Molecular characterization of copper resistance genes from Xanthomonas citri subsp. citri and Xanthomonas alfalfae subsp. citrumelonis

Abstract

Copper sprays have been widely used for control of endemic citrus canker caused by Xanthomonas citri subsp. citri in citrus-growing areas for more than 2 decades. Xanthomonas alfalfae subsp. citrumelonis populations were also exposed to frequent sprays of copper for several years as a protective measure against citrus bacterial spot (CBS) in Florida citrus nurseries. Long-term use of these bactericides has led to the development of copper-resistant (Cu(r)) strains in both X. citri subsp. citri and X. alfalfae subsp. citrumelonis, resulting in a reduction of disease control. The objectives of this study were to characterize for the first time the genetics of copper resistance in X. citri subsp. citri and X. alfalfae subsp. citrumelonis and to compare these organisms to other Cu(r) bacteria. Copper resistance determinants from X. citri subsp. citri strain A44(pXccCu2) from Argentina and X. alfalfae subsp. citrumelonis strain 1381(pXacCu2) from Florida were cloned and sequenced. Open reading frames (ORFs) related to the genes copL, copA, copB, copM, copG, copC, copD, and copF were identified in X. citri subsp. citri A44. The same ORFs, except copC and copD, were also present in X. alfalfae subsp. citrumelonis 1381. Transposon mutagenesis of the cloned copper resistance determinants in pXccCu2 revealed that copper resistance in X. citri subsp. citri strain A44 is mostly due to copL, copA, and copB, which are the genes in the cloned cluster with the highest nucleotide homology (≥ 92%) among different Cu(r) bacteria.

Fig. 1.

Copper resistance determinants in Xanthomonas citri subsp. citri (Xcc) strain A44 and Xanthomonas alfalfae subsp. citrumelonis (Xac) strain 1381. ORF numbers are indicated inside the shapes.

Fig. 2.

Comparison of genes involved in copper metabolism. (A) Comparison of different bacterial strains with regard to the composition of the copper resistance gene cluster. (B) Comparison of copper resistance gene clusters with regard to the identity of nucleotide sequences. Areas with the same color indicate conservation of nucleotide sequences among the strains (an identity of ≥92%). The references in the figure refer to reference numbers 17 and 44. (C) The chromosomal genes cohL, cohA, and cohB are homologous to copL, copA, and copB, respectively, which are present in both copper-sensitive and -resistant strains of Xanthomonas. Xcc, Xanthomonas citri subsp. citri; Stm, Stenotrophomonas maltophilia; Xac, Xanthomonas alfalfae subsp. citrumelonis; Xav, Xanthomonas axonopodis pv. vesicatoria.

Fig. 3.

Transposon insertion sites within the copper resistance determinants of pXccCu2 from Xanthomonas citri subsp. citri strain A44. Black triangles indicate sites of transposon insertion.