MOLECULAR-CLONING, CHROMOSOMAL MAPPING, AND SEQUENCE-ANALYSIS OF COPPER RESISTANCE GENES FROM XANTHOMONAS-CAMPESTRIS PV JUGLANDIS - HOMOLOGY WITH SMALL BLUE COPPER PROTEINS AND MULTICOPPER OXIDASE

Copper-resistant strains of Xanthomonas campestris pv. juglandis occur in walnut orchards throughout northern California. The copper resista nce genes from a copper-resistant strain C5 of X. campestris is pv. ju glandis were cloned and located on a 4.9-kb ClaI fragment, which hybri dized only to DNA of copper-resistant strains of X. campestris pv. jug landis, and was part of an approximately 20-kb region which was conser ved among such strains of X. campestris pv. juglandis. Hybridization a nalysis indicated that the copper resistance genes were located on the chromosome. Plasmids conferring copper resistance were not detected i n copper-resistant strains, nor did mating with copper-sensitive strai ns result in copper-resistant transconjugants. Copper resistance genes from X. campestris pv. juglandis shared nucleotide sequence similarit y with copper resistance genes from Pseudomonas syringae pv. tomato, P . syringae, and X. campestris pv. vesicatoria. DNA sequence analysis o f the 4.9-kh fragment from strain C5 revealed that the sequence had an overall G + C content of 58.7%, and four open reading frames (ORF1 to ORF4), oriented in the same direction. All four ORFs were required fo r full expression of copper resistance, on the basis of Tn3-spice inse rtional inactivation and deletion analysis. The predicted amino acid s equences of ORF1 to ORF4 showed 65, 45, 47, and 40% identity with CopA , CopB, CopC, and CopD, respectively, from P. syringae pv. tomato. The most conserved regions are ORF1 and CopA and the C-terminal region (1 66 amino acids from the C terminus) of ORF2 and CopB. The hydrophobici ty profiles of each pair of predicted polypeptides are similar except for the N terminus of ORF2 and CopB. Four histidine-rich polypeptide r egions in ORF1 and CopA strongly resembled the copper-binding motifs o f small blue copper proteins and multicopper oxidases, such as fungal laccases, plant ascorbate oxidase, and human ceruloplasmin. Putative c opper ligands of the ORF1 polypeptide product are proposed, indicating that the polypeptide of ORF1 might bind four copper ions: one type 1, one type 2, and two type 3.