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

Abstract

Copper-resistant strains of Xanthomonas campestris pv. juglandis occur in walnut orchards throughout northern California. The copper resistance genes from a copper-resistant strain C5 ofX. campestris pv.juglkndis were cloned and located on a 4.9-kb ClaI fragment, which hybridized only to DNA of copper-resistant strains ofX. campestris pv.juglandis, and was part of an approximately 20-kb region which was conserved among such strains of X. campestris pv. juglandis. Hybridization analysis indicated that the copper resistance genes were located on the chromosome. Plasmids conferring copper resistance were not detected in copper-resistant strains, nor did mating with copper-sensitive strains result in copper-resistant transconjugants. Copper resistance genes from X. campestris pv. juglandis shared nucleotide sequence similarity with copper resistance genes from Pseudomonas syringae pv. tomato, P. syringae, and X. campestris pv. vesicatoria. DNA sequence analysis of the 4.9-kb fragment from strain C5 revealed that the sequence had an overall G+C content of 58.7%, and four open reading frames (ORFI to ORF4), oriented in the same direction. All four ORFs were required for full expression of copper resistance, on the basis of Tn3-spice insertional inactivation and deletion analysis. The predicted amino acid sequences of ORF1 to ORF4 showed 65, 45, 47, and 40%o 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 (166 amino acids from the C terminus) of ORF2 and CopB. The hydrophobicity profiles of each pair of predicted polypeptides are similar except for the N terminus of ORF2 and CopB. Four histidine-rich polypeptide regions in ORF1 and CopA strongly resembled the copper-binding motifs of small blue copper proteins and multicopper oxidases, such as fungal laccases, plant ascorbate oxidase, and human ceruloplasmin. Putative copper ligands of the ORFI 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.Walnut blight caused by Xanthomonas campestris pv. juglandis is an important disease of Persian (English) walnuts (Juglans regia L.) in California, Oregon, and other states (4,6,43). Copper-containing bactericides have been used for many decades to reduce the severity of this disease and other plant bacterial diseases (7, 52). However, the efficacy of copper has been reduced by the occurrence of copper-resistant bacterial strains. Plasmid-encoded resistance to copper has been demonstrated for several phytopathogenic bacteria and some other bacteria, including Pseudomonas syringae pv. tomato, a pathogen of tomato (8) non-self-transmissible plasmid approximately 100 kb in size (17). By using low-stringency posthybridization conditions, it was shown (77) that Cur genes in X campestris pv. vesicatoria from Florida, Oklahoma, and California are closely related and that they have similarities to the cop operon of P. syringae pv. tomato.The...

A Multicopper Oxidase Is Required for Copper Resistance in Mycobacterium tuberculosis

“…Multicopper oxidases play a crucial role in copper detoxification in many bacteria, including Escherichia coli (20), Pseudomonas syringae (21), Salmonella enterica (22), and others (23,24). Multicopper oxidases are also required for virulence in Salmonella (22) and Xanthomonas campestris (25).…”

Characterization of a Unique Chromosomal Copper Resistance Gene Cluster from X anthomonas campestris pv. vesicatoria

“…Copper compounds have been used for several decades for the control of bacterial and fungal plant pathogens; however, copper-resistant bacterial pathogens have become prevalent (1,7,8,17,26,29,43) and have reduced the efficacy of copper-based bactericides. Most of the genes associated with copper resistance from plant-pathogenic bacteria (7,9,15,16,41,46) and some other bacteria, including Escherichia coli (44) and Mycobacterium scrofulaceum (20), are plasmid encoded.…”

Role of Plasmids in Microbiology

“…Tomato [49]. There is other similar copper resistance mechanism found in Pseudomonas [50] Xanthomonas [51]. There are reports of other metal resistant bacteria controlled by plasmids such as Cadmium and Chromate and tellurite etc.…”