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We characterized the copper resistance genes in strain XvP26 of Xanthomonas campestris pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in Xanthomonas and Pseudomonas strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in Pseudomonas syringae pv. tomato and Escherichia coli, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in E. coli and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in Sinorhizobium meliloti, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant X. campestris pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in X. campestris pv. vesicatoria.

Genes for copper resistance, located on the chromosome of strain XvP26 of Xanthomonas axonopodis pv. vesicatoria, were transferred by conjugation to a recipient strain of the bacterium. The chromosomal gene transfer was verified by analyses of the genomes of donor, recipient, and putative transconjugants for plasmid profiles, by polymorphism of DNA bands obtained by digesting total genomic DNA by a rare-cutting endonuclease and pulsed-field gel electrophoresis, and by Southern hybridization with a probe containing the copper genes. Transfer of kanamycin resistance to a recipient strain, associated with Tn5 insertion into the chromosome of another strain of the bacterial spot pathogen, was also verified. The frequency of kanamycin resistance transfer to recipient was more than 75 times greater in pepper leaves than in vitro. The transfer of chromosomal sequences containing the hypersensitive reaction and pathogenicity (hrp) genes and pigmentation (pig) genes was linked with transfer of kanamycin resistance (Tn5). Horizontal transfer in planta of the chromosomal genes (i.e., cop, pig, hrp, and Tn5 sequences) among strains of X. axonopodis pv. vesicatoria means that horizontal chromosomal gene transfer is possible in nature. This type of gene transfer may explain the presence of great diversity among strains of the bacterial spot pathogen in terms of DNA polymorphism and may also explain the apparent horizontal transfer of hrp sequences among pathovars of Xanthomonas.

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