Molecular Characterization of Copper Resistance Genes from <i>Xanthomonas arboricola</i> pv. <i>pruni</i>

<i>Xanthomonas arboricola</i> pv. <i>pruni</i> (XAP) causes bacterial spot in <i>Prunus</i>, 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 <i>Prunus laurocerasus</i> 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 <i>copL</i>, <i>copA</i>, <i>copB</i>, <i>copC</i>, <i>copD</i>, <i>copM</i>, <i>copG</i>, <i>copF</i>, <i>cusA</i>, and <i>cusB</i>. 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 <i>copL</i> from <i>copL-D</i> decreased the resistance level to 300 µg/ml of CSP. The <i>copF</i> and <i>cusAB</i> genes alone did not confer copper resistance; however, adding <i>copF-cusB</i> to <i>copL-D</i> 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 <i>Xanthomonas perforans</i> 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.