Functional characterization of copA gene encoding multicopper oxidase in Xanthomonas campestris pv. campestris.

The gram-negative plant pathogenic Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers, a disease causing tremendous loss in agriculture. Copper-containing bactericides have been widely used to control this disease for many years, possibly leading to the development of copper resistance in Xcc. Homologues of copper resistance genes copLAB are present in the Xcc genome, but none has been characterized. In this study, mutations in copL, copA, and copB decreased Xcc copper tolerance. Among them, the copA mutant displayed the most significant reduction. The copA mutant also resulted in a reduction in virulence on the host cabbage. Sequence and mutational analysis demonstrated that copA encodes a multicopper oxidase and that CopA is able to catalyze the oxidation of 2,6-dimethoxyphenol. Alanine substitutions in each of the putative copper binding residues (H538, H583, C584, and H585) of CopA caused a loss of function including copper tolerance and oxidase activity. Furthermore, reporter assays showed that copA transcription is inducible in the presence of copper, subject to catabolite repression, and repressed under conditions of high osmolarity, nitrogen starvation, or oxygen limitation. This is the first time that multicopper oxidase has been characterized in the crucifer pathogen Xcc.