The management of bacterial spot of tomato is challenged by the widespread occurrence of copper resistance among its causal pathogens. In Xanthomonas euvesicatoria pv. perforans , a major causal agent, copper resistance is conferred by cop genes organized in an operon located on either the chromosome or plasmid. In the United States, copper resistance is predominantly chromosomally encoded. Plasmid-borne resistance is limited to a few phylogenetic groups but is prevalent in other bacterial spot pathogens. We hypothesized that plasmid-encoded copper resistance genes exhibit higher expression than chromosomally encoded genes but may be unstable in the absence of selective pressure for resistance. To test this hypothesis, we examined how the chromosomal versus plasmid-borne cop operon influences gene copy number and expression, as well as plasmid stability. Relative to strains with only chromosomal resistance, strains carrying plasmid-encoded resistance exhibited an increased copy number of copB , a key gene in the operon, while strains carrying both chromosomal and plasmid-encoded resistance showed an even greater increase in gene copy number. Correspondingly, copB expression was elevated in both groups. During in vitro growth without copper, partial or complete plasmid loss occurred in a subset of strains, and genome sequencing of colonies with reduced plasmid size revealed transposase-mediated plasmid rearrangements. These findings demonstrate that plasmid-borne copper resistance increases gene copy number and expression of copper resistance genes but can be unstable without copper selection in some strain backgrounds, highlighting the role of mobile genetic elements in shaping Xanthomonas euvesicatoria pv. perforans adaptation to changing copper environments. IMPORTANCE: Plasmids are key players in the dissemination of antimicrobial resistance. Although copper resistance is widespread among Xanthomonas euvesicatoria pv. perforans strains, copper-based bactericides remain at the forefront of disease management. We previously demonstrated that distinct genetic elements, including plasmid- and chromosomally encoded genomic islands, contribute to the acquisition of copper resistance and the occurrence of multicopy resistance in X. euvesicatoria pv. perforans . Here, we show that the localization of copper resistance genes on a plasmid, chromosome, or both influences gene copy number and expression of copper resistance genes. Furthermore, rearrangements between co-resident plasmids affect plasmid stability, leading to the loss of copper resistance in X. euvesicatoria pv. perforans . These findings highlight how plasmid dynamics can shape bacterial evolvability and adaptation to selective environments.

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