Evaluation of the Stability of Synthetic Plasmids in Xanthomonas euvesicatoria Cells

The aim of the present study was to investigate the possibility of obtaining stably transformed cultures of a wild strain of Xanthomonas euvesicatoria (269p). The stability of two commercial synthetic plasmids, pBbB13k-GFP and pWVR5, and one recombinant plasmid constructed in this study (pWG5) was evaluated in the cell of this phytopathogen. Plasmid pWG5 was obtained after ligase-independent assembly of ampli¬fied regions of pBbB13k-GFP and pWVR5. The transformations were carried out via electroporation and heat shock. PCR and RFLP were used for plasmid and species identification. Primer pairs were designed for amplification of genes encoding GFP and gentamicin resistance and for obtaining amplified parts of both commercial plasmids. Transformation by electroporation of strain 269p with pBbB13k – GFP failed. Positive transformation was achieved with pWVR5 but low plasmid stability and loss of ability to grow on antibiotic media was observed. The recombinant plasmid pWG5 was able to transform the wild strain. However, a rapid loss of the gentamicin resistance trait was observed after several passages, indicating that the obtained transformants were unstable. The transformation of E. coli with pWG5 was also successful. The genetic determinants for GFP and gentamicin resistance were detectable in the broth-enriched trans¬formed culture of E. coli, but an outgrowth of spontaneously resistant mutants on agar media prevented the isolation of true transformants. The overall results showed that X. euvesicatoria 269p cannot maintain the studied plasmids. Further finetuning of the synthetic plasmid features (origin of replication, selective marker, and reporter genes) is needed.