Molecular Characterization of Copper Resistance Genes from Xanthomonas arboricola pv. pruni

Xanthomonas arboricola pv. pruni (XAP) causes bacterial spot in Prunus, and copper sprays have been widely used to manage this disease. Copper tolerance (≥150 µg/ml of copper sulfate pentahydrate [CSP]) is commonly found in XAP populations, but copper resistance (>200 µg/ml of CSP) has not been previously reported. This study reports and characterizes the first copper-resistant strain of XAP (XAPCuR), which was isolated from diseased leaves of Prunus laurocerasus in North Carolina in 2017. Whole-genome sequence analysis of XAPCuR revealed an approximately 247-kb plasmid carrying a duplicated 17-kb cluster containing copper resistance candidate genes copL, copA, copB, copC, copD, copM, copG, copF, cusA, and cusB. The two copies of the copper resistance cluster did not increase the level of copper resistance compared with a single copy, but deletion of both copies led to the loss of resistance. Functional analysis of the cluster revealed that copL-D is the major contributor to copper resistance, allowing XAP to grow on nutrient agar containing up to 750 µg/ml of CSP. Removing copL from copL-D decreased the resistance level to 300 µg/ml of CSP. The copF and cusAB genes alone did not confer copper resistance; however, adding copF-cusB to copL-D increased the resistance level of XAP to 1,000 µg/ml of CSP. The resistance genotype and phenotype were able to be transferred from XAP to Xanthomonas perforans via conjugation. This plasmid has up to 99% identity to other copper resistance plasmids of closely related xanthomonads, indicating that horizontal transfer is driving its spread.