains Share Extensive Sequence Identity and Gene Complement with pVEIS01 From the Earthworm Symbiont Verminephrobacter Eiseniae

Authors: Stenger, Drake; Lee, Min Woo; Rogers, Elizabeth; Chen, Jianchi

Submitted to: Physiological and Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/15/2010
Publication Date: 6/7/2010
Citation: Stenger, D.C., Lee, M.W., Rogers, E.E., Chen, J. 2010. Plasmids of Xylella fastidiosa Mulberry-Infecting Strains Share Extensive Sequence Identity and Gene Complement with pVEIS01 From the Earthworm Symbiont Verminephrobacter Eiseniae. Physiological and Molecular Plant Pathology. 74:238-245.

Interpretive Summary: Plasmids are small circular DNA molecules that serve as accessory genetic elements of bacteria. Strains of Xylella fastidiosa (Xf) isolated from mulberry trees affected with leaf scorch disease were found to harbor a plasmid closely related to a plasmid of an unrelated bacterium, Verminephrobacter eiseniae, that lives as a symbiont within earthworms. Sequence analyses indicated that the Xf plasmid encoded genes for DNA transfer and plasmid stability. Fragments of the Xf plasmid were cloned into Escherichia coli plasmids and evaluated for the ability to replicate in both E. coli and X. fastidiosa. Collectively, these results indicated that 1) ancestors of two distantly related bacterial species occupying different niches appear to have exchanged genetic material, 2) accessory genetic elements encode genes for DNA transfer that are absent on the Xf chromosome, and 3) a specific fragment of the Xf plasmid contains the origin of DNA replication that may be used to develop a shuttle vector to deliver foreign genes to Xf. This new information facilitates basic understanding of DNA transfer by Xf, a process through which this plant pathogen may acquire genes with potential to affect pathogenicity to horticultural crops and landscape plants.

Technical Abstract: A ~25 kbp plasmid was present in each of four Californian strains of Xylella fastidiosa from mulberry affected with leaf scorch disease. Fragments of each plasmid were cloned into E. coli, sequenced, and assembled into circular contigs of 25,105 bp (pXF-RIV11 and pXF-RIV16) or 24,372 bp (pXF-RIV19 and pXF-RIV25). The four plasmids shared >99.8% sequence identity, excluding a 732 bp insertion common to pXF-RIV11 and pXF-RIV16. BLAST searches identified seven regions (totaling 19,252 bp) sharing >75% nucleotide sequence identity with pVEIS01, a 31 kbp plasmid from the earthworm symbiont Verminephrobacter eiseniae. Of 33 open reading frames (ORFs) encoded by pXF-RIV11, fourteen were associated with DNA transfer (Type IV secretion), four were associated with plasmid stability (plasmid addiction system), one with protein export (Type II secretion), and the remainder associated with other or unknown functions. ORFs associated with DNA transfer were arranged in two main clusters that may represent operons of a Type IV secretion system. Another cluster of three ORFs may represent an operon encoding a plasmid addiction system consisting of a zeta-family toxin, an antitoxin, and a negative regulator of transcription. E. coli plasmids bearing fragments of pXF-RIV11 and the nptII gene as a selectable marker were tested for replication in X. fastidiosa strain Temecula1. The origin of DNA replication was crudely mapped to a 5319 bp region of pXF-RIV11. Collectively, these results indicate that mulberry strains of X. fastidiosa harbor plasmids encoding genes associated with DNA transfer and plasmid stability not previously identified on the chromosome of sequenced X. fastidiosa strains and that ancestors of two distantly related bacterial species occupying different niches appear to have exchanged genetic material.